research paper about body

How to Write a Body of a Research Paper

The main part of your research paper is called “the body.” To write this important part of your paper, include only relevant information, or information that gets to the point. Organize your ideas in a logical order—one that makes sense—and provide enough details—facts and examples—to support the points you want to make.

Logical Order

Transition words and phrases, adding evidence, phrases for supporting topic sentences.

• Transition Phrases for Comparisons
• Transition Phrases for Contrast
• Transition Phrases to Show a Process
• Phrases to Introduce Examples
• Transition Phrases for Presenting Evidence

How to Make Effective Transitions

Examples of effective transitions, drafting your conclusion, writing the body paragraphs.

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• The third and fourth paragraphs follow the same format as the second:
• Transition or topic sentence.
• Topic sentence (if not included in the first sentence).
• Supporting sentences including a discussion, quotations, or examples that support the topic sentence.
• Concluding sentence that transitions to the next paragraph.

The topic of each paragraph will be supported by the evidence you itemized in your outline. However, just as smooth transitions are required to connect your paragraphs, the sentences you write to present your evidence should possess transition words that connect ideas, focus attention on relevant information, and continue your discussion in a smooth and fluid manner.

You presented the main idea of your paper in the thesis statement. In the body, every single paragraph must support that main idea. If any paragraph in your paper does not, in some way, back up the main idea expressed in your thesis statement, it is not relevant, which means it doesn’t have a purpose and shouldn’t be there.

Each paragraph also has a main idea of its own. That main idea is stated in a topic sentence, either at the beginning or somewhere else in the paragraph. Just as every paragraph in your paper supports your thesis statement, every sentence in each paragraph supports the main idea of that paragraph by providing facts or examples that back up that main idea. If a sentence does not support the main idea of the paragraph, it is not relevant and should be left out.

A paper that makes claims or states ideas without backing them up with facts or clarifying them with examples won’t mean much to readers. Make sure you provide enough supporting details for all your ideas. And remember that a paragraph can’t contain just one sentence. A paragraph needs at least two or more sentences to be complete. If a paragraph has only one or two sentences, you probably haven’t provided enough support for your main idea. Or, if you have trouble finding the main idea, maybe you don’t have one. In that case, you can make the sentences part of another paragraph or leave them out.

Arrange the paragraphs in the body of your paper in an order that makes sense, so that each main idea follows logically from the previous one. Likewise, arrange the sentences in each paragraph in a logical order.

If you carefully organized your notes and made your outline, your ideas will fall into place naturally as you write your draft. The main ideas, which are building blocks of each section or each paragraph in your paper, come from the Roman-numeral headings in your outline. The supporting details under each of those main ideas come from the capital-letter headings. In a shorter paper, the capital-letter headings may become sentences that include supporting details, which come from the Arabic numerals in your outline. In a longer paper, the capital letter headings may become paragraphs of their own, which contain sentences with the supporting details, which come from the Arabic numerals in your outline.

In addition to keeping your ideas in logical order, transitions are another way to guide readers from one idea to another. Transition words and phrases are important when you are suggesting or pointing out similarities between ideas, themes, opinions, or a set of facts. As with any perfect phrase, transition words within paragraphs should not be used gratuitously. Their meaning must conform to what you are trying to point out, as shown in the examples below:

• “Accordingly” or “in accordance with” indicates agreement. For example :Thomas Edison’s experiments with electricity accordingly followed the theories of Benjamin Franklin, J. B. Priestly, and other pioneers of the previous century.
• “Analogous” or “analogously” contrasts different things or ideas that perform similar functions or make similar expressions. For example: A computer hard drive is analogous to a filing cabinet. Each stores important documents and data.
• “By comparison” or “comparatively”points out differences between things that otherwise are similar. For example: Roses require an alkaline soil. Azaleas, by comparison, prefer an acidic soil.
• “Corresponds to” or “correspondingly” indicates agreement or conformity. For example: The U.S. Constitution corresponds to England’s Magna Carta in so far as both established a framework for a parliamentary system.
• “Equals,”“equal to,” or “equally” indicates the same degree or quality. For example:Vitamin C is equally as important as minerals in a well-balanced diet.
• “Equivalent” or “equivalently” indicates two ideas or things of approximately the same importance, size, or volume. For example:The notions of individual liberty and the right to a fair and speedy trial hold equivalent importance in the American legal system.
• “Common” or “in common with” indicates similar traits or qualities. For example: Darwin did not argue that humans were descended from the apes. Instead, he maintained that they shared a common ancestor.
• “In the same way,”“in the same manner,”“in the same vein,” or “likewise,” connects comparable traits, ideas, patterns, or activities. For example: John Roebling’s suspension bridges in Brooklyn and Cincinnati were built in the same manner, with strong cables to support a metallic roadway. Example 2: Despite its delicate appearance, John Roebling’s Brooklyn Bridge was built as a suspension bridge supported by strong cables. Example 3: Cincinnati’s Suspension Bridge, which Roebling also designed, was likewise supported by cables.
• “Kindred” indicates that two ideas or things are related by quality or character. For example: Artists Vincent Van Gogh and Paul Gauguin are considered kindred spirits in the Impressionist Movement. “Like” or “as” are used to create a simile that builds reader understanding by comparing two dissimilar things. (Never use “like” as slang, as in: John Roebling was like a bridge designer.) For examples: Her eyes shone like the sun. Her eyes were as bright as the sun.
• “Parallel” describes events, things, or ideas that occurred at the same time or that follow similar logic or patterns of behavior. For example:The original Ocktoberfests were held to occur in parallel with the autumn harvest.
• “Obviously” emphasizes a point that should be clear from the discussion. For example: Obviously, raccoons and other wildlife will attempt to find food and shelter in suburban areas as their woodland habitats disappear.
• “Similar” and “similarly” are used to make like comparisons. For example: Horses and ponies have similar physical characteristics although, as working farm animals, each was bred to perform different functions.
• “There is little debate” or “there is consensus” can be used to point out agreement. For example:There is little debate that the polar ice caps are melting.The question is whether global warming results from natural or human-made causes.

Other phrases that can be used to make transitions or connect ideas within paragraphs include:

• Use “alternately” or “alternatively” to suggest a different option.
• Use “antithesis” to indicate a direct opposite.
• Use “contradict” to indicate disagreement.
• Use “on the contrary” or “conversely” to indicate that something is different from what it seems.
• Use “dissimilar” to point out differences between two things.
• Use “diverse” to discuss differences among many things or people.
• Use “distinct” or “distinctly” to point out unique qualities.
• Use “inversely” to indicate an opposite idea.
• Use “it is debatable,” “there is debate,” or “there is disagreement” to suggest that there is more than one opinion about a subject.
• Use “rather” or “rather than” to point out an exception.
• Use “unique” or “uniquely” to indicate qualities that can be found nowhere else.
• Use “unlike” to indicate dissimilarities.
• Use “various” to indicate more than one kind.

Writing Topic Sentences

Remember, a sentence should express a complete thought, one thought per sentence—no more, no less. The longer and more convoluted your sentences become, the more likely you are to muddle the meaning, become repetitive, and bog yourself down in issues of grammar and construction. In your first draft, it is generally a good idea to keep those sentences relatively short and to the point. That way your ideas will be clearly stated.You will be able to clearly see the content that you have put down—what is there and what is missing—and add or subtract material as it is needed. The sentences will probably seem choppy and even simplistic.The purpose of a first draft is to ensure that you have recorded all the content you will need to make a convincing argument. You will work on smoothing and perfecting the language in subsequent drafts.

Transitioning from your topic sentence to the evidence that supports it can be problematic. It requires a transition, much like the transitions needed to move from one paragraph to the next. Choose phrases that connect the evidence directly to your topic sentence.

• Consider this: (give an example or state evidence).
• If (identify one condition or event) then (identify the condition or event that will follow).
• It should go without saying that (point out an obvious condition).
• Note that (provide an example or observation).
• Take a look at (identify a condition; follow with an explanation of why you think it is important to the discussion).
• The authors had (identify their idea) in mind when they wrote “(use a quotation from their text that illustrates the idea).”
• The point is that (summarize the conclusion your reader should draw from your research).
• This becomes evident when (name the author) says that (paraphrase a quote from the author’s writing).
• We see this in the following example: (provide an example of your own).
• (The author’s name) offers the example of (summarize an example given by the author).

If an idea is controversial, you may need to add extra evidence to your paragraphs to persuade your reader. You may also find that a logical argument, one based solely on your evidence, is not persuasive enough and that you need to appeal to the reader’s emotions. Look for ways to incorporate your research without detracting from your argument.

Writing Transition Sentences

It is often difficult to write transitions that carry a reader clearly and logically on to the next paragraph (and the next topic) in an essay. Because you are moving from one topic to another, it is easy to simply stop one and start another. Great research papers, however, include good transitions that link the ideas in an interesting discussion so that readers can move smoothly and easily through your presentation. Close each of your paragraphs with an interesting transition sentence that introduces the topic coming up in the next paragraph.

Transition sentences should show a relationship between the two topics.Your transition will perform one of the following functions to introduce the new idea:

• Indicate that you will be expanding on information in a different way in the upcoming paragraph.
• Indicate that a comparison, contrast, or a cause-and-effect relationship between the topics will be discussed.
• Indicate that an example will be presented in the next paragraph.
• Indicate that a conclusion is coming up.

Transitions make a paper flow smoothly by showing readers how ideas and facts follow one another to point logically to a conclusion. They show relationships among the ideas, help the reader to understand, and, in a persuasive paper, lead the reader to the writer’s conclusion.

Each paragraph should end with a transition sentence to conclude the discussion of the topic in the paragraph and gently introduce the reader to the topic that will be raised in the next paragraph. However, transitions also occur within paragraphs—from sentence to sentence—to add evidence, provide examples, or introduce a quotation.

The type of paper you are writing and the kinds of topics you are introducing will determine what type of transitional phrase you should use. Some useful phrases for transitions appear below. They are grouped according to the function they normally play in a paper. Transitions, however, are not simply phrases that are dropped into sentences. They are constructed to highlight meaning. Choose transitions that are appropriate to your topic and what you want the reader to do. Edit them to be sure they fit properly within the sentence to enhance the reader’s understanding.

Transition Phrases for Comparisons:

• We also see
• In addition to
• Notice that
• Beside that,
• In comparison,
• Once again,
• Identically,
• For example,
• Comparatively, it can be seen that
• We see this when
• This corresponds to
• In other words,
• At the same time,
• By the same token,

Transition Phrases for Contrast:

• By contrast,
• On the contrary,
• Nevertheless,
• An exception to this would be …
• Alongside that,we find …
• On one hand … on the other hand …
• [New information] presents an opposite view …
• Conversely, it could be argued …
• Other than that,we find that …
• We get an entirely different impression from …
• One point of differentiation is …
• Further investigation shows …
• An exception can be found in the fact that …

Transition Phrases to Show a Process:

• At the top we have … Near the bottom we have …
• Here we have … There we have …
• Continuing on,
• We progress to …
• Close up … In the distance …
• With this in mind,
• Moving in sequence,
• Proceeding sequentially,
• Moving to the next step,
• First, Second,Third,…
• Examining the activities in sequence,
• Sequentially,
• As a result,
• The end result is …
• To illustrate …
• Subsequently,
• One consequence of …
• If … then …
• It follows that …
• This is chiefly due to …
• The next step …
• Later we find …

Phrases to Introduce Examples:

• For instance,
• Particularly,
• In particular,
• This includes,
• Specifically,
• To illustrate,
• One illustration is
• One example is
• This is illustrated by
• This can be seen when
• This is especially seen in
• This is chiefly seen when

Transition Phrases for Presenting Evidence:

• Another point worthy of consideration is
• At the center of the issue is the notion that
• Before moving on, it should be pointed out that
• Another important point is
• Another idea worth considering is
• Consequently,
• Especially,
• Even more important,
• Getting beyond the obvious,
• In spite of all this,
• It follows that
• It is clear that
• More importantly,
• Most importantly,

How to make effective transitions between sections of a research paper? There are two distinct issues in making strong transitions:

• Does the upcoming section actually belong where you have placed it?
• Have you adequately signaled the reader why you are taking this next step?

The first is the most important: Does the upcoming section actually belong in the next spot? The sections in your research paper need to add up to your big point (or thesis statement) in a sensible progression. One way of putting that is, “Does the architecture of your paper correspond to the argument you are making?” Getting this architecture right is the goal of “large-scale editing,” which focuses on the order of the sections, their relationship to each other, and ultimately their correspondence to your thesis argument.

It’s easy to craft graceful transitions when the sections are laid out in the right order. When they’re not, the transitions are bound to be rough. This difficulty, if you encounter it, is actually a valuable warning. It tells you that something is wrong and you need to change it. If the transitions are awkward and difficult to write, warning bells should ring. Something is wrong with the research paper’s overall structure.

After you’ve placed the sections in the right order, you still need to tell the reader when he is changing sections and briefly explain why. That’s an important part of line-by-line editing, which focuses on writing effective sentences and paragraphs.

Effective transition sentences and paragraphs often glance forward or backward, signaling that you are switching sections. Take this example from J. M. Roberts’s History of Europe . He is finishing a discussion of the Punic Wars between Rome and its great rival, Carthage. The last of these wars, he says, broke out in 149 B.C. and “ended with so complete a defeat for the Carthaginians that their city was destroyed . . . .” Now he turns to a new section on “Empire.” Here is the first sentence: “By then a Roman empire was in being in fact if not in name.”(J. M. Roberts, A History of Europe . London: Allen Lane, 1997, p. 48) Roberts signals the transition with just two words: “By then.” He is referring to the date (149 B.C.) given near the end of the previous section. Simple and smooth.

Michael Mandelbaum also accomplishes this transition between sections effortlessly, without bringing his narrative to a halt. In The Ideas That Conquered the World: Peace, Democracy, and Free Markets , one chapter shows how countries of the North Atlantic region invented the idea of peace and made it a reality among themselves. Here is his transition from one section of that chapter discussing “the idea of warlessness” to another section dealing with the history of that idea in Europe.

The widespread aversion to war within the countries of the Western core formed the foundation for common security, which in turn expressed the spirit of warlessness. To be sure, the rise of common security in Europe did not abolish war in other parts of the world and could not guarantee its permanent abolition even on the European continent. Neither, however, was it a flukish, transient product . . . . The European common security order did have historical precedents, and its principal features began to appear in other parts of the world. Precedents for Common Security The security arrangements in Europe at the dawn of the twenty-first century incorporated features of three different periods of the modern age: the nineteenth century, the interwar period, and the ColdWar. (Michael Mandelbaum, The Ideas That Conquered the World: Peace, Democracy, and Free Markets . New York: Public Affairs, 2002, p. 128)

It’s easier to make smooth transitions when neighboring sections deal with closely related subjects, as Mandelbaum’s do. Sometimes, however, you need to end one section with greater finality so you can switch to a different topic. The best way to do that is with a few summary comments at the end of the section. Your readers will understand you are drawing this topic to a close, and they won’t be blindsided by your shift to a new topic in the next section.

Here’s an example from economic historian Joel Mokyr’s book The Lever of Riches: Technological Creativity and Economic Progress . Mokyr is completing a section on social values in early industrial societies. The next section deals with a quite different aspect of technological progress: the role of property rights and institutions. So Mokyr needs to take the reader across a more abrupt change than Mandelbaum did. Mokyr does that in two ways. First, he summarizes his findings on social values, letting the reader know the section is ending. Then he says the impact of values is complicated, a point he illustrates in the final sentences, while the impact of property rights and institutions seems to be more straightforward. So he begins the new section with a nod to the old one, noting the contrast.

In commerce, war and politics, what was functional was often preferred [within Europe] to what was aesthetic or moral, and when it was not, natural selection saw to it that such pragmatism was never entirely absent in any society. . . . The contempt in which physical labor, commerce, and other economic activity were held did not disappear rapidly; much of European social history can be interpreted as a struggle between wealth and other values for a higher step in the hierarchy. The French concepts of bourgeois gentilhomme and nouveau riche still convey some contempt for people who joined the upper classes through economic success. Even in the nineteenth century, the accumulation of wealth was viewed as an admission ticket to social respectability to be abandoned as soon as a secure membership in the upper classes had been achieved. Institutions and Property Rights The institutional background of technological progress seems, on the surface, more straightforward. (Joel Mokyr, The Lever of Riches: Technological Creativity and Economic Progress . New York: Oxford University Press, 1990, p. 176)

Note the phrase, “on the surface.” Mokyr is hinting at his next point, that surface appearances are deceiving in this case. Good transitions between sections of your research paper depend on:

• Getting the sections in the right order
• Moving smoothly from one section to the next
• Signaling readers that they are taking the next step in your argument
• Explaining why this next step comes where it does

Every good paper ends with a strong concluding paragraph. To write a good conclusion, sum up the main points in your paper. To write an even better conclusion, include a sentence or two that helps the reader answer the question, “So what?” or “Why does all this matter?” If you choose to include one or more “So What?” sentences, remember that you still need to support any point you make with facts or examples. Remember, too, that this is not the place to introduce new ideas from “out of the blue.” Make sure that everything you write in your conclusion refers to what you’ve already written in the body of your paper.

Back to How To Write A Research Paper .

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• A Research Guide
• Research Paper Guide

Research Paper Body Paragraph Structure

Introduction.

• Referrences
• Ways to start paragraph
• Step by step guide
• Research paragraph examples

Learning the basics of a paragraph structure

• Title (cover page).
• Introduction.
• Literature review.
• Research methodology.
• Data analysis.
• Conclusion.
• Reference page.

5 winning ways to start a body paragraph

• Topic Sentence : it should provide a clear focus and introduce the specific aspect you will discuss. For example, “One key factor influencing climate change is…”.
• Opening Statement: grab your readers’ attention with a thought-provoking or surprising statement related to your topic. For instance, “The alarming increase in global temperatures has reached a critical point, demanding immediate action.”
• Quotation: find a relevant quote from a reputable source. It won’t only add credibility to your research but will also engage the reader right from the start.
• Anecdote or example: start your academic paragraph with a funny story or a real-world example that illustrates the significance of your research topic.
• Background information : provide a brief background or context for the topic you are about to discuss. For example, “In recent years, the prevalence of cyber-attacks has skyrocketed, posing a severe threat to individuals, organizations, and even national security.”

A step-by-step guide to starting a concise body paragraph

Step 1: introduce the main point or argument., step 2: provide evidence or examples., step 3: explain and analyze., step 4: connect to the main argument., step 5: review and revise., flawless body paragraph example: how does it look.

• Topic Sentence: Rising global temperatures have significant implications for ecosystems and biodiversity.
• Evidence/Example 1: According to a study by the Intergovernmental Panel on Climate Change (IPCC), global average temperatures have increased by 1.1 degrees Celsius since pre-industrial times (IPCC, 2021). This temperature rise has led to melting polar ice caps and glaciers, rising sea levels, and coastal erosion (Smith et al., 2019).
• Explanation/Analysis 1: The significant increase in global temperatures has caused observable changes in the Earth’s physical environment. The melting of polar ice caps not only contributes to the rise in sea levels but also disrupts marine ecosystems.
• Evidence/Example 2: In addition to the loss of coastal habitats, higher temperatures have also resulted in shifts in the geographical distribution of species. Research by Parmesan and Yohe (2019) indicates that many plant and animal species have altered their ranges and migration patterns in response to changing climate conditions.
• Explanation/Analysis 2: The observed shifts in species distribution highlight the vulnerability of ecosystems to climate change. As temperature zone modification, species that cannot adapt or migrate to suitable habitats may face reduced reproductive success and increased risk of extinction.
• Connect to the main argument: These examples demonstrate that the rising global temperatures associated with climate change have profound implications for ecosystems and biodiversity.

The bottom line

• Writing a Research Paper
• Research Paper Title
• Research Paper Sources
• Research Paper Problem Statement
• Research Paper Thesis Statement
• Hypothesis for a Research Paper
• Research Question
• Research Paper Outline
• Research Paper Summary
• Research Paper Prospectus
• Research Paper Proposal
• Research Paper Format
• Research Paper Styles
• AMA Style Research Paper
• MLA Style Research Paper
• Chicago Style Research Paper
• APA Style Research Paper
• Research Paper Structure
• Research Paper Cover Page
• Research Paper Abstract
• Research Paper Introduction
• Research Paper Body Paragraph
• Research Paper Literature Review
• Research Paper Background
• Research Paper Methods Section
• Research Paper Results Section
• Research Paper Discussion Section
• Research Paper Conclusion
• Research Paper Appendix
• Research Paper Bibliography
• APA Reference Page
• Annotated Bibliography
• Bibliography vs Works Cited vs References Page
• Research Paper Types
• What is Qualitative Research

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Body Paragraphs

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This resource outlines the generally accepted structure for introductions, body paragraphs, and conclusions in an academic argument paper. Keep in mind that this resource contains guidelines and not strict rules about organization. Your structure needs to be flexible enough to meet the requirements of your purpose and audience.

Body paragraphs: Moving from general to specific information

Your paper should be organized in a manner that moves from general to specific information. Every time you begin a new subject, think of an inverted pyramid - The broadest range of information sits at the top, and as the paragraph or paper progresses, the author becomes more and more focused on the argument ending with specific, detailed evidence supporting a claim. Lastly, the author explains how and why the information she has just provided connects to and supports her thesis (a brief wrap-up or warrant).

Moving from General to Specific Information

The four elements of a good paragraph (TTEB)

A good paragraph should contain at least the following four elements: T ransition, T opic sentence, specific E vidence and analysis, and a B rief wrap-up sentence (also known as a warrant ) –TTEB!

• A T ransition sentence leading in from a previous paragraph to assure smooth reading. This acts as a hand-off from one idea to the next.
• A T opic sentence that tells the reader what you will be discussing in the paragraph.
• Specific E vidence and analysis that supports one of your claims and that provides a deeper level of detail than your topic sentence.
• A B rief wrap-up sentence that tells the reader how and why this information supports the paper’s thesis. The brief wrap-up is also known as the warrant. The warrant is important to your argument because it connects your reasoning and support to your thesis, and it shows that the information in the paragraph is related to your thesis and helps defend it.

Supporting evidence (induction and deduction)

Induction is the type of reasoning that moves from specific facts to a general conclusion. When you use induction in your paper, you will state your thesis (which is actually the conclusion you have come to after looking at all the facts) and then support your thesis with the facts. The following is an example of induction taken from Dorothy U. Seyler’s Understanding Argument :

There is the dead body of Smith. Smith was shot in his bedroom between the hours of 11:00 p.m. and 2:00 a.m., according to the coroner. Smith was shot with a .32 caliber pistol. The pistol left in the bedroom contains Jones’s fingerprints. Jones was seen, by a neighbor, entering the Smith home at around 11:00 p.m. the night of Smith’s death. A coworker heard Smith and Jones arguing in Smith’s office the morning of the day Smith died.

Conclusion: Jones killed Smith.

Here, then, is the example in bullet form:

• Conclusion: Jones killed Smith
• Support: Smith was shot by Jones’ gun, Jones was seen entering the scene of the crime, Jones and Smith argued earlier in the day Smith died.
• Assumption: The facts are representative, not isolated incidents, and thus reveal a trend, justifying the conclusion drawn.

When you use deduction in an argument, you begin with general premises and move to a specific conclusion. There is a precise pattern you must use when you reason deductively. This pattern is called syllogistic reasoning (the syllogism). Syllogistic reasoning (deduction) is organized in three steps:

• Major premise
• Minor premise

In order for the syllogism (deduction) to work, you must accept that the relationship of the two premises lead, logically, to the conclusion. Here are two examples of deduction or syllogistic reasoning:

• Major premise: All men are mortal.
• Minor premise: Socrates is a man.
• Conclusion: Socrates is mortal.
• Major premise: People who perform with courage and clear purpose in a crisis are great leaders.
• Minor premise: Lincoln was a person who performed with courage and a clear purpose in a crisis.
• Conclusion: Lincoln was a great leader.

So in order for deduction to work in the example involving Socrates, you must agree that (1) all men are mortal (they all die); and (2) Socrates is a man. If you disagree with either of these premises, the conclusion is invalid. The example using Socrates isn’t so difficult to validate. But when you move into more murky water (when you use terms such as courage , clear purpose , and great ), the connections get tenuous.

For example, some historians might argue that Lincoln didn’t really shine until a few years into the Civil War, after many Union losses to Southern leaders such as Robert E. Lee.

The following is a clear example of deduction gone awry:

• Major premise: All dogs make good pets.
• Minor premise: Doogle is a dog.
• Conclusion: Doogle will make a good pet.

If you don’t agree that all dogs make good pets, then the conclusion that Doogle will make a good pet is invalid.

When a premise in a syllogism is missing, the syllogism becomes an enthymeme. Enthymemes can be very effective in argument, but they can also be unethical and lead to invalid conclusions. Authors often use enthymemes to persuade audiences. The following is an example of an enthymeme:

If you have a plasma TV, you are not poor.

The first part of the enthymeme (If you have a plasma TV) is the stated premise. The second part of the statement (you are not poor) is the conclusion. Therefore, the unstated premise is “Only rich people have plasma TVs.” The enthymeme above leads us to an invalid conclusion (people who own plasma TVs are not poor) because there are plenty of people who own plasma TVs who are poor. Let’s look at this enthymeme in a syllogistic structure:

• Major premise: People who own plasma TVs are rich (unstated above).
• Minor premise: You own a plasma TV.
• Conclusion: You are not poor.

To help you understand how induction and deduction can work together to form a solid argument, you may want to look at the United States Declaration of Independence. The first section of the Declaration contains a series of syllogisms, while the middle section is an inductive list of examples. The final section brings the first and second sections together in a compelling conclusion.

Research Paper

How to write a body of a research paper.

The main part of your research paper is called “the body.” To write this important part of your paper, include only relevant information, or information that gets to the point. Organize your ideas in a logical order—one that makes sense—and provide enough details—facts and examples—to support the points you want to make.

The first sentence of your second paragraph should continue the transition from the end of your introduction to present your first topic.Often too, your first sentence will be your “topic sentence,” the sentence that presents the topic, point, or argument that will be presented in the paragraph. The body of the paragraph should contain evidence, in the form of a discussion using quotations and examples, that supports or “proves” the topic. The final sentence of the paragraph should provide a transition to the third paragraph of the paper where the second topic will be presented.

• The third and fourth paragraphs follow the same format as the second:
• Transition or topic sentence.
• Topic sentence (if not included in the first sentence).
• Supporting sentences including a discussion, quotations, or examples that support the topic sentence.
• Concluding sentence that transitions to the next paragraph.

The topic of each paragraph will be supported by the evidence you itemized in your outline. However, just as smooth transitions are required to connect your paragraphs, the sentences you write to present your evidence should possess transition words that connect ideas, focus attention on relevant information, and continue your discussion in a smooth and fluid manner.

You presented the main idea of your paper in the thesis statement. In the body, every single paragraph must support that main idea. If any paragraph in your paper does not, in some way, back up the main idea expressed in your thesis statement, it is not relevant, which means it doesn’t have a purpose and shouldn’t be there.

Each paragraph also has a main idea of its own. That main idea is stated in a topic sentence, either at the beginning or somewhere else in the paragraph. Just as every paragraph in your paper supports your thesis statement, every sentence in each paragraph supports the main idea of that paragraph by providing facts or examples that back up that main idea. If a sentence does not support the main idea of the paragraph, it is not relevant and should be left out.

A paper that makes claims or states ideas without backing them up with facts or clarifying them with examples won’t mean much to readers. Make sure you provide enough supporting details for all your ideas. And remember that a paragraph can’t contain just one sentence. A paragraph needs at least two or more sentences to be complete. If a paragraph has only one or two sentences, you probably haven’t provided enough support for your main idea. Or, if you have trouble finding the main idea, maybe you don’t have one. In that case, you can make the sentences part of another paragraph or leave them out.

Logical order

Arrange the paragraphs in the body of your paper in an order that makes sense, so that each main idea follows logically from the previous one. Likewise, arrange the sentences in each paragraph in a logical order.

If you carefully organized your notes and made your outline, your ideas will fall into place naturally as you write your draft. The main ideas, which are building blocks of each section or each paragraph in your paper, come from the Roman-numeral headings in your outline. The supporting details under each of those main ideas come from the capital-letter headings. In a shorter paper, the capital-letter headings may become sentences that include supporting details, which come from the Arabic numerals in your outline. In a longer paper, the capital letter headings may become paragraphs of their own, which contain sentences with the supporting details, which come from the Arabic numerals in your outline.

Transition Words and Phrases

In addition to keeping your ideas in logical order, transitions are another way to guide readers from one idea to another. Transition words and phrases are important when you are suggesting or pointing out similarities between ideas, themes, opinions, or a set of facts. As with any perfect phrase, transition words within paragraphs should not be used gratuitously. Their meaning must conform to what you are trying to point out, as shown in the examples below:

• “Accordingly” or “in accordance with” indicates agreement. For example :Thomas Edison’s experiments with electricity accordingly followed the theories of Benjamin Franklin, J. B. Priestly, and other pioneers of the previous century.
• “Analogous” or “analogously” contrasts different things or ideas that perform similar functions or make similar expressions. For example: A computer hard drive is analogous to a filing cabinet. Each stores important documents and data.
• “By comparison” or “comparatively”points out differences between things that otherwise are similar. For example: Roses require an alkaline soil. Azaleas, by comparison, prefer an acidic soil.
• “Corresponds to” or “correspondingly” indicates agreement or conformity. For example: The U.S. Constitution corresponds to England’s Magna Carta in so far as both established a framework for a parliamentary system.
• “Equals,”“equal to,” or “equally” indicates the same degree or quality. For example:Vitamin C is equally as important as minerals in a well-balanced diet.
• “Equivalent” or “equivalently” indicates two ideas or things of approximately the same importance, size, or volume. For example:The notions of individual liberty and the right to a fair and speedy trial hold equivalent importance in the American legal system.
• “Common” or “in common with” indicates similar traits or qualities. For example: Darwin did not argue that humans were descended from the apes. Instead, he maintained that they shared a common ancestor.
• “In the same way,”“in the same manner,”“in the same vein,” or “likewise,” connects comparable traits, ideas, patterns, or activities. For example: John Roebling’s suspension bridges in Brooklyn and Cincinnati were built in the same manner, with strong cables to support a metallic roadway. Example 2: Despite its delicate appearance, John Roebling’s Brooklyn Bridge was built as a suspension bridge supported by strong cables. Example 3: Cincinnati’s Suspension Bridge, which Roebling also designed, was likewise supported by cables.
• “Kindred” indicates that two ideas or things are related by quality or character. For example: Artists Vincent Van Gogh and Paul Gauguin are considered kindred spirits in the Impressionist Movement. “Like” or “as” are used to create a simile that builds reader understanding by comparing two dissimilar things. (Never use “like” as slang, as in: John Roebling was like a bridge designer.) For examples: Her eyes shone like the sun. Her eyes were as bright as the sun.
• “Parallel” describes events, things, or ideas that occurred at the same time or that follow similar logic or patterns of behavior. For example:The original Ocktoberfests were held to occur in parallel with the autumn harvest.
• “Obviously” emphasizes a point that should be clear from the discussion. For example: Obviously, raccoons and other wildlife will attempt to find food and shelter in suburban areas as their woodland habitats disappear.
• “Similar” and “similarly” are used to make like comparisons. For example: Horses and ponies have similar physical characteristics although, as working farm animals, each was bred to perform different functions.
• “There is little debate” or “there is consensus” can be used to point out agreement. For example:There is little debate that the polar ice caps are melting.The question is whether global warming results from natural or human-made causes.

Other phrases that can be used to make transitions or connect ideas within paragraphs include:

• Use “alternately” or “alternatively” to suggest a different option.
• Use “antithesis” to indicate a direct opposite.
• Use “contradict” to indicate disagreement.
• Use “on the contrary” or “conversely” to indicate that something is different from what it seems.
• Use “dissimilar” to point out differences between two things.
• Use “diverse” to discuss differences among many things or people.
• Use “distinct” or “distinctly” to point out unique qualities.
• Use “inversely” to indicate an opposite idea.
• Use “it is debatable,” “there is debate,” or “there is disagreement” to suggest that there is more than one opinion about a subject.
• Use “rather” or “rather than” to point out an exception.
• Use “unique” or “uniquely” to indicate qualities that can be found nowhere else.
• Use “unlike” to indicate dissimilarities.
• Use “various” to indicate more than one kind.

Writing Topic Sentences

Remember, a sentence should express a complete thought, one thought per sentence—no more, no less. The longer and more convoluted your sentences become, the more likely you are to muddle the meaning, become repetitive, and bog yourself down in issues of grammar and construction. In your first draft, it is generally a good idea to keep those sentences relatively short and to the point. That way your ideas will be clearly stated.You will be able to clearly see the content that you have put down—what is there and what is missing—and add or subtract material as it is needed. The sentences will probably seem choppy and even simplistic.The purpose of a first draft is to ensure that you have recorded all the content you will need to make a convincing argument. You will work on smoothing and perfecting the language in subsequent drafts.

Adding Evidence

Transitioning from your topic sentence to the evidence that supports it can be problematic. It requires a transition, much like the transitions needed to move from one paragraph to the next. Choose phrases that connect the evidence directly to your topic sentence.

Phrases for Supporting Topic Sentences

• Consider this: (give an example or state evidence).
• If (identify one condition or event) then (identify the condition or event that will follow).
• It should go without saying that (point out an obvious condition).
• Note that (provide an example or observation).
• Take a look at (identify a condition; follow with an explanation of why you think it is important to the discussion).
• The authors had (identify their idea) in mind when they wrote “(use a quotation from their text that illustrates the idea).”
• The point is that (summarize the conclusion your reader should draw from your research).
• This becomes evident when (name the author) says that (paraphrase a quote from the author’s writing).
• We see this in the following example: (provide an example of your own).
• (The author’s name) offers the example of (summarize an example given by the author).

If an idea is controversial, you may need to add extra evidence to your paragraphs to persuade your reader. You may also find that a logical argument, one based solely on your evidence, is not persuasive enough and that you need to appeal to the reader’s emotions. Look for ways to incorporate your research without detracting from your argument.

Writing Transition Sentences

It is often difficult to write transitions that carry a reader clearly and logically on to the next paragraph (and the next topic) in an essay. Because you are moving from one topic to another, it is easy to simply stop one and start another. Great research papers, however, include good transitions that link the ideas in an interesting discussion so that readers can move smoothly and easily through your presentation. Close each of your paragraphs with an interesting transition sentence that introduces the topic coming up in the next paragraph.

Transition sentences should show a relationship between the two topics.Your transition will perform one of the following functions to introduce the new idea:

• Indicate that you will be expanding on information in a different way in the upcoming paragraph.
• Indicate that a comparison, contrast, or a cause-and-effect relationship between the topics will be discussed.
• Indicate that an example will be presented in the next paragraph.
• Indicate that a conclusion is coming up.

Transitions make a paper flow smoothly by showing readers how ideas and facts follow one another to point logically to a conclusion. They show relationships among the ideas, help the reader to understand, and, in a persuasive paper, lead the reader to the writer’s conclusion.

Each paragraph should end with a transition sentence to conclude the discussion of the topic in the paragraph and gently introduce the reader to the topic that will be raised in the next paragraph. However, transitions also occur within paragraphs—from sentence to sentence—to add evidence, provide examples, or introduce a quotation.

The type of paper you are writing and the kinds of topics you are introducing will determine what type of transitional phrase you should use. Some useful phrases for transitions appear below. They are grouped according to the function they normally play in a paper. Transitions, however, are not simply phrases that are dropped into sentences. They are constructed to highlight meaning. Choose transitions that are appropriate to your topic and what you want the reader to do. Edit them to be sure they fit properly within the sentence to enhance the reader’s understanding.

Transition Phrases for Comparisons:

• We also see
• In addition to
• Notice that
• Beside that,
• In comparison,
• Once again,
• Identically,
• For example,
• Comparatively, it can be seen that
• We see this when
• This corresponds to
• In other words,
• At the same time,
• By the same token,

Transition Phrases for Contrast:

• By contrast,
• On the contrary,
• Nevertheless,
• An exception to this would be …
• Alongside that,we find …
• On one hand … on the other hand …
• [New information] presents an opposite view …
• Conversely, it could be argued …
• Other than that,we find that …
• We get an entirely different impression from …
• One point of differentiation is …
• Further investigation shows …
• An exception can be found in the fact that …

Transition Phrases to Show a Process:

• At the top we have … Near the bottom we have …
• Here we have … There we have …
• Continuing on,
• We progress to …
• Close up … In the distance …
• With this in mind,
• Moving in sequence,
• Proceeding sequentially,
• Moving to the next step,
• First, Second,Third,…
• Examining the activities in sequence,
• Sequentially,
• As a result,
• The end result is …
• To illustrate …
• Subsequently,
• One consequence of …
• If … then …
• It follows that …
• This is chiefly due to …
• The next step …
• Later we find …

Phrases to Introduce Examples:

• For instance,
• Particularly,
• In particular,
• This includes,
• Specifically,
• To illustrate,
• One illustration is
• One example is
• This is illustrated by
• This can be seen when
• This is especially seen in
• This is chiefly seen when

Transition Phrases for Presenting Evidence:

• Another point worthy of consideration is
• At the center of the issue is the notion that
• Before moving on, it should be pointed out that
• Another important point is
• Another idea worth considering is
• Consequently,
• Especially,
• Even more important,
• Getting beyond the obvious,
• In spite of all this,
• It follows that
• It is clear that
• More importantly,
• Most importantly,

See the examples of effective transitions for more.

Draft Your Conclusion

Every good paper ends with a strong concluding paragraph. To write a good conclusion, sum up the main points in your paper. To write an even better conclusion, include a sentence or two that helps the reader answer the question, “So what?” or “Why does all this matter?” If you choose to include one or more “So What?” sentences, remember that you still need to support any point you make with facts or examples. Remember, too, that this is not the place to introduce new ideas from “out of the blue.” Make sure that everything you write in your conclusion refers to what you’ve already written in the body of your paper.

Read more on How to Write a Research Paper .

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TOPIC SENTENCE/ In his numerous writings, Marx critiques capitalism by identifying its flaws. ANALYSIS OF EVIDENCE/ By critiquing the political economy and capitalism, Marx implores his reader to think critically about their position in society and restores awareness in the proletariat class. EVIDENCE/ To Marx, capitalism is a system characterized by the “exploitation of the many by the few,” in which workers accept the exploitation of their labor and receive only harm of “alienation,” rather than true benefits ( MER 487). He writes that “labour produces for the rich wonderful things – but for the worker it produces privation. It produces palaces—but for the worker, hovels. It produces beauty—but for the worker, deformity” (MER 73). Marx argues capitalism is a system in which the laborer is repeatedly harmed and estranged from himself, his labor, and other people, while the owner of his labor – the capitalist – receives the benefits ( MER 74). And while industry progresses, the worker “sinks deeper and deeper below the conditions of existence of his own class” ( MER 483).  ANALYSIS OF EVIDENCE/ But while Marx critiques the political economy, he does not explicitly say “capitalism is wrong.” Rather, his close examination of the system makes its flaws obvious. Only once the working class realizes the flaws of the system, Marx believes, will they - must they - rise up against their bourgeois masters and achieve the necessary and inevitable communist revolution.

Not every paragraph will be structured exactly like this one, of course. But as you draft your own paragraphs, look for all three of these elements: topic sentence, evidence, and analysis.

• picture_as_pdf Anatomy Of a Body Paragraph

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13.1 Formatting a Research Paper

Learning objectives.

• Identify the major components of a research paper written using American Psychological Association (APA) style.
• Apply general APA style and formatting conventions in a research paper.

In this chapter, you will learn how to use APA style , the documentation and formatting style followed by the American Psychological Association, as well as MLA style , from the Modern Language Association. There are a few major formatting styles used in academic texts, including AMA, Chicago, and Turabian:

• AMA (American Medical Association) for medicine, health, and biological sciences
• APA (American Psychological Association) for education, psychology, and the social sciences
• Chicago—a common style used in everyday publications like magazines, newspapers, and books
• MLA (Modern Language Association) for English, literature, arts, and humanities
• Turabian—another common style designed for its universal application across all subjects and disciplines

While all the formatting and citation styles have their own use and applications, in this chapter we focus our attention on the two styles you are most likely to use in your academic studies: APA and MLA.

If you find that the rules of proper source documentation are difficult to keep straight, you are not alone. Writing a good research paper is, in and of itself, a major intellectual challenge. Having to follow detailed citation and formatting guidelines as well may seem like just one more task to add to an already-too-long list of requirements.

Following these guidelines, however, serves several important purposes. First, it signals to your readers that your paper should be taken seriously as a student’s contribution to a given academic or professional field; it is the literary equivalent of wearing a tailored suit to a job interview. Second, it shows that you respect other people’s work enough to give them proper credit for it. Finally, it helps your reader find additional materials if he or she wishes to learn more about your topic.

Furthermore, producing a letter-perfect APA-style paper need not be burdensome. Yes, it requires careful attention to detail. However, you can simplify the process if you keep these broad guidelines in mind:

• Work ahead whenever you can. Chapter 11 “Writing from Research: What Will I Learn?” includes tips for keeping track of your sources early in the research process, which will save time later on.
• Get it right the first time. Apply APA guidelines as you write, so you will not have much to correct during the editing stage. Again, putting in a little extra time early on can save time later.
• Use the resources available to you. In addition to the guidelines provided in this chapter, you may wish to consult the APA website at http://www.apa.org or the Purdue University Online Writing lab at http://owl.english.purdue.edu , which regularly updates its online style guidelines.

General Formatting Guidelines

This chapter provides detailed guidelines for using the citation and formatting conventions developed by the American Psychological Association, or APA. Writers in disciplines as diverse as astrophysics, biology, psychology, and education follow APA style. The major components of a paper written in APA style are listed in the following box.

These are the major components of an APA-style paper:

Body, which includes the following:

• Headings and, if necessary, subheadings to organize the content
• In-text citations of research sources
• References page

All these components must be saved in one document, not as separate documents.

The title page of your paper includes the following information:

• Title of the paper
• Author’s name
• Name of the institution with which the author is affiliated
• Header at the top of the page with the paper title (in capital letters) and the page number (If the title is lengthy, you may use a shortened form of it in the header.)

List the first three elements in the order given in the previous list, centered about one third of the way down from the top of the page. Use the headers and footers tool of your word-processing program to add the header, with the title text at the left and the page number in the upper-right corner. Your title page should look like the following example.

The next page of your paper provides an abstract , or brief summary of your findings. An abstract does not need to be provided in every paper, but an abstract should be used in papers that include a hypothesis. A good abstract is concise—about one hundred fifty to two hundred fifty words—and is written in an objective, impersonal style. Your writing voice will not be as apparent here as in the body of your paper. When writing the abstract, take a just-the-facts approach, and summarize your research question and your findings in a few sentences.

In Chapter 12 “Writing a Research Paper” , you read a paper written by a student named Jorge, who researched the effectiveness of low-carbohydrate diets. Read Jorge’s abstract. Note how it sums up the major ideas in his paper without going into excessive detail.

Write an abstract summarizing your paper. Briefly introduce the topic, state your findings, and sum up what conclusions you can draw from your research. Use the word count feature of your word-processing program to make sure your abstract does not exceed one hundred fifty words.

Depending on your field of study, you may sometimes write research papers that present extensive primary research, such as your own experiment or survey. In your abstract, summarize your research question and your findings, and briefly indicate how your study relates to prior research in the field.

Margins, Pagination, and Headings

APA style requirements also address specific formatting concerns, such as margins, pagination, and heading styles, within the body of the paper. Review the following APA guidelines.

Use these general guidelines to format the paper:

• Set the top, bottom, and side margins of your paper at 1 inch.
• Use double-spaced text throughout your paper.
• Use a standard font, such as Times New Roman or Arial, in a legible size (10- to 12-point).
• Use continuous pagination throughout the paper, including the title page and the references section. Page numbers appear flush right within your header.
• Section headings and subsection headings within the body of your paper use different types of formatting depending on the level of information you are presenting. Additional details from Jorge’s paper are provided.

Begin formatting the final draft of your paper according to APA guidelines. You may work with an existing document or set up a new document if you choose. Include the following:

• Your title page
• The abstract you created in Note 13.8 “Exercise 1”
• Correct headers and page numbers for your title page and abstract

APA style uses section headings to organize information, making it easy for the reader to follow the writer’s train of thought and to know immediately what major topics are covered. Depending on the length and complexity of the paper, its major sections may also be divided into subsections, sub-subsections, and so on. These smaller sections, in turn, use different heading styles to indicate different levels of information. In essence, you are using headings to create a hierarchy of information.

The following heading styles used in APA formatting are listed in order of greatest to least importance:

• Section headings use centered, boldface type. Headings use title case, with important words in the heading capitalized.
• Subsection headings use left-aligned, boldface type. Headings use title case.
• The third level uses left-aligned, indented, boldface type. Headings use a capital letter only for the first word, and they end in a period.
• The fourth level follows the same style used for the previous level, but the headings are boldfaced and italicized.
• The fifth level follows the same style used for the previous level, but the headings are italicized and not boldfaced.

Visually, the hierarchy of information is organized as indicated in Table 13.1 “Section Headings” .

Table 13.1 Section Headings

A college research paper may not use all the heading levels shown in Table 13.1 “Section Headings” , but you are likely to encounter them in academic journal articles that use APA style. For a brief paper, you may find that level 1 headings suffice. Longer or more complex papers may need level 2 headings or other lower-level headings to organize information clearly. Use your outline to craft your major section headings and determine whether any subtopics are substantial enough to require additional levels of headings.

Working with the document you developed in Note 13.11 “Exercise 2” , begin setting up the heading structure of the final draft of your research paper according to APA guidelines. Include your title and at least two to three major section headings, and follow the formatting guidelines provided above. If your major sections should be broken into subsections, add those headings as well. Use your outline to help you.

Because Jorge used only level 1 headings, his Exercise 3 would look like the following:

Citation Guidelines

In-text citations.

Throughout the body of your paper, include a citation whenever you quote or paraphrase material from your research sources. As you learned in Chapter 11 “Writing from Research: What Will I Learn?” , the purpose of citations is twofold: to give credit to others for their ideas and to allow your reader to follow up and learn more about the topic if desired. Your in-text citations provide basic information about your source; each source you cite will have a longer entry in the references section that provides more detailed information.

In-text citations must provide the name of the author or authors and the year the source was published. (When a given source does not list an individual author, you may provide the source title or the name of the organization that published the material instead.) When directly quoting a source, it is also required that you include the page number where the quote appears in your citation.

This information may be included within the sentence or in a parenthetical reference at the end of the sentence, as in these examples.

Epstein (2010) points out that “junk food cannot be considered addictive in the same way that we think of psychoactive drugs as addictive” (p. 137).

Here, the writer names the source author when introducing the quote and provides the publication date in parentheses after the author’s name. The page number appears in parentheses after the closing quotation marks and before the period that ends the sentence.

Addiction researchers caution that “junk food cannot be considered addictive in the same way that we think of psychoactive drugs as addictive” (Epstein, 2010, p. 137).

Here, the writer provides a parenthetical citation at the end of the sentence that includes the author’s name, the year of publication, and the page number separated by commas. Again, the parenthetical citation is placed after the closing quotation marks and before the period at the end of the sentence.

As noted in the book Junk Food, Junk Science (Epstein, 2010, p. 137), “junk food cannot be considered addictive in the same way that we think of psychoactive drugs as addictive.”

Here, the writer chose to mention the source title in the sentence (an optional piece of information to include) and followed the title with a parenthetical citation. Note that the parenthetical citation is placed before the comma that signals the end of the introductory phrase.

David Epstein’s book Junk Food, Junk Science (2010) pointed out that “junk food cannot be considered addictive in the same way that we think of psychoactive drugs as addictive” (p. 137).

Another variation is to introduce the author and the source title in your sentence and include the publication date and page number in parentheses within the sentence or at the end of the sentence. As long as you have included the essential information, you can choose the option that works best for that particular sentence and source.

Citing a book with a single author is usually a straightforward task. Of course, your research may require that you cite many other types of sources, such as books or articles with more than one author or sources with no individual author listed. You may also need to cite sources available in both print and online and nonprint sources, such as websites and personal interviews. Chapter 13 “APA and MLA Documentation and Formatting” , Section 13.2 “Citing and Referencing Techniques” and Section 13.3 “Creating a References Section” provide extensive guidelines for citing a variety of source types.

Writing at Work

APA is just one of several different styles with its own guidelines for documentation, formatting, and language usage. Depending on your field of interest, you may be exposed to additional styles, such as the following:

• MLA style. Determined by the Modern Languages Association and used for papers in literature, languages, and other disciplines in the humanities.
• Chicago style. Outlined in the Chicago Manual of Style and sometimes used for papers in the humanities and the sciences; many professional organizations use this style for publications as well.
• Associated Press (AP) style. Used by professional journalists.

References List

The brief citations included in the body of your paper correspond to the more detailed citations provided at the end of the paper in the references section. In-text citations provide basic information—the author’s name, the publication date, and the page number if necessary—while the references section provides more extensive bibliographical information. Again, this information allows your reader to follow up on the sources you cited and do additional reading about the topic if desired.

The specific format of entries in the list of references varies slightly for different source types, but the entries generally include the following information:

• The name(s) of the author(s) or institution that wrote the source
• The year of publication and, where applicable, the exact date of publication
• The full title of the source
• For books, the city of publication
• For articles or essays, the name of the periodical or book in which the article or essay appears
• For magazine and journal articles, the volume number, issue number, and pages where the article appears
• For sources on the web, the URL where the source is located

The references page is double spaced and lists entries in alphabetical order by the author’s last name. If an entry continues for more than one line, the second line and each subsequent line are indented five spaces. Review the following example. ( Chapter 13 “APA and MLA Documentation and Formatting” , Section 13.3 “Creating a References Section” provides extensive guidelines for formatting reference entries for different types of sources.)

In APA style, book and article titles are formatted in sentence case, not title case. Sentence case means that only the first word is capitalized, along with any proper nouns.

Key Takeaways

• Following proper citation and formatting guidelines helps writers ensure that their work will be taken seriously, give proper credit to other authors for their work, and provide valuable information to readers.
• Working ahead and taking care to cite sources correctly the first time are ways writers can save time during the editing stage of writing a research paper.
• APA papers usually include an abstract that concisely summarizes the paper.
• APA papers use a specific headings structure to provide a clear hierarchy of information.
• In APA papers, in-text citations usually include the name(s) of the author(s) and the year of publication.
• In-text citations correspond to entries in the references section, which provide detailed bibliographical information about a source.

Writing for Success Copyright © 2015 by University of Minnesota is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

How to Write a Research Paper: Parts of the Paper

• Choosing Your Topic
• Citation & Style Guides This link opens in a new window
• Critical Thinking
• Evaluating Information
• Parts of the Paper
• Writing Tips from UNC-Chapel Hill
• Librarian Contact

Parts of the Research Paper Papers should have a beginning, a middle, and an end. Your introductory paragraph should grab the reader's attention, state your main idea, and indicate how you will support it. The body of the paper should expand on what you have stated in the introduction. Finally, the conclusion restates the paper's thesis and should explain what you have learned, giving a wrap up of your main ideas.

1. The Title The title should be specific and indicate the theme of the research and what ideas it addresses. Use keywords that help explain your paper's topic to the reader. Try to avoid abbreviations and jargon. Think about keywords that people would use to search for your paper and include them in your title.

2. The Abstract The abstract is used by readers to get a quick overview of your paper. Typically, they are about 200 words in length (120 words minimum to  250 words maximum). The abstract should introduce the topic and thesis, and should provide a general statement about what you have found in your research. The abstract allows you to mention each major aspect of your topic and helps readers decide whether they want to read the rest of the paper. Because it is a summary of the entire research paper, it is often written last. 

3. The Introduction The introduction should be designed to attract the reader's attention and explain the focus of the research. You will introduce your overview of the topic,  your main points of information, and why this subject is important. You can introduce the current understanding and background information about the topic. Toward the end of the introduction, you add your thesis statement, and explain how you will provide information to support your research questions. This provides the purpose and focus for the rest of the paper.

4. Thesis Statement Most papers will have a thesis statement or main idea and supporting facts/ideas/arguments. State your main idea (something of interest or something to be proven or argued for or against) as your thesis statement, and then provide your supporting facts and arguments. A thesis statement is a declarative sentence that asserts the position a paper will be taking. It also points toward the paper's development. This statement should be both specific and arguable. Generally, the thesis statement will be placed at the end of the first paragraph of your paper. The remainder of your paper will support this thesis.

Students often learn to write a thesis as a first step in the writing process, but often, after research, a writer's viewpoint may change. Therefore a thesis statement may be one of the final steps in writing. 

Examples of Thesis Statements from Purdue OWL

5. The Literature Review The purpose of the literature review is to describe past important research and how it specifically relates to the research thesis. It should be a synthesis of the previous literature and the new idea being researched. The review should examine the major theories related to the topic to date and their contributors. It should include all relevant findings from credible sources, such as academic books and peer-reviewed journal articles. You will want  to:

• Explain how the literature helps the researcher understand the topic.
• Try to show connections and any disparities between the literature.
• Identify new ways to interpret prior research.
• Reveal any gaps that exist in the literature.

More about writing a literature review. . .

6. The Discussion ​The purpose of the discussion is to interpret and describe what you have learned from your research. Make the reader understand why your topic is important. The discussion should always demonstrate what you have learned from your readings (and viewings) and how that learning has made the topic evolve, especially from the short description of main points in the introduction.Explain any new understanding or insights you have had after reading your articles and/or books. Paragraphs should use transitioning sentences to develop how one paragraph idea leads to the next. The discussion will always connect to the introduction, your thesis statement, and the literature you reviewed, but it does not simply repeat or rearrange the introduction. You want to: 

• Demonstrate critical thinking, not just reporting back facts that you gathered.
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Normal Body Temperature: A Systematic Review

Ivayla i geneva.

1 State University of New York Upstate Medical University, Syracuse, NY, USA

2 Department of Internal Medicine, Icahn School of Medicine at Mount Sinai, New York, NY

Brian Cuzzo

Tasaduq fazili.

3 Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY

Waleed Javaid

4 Icahn School of Medicine at Mount Sinai, New York, NY

PubMed was searched from 1935 to December 2017 with a variety of search phrases among article titles. The references of the identified manuscripts were then manually searched. The inclusion criteria were as follows: (1) the paper presented data on measured normal body temperature of healthy human subjects ages 18 and older, (2) a prospective design was used, and (3) the paper was written in or translated into the English language. Thirty-six articles met the inclusion criteria. This comprised 9227 measurement sites from 7636 subjects. The calculated ranges (mean ± 2 standard deviations) were 36.32–37.76 (rectal), 35.76–37.52 (tympanic), 35.61–37.61 (urine), 35.73–37.41 (oral), and 35.01–36.93 (axillary). Older adults (age ≥60) had lower temperature than younger adults (age <60) by 0.23°C, on average. There was only insignificant gender difference. Compared with the currently established reference point for normothermia of 36.8°C, our means are slightly lower but the difference likely has no physiological importance. We conclude that the most important patient factors remain site of measurement and patient’s age.

Human body temperature is well established as one of the key vital signs. It is measured at regular intervals in the medical setting and often at home to try estimate the degree of “sickness” of an individual [ 1 ]. It had been used since antiquity [ 2–5 ], yet its interpretation had been, and still is, actively debated in the clinical setting [ 1 , 6 , 7 ]. The first step towards understanding the relationship between temperature and disease is to define “normal” body temperature, from where deviations can be measured. Indeed, many attempts had been made to this end, including the 1868 seminal paper by Wunderlich [ 8 ], who is believed to be the first to establish a link between fever and clinical diagnosis. He was also the first to apply a thermometer experimentally to measure human body temperature. Using a large sample size, Wunderlich [ 8 ] concluded that the average axillary temperature was 37.0°C, with the upper limit of normal defined as 38.0°C. However, newer studies challenged Wunderlich’s [ 8 ] “normothermia” [ 6 ]. Furthermore, research had shown that body temperature is a nonlinear function of several variables such as age, state of health, gender, environmental temperature, time of the diurnal cycle, among many others [ 9 , 10 ]. To make the best use of the currently available literature, we reviewed and herein present an analysis of previously published human body temperature studies using healthy individuals, with the goal of better understanding the variables that determine normal body temperature.

The peer-reviewed literature was searched using PubMed ( Table 1 ). The time period ranged from 1935 to December 2017. The following search phrases among article titles were used: “normal body temperature”, “body temperature AND review”, “body temperature AND adult”, “body temperature AND gender”, “human body temperature”, “core body temperature”, “hypothermia AND elderly”, “body temperature AND measurement”, “tympanic body temperature AND measurement”, “rectal body temperature AND measurement”, and “oral body temperature AND measurement”. Furthermore, the references of the above-identified papers were manually searched for additional useful articles. To be included in our analysis, papers had to meet the following inclusion criteria: (1) the paper presented data on measured normal body temperature of healthy human subjects ages 18 and older, (2) a prospective design was used, and (3) the paper was written in or translated into the English language. Using the data from the articles that met our inclusion criteria, we calculated mean temperatures and ranges before and after stratifying the data by gender, age (less than 60 years old vs 60 years old or older), and site of measurement (oral, axillary, temporal, rectal, urine) or by both variables.

Summary of the Literature Data Search Grouped by Search Phrase

Pooled standard deviations were calculated using the pooled standard deviation formula:

For equal sample sizes, the formula was simplified as follows:

For the data in which standard deviation for the measured temperatures was not reported in the original articles, the standard deviation was estimated via extrapolation from a plot of the known standard deviations and the corresponding sample sizes. Table 2 shows the available and missing standard deviations (8 of the 36 articles that met our inclusion criteria did not report standard deviations for at least some portion of their data).

Data Summary From the Articles That Met the Inclusion Criteria

Abbreviations: N, number of participants; NH, New Hampshire; SD, standard deviation.

The search hits are summarized in Table 1 . A total of 36 articles met our inclusion criteria and the extracted raw data is shown in Table 2 . The sample sizes for all of these studies were plotted against the year in which the studies were published in Figure 1A . Of the identified articles, 33 reported oral temperatures, 13 reported rectal temperatures, 9 reported tympanic temperatures, 6 reported urine temperatures, and 5 reported axillary temperatures. Seventeen of the studies reported temperatures in younger adults (age <60 years) and 19 reported temperatures in older adults (age ≥60 years). There were a total of 7636 healthy subjects, 1992 of which were identified as female and 2102 were identified as male, and the rest did not have their gender reported. There were a total of 9227 individual measurement sites used, where 5257 adults provided oral measurements, 2462 provided tympanic measurements, 618 provided rectal measurements, 551 provided axillary measurements, and 339 adults provided urine measurements. Our statistical analysis ( Table 3 ) showed that the average body temperature among all subjects in all 36 studies and combining the data from all measurement sites was 36.59 ± 0.43 (standard deviation).

Summary of Normal Body Temperature Ranges Stratified by the Modifying Factors Measurement Site, Age, and Gender

Literature search results and the determinants of normothermia. (A) Number of studies and their sizes over the search time period. (B) The dependence of body temperature on measurement site. (C) The dependence of body temperature on age, shown stratified by measurement site. (D) The dependence of body temperature on gender, shown stratified by measurement site.

The average temperatures per measurement site, in decreasing order, were rectal at 37.04 ± 0.36, tympanic at 36.64 ± 0.44, urine at 36.61 ± 0.5, oral at 36.57 ± 0.42, and axillary at 35.97 ± 0.48 ( Figure 1B , Table 3 ). Overall, when using the data from all of the measurement sites, the average body temperature of younger adults (<60 years of age) was higher (36.69 ± 0.34) than the average body temperature of older adults ( ≥60 years of age), which was 36.5 ± 0.48. The same age-related trend held true for all individual measurement sites ( Figure 1C , Table 3 ). When looking at gender differences, we found that when using all reported measurements, the average body temperature of females was slightly lower (36.65 ± 0.46) compared with males (36.69 ± 0.43), but this trend was not pronounced when looking at the individual measurement sites, except for the urine measurement site ( Figure 1D , Table 3 ).

The quest for understanding human body temperature and defining normothermia is ongoing, as is evidenced by the steady number of published prospective studies depicted in Figure 1A . To the best of our knowledge, our systematic review, where we analyzed 36 separate prospective studies, is the largest of its kind. When using the data from all measurement sites and all included studies, we calculated the overall mean body temperature to be 36.59°C, which is lower than the currently acceptable mean of 36.8, as published in one of the most respected medical reference books, Harrison’s Principles of Internal Medicine [ 46 ]. However, the latter number from the reference book is not based on an all-inclusive meta-analysis, and therefore our average is likely more accurate. Of course, it should be kept in mind that there is no single number that defines normothermia; instead, there is a range for normal temperature, with corresponding standard deviation and standard error. As such, the 0.2°C difference in the mean when we compare our mean temperature with the Harrrison’s is likely not of much physiological relevance. In that respect, our calculated overall range (mean ± 2 standard deviations) is 36.16–37.02°C, which is narrower than the range of 33.2–38.3°C reported by Sund-Levander et al [ 42 ], which is an older systematic review comprising of only 20 studies, all of which were also part of our analysis. The tighter range is most likely due to bigger sample size used in our report, which validates our results further.

Knowing that body temperature is influenced by the measurement site, we calculated average temperatures, in decreasing order, rectal at 37.04°C, tympanic at 36.64°C, urine at 36.61°C, oral at 36.57°C, and axillary at 35.97°C. The trend is similar to the one reported by Sund-Levander et al [ 42 ]; however, the latter systematic review did not contain measurements of urine temperature. In addition, all of our site-specific calculated temperatures, except for axillary, were higher compared with the Sund-Levander et al [ 42 ] report. Furthermore, it is intriguing that we found such a large difference between what is considered the body core temperatures: rectal (37.04°C) and urine (urine at 36.61°C). This likely reflects a fault in the measurement in earlier studies from the 1970s and 1980s, which constitute a significant portion of the analyzed data and in which the measurements of urine temperature were not done invasively, eg, via a monotherm system. Therefore, these urine temperatures are fundamentally different from what we should consider core body temperature, which is temperature measured inside the human body.

With regards to age, our analysis confirmed that, on average, healthy elderly people have lower body temperature ( Table 3 and Figure 1B ) compared with younger adults. This was true for both the total average as well as for the individual measurements sites, except for urine temperatures because there were no studies reporting such measurements among younger adults. The decrease in body temperature with age is believed to be a phenomenon arising from a slowing of the human metabolic rate coupled with a decline in the ability to regulate body temperature in response to environmental changes such as seasonal changes, which had been previously studied [ 17 , 19 , 22 , 47 , 48 ]. These age-related changes are of particular clinical importance because elderly patients are often not capable of mounting a strong inflammatory response to infection and disease, with their temperature failing to reach the temperature range of what is traditionally considered the fever temperature range. Moreover, there is evidence to suggest that the presence of a robust fever response carries prognostic value when considering such infectious disease processes [ 49 ]. In the elderly, who may not be able to mount such a thermal response, we may similarly have to readjust our outlook on temperature-based prognostication. However, until we have research data to specifically address this question, clinicians should use lower normal temperature ranges as reference in the elderly, such as the ones presented in our systematic review.

Finally, our analysis demonstrated only a trivial difference in body temperature between the genders ( Table 2 and Figure 1C ), with women’s temperature being slightly lower when using all measurements from all measurement sites. However, when grouping the results by measurement site, in some cases (tympanic site) females’ body temperature is in fact higher compared with their male counterparts, whereas in other cases there is no difference (oral and rectal sites). There had been a disagreement in the literature as well, with some studies reporting that females have higher body temperature [ 6 , 8 , 16 , 31 ], whereas others reported no differences among the genders [ 39 ]. Gender differences in body temperature had been suspected to relate to a difference in body fat percentage between women and men. Those studies revealed that women have a comparably larger percentage of body fat distribution subcutaneously, which in turn correlates with lower average skin temperatures [ 50 , 51 ]. It had also been theorized that body temperature differences relate to female hormone levels, and yet, even in the studies that report statistically significant differences, the actual difference is fairly small and thus not likely to be of any clinical significance. Our large sample size from 36 individual studies is expected to reflect the true temperature variable in the human population and supports the lack of clinical significance of gender-based body temperature difference even if it could be measured.

CONCLUSIONS

Human body temperature is a highly variable vital sign and known to be influenced by several variables, most prominently the person’s age and the site of measurement. Our systematic review is the largest of its kind and provides clinicians with evidence-based normal temperature ranges to guide their evaluation of patients with possible fever or hypothermia.

Acknowledgments

Potential conflicts of interest.   All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

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• Published: 14 March 2024

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit

• Vayu Maini Rekdal 1 , 2 , 3 ,
• Casper R. B. van der Luijt   ORCID: orcid.org/0000-0001-5978-7731 3 , 4 , 5 , 6 ,
• Yan Chen 3 , 6 ,
• Ramu Kakumanu 3 , 6 ,
• Edward E. K. Baidoo 3 , 6 ,
• Christopher J. Petzold   ORCID: orcid.org/0000-0002-8270-5228 3 , 6 ,
• Pablo Cruz-Morales 4 &
• Jay D. Keasling   ORCID: orcid.org/0000-0003-4170-6088 1 , 3 , 4 , 6 , 7 , 8  

Nature Communications volume  15 , Article number:  2099 ( 2024 ) Cite this article

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• Food microbiology
• Genetic engineering
• Metabolic engineering
• Synthetic biology

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae , an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

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Introduction

The global food system has been identified as one of the major contributors to climate change. Food production is responsible for an estimated one-third of global greenhouse gas emissions and contributes to widespread environmental degradation, biodiversity loss, and the emergence of new diseases 1 , 2 , 3 . Transitioning food production away from resource-intensive industrial animal agriculture toward alternative methods, including microbial processes, is critical for mitigating these negative planetary impacts and sustainably feeding a growing global population that is estimated to reach over 9 billion by 2050 1 , 2 , 4 , 5 , 6 , 7 . Among other applications, microbes can be used for upcycling byproducts 8 , as hosts for production of environmentally taxing small molecules and proteins 9 , 10 , and for producing nutritious biomass that can be consumed directly 11 . Compared to animal agriculture, microbial food production can offer increased resource efficiency and safety, more precise control of production, reduced animal suffering, and a reduced environmental footprint 5 .

Filamentous fungi, a diverse group of microorganisms that includes molds and mushrooms, have several advantages compared to other hosts for microbially based food production 12 (Fig.  1A ). In addition to the historical use of many fungi for safe and delicious fermented foods 13 , the naturally high secretory capacity of these organisms makes them powerful hosts for production of proteins for food and other uses 14 . Additionally, many fungi rapidly degrade and grow on complex substrates such as food byproducts or lignocellulose, which can alleviate the cost and environmental burden associated with highly purified substrates such as glucose 9 , 15 . Finally, owing to its filamentous morphology which mimics the structure of animal muscle, fungal biomass (mycelium) can be formulated into meat alternatives with convincing textures (mycoprotein), and used as scaffolds for adherent animal cells in cellular agriculture 11 . A recent Life Cycle Assessment revealed that substituting 20% of animal protein with mycoprotein by 2050 could lower methane emissions as well as reduce deforestation and associated CO 2 emissions by half, underscoring the concrete environmental benefits of fungal foods 6 .

A Fungal applications in sustainable food production. The figure was created was created using BioRender ( http://BioRender.com ). B Strategy for RNP-based CRISPR-Cas9 editing. Upon integration at the correct locus, the pyrG selection marker becomes flanked by two identical 300-bp sequences. Counter-selection using 5-fluoroorotic acid (5-FOA) allows locus-specific marker excision. C Strategy for integration of a GFP-expression cassette (pAmyB promoter) at the wA locus, which controls spore pigmentation, in A. oryzae RIB40. The two squiggly lines indicate that not the whole 6.7-kb wA gene is shown. Primers TamyB-DC-F (on fixing template) and wA-in-1R (on chromosome) were used to confirm successful insertion. Ladder is Generuler 1 kb ladder (Thermo Scientific). D The wA : gfp transformant has white conidia instead of the yellow-green conidial pigmentation of the RIB40 strain (grown on PDA medium). E Efficiency of gene integration at the wA locus as a function of the homology arm length in the fixing template. 10 colonies were randomly selected at each length and subjected to colony PCR. F Following pyrG marker recycling from the wA locus the strain displays uracil/uridine auxotrophy when grown on CDA medium. UU = uracil and uridine supplementation G The auxotrophy was accompanied by clear marker loop out from the wA locus. Ladder is Generuler 1 kb ladder (Thermo Scientific). H The looped-out wA:gfp strain (Δ pyrG) was transformed with an mCherry expression cassette targeted at the niaD locus, which controls nitrate assimilation. Microscopy in the wA:gfp and niaD:mCherry strain confirmed the expected pattern of protein expression. Scale bar = 25 µm. I The resulting transformant only grows with leucine (CDA-Leu) as the nitrogen source instead of nitrate (CDA), as indicated by the colony radiating from the center of the plate. J The wA:gfp and niaD:mCherry strain was subjected to marker recycling from the niaD locus. PCR confirmation indicates that marker recycling was successful from the niaD locus (see Supplementary Fig.  3 for details), while the wA locus remained unchanged. Ladder is Generuler 1 kb ladder (Thermo Scientific).

Fungal food production is a rapidly growing area with vast commercial interest and potential, and a growing number of meat and dairy substitutes based on fungi are now available on the market across Europe, U.S., and Asia 12 , 16 . While these products showcase the astonishing versatility and commercial promise of fungi for sustainable food production, most current products are based on a limited group of non-engineered strains, which have inherent limitations in their metabolism, structure, and industrial capacity. Genetic engineering could overcome these limitations and further expand beyond naturally occurring biodiversity, allowing new uses and applications of fungi in human food production 17 . For instance, a synthetic gene expression tool in Trichoderma reesei , an industrial fungus traditionally used for enzyme production, recently enabled production of gram-scale quantities of egg white and milk protein 14 , 18 . However, like many other industrial fungi, T. reesei has no history of safe or palatable consumption by humans, which limits the possible food contexts in which the fungus can be utilized, such as the highly efficient and sustainable production of fungal biomass for human food. Extending such synthetic biology tools and approaches to historically consumed, food-safe, edible fungi could expand the engineering possibilities for fungal food production, including enhancing fermented foods and altering the properties of mycoprotein to better suit human dietary needs and preferences. However, synthetic biology tools and demonstrated use cases for bioengineered food production by historically consumed food-safe filamentous fungi are lacking.

Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae , an edible fungus with a long history of safe and palatable human consumption, and demonstrate its applicability for enhancing fungal foods. Our toolkit includes a CRISPR-Cas9 method for precise and efficient gene modification, neutral loci for targeted gene insertion, and tunable promoters, including bidirectional promoters as well as a synthetic expression system that offers strong gene expression independent of the medium composition. We use these tools to engineer the nutritional value and sensory appeal of the edible fungal biomass for alternative meat applications. We overproduce ergothioneine, a potent antioxidant, at levels that are higher than in mushrooms, the largest source of this molecule in the human diet. Additionally, we engineer the eight-step heme biosynthetic pathway to create an edible biomass that contains heme at levels approaching those found in leading plant-based meats incorporating heme for flavor and color. In contrast to plant-based protein, the engineered fungal biomass can be readily formulated into meat-like patties without the need for extensive processing, protein purification, or ingredient addition. In addition to demonstrating the potential of bioengineering edible fungi, this work provides synthetic biology tools and approaches that could be useful for fungi across diverse applications and industries.

A recyclable CRISPR-Cas9 method for efficient gene integration and expression

We selected Aspergillus oryzae (koji mold) as our model edible fungus and engineering target, as this fungus has a long history of safe use and human acceptance in fermented foods 13 , has a biomass with a palatable and umami-rich flavor that is commercially available as mycoprotein 19 , 20 , secretes high amounts of protein 21 , is used industrially for enzyme production 22 , and has demonstrated promise as a scaffold for animal cells in cellular agriculture 23 . To enable synthetic biology efforts across the diverse food applications of this edible fungus, we first set out to create a comprehensive genetic toolkit, including a method for efficient gene integration, neutral loci for high expression, and tunable promoters.

In designing our toolkit, we first considered the challenge of efficiently integrating heterologous genes in desired genomic locations. Filamentous fungi are notoriously poor at homology-based recombination and thus transformation with linear DNA templates often results in off-target, ectopic integrations 24 . To overcome this, strains deficient in non-homologous end-joining (NHEJ) have historically been used 25 . However, disruption of NHEJ presents potential issues with genomic instability or increased risk of DNA damage 26 . Recently, CRISPR-Cas9 has revolutionized the ability to transform and genetically modify fungi, including A. oryzae 18 , 27 , 28 , 29 . For example, the recently developed state-of-the-art CRISPR-Cas9 method for A. oryzae allows high-efficiency modification of strains proficient in non-homologous end-joining (NHEJ) 28 . The method uses plasmids to drive constitutive Cas9 and sgRNA expression, and the plasmid can be readily removed using selection, which enables sequential rounds of transformation and genome modification 28 .

To efficiently engineer A. oryzae , we sought to develop an alternative, easy-to-use CRISPR-Cas9 approach that is compatible with readily available commercial reagents 26 , minimizes the possibility of off-target effects and toxicity resulting from constitutive Cas9 expression 30 , 31 , 32 and incorporates a straight-forward phenotypic screen to verify that integration at the locus of interest has taken place 23 . Rather than encoding the Cas9 and sgRNAs from a plasmid, our method involves direct transformation of CRISPR-Cas9 Ribonucleoprotein complexes (RNPs), which can be formed in vitro from commercially available Cas9 protein and sgRNAs. At the start of our study, the RNP-based approach had been demonstrated as a strategy for rapid and precise genome editing compatible with high-throughput screening in diverse filamentous fungi 29 , 33 , 34 , 35 , 36 but had not been experimentally validated for gene integration in A. oryzae .

In our method, the DNA template introduced to fix the Double-Stranded Breaks (DSBs) harbors a pyrG marker to allow for both positive selection using uracil/uridine auxotroph and negative selection using media with 5-fluoroorotic acid (5-FOA). Although pyrG marker recycling has been demonstrated in A. oryzae , previous approaches did not incorporate CRISPR-Cas9 and required an NHEJ-deficient strain to avoid off-target integrations 37 . To overcome potential issues with ectopic integrations in wild-type A. oryzae , we designed the system such that a successful loop out of the pyrG marker can only occur if the fixing template is integrated at the locus of interest, in which case it will be flanked by two identical 300 bp sequences. In this system, ectopic integrations resulting from NHEJ are unable to loop out and survive on media supplemented with 5-FOA (Fig.  1B ). This phenotypic screening approach to locus-specific gene integration and pyrG marker recycling was first established using a plasmid-based CRISPR-Cas9 system in the related A. niger , where it allowed precise and efficient genome modification 27 . In A. niger , all colonies surviving on 5-FOA had the expected genome modification, highlighting the robustness of this approach. The A. niger method was used for mutation and gene deletion but was not utilized for integration and expression of proteins 27 .

To evaluate our RNP-based method for integration and expression in A. oryzae , we first targeted a GFP-expression cassette to the wA locus, which controls spore pigmentation, into a Δ pyrG mutant of the common laboratory strain RIB40 28 (Supplementary Table  1 ). This experiment yielded strains displaying the expected white spore phenotype, GFP expression, and fixing template insert (Fig.  1C, D , and Supplementary Fig.  1A, B ). To explore whether this method works beyond the model laboratory strain RIB40, we collected a group of A. oryzae strains with distinct industrial uses and geographical origins (Supplementary Fig.  2 ). Whole-genome sequencing of these strains revealed that these strains are phylogenetically distinct from one another and display minor variations in both the number of coding genes and biosynthetic gene clusters (Supplementary Table  2 ). To enable gene editing, we first generated Δ pyrG strains by targeting two RNPs to the pyrG locus and plating on agar supplemented with 5-FOA, uracil, and uridine to select mutants. PCR amplification of the region revealed clear mutations at the predicted sgRNA cut sites, including deletions and insertions, likely resulting from erroneous fixing by A. oryae . We observed no off-target effect on the surrounding genes despite not providing a fixing template, highlighting the precision of the RNP-based editing method (Supplementary Fig.  3 ). We then successfully introduced a GFP-expression cassette at the wA locus to alter the spore phenotype and establish heterologous protein production across the strain collection (Supplementary Fig.  4 ). These strains had not previously been genome sequenced or edited, suggesting that “wild” A. oryzae strains with potentially favorable phenotypes could be efficiently modified using our method. Moreover, these results indicate that the Δ pyrG mutants required for our method are readily generated in a single-step transformation.

The RNP-based CRISPR-Cas9 method was highly efficient. PCR amplification of RIB40 transformants at the wA locus revealed a 90% integration efficiency with 950 bp homology arms (Fig.  1E ), similar to the high targeting efficiency of the previously developed plasmid-based CRISPR-Cas9 system 28 . Even with homology arms as short as 25 bp, the method proved highly efficient (70%), suggesting that PCR primer overhangs could be used to specify the integration locus of interest (Fig.  1E ). The high efficiency with such small homology arms is consistent with findings from other filamentous fungi 34 , 38 . We also found that we could miniaturize the transformation to smaller volumes and remove the final top agar step without major decreases in integration efficiency, making the transformation process quicker and easier compared to the standard A. oryzae protoplast transformation protocol and compatible with a microplate format 28 , 35 (Supplementary Fig.  1E ). Although our method utilizes two RNP complexes for each locus to maximize the likelihood of DSB based on previous successes in fungi 27 , 39 , 40 , 41 , we found no major difference in integration efficiency between using one and two RNP complexes across two distinct genomic loci in A. oryzae ( wA and niaD ) (Supplementary Table  3 ).

A core design feature of our method is the ability to recycle the pyrG marker upon insertion to the correct locus, as transformants should undergo marker excision in the presence of 5-FOA. We first confirmed successful recycling from the wA locus. Consistent with previous results in A. niger 27 , surviving colonies displayed uracil-uridine auxotrophy and marker excision, indicating successful pyrG removal. We observed this across three out of three colonies analyzed, highlighting the robustness of the growth-based method to assess locus-specific marker recycling (Fig.  1F, G , and Supplementary Fig.  1C, D ). Finally, we successfully integrated GFP-expression cassettes and excised pyrG markers at the niaD locus, which controls nitrate assimilation, and the yA locus, which contributes to spore coloration (Supplementary Fig.  5 and Supplementary Table  1 ) 28 .

The recyclability of the pyrG marker allows for potentially endless rounds of sequential engineering. To evaluate this possibility, we transformed the looped-out wA : gfp strains with an mCherry cassette targeted to the niaD locus. Positive transformants demonstrated the expected phenotype and protein expression (Fig.  1H, I ). We then successfully recycled the pyrG marker from the niaD locus, enabling sequential engineering using our marker recycling approach (Fig.  1J ). Finally, we also targeted the wA and niaD loci simultaneously in a single experiment. However, in contrast to the high efficiency observed with single integration at the wA locus, simultaneous modification at the niaD and wA loci was only 30% efficient with 950 bp homology arms (Supplementary Fig.  6 ). This is consistent with previous findings of reduced efficiency with multiple RNP complexes and fixing templates in fungi 35 . Overall, these results establish the RNP-based method as a method for genome modification and protein expression in diverse strains of the edible fungus A. oryzae . This method displays a comparable high efficiency and scope as the plasmid-based method for genetically engineering this fungus 28 . The use of commercially available reagents and the ability to phenotypically screen for insertion at the locus of interest makes the protocol easy to use.

Identification and evaluation of neutral loci for gene expression

After establishing the RNP-based CRISPR-Cas9 method for gene modification, we considered another challenge in genetic engineering of filamentous fungi: where to integrate genes for overexpression. While multi-gene expression has been achieved in A. oryzae for natural products biosynthesis, the historically preferred method involves plasmids that integrate randomly throughout the genome 42 . These can cause unintended pleiotropic effects or genomic instability and make it challenging to compare phenotypes between constructs and strains 43 . In contrast, neutral loci, intergenic regions, and genomic safe havens that allow targeted expression without interfering with host physiology, is a standard feature of engineering for many bacteria and yeasts such as S. cerevisae 44 , 45 . Recently, genome sequencing of A. oryzae transformed with randomly integrated plasmids revealed two intergenic regions (called “hot-spots”) that were successfully targeted with CRISPR-Cas9 for expression of natural products genes 46 . However, to this date, neutral loci have not been systematically identified and evaluated for the efficiency of gene integration and level of protein expression across the A. oryzae genome. This information, along with readily available plasmids and DNA parts targeted to characterized loci, is critical to advance engineering efforts, as has been shown in S. cerevisae 45 .

We took a computational approach to identify candidate-neutral loci in A. oryzae . We first identified intergenic regions in the A. oryzae RIB40 genome. Using publicly available RNA-sequencing data across diverse conditions and growth stages, we ranked the expression level of the two genes immediately surrounding the intergenic region, thus generating a list of candidate loci predicted to enable high gene expression (Fig.  2A and Supplementary Data file  1 ). From this set, we selected 10 promising high-expression regions (>4.8 kb) spread across A. oryza e chromosomes for further evaluation (Fig.  2B , Supplementary Tables  4 and 5 ). We then integrated cassettes harboring GFP under control of the strong, constitutive pTEF1 promoter, and assessed fluorescence using flow cytometry on the conidia of looped-out strains 47 (Fig.  2B, C and Supplementary Fig.  7 ).

A A computational approach was used to identify intergenic regions with high expression of surrounding genes. The highest expressing regions were selected as promising neutral loci for further experimental evaluation. B Targeting plasmids were designed with the 5′ and 3′ homology arms as well as the specific 300 bp sequence for the locus of interest. The plasmid harbors a GFP-expression cassette driven by the constitutive pTEF1 promoter and terminated by the commonly used TamyB terminator, both from A. oryzae . The plasmids were cloned in E. coli and were linearized using PCR to create linear fixing templates that target the locus of interest. C Flow cytometry of conidia constitutively expressing pTEF1 was used to evaluate expression strength at the neutral locus of interest and determine their suitability for engineering efforts. A representative microscopy image showing GFP expression from A. oryzae conidia is shown. Scale bar = 25 µm. The flow cytometry figure was created was created using BioRender ( http://BioRender.com ). D Integration efficiency of GFP-expression cassette at neutral loci. All loci except for chro3_1 displayed a high efficiency of integration (>50%), as assessed by PCR. We could not detect insertion at chro3_1 by PCR. E GFP expression (expressed as Mean Equivalents of Fluorescein, or MEFL) across neutral loci. All loci except for chro4_2 displayed expression levels above the background strain (RIB40) and were higher than the amyA locus, which was included as a positive control to validate the method. Results are average and standard error of the mean (SEM) of three biological replicates.

Out of 10 tested loci, 9 showed high-efficiency integration (>50%) (Fig.  2D ). We could not detect successful gene insertion at chro3_1 by PCR amplification, suggesting issues with PCR amplification or the integration itself. Following marker loop out, we detected GFP expression above background levels from 8 of the remaining 9 loci, with chro4_2 displaying no clear GFP expression (Fig.  2E ). Expression levels were largely consistent across the loci, with the highest (chro6-1) showing ~25% higher expression than the lowest (chro7-1) (Fig.  2E ). All loci displayed higher expression than the amyA locus, which was included as a positive control to validate the method. Finally, colony growth and morphology were consistent between all strains and similar to those of the background strain, suggesting no gross effects of gene integration and expression on fungal growth (Supplementary Fig.  8 ). Overall, these efforts identified not only neutral loci, but also a set of plasmids and sgRNAs that facilitates easy transformation and expression for diverse purposes (Supplementary Table  5 ). Our computational identification and experimental evaluation using flow cytometry provides a framework for how to identify and evaluate promising candidate loci across fungal hosts.

Expansion of the promoter toolkit using a synthetic expression system and bidirectional promoters

An additional challenge in engineering edible filamentous fungi is the narrow set of characterized parts available for gene regulation, as fungal promoters remain limited in both sophistication and scope. For example, only a handful of endogenous promoters have been used for gene expression in A. oryzae , and these are either regulated by the nutrient source (such as the amylase or glucoamylase promoters), have a limited dynamic range, or a poorly understood mode of regulation 48 , 49 , 50 . Synthetic expression systems (SES), which are widely available in yeast and bacteria and increasingly in mammalian cells and plants, could address the technical limitations of current fungal expression tools and expand engineering opportunities in A. oryzae 51 , 52 , 53 , 54 , 55 , 56 , 57 . SES couple synthetic transcription factors (sTF) with minimal core promoters (Cp) and DNA binding sites (UAS) and offer an orthogonal and highly programmable mode of gene expression 18 (Fig.  3A ). The Tet-On SES has shown promise in A. niger and A. fumigatus for inducible and titratable gene expression 58 , 59 , but food applications are limited by the cost and potential food incompatibility of the small molecule inducer. In contrast, a constitutive SES based on the Bm3R1 DNA binding domain and the VP16 activation domain was recently established in the two non-edible, industrial filamentous fungi A. niger and T. reesei . The highly modular SES showed high programmability and stability across the two hosts and afforded high secreted protein expression independent of the composition of the growth medium 18 .

A Design of synthetic expression system (SES). Coupling a synthetic transcription factor (sTF, composed of an Activating Domain = AD and DNA binding domain = DBD) and Upstream Activating Sequences (UAS) enables orthogonal and highly programmable gene expression from core promoters (Cp). B Confirmation of the Bm3R1-VP16-based SES in A. oryzae using the An_201205 core promoter. Fluorescence imaging that the SES in A. oryzae requires both the sTF and the UAS for expression. Scale bar = 50 µm for −UAS, 25 µm for other strains. C Conidia from strains shown in ( B ) were subjected to flow cytometry for fluorescence quantification of the constitutively expressed mCherry (expressed as Mean Equivalents of Texas Red, or METR). Results are average and SEM of three biological replicates. D Core promoter screen using the SES in A. oryzae . 200-bp sequences were cloned upstream of mCherry and fluorescence intensity was quantified using flow cytometry of conidia. The full-length, constitutively expressed promoter pTEF1 was included as a benchmark for promoter strength. Results are average and SEM of three biological replicates. E Proteomic comparison of intracellular mCherry abundance between the core promoter, Ao_0583, and the full-length starch-inducible endogenous promoter pAmyB from A. oryzae . Proteomics was conducted on lyophilized mycelia grown in liquid cultures (CDA medium with dextrin or glucose as the sole carbon source). Results are average and SEM of three biological replicates. F A minimal bidirectional promoter (Syn-BD) constructed of 2x UAS binding sites and the gpdA and hhfA core promoters can drive dual mCherry and GFP expression. Scale bar = 25 µm for RIB40, 50 µm for engineered strain. G Identification and evaluation of endogenous bidirectional promoters from A. oryzae . Flow cytometry quantification indicated that two promoters, p2-1 and p4-2, could drive bidirectional gene expression at varying levels. p4-2 was similar to Syn-BD in terms of expression. A concatenated sequence of pAmyB and pTEF1 pointing in opposite directions was included as a positive control. MEFL = mean equivalents of fluorescein. METR = mean equivalents of Texas red. Results are average and SEM of three biological replicates.

We sought to expand the engineering possibilities in the edible A. oryzae by building on these advances in the industrial workhorses A. niger and T. reesei . To first establish SES as a mode for gene regulation in A. oryzae , we initially evaluated the ability of the previously characterized Bm3R1-NLS-VP16 sTF to drive mCherry expression from a core promoter. Using the RNP-based CRISPR-Cas9 integration tools and neutral loci, we genetically integrated the sTF and drove low levels of basal expression of this transcription factor using a characterized core promoter from A. niger (An008). In a separate genomic location, we integrated an mCherry cassette harboring 6x UAS upstream of the A. niger An201205 core promoter. There was clear expression of mCherry in mycelia and conidia using the full system. The UAS and the sTF were both necessary for activity, validating the predicted function of the SES in A. oryzae 18 (Fig.  3B, C ).

To explore the programmability of this modular SES in A. oryzae , we initially focused on core promoters, as the identity of these short 200-bp sequences influences the level of gene expression upon sTF binding 18 , 57 . Using available transcriptome data and a curated list of promoters from highly expressed A. oryzae and Aspergillus flavus genes, we first assembled an initial library of twelve 200-bp core promoters and evaluated their ability to drive mCherry expression in the SES (Supplementary Table  6 ). We used flow cytometry of conidia 47 as an initial screening approach to assess expression and used the strong, constitutive pTEF1 promoter as a benchmark for comparison. Three of twelve selected core promoters did not drive mCherry expression at levels above the background strain (Fig.  3D ). However, across strains producing detectable mCherry, mean expression across the core promoter library displayed a 14-fold expression range, from 0.25 to more than 5-fold pTEF1. These results indicate that, like full-length promoters, core promoter sequences can drive divergent transcriptional outputs in A. oryzae (Fig.  3D ). Proteomics analysis of mCherry in biomass grown in submerged fermentations confirmed that core promoters could also drive protein expression in mycelia, including at levels that were several-fold higher than pTEF1 (Supplementary Fig.  9A ). There was a significant correlation between the flow cytometry and proteomics data ( r  = 0.82, R 2  = 0.67, p  < 0.01, Supplementary Fig.  9B ). This suggests that flow cytometry is a useful screening approach to identify constitutive promoters. Nonetheless, following up on flow cytometry screening results in mycelia may be needed for establishing the precise promoter strength for submerged fermentations.

To further benchmark the SES system we used proteomics to compare the strength of the SES promoter Ao_0583, identified as >4-fold stronger than pTEF1 in both conidia and mycelia, with the starch-inducible pAmyB promoter, one of the strongest known endogenous A. oryzae promoters that is frequently used for high protein expression and secretion from submerged cultures 21 , 50 . Strikingly, Ao_0583 was approximately 6-fold stronger than that of the pAmyB promoter when the strain harboring it was grown under inducing conditions. mCherry levels were estimated to comprise approximately 13% intracellular protein under the Ao_0583 system, and only 1–2% in the pAmyB expression strain (Fig.  3E and Supplementary Fig.  10 ). While mCherry levels did not differ between glucose and dextrin when using the SES, pAmyB expression increased on dextrin, the predicted inducer of pAmyB (Fig.  3E and Supplementary Fig.  10 ). Thus, the SES permits high protein expression independently of the carbon source and avoids the complex multi-step regulation and global metabolic changes involved in pAmyB-driven expression in A. oryzae 60 . To our knowledge, the expression levels afforded by the SES far outperform any characterized promoter in A. oryzae .

In addition to the limited set of available mono-directional promoters, there is a lack of bidirectional promoters for filamentous fungi. Bidirectional promoters, which are available in yeast, could accelerate genetic engineering in edible filamentous fungi by enabling assembly of multi-step metabolic pathways or multi-protein complexes through fewer transformations 61 , 62 . We addressed this challenge in two ways. First, we created a synthetic bidirectional promoter (Syn-BD), as the modular nature of the SES enables different parts to be combined to create highly programmable modes of gene expression 63 . By combining two core promoters ( gpdA and hhfA ) with 2× UAS binding sites, we created a 485-bp bidirectional promoter was sufficient to drive bidirectional gene expression using the SES (Fig.  3F ). Second, we computationally identified candidate endogenous bidirectional promoters using publicly available RNAseq data collected across diverse growth conditions (Supplementary Data file  2 ). Out of five computationally identified bidirectional promoter candidates (Supplementary Table  7 ), two (p2-1 and p4-2) could drive mCherry and GFP expression in a bidirectional fashion (Supplementary Fig.  11 ). The p4-2 promoter displayed similar levels of expression as the rationally designed Syn-BD and a control bidirectional promoter composed of concatenated pAmyB-pTEF1 sequences pointing in separate directions (Fig.  3G ). Interestingly, the sequence of the identified p4-2 endogenous promoter in A. oryzae has the same length and genomic context as the H3/H4 histone promoter which was previously identified and evaluated in a range of Aspergilli , but not in A. oryzae 64 . This suggests that our computational pipeline might be broadly useful to identify bidirectional promoters in filamentous fungi. Overall, these results expand the set of gene regulation tools and promoters available for engineering the edible A. oryzae .

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal

Having established a synthetic biology toolkit for A. oryzae , we next sought to deploy our tools to bioengineer its edible mycelium, as a first step toward enhancing its value as mycoprotein. We were inspired by the recent commercial success of bioengineered Saccharomyces used for brewing, which have been modified for improved cost savings, sustainability, and sensory profiles 17 . To explore whether genetic modification could similarly enhance foods made with filamentous fungi, we set out to modify endogenous biosynthetic pathways that could potentially improve the nutritional value and sensory appeal of the edible A. oryzae mycelium for alternative meat applications.

We initially focused our engineering efforts on ergothioneine, a bioactive amino acid and powerful antioxidant. Low plasma levels of ergothioneine are correlated with cardiovascular disease and neurological decline, and humans encode a specific ergothioneine transporter that uptakes ergothioneine from the diet, underscoring the potential importance of this molecule in human health 65 . While many foods contain low levels of ergothioneine, fungi are the major dietary source 66 . Work in the model ascomycete mold Neurospora crassa has revealed that fungal ergothioneine biosynthesis involves two enzymes, Egt1 and Egt2, that convert cysteine, S-adenosylmethionine, and histidine, to ergothioneine 67 , 68 , 69 (Fig.  4A ). N. crassa Egt1 and Egt2 were recently co-expressed in A. oryzae using plasmid-based random integration 70 . Rice cultured with transformants in solid-state fermentation had elevated ergothioneine levels, but the levels in the biomass alone were not investigated. Untransformed A. oryzae produced low levels of ergothioneine, suggesting that this fungus may harbor endogenous pathways for production 70 .

A Fungal biosynthesis of ergothioneine, a powerful antioxidant associated with several health benefits in humans. The characterized biosynthetic pathway from the fungus Neurospora crassa involves the enzymes Egt1 and Egt2. B A. oryzae homologs of N. crassa Egt1 and Egt2 were identified bioinformatically (see Supplementary Table  7 and Supplementary Fig.  12 for details) and expressed from neutral loci using a bidirectional promoter (strain VMR-Eg1-2) or as two separate genes at two different genomic locations, with each gene under the control of its own promoter (strain VMR-Eg1_2). The strategy is described in Supplementary Fig.  12 . Oyster mushroom, the dietary mushroom with the highest ergothioneine content, was included for comparison. Biomass was analyzed by LC–MS. Results are average and SEM of three biological replicates. C Engineering of heme biosynthesis in A. oryzae biomass. The strategy is described in Supplementary Fig.  15 . Heme was quantified using LC–MS in the biomass. The intracellular heme levels in the engineered strain were 4-fold higher than in the background strain, RIB40, and 40% of those found in IMPOSSIBLE™ burger made from plants, a leading plant-based meat product incorporating heme for flavor and color, was included for comparison. Results are average and SEM of three biological replicates. D Color of harvested background and engineered heme strain after culturing. The engineered strain overproducing heme (VMR-HEM_v1) was distinctly red in color, while RIB40 was off-white. The harvested fungal biomass could be readily formulated into an imitation meat patty with minimal processing. The color difference remained upon cooking, further enhancing the meat-like appearance of the naturally textured fibrous biomass.

Instead of introducing foreign genes, we hypothesized that by changing the expression of potential endogenous A. oryzae genes involved in ergothioneine biosynthesis, we could elevate production in the edible biomass to levels found in dietary mushrooms. To identify candidates, we searched the A. oryzae genome for homologs of N. crassa Egt1 and Egt2. We found two A. oryzae ortholog candidates sharing 49.2 and 45.1% amino acid sequence (Supplementary Table  8 ). Sequence alignment indicated conservation of key residues or functional groups bioinformatically predicted to be involved in substrate binding in Egt1, as well as residues structurally confirmed to participate in catalysis in Egt2 68 (Supplementary Fig.  12 ). We then integrated the A. oryzae homologs (named AO_Egt1 and AO_Egt2) at neutral loci and drove expression of both genes from either a bidirectional promoter (strain VMR-Eg1-2), or as two separate genes in two separate genomic locations (strain VMR-Eg1_2) (Supplementary Fig.  12 ). High-resolution Liquid Chromatography-Mass Spectrometry (LC–MS) was used to detect ergothioneine in samples (Supplementary Fig.  13 ). Consistent with previous observations 70 , we detected low levels of ergothioneine in the mycelium in the background strain RIB40 (Fig.  4B and Supplementary Fig.  13 ). However, the bidirectional promoter and separate promoter strains elevated ergothioneine 11-fold and 21-fold, respectively, over RIB40 (Fig.  4B ). While the ergothioneine levels in VMR-Eg1-2 was similar to those found in oyster mushroom, the highest known dietary ergothioneine source 66 , the mean levels in strain VMR-Eg1_2 were 1.5-fold higher. We observed no major difference in protein content between the wild-type and engineered strains; however, ergothioneine overproduction was associated with a slight growth defect, suggesting a metabolic burden of ergothioneine production under the growth conditions (Supplementary Fig.  14 ). Overall, these results implicate the endogenous genes AO_Egt1 and AO_Egt2 in ergothioneine biosynthesis in A. oryzae and validate the metabolic engineering approach to alter the molecular composition of mycoprotein.

Having validated our tools to increase levels of bioactive molecules for enhanced nutritional value, we asked whether a similar approach could be applied to sensory properties of the edible biomass to more closely mimic animal meat. For example, even though the A. oryzae biomass has a meat-like fibrous texture owing to its microscopic morphology, the biomass, which is off-white, would necessitate color addition for many meat applications. As a first step toward improving the meat-like flavor composition and appearance of the edible biomass using bioengineering, we initially targeted the biosynthesis of heme, an essential cofactor that catalyzes a wide range of reactions across all domains of life and gives red meat its color and contributes to flavor upon cooking 71 . IMPOSSIBLE Foods, a leading plant-based meat producer, has taken advantage of these properties of heme and adds a purified soy Leghemoglobin (LegH) produced with the yeast Pichia pastoris to its products based on plant protein isolates to create realistic alternatives that look like red meat 72 , 73 . Other plant-based meat producers have now followed suit with similar hemoglobin addition strategies 74 .

We reasoned that by modulating the expression of key heme biosynthetic enzymes, we could elevate intracellular heme in the edible fungal biomass to levels found in leading meat alternatives incorporating heme for flavor and color. Fungal heme biosynthesis is carefully regulated at the transcriptional and post-translational levels and involves eight dedicated enzymes, which are split between the mitochondria and the cytosol 75 (Supplementary Fig.  15 ). We identified potential heme biosynthesis proteins in A. oryzae by searching the genome for sequences found in S. cerevisiae 76 (Supplementary Table  9 ). There is limited experimental information about heme biosynthesis in filamentous fungi, but based on successful engineering efforts from yeast 77 , 78 , and studies of individual heme biosynthetic enzymes in different Aspergilli 75 , 79 , 80 , 81 , we initially targeted expression of predicted rate-limiting enzymes, including ALAS (biosynthetic enzyme#1), PBGD (#3), UROD (#4), and CPO (#5). Additionally, we mutated key cysteine residues in the Heme Regulatory Motif (HRM) of ALAS to remove potential feedback inhibition by heme 82 (Supplementary Fig.  15 ). Importantly, high levels of free heme and the porphyrin intermediates can be toxic to the cell, causing oxidative damage and hampering growth 77 , 83 . To address this potential challenge, we expressed two copies of Soy Leghemoglobin, the FDA-approved protein used in IMPOSSIBLE meat 72 , as a potential heme sink, using both the SES and the significantly weaker pTEF1 promoter. Though the regulation of heme biosynthesis has not been characterized in detail in filamentous fungi, simultaneous elevation of biosynthetic enzymes and a heme-binding protein was necessary to increase heme levels without causing excessive toxicity in S. cerevisiae 77 , 78 . The final engineered strain A. oryzae contained a total of separate six modifications (Supplementary Fig.  15 ).

We used high-resolution LC–MS to detect heme across all samples (Supplementary Fig.  16 ). The biomass of the engineered strain contained 4-fold higher levels of heme compared to the non-engineered strain, on a dry weight basis. These levels of heme in the engineered strain were nearly half (40%) of those found in IMPOSSIBLE meat (Fig.  4C ). Increasing levels further may require tuning pathway flux or making additional modifications beyond biosynthetic enzyme expression levels, as was recently shown in S. cerevisae 76 . However, to our knowledge, this is the highest levels of intracellular heme in fungal mycelium and a rare example of heme biosynthesis engineering in filamentous fungi. Given the importance of heme for enzyme production for biofuels and medical applications 78 , 84 , we envision that these strains and approaches could have broad applicability for engineering efforts beyond food.

Upon harvesting the biomass of the heme overproducer, we noticed that it was red in color, compared to the off-white color of the background strain. In contrast to other plant-based meat alternatives, which require extensive processing and ingredient addition to transform off-flavor plant protein isolates (such as soy or pea) to meat alternatives, this bioengineered mycoprotein required minimal post-harvest processing for formulation into an imitation red meat patty following a standard mycoprotein production protocol 11 (Fig.  4D ). The only processing needed was removing excess liquid from the biomass prior to grinding and cooking. The color difference between the background and engineered strains remained after cooking, enhancing the meat-like appearance of the naturally textured, fibrous fungal biomass (Fig.  4D ). There was no decrease in the growth yield or protein content (46%, on a dry weight basis) in the engineered heme strain relative to the background strain (Supplementary Fig.  17 ). The engineered mycoprotein also contained all the essential amino acids, suggesting a promising nutritional profile (Supplementary Fig.  17 ). Taken together, these data suggest that the engineered edible fungal mycelium could have promise in meat alternative applications.

Filamentous fungi are widely used for the industrial production of enzymes and metabolites and recently have found more widespread use in both sustainable materials and foods 12 . However, genetic tools for these organisms have historically been limited in both sophistication and scope, preventing both engineering efforts and fundamental studies. Recent advances in CRISPR-Cas9 technology have dramatically improved the possibilities of modifying diverse mushrooms and molds, including the food-safe, edible fungus A. oryzae 27 , 28 , 29 , 34 . In contrast to industrial strains such as T. reesei , which was recently used to produce milk and egg proteins at lab scale 14 , the historically consumed A. oryzae has potential uses across fermented foods, food protein production, cellular agriculture, and mycoprotein 13 , 20 , 22 , 23 .

To enable bioengineering for these diverse applications, we developed a ready-to-use toolkit that is now available to the research community and includes DNA parts for integration and regulation of genes and pathways. Similar toolkits are available in S. cerevisiae , where they have significantly expanded opportunities for genetic engineering 45 . We hope that our tools will be similarly useful for expanding the engineering possibilities in A. oryzae , alongside other recently developed genome modification methods such as base editing 85 , in vivo DNA assembly in NHEJ-deficient strains 86 , and protein expression screening 87 . Additionally, we envision that the computational and experimental approaches used here – for identification and evaluation of neutral loci and design and identification of promoters – could be broadly useful for constructing genetic toolkits and engineering diverse fungal hosts.

We used our tools to enhance the molecular composition and appearance of the mycelium as a first step toward improving its nutritional and sensory properties. First, we engineered A. oryzae mycoprotein to overproduce the nutraceutical ergothioneine at levels that are higher than those in mushrooms, the highest known natural source from the diet. While ergothioneine has been produced in a range of microbial hosts for the purpose of isolating the nutraceutical 88 , 89 , our work represents a proof of concept of modifying endogenous ergothioneine biosynthesis for mycoprotein applications. Separately, we engineered the A. oryzae mycelium to overproduce heme, a key flavor-and-color molecule in red meat, at levels that are close to half those found in leading plant-based meats. Our engineering of the edible fungal biomass for alternative meat presents an alternative approach to fungal food beyond the production of secreted animal proteins, which is a less efficient fermentation process and has a higher environmental footprint than biomass production 6 , 9 . Future engineering targets for edible fungal biomass could include lipid pathways for flavor, amino acids for nutrition, structural alteration for texture improvement, or enzymes for improved growth on affordable, complex feedstocks. However, it is important to note that our work represents early prototypes, and further assessment of the sensory attributes, consumer acceptability, potential food safety concerns, and the regulatory landscape around genetically modified organisms (GMO), is needed to bring engineered edible fungi from lab bench to the table.

A. oryzae , like many of the strains that form the basis of fungal foods available on the market, has been genetically modified through extensive selection and breeding throughout human history 90 , 91 . Genetic modification using contemporary gene editing tools such as CRISPR-Cas9 represents a natural next step in this long history of microbial gene modification to suit human needs and holds promise to further expand fungal strain diversity and accelerate the adaptation of fungal strains to the demands of current production methods and consumer preferences. Bioengineered edible plants and yeasts have demonstrated reduced environmental impact, improved nutrition, and improved flavor profiles compared to their non-engineered counterparts and are already available on the market 17 , 92 , 93 . We anticipate similar possibilities with genetic modification of edible filamentous fungi, as synthetic biology in these organisms is uniquely positioned to address the pressing environmental, ethical, and public health challenges of industrial animal agriculture.

All primers used for genome modification are shown in Supplementary Table  10 . All strains and plasmids used for strain construction are listed and described in Supplementary Tables  11 - 12 . The sequence files corresponding to each strain and plasmid can be found in the JBEI Public Registry ( https://public-registry.jbei.org/ ) 94 . All plasmids were propagated in Escherichia coli strain DH10B and purified by Miniprep (Qiagen). The plasmids generated in this study were based on the pTWIST_amp backbone (TWIST biosciences) and were constructed by Gibson assembly 95 using Gibson assembly master mix (New England Biolabs). PCR amplification was performed using NEB Q5 polymerase according to the manufacturer’s instructions (New England Biolabs). All genes were codon optimized for A. oryzae and ordered either as G-blocks from IDT or as complete, sequence-verified genes from IDT or TWIST biosciences. The coding sequences of heterologous genes in all plasmids were validated by Sanger sequencing (Azenta) or whole-plasmid sequencing (Primordium).

Growth conditions

A. oryzae strains were always grown at 30 °C. A variety of media were used in the transformation and cultivation of A. oryzae , and these are indicated below. They are referenced in the materials and methods section. Media were supplemented with 5 g/L uridine (Sigma–Aldrich, #U6381) or 2 g/L uracil (Sigma–Aldrich, #U1128) when supplementation to support growth of pyrG mutants was needed. Supplementation is indicated as UU throughout the manuscript.

GP medium (per 1 L of medium)

5 g yeast extract, 10 g polypeptone, 0.5 g MgSO 4 ⋅ 7H 2 O, 5 g KH 2 PO 4 . 20 g glucose was used as the carbon source unless otherwise indicated. Alternatively, 20 g dextrin (Sigma–Aldrich, #31400) was used as the carbon source.

PDA + 5-FOA + Uridine + Uracil (PDA + 5-FOA + UU) medium (per 1 L of medium)

39 g Potato Dextrose Agar (PDA, Sigma–Aldrich, #70139-500 G), 5 g uridine,2 g uracil, and 1 mg/mL 5-fluoroorotic acid (ThermoFisher, #R0812).

Bottom Agar + Methionine (BA + Met, per 0.5 L of medium)

1 g NH 4 Cl, 0.5 g (NH 4 ) 2 SO 4 , 0.25 g KCl, 0.25 g NaCl, 0.5 g KH 2 PO 4 , 0.25 g MgSO 4 •7H 2 O, 0.01 g FeSO 4 , 109.3 g sorbitol, 7.5 g agar, 10 g glucose, 0.75 g methionine. pH was adjusted to 5.5 prior to autoclaving.

Top Agar + Methionine (TA + Met, per 0.5 L of medium)

Same as BA + Met, but 4 g agar instead of 7.5 g agar per 0.5 L.

Minimal Medium Agar + Methionine (MMA + Met, per 0.5 L of medium)

Same as BA + Met, but no sorbitol added as the osmotic stabilizer.

CDA medium (per 1 L of medium)

3 g NaNO 3, 2 g KCl, 1 g KH 2 PO 4 , 0.5 g MgSO 4  × 7 H 2 O, 0.02 g FeSO 4 ·7H 2 O, 15 g agar. 20 g glucose was used as the carbon source unless otherwise indicated. Alternatively, 20 g dextrin (Sigma–Aldrich, #31400) was used as the carbon source.

CDA(Leu) medium

same as CDA medium but containing 10 mM leucine as the sole nitrogen source instead of the 3 g/L NaNO 3 .

Strain construction

A. oryzae strains were genetically modified using protoplast transformation (see standard transformation protocol below). All A. oryzae strains are described in Supplementary Table  12 . Regenerated protoplasts were restreaked onto MMA + Met plates to obtain single colonies and purify the potentially heterokaryotic conidia. Following 48 h of growth at 30 °C, the conidia of individual, single colonies were transferred to MMA + Met slants for growth for 48–72 h at 30 °C. These purified strains represented the strains used in all assays and characterizations. To confirm the insertion of genes at the correct locus, colony PCR was performed on conidia on slants using PHIRE direct plant PCR kit (ThermoFisher, #F130WH) by boiling conidia in 20 µL of dilution buffer for 10 min at 95 °C and using 1 µL of the conidial spore suspension as the template for PCR, which was set up according to the manufacturer’s instructions. Strains harboring the correct insertions were saved as glycerol stocks by suspending conidia in 30% glycerol (v/v). For the simultaneous targeting of wA and niaD loci in a single transformation, DNA templates of plasmids were prepared as described below and 10 µg of each plasmid, along 5 µL of each RNP complex (four total, two per locus) were added at the DNA-RNP incubation step. To check for spore coloration ( wA and yA mutants), strains were grown on PDA medium for 5 days at 30 °C. To check for nitrate assimilation ( niaD mutant), strains were grown on CDA and CDA-Leu for 5 days at 30 °C. To check for pyrG mutation and the associated uridine/uracil auxotrophy, strains were grown on CDA and CDA supplemented with 2 g/L uracil and 5 g/L uridine for 5 days at 30 °C. To assess the targeting efficiency at individual loci, and to evaluate the effect of homology arm length on integration at the wA locus, colony PCR of 10 individual strains was performed, and those displaying the correct band by PCR were deemed successful integrations. Varying homology arm lengths of the wA fixing template were obtained by linearizing the full-length template with different primers (see primer table, Supplementary Table  10 ). Flow cytometry or microscopy assays were used to assess the expression of fluorescent proteins (see below for details). To create the engineered strain VMR-Eg1-2, A. oryzae RIB40 pyrG mutant was transformed with the linear DNA template originating from JBx_250940 and the two 5′ and 3′ RNP complexes targeting the chro1-3 neutral locus. To create the engineered strain VMR-Eg1_2, A. oryzae RIB40 pyrG mutant was transformed with the linear DNA template originating from JBx_250936 and the two 5′ and 3′ RNP complexes targeting the chro1-3 neutral locus, and subsequently with the linear DNA template originating from JBx_250938 and the two 5′ and 3′ RNP complexes targeting the chro2-2 neutral locus. For the engineered strain overproducing heme (VMR-HEM_v1), A. oryzae RIB40 pyrG was sequentially transformed with linearized DNA originating from plasmids JBx_250942, JBx_250944, JBx_250946, JBx_250948, JBx_250950, JBx_236225, as well as the specific 5′ and 3′ RNP complexes associated with the target locus for integration. For the cultivation of RIB40, and the engineered strains overproducing ergothioneine and heme, 5 × 10 5 conidia were inoculated into 50 mL of GP-glucose medium (ergothioneine strains and corresponding RIB40 control) or 50 mL GP-dextrin medium (heme strain and corresponding RIB40 control) in 250 mL Erlenmeyer flasks. The strains were grown for 96 h at 30 °C, shaking at 160 rpm. Biomass was harvested by vacuum filtration over Miracloth. Biomass was lyophilized for extraction of metabolites and was dried for 7 days at 50 °C prior to recording of the dry mass.

Transformation of A. oryzae

Preparation of linearized dna fixing templates for transformation.

To generate linear DNA to be transformed into A. oryzae as fixing templates alongside CRISPR-Cas9 RNP complexes, the DNA was linearized using PCR from the corresponding plasmids harboring the DNA fixing template of interest. Briefly, 1 ng of plasmid DNA was used as the template for a 60 µL PCR reaction using the Q5 high-fidelity polymerase master mix (New England Biolabs, #M0492S) and following the manufacturer’s instructions for the PCR protocol. For each DNA template, five 60 µL reactions were set up in parallel to obtain sufficient DNA for transformation. The PCR reactions were then combined and purified using the QIAquick PCR purification kit (Qiagen) and were eluted at the final step in 25 µL of sterile water. This typically gave sufficient quantities of the large amount of DNA needed for protoplast transformation (>10 µg in 20 µL).

Preparation of RNP complexes for transformation

All CRISPR-Cas9 reagents were obtained from IDT, including the Alt-R S.p. HiFi Cas9 Nuclease V3 (IDT, #1081061), Alt-R CRISPR-Cas9 crRNA XT 2 nmol (customized sequence), and Alt-R CRISPR-Cas9 tracrRNA 100 nmol (IDT, #1072534). RNA duplex buffer was included as part of the tracrRNA. The crRNA, which is the sequence-specific RNA that targets the region of interest, was resuspended in 20 µL water for 100 µM final concentration. To hybridize the sequence-specific crRNA to the universal tracrRNA to generate the final sgRNA, 0.5 µL of crRNA (5 µM final concentration) was mixed with 0.5 µL tracrRNA (5 µM final concentration) in 9 µL RNA duplex buffer and the mixture was then heated for 5 min at 95 °C and was then left to cool. This hybridized mixture represents the final sgRNA which is ready to bind to the Cas9 protein to form the RNP complex. To create the final RNP complexes for transformation, 2.16 µL of the hybridized crRNA-tracrRNA (the sgRNA, final concentration 540 nM) was mixed with 0.18 µL of Alt-R S.p. HiFi Cas9 Nuclease V3 (final concentration 540 nM) and 17.66 µL buffer (9 mM HEPES, 67 mM KCl, pH 7.5). The mixture was incubated at room temperature for 20 min to form the RNP complex and was then transferred to ice. As specified below in the transformation protocol, 5 µL of each RNP complex was used per 200 µL protoplast transformation. The RNP complexes were always prepared fresh on the day of the transformation and were never subjected to freeze-thaw.

Standard transformation protocol

Protoplast-mediated transformation was used to transform Aspergillus oryzae . We followed the protocol from 28 , with minor modifications. To generate mycelial biomass for protoplast generation, pyrG mutant strains were grown in duplicate in 50 mL GP medium supplemented with uracil and uridine in 250 mL flasks at 30 °C, shaking at 160 rpm. Following 72 h of growth, mycelia were harvested by pressing the liquid from the mycelia in a 20 mL syringe harboring a sterile cotton ball.

Dry mycelia from one flask were then put in 10 mL of TF1 solution (Per 500 mL of water: 2.9 g maleic acid, 39.5 g (NH 4 ) 2 SO 4 , pH adjusted to 5.5 and filter sterilized) harboring 0.1 grams of YATALASE enzyme (Takara Bio, #T017) which was used to digest the cell wall. This was incubated shaking for two and a half hours at 30 °C and 160 RPM, and the tissue was pressed within a sterile syringe with cotton to collect the protoplasts within the flow-through. The flow-through was checked for cloudiness, which indicated the generation of protoplasts. The resulting protoplast solution (7–8 mL usually, as sometimes not all protoplast solution could be pushed through the cotton ball due to clogging) was centrifuged at 475 g for 10 min, and the supernatant was then discarded. The protoplasts were then gently resuspended in 10 mL of TF2 solution pre-warmed at 30 °C (Per 1 L of water: 218.5 g sorbitol, 10.95 g CaCl 2 •6H 2 O, 2.05 g NaCl, 1.21 g Tris buffer, pH adjusted to 7.5 and filter sterilized). The protoplast solution resuspended in TF2 was centrifuged at 475 g for 10 min to discard the supernatant. The protoplasts were resuspended in 1–2 mL of TF2 solution and 200 μL of protoplast solution was placed into 15 mL volume centrifuge tubes. At least 1.2 × 10 7 protoplasts/mL was needed, so it is useful to consider this concentration when resuspending in the 1–2 mL TF2 solution. Concentrations of ~10 8 protoplasts per mL were typically obtained in the standard digestion protocol.

To transform the protoplasts, a total of 10 µg of PCR-linearized DNA fixing template (in 20 µL sterile water) was added to the 200 μL protoplast solution. Then 5 µL of each of the two pre-formed sgRNA-Cas9 RNP complexes were added and the protoplast-DNA-RNP mixture was incubated on ice for 30 min. After 30 min, sequentially and slowly, 250 μL, 250 μL, and 850 μL aliquots of TF3 solution were added (TF3 solution: per 1 L of water: 600 g Polyethylene glycol (4000), 10.95 g CaCl 2 •6H 2 O, 1.21 g Tris buffer, pH adjusted to 7.5 followed by autoclaving) and left to incubate at room temperature for 30 min. A total of 5 mL of TF2 solution was then added to each protoplast solution then centrifuged for 10 min at 475 g to discard the supernatant. The protoplast pellet was resuspended in 500 μL of TF2. At this point, a bottom agar plate pre-warmed at 30 °C already brought out from its heating location (see above for bottom agar recipe). The 500 μL of protoplast suspension was then mixed with 5 mL of a liquid layer of top agar (see above for recipe) pre-warmed to 50 °C, then quickly spread uniformly across the bottom agar and left to solidify at room temperature. Transformants were left for 72 h to regenerate the protoplasts. Transformants were then restreaked according to the procedure described in “strain construction” above.

Miniaturized transformation protocol

To speed up and miniaturize the transformation protocol to make it compatible with a 96-well plate format, it was modified according to the process below. Protoplasts were generated by digestion according to the standard protocol. However, only 50 µL protoplasts were used for the transformation, and 1.75 µL of each RNP complex was added alongside 2.5 µg DNA to these protoplasts and the mixture was incubated on ice for 30 min as in the standard protocol. Then, only 212.5 µL TF3 solution was added to the DNA-RNP-protoplast solution, and the entire mixture was spread on a bottom agar plate following the standard washing, using an L-shaped spreader. No top agar was used in the regeneration of the protoplasts. All steps following the plating of protoplasts were the same as described above in the standard protocol. All results reported in the paper followed the standard transformation protocol unless otherwise indicated.

Generation of pyrG mutants

The two transformation protocols above describe how to transform pyrG strains using a linear DNA fixing template. To generate pyrG mutants in the first place, as we demonstrated with five different A. oryzae strains obtained from NRRL, the standard protocol was changed slightly according to the following modifications. No linear DNA was transformed. Only the two RNP complexes targeting the pyrG gene were added to incubate on ice with the protoplasts. At the final step, strains were plated onto top and bottom agar supplemented with 1 mg/mL 5-FOA 2 g/L uracil, and 5 g/L uridine. Following the regeneration of protoplasts, instead of being restreaked on MMA + Met plates for single colonies and then transferred to MMA + Met slants, strains were restreaked on MMA + Met supplemented with 1 mg/mL 5-FOA and 2 g/L uracil and 5 g/L uridine. Surviving colonies were transferred to slants with the same medium. Between two and three colonies of each strain were analyzed for auxotrophy by plating on CDA, CDA + Uracil + Uridine. One of these strains was subjected to further analysis by amplification of the pyrG gene and flanking regions using primers pyrG-2F and pyrG-2R. The 2640-bp amplicon was purified and subjected to sequencing. The sequences were aligned using Snapgene.

Excision of pyrG marker

To set up the excision to remove the pyrG marker via locus-specific recycling, we followed the protocol described in ref. 96 with minor modifications: spore suspensions were generated by adding 0.5–1 mL sterile water to MMA + Met slants harboring single colonies subjected to colony PCR. Slants were vortexed to resuspend conidia, and then 500 µL of suspension harboring between 10 5 and 10 6 conidia/mL was spread onto PDA + 5-FOA + Uridine + Uracil plates. The conidia were spread using an L-shaped spreader and plates were left to dry for 1–2 h. The plates were then incubated at 30 °C for 5–7 days, at which point healthy, robustly growing colonies appeared on the plates. Conidia from individual colonies that appeared healthy were transferred to PDA + 5-FOA + uridine + uracil slants, whereby they were subject to an additional 4–5 days of growth. These strains had the pyrG marker excised. To verify the marker excision, conidia from slants were subjected to colony PCR (as described in standard transformation protocol), or PCR on extracted genomic DNA. To extract genomic DNA, a small amount of conidia was transferred to 300 µL lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 1 mM EDTA, 10 mM Tris pH 8) in a bead-beating tube (Lysing Matrix Z, MP Biomedicals, catalog#: 116961050-CF). Bead beating was performed for 1 min. Then, samples were incubated at 65 °C for 30 min, vortexing every 10 min. 300 µL of phenol:chloroform:isoamyl alcohol 25:24:1 reagent was then added (Sigma–Aldrich, #P3803) and tubes were vortexed for 5 min and were then centrifuged at max speed for 10 min to separate the layers. 120 µL of the top aqueous layer was transferred to a new tube, and 210 µL of ice-cold 100% ethanol was added to precipitate the DNA. The DNA pellet was washed twice with 70% and was resuspended in 30 µL sterile water. 1 µL was used as the template for PCR reactions using the PHIRE direct plant PCR kit (ThermoFisher, #F130WH).

Whole-genome sequencing, assembly, annotation, and phylogenetic analysis of diverse A. oryzae strains obtained from NRRL

A. oryzae strains subjected to sequencing were obtained from NRRL (NRRL numbers: #2215, #5592, #32614, #1911, #6574). They were grown in GP-glucose medium (50 mL medium in 250 mL flasks) for 72 h prior to harvesting by vacuum filtration and flash-freezing in liquid nitrogen. gDNA extraction, sample quality assessment, DNA library preparation, sequencing, and bioinformatics analysis were conducted at Azenta Life Sciences. Genomic DNA was extracted using DNeasy Plant Mini Kit following manufacturer’s instructions (Qiagen). Genomic DNA was quantified using the Qubit 2.0 Fluorometer (ThermoFisher Scientific). NEBNext® Ultra™ II DNA Library Prep Kit for Illumina, clustering, and sequencing reagents was used throughout the process following the manufacturer’s recommendations. Briefly, the genomic DNA was fragmented by acoustic shearing with a Covaris S220 instrument. Fragmented DNA was cleaned up and end repaired. Adapters were ligated after adenylation of the 3′ends followed by enrichment by limited cycle PCR. DNA libraries were validated using a High Sensitivity D1000 ScreenTape on the Agilent TapeStation (Agilent Technologies) and were quantified using Qubit 2.0 Fluorometer. The DNA libraries were also quantified by real-time PCR (Applied Biosystems). The sequencing library was clustered onto lanes of an Illumina HiSeq 4000 (or equivalent) flow cell. After clustering, the flow cell was loaded onto the Illumina HiSeq instrument according to the manufacturer’s instructions. The samples were sequenced using a 2 × 150 bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into FastQ files and de-multiplexed using Illumina bcl2FastQ 2.17 software. One mismatch was allowed for index sequence identification.

Assembly, annotation, and prediction of biosynthetic gene clusters

The reads were filtered with TrimmomaticPE version 0.39 97 with the following parameters: LEADING:30 TRAILING:30 MINLEN:120. The filtered reads were used for de novo assembly using the SPAdes 98 genome assembler v3.13.1-1 with the following parameters --careful --cov-cutoff 100. The resulting assemblies were then processed with AUGUSTUS 99 v3.4.0, to obtain coding sequences and protein predictions. Augustus was executed using a gene model for Aspergillus oryzae to identify start and stop codons, introns, and exons. For the prediction of natural product production repertoire of the strains, the assembled genomes and their gene calling files were used for functional annotation and mining for natural products biosynthetic gene clusters using antiSMASH version 7 100 .

Phylogenetic analysis

The taxonomic affiliation of the A. oryzae strains used in this study (tree in Supplementary Fig.  2 ) was defined using a multilocus phylogenetic tree constructed with the genomes of 59 Aspergillus spp. strains which were obtained from the GenBank database. These genomes were processed with AUGUSTUS 99 v3.4.0, to obtain coding sequences and protein predictions. The predicted proteomes of the Aspergillus dataset. Given the closeness of the strains, a genome from a distantly related taxonomic group ( Trichoderma atroviridae ) was added to the dataset to reduce the number of shared orthologs. The core genome was then calculated using BPGA 101 this analysis led to a set 237 conserved proteins that were sorted, aligned 102 , and trimmed 103 , after this process 167 protein sequences remained. They were then concatenated, and an evolutionary model was calculated for each of the 167 protein partitions. Then a phylogenetic tree was calculated with IQtree2 v2.0.7 104 using maximum likelihood with 10,000 bootstrap replicates. The entire process was executed automatically using a script available at https://github.com/WeMakeMolecules/Core-to-Tree .

Computational identification of candidate neutral, highly transcribed integration sites for protein expression

These sites were identified from a dataset deposited under BioProject accession: “ PRJDB8293 ”. This set of Illumina RNAseq data included 18 libraries which were obtained from A. oryzae RIB40 growing in 50 mL cultures in Czapek–Dox liquid medium supplemented with 1% (w/v) Triton X-100 at 30 °C. This dataset was deposited previously by Wong et al. 105 . The specific datasets that were used are shown in Supplementary Table  13 . The reads were downloaded from the GenBank FTP using fastq-dump v2.113, the reads were then aligned to the A. oryzae reference genome (NCBI RefSeq assembly GCF_000184455.2) using subread package v 2.0.3 106 . To select highly expressed genes, we counted the number of reads that were mapped to each gene in the A. oryzae RIB40 genome using featureCounts v2.0.3 107 . The read counts were calculated independently for each run. As the read counts depend on the depth and processing of each sample, a single cutoff cannot be established. Instead, we used this value to rank genes from most expressed to not expressed using the numbers of reads mapped per library (Supplementary Table  13 ). Then we reasoned that gene that ranked top in all libraries, could be safely considered highly expressed. For selection of neutral, highly transcribed integration sites, we first identified all the intergenic regions in the genome and calculated the average read count of the genes flaking them. Then, we sorted these regions from highest to lowest by their average flanking gene read count. For each of the 18 libraries we selected the top 500 intergenic regions and filtered out those that were not found in all conditions; therefore, we selected intergenic regions that are flanked by constitutive, highly expressed genes. This led to a set of 334 regions that were filtered by length (>4.8 kb). Finally, we selected 10 promising intergenic regions spread across A. oryza e (Fig.  2 , Supplementary Table  4 ).

Computational identification of candidate endogenous bidirectional promoters

For the development of new bidirectional promoters for A. oryzae , we used the annotated genome of strain RIB40 to mine for all the coding sequences whose start codons are in opposite directions. Then we selected the gene pairs that were highly expressed in most conditions using the Wong et al dataset 105 described above. We then identified the promoter region. We used the same dataset or read counts used for the identification of neutral, highly transcribed integration sites.

Identification of core promoters for expression in A. oryzae using the synthetic expression system

To identify candidate core promoters from the A. oryzae genome, we searched for highly expressed genes from publicly available transcriptome data 108 . The gene list of A. oryzae RIB40 grown in CD-glucose in liquid cultures without ER stress was sorted by RPKM to generate a list of the top most highly expressed genes. The top eight most highly expressed genes (unique genes, no duplicates) were selected as candidate strong promoters. Additionally, pdcA , which appeared at #15 in the rank of this list was selected as other studies suggest this is one of the most highly expressed genes in A. oryzae 109 . thiA , which was ranked #20 in this list, was also selected, because it has successfully been used for overexpression in A. oryzae previously 49 . Finally, hhfA , which ranked #55 in the list, was selected, as it was part of the p4-2 bidirectional promoter used in this study. For each of these genes, the genetic DNA 200 bp upstream of the start codon was used as core promoter sequence and ordered as synthetic dsDNA for downstream cloning and transformation. Two additional core promoters that did not come from the transcriptome rank analysis and were not native sequences to A. oryzae were also included. These were An_201205 from A. niger , which was used previously in the development of a Synthetic expression system in A. niger and T. reesei 18 , as well as the core promoter for Afl_ecm33 from gene AFL2G_04718 in Aspergillus flavus . Afl_ecm33 has been successfully used before to express a secondary metabolite in A. oryzae (promoter P4 in this study 48 ). For the An_201205 core promoter, the sequence was identical to the one used previously 18 , but for Afl_ecm33, the 200 bp upstream of the start codon were selected as the core promoter.

Flow cytometry assays of conidia for fluorescence quantification

The overall approach followed the method for promoter evaluation described in ref. 47 but with minor modifications. For all flow cytometry assays, strains were grown on PDA + 5-FOA + UU slants (excised neutral loci strains) or PDA slants (all others) for 5–6 days at 30 °C to allow robust development of conidia. Conidia were harvested by the addition of 1 mL of sterile water, followed by vortexing. 250 µL conidial suspension was then transferred to a 96-well plate (Corning, Falcon Tissue Culture Plate, #353072). Flow cytometry assays were performed on the BD Accuri C6 instrument (BD Biosciences) using the following settings: Run limit = 50,000 events, FSC-H threshold <80,000, agitation = 1 cycle every 1 wells. Raw fluorescence data were converted into MEFL (mean equivalents of Fluorescein, for GFP) or METR (mean equivalents of Texas Red, for mCherry), using a fluorescence beads standard (Spherotech, #RCP-30-5A). At least three biological replicates were run for each sample. FlowJo software (version 10) was used to analyze the data.

Fluorescence microscopy

Strains were grown on either CDA, CDA-Leu, or CDA-dextrin for 4–5 days at 30 °C. Fluorescent protein expression was then imaged in mycelia (edge of the colony) using Leica Microscope DM6B (Leica) and the associated Leica LAS X software (v.5.1.0).

Proteomic comparison of mCherry expression across media and promoters

to compare the expression of mCherry under endogenous promoters and the core promoters in the synthetic expression system, conidia from three different transformants per construct were inoculated at 5 × 10 5 conidia in 50 mL of either CD-dextrin or CD-glucose medium in 250 mL Erlenmeyer flasks. Strains were grown for 96 h at 30 °C, 160 rpm shaking. Biomass was harvested by vacuum filtration over Miracloth and was then lyophilized prior to proteomics.

Proteomics analysis

Protein was extracted and tryptic peptides were prepared by following established proteomic sample preparation protocol 110 . Briefly, cell pellets were resuspended in Qiagen P2 Lysis Buffer (Qiagen, Hilden, Germany, Cat.#19052) to promote cell lysis. Proteins were precipitated with addition of 1 mM NaCl and 4× vol acetone, followed by two additional wash with 80% acetone in water. The recovered protein pellet was homogenized by pipetting mixing with 100 mM Ammonium bicarbonate in 20% Methanol. Protein concentration was determined by the DC protein assay (BioRad, Hercules, CA). Protein reduction was accomplished using 5 mM tris 2-(carboxyethyl)phosphine (TCEP) for 30 min at room temperature, and alkylation was performed with 10 mM iodoacetamide (IAM; final concentration) for 30 min at room temperature in the dark. Overnight digestion with trypsin was accomplished with a 1:50 trypsin:total protein ratio. The resulting peptide samples were analyzed on an Agilent 1290 UHPLC system coupled to a Thermo Scientific Orbitrap Exploris 480 mass spectrometer for discovery proteomics 111 . Briefly, peptide samples were loaded onto an Ascentis® ES-C18 Column (Sigma–Aldrich, St. Louis, MO) and separated with a 10 min LC gradient from 98% solvent A (0.1% FA in H2O) and 2% solvent B (0.1% FA in ACN) to 65% solvent A and 35% solvent B. Eluting peptides were introduced to the mass spectrometer operating in positive-ion mode and were measured in data-independent acquisition (DIA) mode with a duty cycle of 3 survey scans from m/z 380 to m/z 985 and 45 MS2 scans with precursor isolation width of 13.5  m/z to cover the mass range. DIA raw data files were analyzed by an integrated software suite DIA-NN 112 . The database used in the DIA-NN search (library-free mode) is the latest A. oryzae UniProt proteome FASTA sequences plus the protein sequences of the heterologous proteins and common proteomic contaminants. DIA-NN determines mass tolerances automatically based on first-pass analysis of the samples with automated determination of optimal mass accuracies. The retention time extraction window was determined individually for all MS runs analyzed via the automated optimization procedure implemented in DIA-NN. Protein inference was enabled, and the quantification strategy was set to Robust LC = High Accuracy. Output main DIA-NN reports were filtered with a global FDR = 0.01 on both the precursor level and protein group level. The total peak area of tryptic peptides of identified proteins was used to plot the quantity of the targeted proteins in the samples.

Extraction and LC–MS analysis of ergothioneine and heme in fungal mycelium and reference samples

Extraction and analysis of heme.

Extraction was performed according to the protocol specified in ref. 113 , with minor modifications. Lyophilized fungal biomass was ground into a homogeneous powder using a mortar and pestle. Approximately 30 mg of the powder was then transferred to a bead-beating tube (Lysing Matrix Z, MP Biomedicals, catalog#: 116961050-CF) and 1 mL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8) was added. The tube was vortexed to suspend the powder and was then subjected to bead beating for 2 × 1 min using the Biospec Mini Beadbeater. 750 µL of the bead-beaten solution was then transferred to 15 mL conical tubes containing 4 mL of 8:2 acetonitrile:1.7 M HCl. The tubes were then vortexed for 20 min. Then, 1 mL of saturated 0.25 g MgSO 4 •7H 2 O was added to each tube, followed by the addition of 0.1 g NaCl. This created a separation of the aqueous and organic layers. The tubes were then vortexed for 10 min, followed by spinning down at 5000 rcf for 10 min to separate the layers. 100 µL of the top layer was transferred to an LC–MS vial for analysis. In addition to the wild-type and engineered biomass, we extracted heme from lyophilized plant-based ground beef (IMPOSSIBLE Foods Inc, 12 oz IMPOSSIBLE™ burger made from plants).

LC–MS analysis of heme

For the LC–MS analysis, analytes were chromatographically separated with a Kinetex XB-C18 column (50-mm length, 2.1-mm internal diameter, 2.6-µm particle size; Phenomenex, Torrance, CA) at 50 °C using a 1260 Infinity HPLC system (Agilent Technologies). The injection volume for each measurement was 5 µL. The mobile phase was composed of 0.1% formic acid in water (as mobile phase A) and 0.1% formic acid in acetonitrile (as mobile phase B). Analytes were separated via the following gradient: linearly increased from 20%B to 45.5%B in 1.7 min, linearly increased from 45.5%B to 95%B in 0.2 min, held at 95%B for 1.6 min, linearly decreased from 95%B to 20%B in 0.2 min, held at 20%B for 1.3 min. A flow rate of 1 mL/min was used throughout. The total run time was 5 min. The HPLC system was coupled to an Agilent Technologies 6520 Quadrupole Time-of-Flight Mass Spectrometer (QTOF-MS) with a 1:4 post-column split. Nitrogen gas was used as both the nebulizing and drying gas to facilitate the production of gas-phase ions. Drying and nebulizing gases were set to 10 L/min and 30 psi, respectively, and a drying gas temperature of 330 °C was used throughout. Fragmentor, skimmer, and OCT1 RF voltages were set to 250 V, 65 V, and 400 V, respectively. Electrospray ionization (ESI) was conducted in the positive-ion mode with a capillary voltage of 3.5 kV. MS experiments were carried out in the full-scan mode ( m/z 60–1100) at 0.86 spectra per second for the detection of [M + H] + ions. The instrument was tuned for a range of m/z 50–1700. Prior to LC-ESI-TOF MS analysis, the TOF MS was calibrated with the Agilent ESI-Low TOF tuning mix. Mass accuracy was maintained via reference ion mass correction, which was performed with purine and HP-0921 (Agilent Technologies). Data acquisition was carried out by MassHunter Workstation Software version B.08.00 (Agilent Technologies). Data processing was carried out by MassHunter Workstation Qualitative Analysis version B.06.00 and MassHunter Quantitative Analysis version 10.00. External calibration curves were used to quantify the analytes. Hemin chloride (Sigma–Aldrich, #3741) was used as the standard. The mass spectrum in the standards and samples corresponded to that from other experimentally validated studies of intracellular heme 114 . Calculated concentrations obtained from LC–MS analysis were normalized to the initial dry sample weight used for extraction.

Extraction and analysis of ergothioneine

All samples were lyophilized prior to analysis. For extraction from solid, samples were ground into a fine powder using a mortar and pestle and then approximately 30 mg was transferred to tubes for homogenization (Lysing Matrix Z, MP Biomedicals, catalog#: 116961050-CF). 1 mL of 20% methanol with 0.1% formic acid was added and samples were subjected to bead beating for 2 × 1 min. Following bead-beating, samples were spun down at 12,000 RCF for 10 min to separate the solids. 500 µL supernatant was transferred to a centrifugal spin filter to remove any particulates larger molecules (3 kDa cutoff) (Amicon Ultra, Sigma–Aldrich, Catalog # UFC500324). The flow-through was collected and subjected to analysis by LC–MS. In addition to wild-type and engineered fungal biomass, we extracted ergothioneine from the fruiting body of the oyster mushroom ( Pleurotus ostreatus ). The mushroom was purchased fresh (from Berkeley Bowl in Berkeley, CA) and subjected to lyophilization prior to extraction using the procedure above.

LC–MS analysis of ergothioneine

For LC–MS analysis, analytes were chromatographically separated with a Kinetex HILIC column (100-mm length, 4.6-mm internal diameter, 2.6-µm particle size; Phenomenex, Torrance, CA) at 20 °C using a 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA). The injection volume for each measurement was 2 µL. The mobile phase was composed of 10 mM ammonium formate (prepared from a pre-made solution from Sigma–Aldrich, St. Louis, MO, USA) and 0.2% formic acid (from an original stock at ≥98% chemical purity from Sigma–Aldrich) in water (as mobile phase A) and 10 mM ammonium formate and 0.2% formic acid in 90% acetonitrile with the remaining solvent being water (as mobile phase B). The solvents used were of LC–MS grade and purchased from Honeywell Burdick & Jackson, CA, USA. Analytes were separated via the following gradient: linearly decreased from 90%B to 70%B in 4 min, held at 70%B for 1.5 min, linearly decreased from 70%B to 40%B in 0.5 min, held at 40%B for 2.5 min, linearly increased from 40%B to 90%B in 0.5 min, held at 90%B for 2 min. The flow rate was changed as follows: 0.6 mL/min for 6.5 min, linearly increased from 0.6 mL/min to 1 mL/min for 0.5 min, held at 1 mL/min for 4 min. The total run time was 11 min. The HPLC system was coupled to an Agilent Technologies 6520 Quadrupole Time-of-Flight Mass Spectrometer (QTOF-MS) with a 1:4 post-column split. Nitrogen gas was used as both the nebulizing and drying gas to facilitate the production of gas-phase ions. Drying and nebulizing gases were set to 12 L/min and 25 psi, respectively, and a drying gas temperature of 350 °C was used throughout. Fragmentor, skimmer, and OCT1 RF voltages were set to 100 V, 50 V, and 250 V, respectively. Electrospray ionization (ESI) was conducted in the positive-ion mode with a capillary voltage of 3.5 kV. MS experiments were carried out in the full-scan mode ( m/z 70–1100) at 0.86 spectra per second for the detection of [M + H] + ions. The instrument was tuned for a range of m/z 50–1700. Prior to LC-ESI-TOF MS analysis, the TOF MS was calibrated with the Agilent ESI-Low TOF tuning mix. Mass accuracy was maintained via reference ion mass correction, which was performed with purine and HP-0921 (Agilent Technologies). Data acquisition was carried out by MassHunter Workstation Software version B.08.00 (Agilent Technologies). Data processing was carried out by MassHunter Workstation Qualitative Analysis version B.06.00 and MassHunter Quantitative Analysis version 10.00. External calibration curves were used to quantify the analytes. Ergothioneine (Sigma–Aldrich, #7521-25MG) was used as the standard. The mass spectrum in the standards and samples corresponded to that from other experimentally validated studies of intracellular ergothioneine 115 . Calculated concentrations obtained from LC–MS analysis were normalized to the initial dry sample weight used for extraction.

Protein and amino acid analysis in fungal mycelium

Protein content was analyzed by combustion method, directly following the Method 990.03 described in ref. 116 . The combustion was performed using Leco FP-528 Nitrogen Combustion Analyzer (Leco). Crude Protein was calculated as Nitrogen × 6.25. Amino acid composition was analyzed using acid hydrolysis of lyophilized fungal biomass, directly following the protocols of Method 994.12 described in ref. 116 , Method 982.30 in ref. 117 , as well as the methods described in ref. 118 .

Statistics and Reproducibility

No statistical method was used to predetermine sample size. n  = 3 was chosen as the minimal number of replicates for experimental characterization. We determined this to be sufficient based on internal controls (using previously characterized promoters and fluorescent genes) to capture biological variability between transformants/strains. All microscopy images and PCR results for confirming insertion/excision displayed are representative of at least three biological replicates. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper, supplementary information, the supplementary data files, and in the source data file. Source data are provided as a Source Data file. Strains and plasmids (and their associated sequences) generated in this study have been deposited in the JBEI Public Registry ( https://public-registry.jbei.org/ ). See Supplementary Tables  11 – 12 for plasmids and strain information. Outputs of computational analysis for identification of candidate endogenous neutral loci and bidirectional promoters are available in Supplementary data files 1 and 2. Output of mass spectrometry data are available as source data. The generated mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 94 partner repository with the dataset identifier “ PXD043152 ”. The genome sequences of the A. oryzae strains obtained from NRRL and sequenced as part of this study have been deposited to the Sequence Read Archive under Bioproject “ PRJNA987873 ”. The previously published transcriptome data used to identify endogenous neutral loci and bidirectional promoters in Aspergillus oryzae can be found in GenBank under BioProject accession: “ PRJDB8293 ”. CAoGD (Comprehensive Aspergillus oryzae Genome Database) v.2.4 was used to identify the genomic location and sequence identity of endogenous promoters and genes targeted in transformations ( https://nribf21.nrib.go.jp/CAoGD/ ).  Source data are provided with this paper.

Code availability

No custom code was used in the analysis of data, and all the previously published software used as well as the relevant commands has been cited in the methods. The automatic phylogenomic analysis from the core genomes of Aspergillus oryzae strains was executed using the script available at https://github.com/WeMakeMolecules/Core-to-Tree . DIA-NN is freely available for download from https://github.com/vdemichev/DiaNN .

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Acknowledgements

V.M.R. was supported by the Miller Institute at the University of California, Berkeley. P.C.M. and J.D.K. were supported by Novo Nordisk Foundation grant no. NNF20CC0035580. C.V.D.L. was supported by Novo Nordisk Foundation grant NNF21OC0065495. This work was part of the DOE Joint BioEnergy Institute ( https://www.jbei.org ) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.

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Vayu Maini Rekdal & Jay D. Keasling

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Vayu Maini Rekdal

Joint BioEnergy Institute, Emeryville, CA, 94608, USA

Vayu Maini Rekdal, Casper R. B. van der Luijt, Yan Chen, Ramu Kakumanu, Edward E. K. Baidoo, Christopher J. Petzold & Jay D. Keasling

Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark

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Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, 94720, USA

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Contributions

VMR and JDK conceptualized the study. VMR developed and optimized transformation methods, established the neutral loci, endogenous promoters, and vectors for transformation, and engineered the final strains. CVDL evaluated parts for the Synthetic expression system and conducted transformation of A. oryzae NRRL strains. PCM conducted bioinformatics analysis, including genome annotation and assembly, phylogenetic analysis, and identification of endogenous neutral loci and bidirectional promoters. YC and CJP conducted proteomics experiments and analysis. RK and EEKB conducted targeted LC–MS experiments and analysis. All authors approved the final manuscript.

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Correspondence to Jay D. Keasling .

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Competing interests.

J.D.K. has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. V.M.R. and J.D.K. are listed as inventors on a provisional patent (US22/42816) which relates to the methods composition described in the engineering of heme metabolism in edible fungal biomass. The other authors declare no competing interests.

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Maini Rekdal, V., van der Luijt, C.R.B., Chen, Y. et al. Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit. Nat Commun 15 , 2099 (2024). https://doi.org/10.1038/s41467-024-46314-8

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Scientific Consensus

It’s important to remember that scientists always focus on the evidence, not on opinions. Scientific evidence continues to show that human activities ( primarily the human burning of fossil fuels ) have warmed Earth’s surface and its ocean basins, which in turn have continued to impact Earth’s climate . This is based on over a century of scientific evidence forming the structural backbone of today's civilization.

NASA Global Climate Change presents the state of scientific knowledge about climate change while highlighting the role NASA plays in better understanding our home planet. This effort includes citing multiple peer-reviewed studies from research groups across the world, 1 illustrating the accuracy and consensus of research results (in this case, the scientific consensus on climate change) consistent with NASA’s scientific research portfolio.

With that said, multiple studies published in peer-reviewed scientific journals 1 show that climate-warming trends over the past century are extremely likely due to human activities. In addition, most of the leading scientific organizations worldwide have issued public statements endorsing this position. The following is a partial list of these organizations, along with links to their published statements and a selection of related resources.

American Scientific Societies

Statement on climate change from 18 scientific associations.

"Observations throughout the world make it clear that climate change is occurring, and rigorous scientific research demonstrates that the greenhouse gases emitted by human activities are the primary driver." (2009) 2

American Association for the Advancement of Science

"Based on well-established evidence, about 97% of climate scientists have concluded that human-caused climate change is happening." (2014) 3

American Chemical Society

"The Earth’s climate is changing in response to increasing concentrations of greenhouse gases (GHGs) and particulate matter in the atmosphere, largely as the result of human activities." (2016-2019) 4

American Geophysical Union

"Based on extensive scientific evidence, it is extremely likely that human activities, especially emissions of greenhouse gases, are the dominant cause of the observed warming since the mid-20th century. There is no alterative explanation supported by convincing evidence." (2019) 5

American Medical Association

"Our AMA ... supports the findings of the Intergovernmental Panel on Climate Change’s fourth assessment report and concurs with the scientific consensus that the Earth is undergoing adverse global climate change and that anthropogenic contributions are significant." (2019) 6

American Meteorological Society

"Research has found a human influence on the climate of the past several decades ... The IPCC (2013), USGCRP (2017), and USGCRP (2018) indicate that it is extremely likely that human influence has been the dominant cause of the observed warming since the mid-twentieth century." (2019) 7

American Physical Society

"Earth's changing climate is a critical issue and poses the risk of significant environmental, social and economic disruptions around the globe. While natural sources of climate variability are significant, multiple lines of evidence indicate that human influences have had an increasingly dominant effect on global climate warming observed since the mid-twentieth century." (2015) 8

The Geological Society of America

"The Geological Society of America (GSA) concurs with assessments by the National Academies of Science (2005), the National Research Council (2011), the Intergovernmental Panel on Climate Change (IPCC, 2013) and the U.S. Global Change Research Program (Melillo et al., 2014) that global climate has warmed in response to increasing concentrations of carbon dioxide (CO2) and other greenhouse gases ... Human activities (mainly greenhouse-gas emissions) are the dominant cause of the rapid warming since the middle 1900s (IPCC, 2013)." (2015) 9

Science Academies

International academies: joint statement.

"Climate change is real. There will always be uncertainty in understanding a system as complex as the world’s climate. However there is now strong evidence that significant global warming is occurring. The evidence comes from direct measurements of rising surface air temperatures and subsurface ocean temperatures and from phenomena such as increases in average global sea levels, retreating glaciers, and changes to many physical and biological systems. It is likely that most of the warming in recent decades can be attributed to human activities (IPCC 2001)." (2005, 11 international science academies) 1 0

U.S. National Academy of Sciences

"Scientists have known for some time, from multiple lines of evidence, that humans are changing Earth’s climate, primarily through greenhouse gas emissions." 1 1

U.S. Government Agencies

U.s. global change research program.

"Earth’s climate is now changing faster than at any point in the history of modern civilization, primarily as a result of human activities." (2018, 13 U.S. government departments and agencies) 12

Intergovernmental Bodies

Intergovernmental panel on climate change.

“It is unequivocal that the increase of CO 2 , methane, and nitrous oxide in the atmosphere over the industrial era is the result of human activities and that human influence is the principal driver of many changes observed across the atmosphere, ocean, cryosphere, and biosphere. “Since systematic scientific assessments began in the 1970s, the influence of human activity on the warming of the climate system has evolved from theory to established fact.” 1 3-17

Other Resources

List of worldwide scientific organizations.

The following page lists the nearly 200 worldwide scientific organizations that hold the position that climate change has been caused by human action. http://www.opr.ca.gov/facts/list-of-scientific-organizations.html

U.S. Agencies

The following page contains information on what federal agencies are doing to adapt to climate change. https://www.c2es.org/site/assets/uploads/2012/02/climate-change-adaptation-what-federal-agencies-are-doing.pdf

Technically, a “consensus” is a general agreement of opinion, but the scientific method steers us away from this to an objective framework. In science, facts or observations are explained by a hypothesis (a statement of a possible explanation for some natural phenomenon), which can then be tested and retested until it is refuted (or disproved).

As scientists gather more observations, they will build off one explanation and add details to complete the picture. Eventually, a group of hypotheses might be integrated and generalized into a scientific theory, a scientifically acceptable general principle or body of principles offered to explain phenomena.

1. K. Myers, et al, "Consensus revisited: quantifying scientific agreement on climate change and climate expertise among Earth scientists 10 years later", Environmental Research Letters Vol.16 No. 10, 104030 (20 October 2021); DOI:10.1088/1748-9326/ac2774 M. Lynas, et al, "Greater than 99% consensus on human caused climate change in the peer-reviewed scientific literature", Environmental Research Letters Vol.16 No. 11, 114005 (19 October 2021); DOI:10.1088/1748-9326/ac2966 J. Cook et al., "Consensus on consensus: a synthesis of consensus estimates on human-caused global warming", Environmental Research Letters Vol. 11 No. 4, (13 April 2016); DOI:10.1088/1748-9326/11/4/048002 J. Cook et al., "Quantifying the consensus on anthropogenic global warming in the scientific literature", Environmental Research Letters Vol. 8 No. 2, (15 May 2013); DOI:10.1088/1748-9326/8/2/024024 W. R. L. Anderegg, “Expert Credibility in Climate Change”, Proceedings of the National Academy of Sciences Vol. 107 No. 27, 12107-12109 (21 June 2010); DOI: 10.1073/pnas.1003187107 P. T. Doran & M. K. Zimmerman, "Examining the Scientific Consensus on Climate Change", Eos Transactions American Geophysical Union Vol. 90 Issue 3 (2009), 22; DOI: 10.1029/2009EO030002 N. Oreskes, “Beyond the Ivory Tower: The Scientific Consensus on Climate Change”, Science Vol. 306 no. 5702, p. 1686 (3 December 2004); DOI: 10.1126/science.1103618

2. Statement on climate change from 18 scientific associations (2009)

3. AAAS Board Statement on Climate Change (2014)

4. ACS Public Policy Statement: Climate Change (2016-2019)

5. Society Must Address the Growing Climate Crisis Now (2019)

6. Global Climate Change and Human Health (2019)

7. Climate Change: An Information Statement of the American Meteorological Society (2019)

8. American Physical Society (2021)

9. GSA Position Statement on Climate Change (2015)

10. Joint science academies' statement: Global response to climate change (2005)

11. Climate at the National Academies

12. Fourth National Climate Assessment: Volume II (2018)

13. IPCC Fifth Assessment Report, Summary for Policymakers, SPM 1.1 (2014)

14. IPCC Fifth Assessment Report, Summary for Policymakers, SPM 1 (2014)

15. IPCC Sixth Assessment Report, Working Group 1 (2021)

16. IPCC Sixth Assessment Report, Working Group 2 (2022)

17. IPCC Sixth Assessment Report, Working Group 3 (2022)

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Explore Earth Science

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Wellbeing research

An increasing body of evidence demonstrates that wellbeing profoundly influences our ability to learn, thrive and lead fulfilling lives. In an ever-evolving education landscape, prioritizing and fostering wellbeing becomes a vital investment in the future of education.

This page highlights research into the existing scientific landscape, emphasizing the foundational evidence that positions wellbeing at the core of the educational ecosystem. Moving beyond a narrow focus on academic achievement, a systemic approach to wellbeing necessitates creating a holistic, interconnected and supportive learning environment.

Foundational research

The Wellbeing Research Centre at the University of Oxford conducted a series of in-depth literature reviews to understand the science behind student, teacher and school community wellbeing. Based on these studies the IB can develop supports for teachers and schools to define wellbeing in their own context and to learn how best to improve it.

Wellbeing in education in childhood and adolescence

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Dr Laura Taylor, Dr Wanying Zhou, Leoni Boyle, Sabina Funk and Prof Jan-Emmanuel De Neve— Wellbeing Research Centre, University of Oxford This foundational literature review provides an overview of the latest research into teacher wellbeing and its importance for teachers themselves, students and the school community. The report is intended to give the International Baccalaureate, policymakers and educational leaders an understanding of the definitions of adult wellbeing, what influences teacher wellbeing and what interventions might be used to improve teacher wellbeing. The researchers also present a teacher wellbeing framework which identifies drivers of teacher wellbeing. Coming soon

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Making the abstract explicit: the role of metacognition in teaching and learning

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Research Paper Format | APA, MLA, & Chicago Templates

Published on November 19, 2022 by Jack Caulfield . Revised on January 20, 2023.

The formatting of a research paper is different depending on which style guide you’re following. In addition to citations , APA, MLA, and Chicago provide format guidelines for things like font choices, page layout, format of headings and the format of the reference page.

Scribbr offers free Microsoft Word templates for the most common formats. Simply download and get started on your paper.

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Table of contents

Formatting an apa paper, formatting an mla paper, formatting a chicago paper, frequently asked questions about research paper formatting.

The main guidelines for formatting a paper in APA Style are as follows:

• Use a standard font like 12 pt Times New Roman or 11 pt Arial.
• Set 1 inch page margins.
• Apply double line spacing.
• If submitting for publication, insert a APA running head on every page.
• Indent every new paragraph ½ inch.

Watch the video below for a quick guide to setting up the format in Google Docs.

The image below shows how to format an APA Style title page for a student paper.

Running head

If you are submitting a paper for publication, APA requires you to include a running head on each page. The image below shows you how this should be formatted.

For student papers, no running head is required unless you have been instructed to include one.

APA provides guidelines for formatting up to five levels of heading within your paper. Level 1 headings are the most general, level 5 the most specific.

Reference page

APA Style citation requires (author-date) APA in-text citations throughout the text and an APA Style reference page at the end. The image below shows how the reference page should be formatted.

Note that the format of reference entries is different depending on the source type. You can easily create your citations and reference list using the free APA Citation Generator.

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The main guidelines for writing an MLA style paper are as follows:

• Use an easily readable font like 12 pt Times New Roman.
• Use title case capitalization for headings .

Check out the video below to see how to set up the format in Google Docs.

On the first page of an MLA paper, a heading appears above your title, featuring some key information:

• Your full name
• Your instructor’s or supervisor’s name
• The course name or number
• The due date of the assignment

Page header

A header appears at the top of each page in your paper, including your surname and the page number.

Works Cited page

MLA in-text citations appear wherever you refer to a source in your text. The MLA Works Cited page appears at the end of your text, listing all the sources used. It is formatted as shown below.

You can easily create your MLA citations and save your Works Cited list with the free MLA Citation Generator.

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The main guidelines for writing a paper in Chicago style (also known as Turabian style) are:

• Use a standard font like 12 pt Times New Roman.
• Use 1 inch margins or larger.
• Place page numbers in the top right or bottom center.

Chicago doesn’t require a title page , but if you want to include one, Turabian (based on Chicago) presents some guidelines. Lay out the title page as shown below.

Bibliography or reference list

Chicago offers two citation styles : author-date citations plus a reference list, or footnote citations plus a bibliography. Choose one style or the other and use it consistently.

The reference list or bibliography appears at the end of the paper. Both styles present this page similarly in terms of formatting, as shown below.

To format a paper in APA Style , follow these guidelines:

• Use a standard font like 12 pt Times New Roman or 11 pt Arial
• Set 1 inch page margins
• Apply double line spacing
• Include a title page
• If submitting for publication, insert a running head on every page
• Indent every new paragraph ½ inch
• Apply APA heading styles
• Cite your sources with APA in-text citations
• List all sources cited on a reference page at the end

The main guidelines for formatting a paper in MLA style are as follows:

• Use an easily readable font like 12 pt Times New Roman
• Include a four-line MLA heading on the first page
• Center the paper’s title
• Use title case capitalization for headings
• Cite your sources with MLA in-text citations
• List all sources cited on a Works Cited page at the end

The main guidelines for formatting a paper in Chicago style are to:

• Use a standard font like 12 pt Times New Roman
• Use 1 inch margins or larger
• Place page numbers in the top right or bottom center
• Cite your sources with author-date citations or Chicago footnotes
• Include a bibliography or reference list

To automatically generate accurate Chicago references, you can use Scribbr’s free Chicago reference generator .

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