Researchers have identified key ingredients for producing high-value chemical compounds in an environmentally friendly fashion: repurposed enzymes, curiosity, and a little bit of light.
A paper published in Nature describes a study led by Xiaoqiang Huang (pictured), a postdoctoral researcher in the University of Illinois at Urbana-Champaign’s Department of Chemical and Biomolecular Engineering (ChBE) and the Carl R. Woese Institute for Genomic Biology (IGB). Huang works in the lab of ChBE Professor Huimin Zhao, Conversion Theme Leader at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a U.S. Department of Energy-funded Bioenergy Research Center (BRC).
Catalysts are substances used to speed up chemical reactions; in living organisms, protein molecules called enzymes catalyze reactions in a process called biocatalysis.
Biocatalysis is rapidly emerging as a nuanced, agile way to synthesize valuable compounds. Scientists are investigating the ability of enzymes to catalyze diverse reactions, and for good reason: biocatalytic reactions are highly selective, meaning that scientists can use enzymes to act on specific substrates and create target products.
Enzymatic reactions are also highly sustainable as they are relatively inexpensive, consume low levels of energy, and do minimal damage to the environment: while chemical catalysts typically require organic solvents, heat, and high pressure to function, biocatalysts work in aqueous solutions, operating at room-temperature and normal-pressure conditions.
Despite their value to science and sustainability, enzymes can be complicated to work with. Reactions enzymes can catalyze are limited to those found in nature; this means that scientists often struggle to track down the perfect biocatalyst to meet their need.
The process is similar to mixing paint: How can an artist creatively combine the colors already on a palette to produce the right shade? In the language of a chemical reaction: How can scientists leverage enzymes already existing in nature to create the products they need?
The research team developed a solution: a visible-light-induced reaction that uses the enzyme family ene-reductase (ER) as a biocatalyst and can produce high yields of valuable chiral carbonyl compounds.
Scientists engineering valuable microbes for renewable fuels and bioproducts have developed a fast, efficient way to identify the most promising varieties.
Researchers at the University of Illinois at Urbana-Champaign developed a novel mass spectrometry-based screening technique to rapidly profile medium-chain fatty acids produced in yeast — part of a larger group of free fatty acids that are key components in essential nutrients, soaps, industrial chemicals, and fuels. They also identified seven new genetically engineered mutants of the yeast Saccharomyces cerevisiae that produce higher levels of those fatty acids.
The study is detailed in a paper published in the journal Biotechnology and Bioengineering. The research was performed at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a U.S. Department of Energy-funded Bioenergy Research Center, by two U of I professors: Huimin Zhao, Professor of Chemical and Biomolecular Engineering (ChBE), and Jonathan Sweedler, Professor of Chemistry and Director of the School of Chemical Sciences. The lead author is Pu Xue (above), a ChBE doctoral student with Zhao’s lab at CABBI.
Zhao’s group genetically engineers tiny yeast cells to increase production of fatty acids, crucial components of biodiesel, fatty alcohols, waxes and olefins — the building blocks for detergents, adhesives, and plastics. CABBI’s goal is to develop robust yeasts that can convert renewable plant biomass to fuels and chemicals, as an environmentally friendly and sustainable alternative to petroleum-based chemical manufacturing processes.
Scientists can create a large library of engineered yeast strains, or mutants, producing various MCFAs very quickly, Xue said. But their ability to control the exact composition of MCFAs produced in these microbial cell factories is limited, with no “high-throughput” way to quickly analyze large numbers of samples.
To overcome this limitation, Xue and other researchers worked with Sweedler’s group to develop a high-throughput screening tool, a chemical characterization approach based on MALDI-ToF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). Mass spectrometry is an information-rich way to analyze complex samples by measuring their mass-to-charge ratio of ions. A laser is shot at a colony of yeast on a slide; the instrument measures the molecular masses of the lipids in the yeast, and thus is able to identify them.
Most existing processes for detecting or analyzing free fatty acids rely on more complex gas or liquid chromatography-mass spectrometry, considered the gold standard methods. But those methods have limitations when dealing with a large pool of variations, and preparing samples is time-consuming and labor-intensive.
The overall approach using MALDI-ToF MS system is faster and has been used successfully with proteins and peptides. The ToF mass analyzer is fast, relatively low cost and has a large detection window, well-suited for screening complex biological targets.
But medium-chain fatty acids are highly volatile and lightweight, making them harder to detect and quantify with this approach. Moreover, the thick cell wall of yeast creates another challenge for researchers to decompose cellular compositions efficiently.
So the CABBI researchers optimized the sample preparation steps with various solvents and matrices. And instead of trying to directly detect the MCFAs, they used a proxy: membrane lipids in yeast cells. They hypothesized that increased levels of membrane lipids with shorter acyl chain phosphatidylcholines (PCs), a class of phospholipids, would correlate with a greater capacity to produce MCFAs, which are shorter in length than the abundant fatty acids commonly found in S. cerevisiae.
To validate that hypothesis, U of I scientists compared the MALDI-ToF MS profile of the naturally occurring yeast with two genetically engineered strains previously found to produce higher levels of MCFAs. The data showed that these two mutants had more of the shorter acyl chain PCs than the naturally occurring yeast. Those preliminary findings were then confirmed by the more exact liquid chromatography and fragment mass spectrometry processes.
With their established screening method in hand, CABBI team members set out to find more mutant strains with higher production of MCFAs. They found two prominent peaks on the mass spectrum that were correlated with the phospholipids, an indication of fatty acids. Those were then used as a sign of MCFA production.
As Sweedler points out, “the mass spectrometry measurement is fast — analyzing up to 2,000 yeast colonies per hour (approximately one sample every two seconds) compared to 30 minutes per sample under traditional methods.” The processing time is also significantly shortened: two to three minutes vs. three to four hours per sample. Overall, the MALDI-ToF MS screening tool allows scientists to quickly identify strains that warrant more detailed analysis.
“Our method allows us to screen tons of mutants in a short time. We can identify the good candidates for further study,” Xue said.
In the future, the method can be modified and used for high-throughput screening of other types of products, such as longer-chain fatty acids or fatty alcohols, saving time and labor. The researchers hope to build on their work at CABBI’s iBioFAB — the Illinois Biological Foundry for Advanced Biomanufacturing. Its robotic system can speed up sample preparation and provide faster, more accurate results, helping scale up the project to test many more mutant strains, Xue said. With about 6,000 genes in S. cerevisiae, millions of potential synergistic effects and features could be discovered.
Beyond science, these C6 to C12 fatty acids are important in human health, providing critical nutrients and useful products, such as Omega-3 fatty acids.
“In the future if we can directly generate biofuels and bioproducts such as fatty acids from microbial cells like yeast in large scale, that means we don’t need to use petrol,” Xue said. “We can save the environment and save a lot of money as well.”
Co-authors on the paper included CABBI researchers Shekhar Mishra (Chemical and Biomolecular Engineering) and Kisurb Choe (Biochemistry); former CABBI researcher Tong Si (ChBE); and Linzixuan Zhang, an undergraduate student in ChBE. All are also affiliated with the Carl R. Woese Institute for Genomic Biology at Illinois.
University of Illinois researchers achieved the highest reported rates of inserting genes into human cells with the CRISPR-Cas9 gene-editing system, a necessary step for harnessing CRISPR for clinical gene-therapy applications.
By chemically tweaking the ends of the DNA to be inserted, the new technique is up to five times more efficient than current approaches. The researchers saw improvements at various genetic locations tested in a human kidney cell line, even seeing 65% insertion at one site where the previous high had been 15%.
Led by chemical and biomolecular engineering professor Huimin Zhao, the researchers published their work in the journal Nature Chemical Biology.
Researchers have found CRISPR to be an efficient tool to turn off, or “knock out,” a gene. However, in human cells, it has not been a very efficient way to insert or “knock in” a gene.
“A good knock-in method is important for both gene-therapy applications and for basic biological research to study gene function,” said Zhao, who leads the biosystems design theme at the Carl R. Woese Institute for Genomic Biology at Illinois. “With a knock-in method, we can add a label to any gene, study its function and see how gene expression is affected by cancer or changes in chromosome structure. Or for gene-therapy applications, if someone has a disease caused by a missing gene, we want to be able to insert it.”
Searching for a way to increase efficiency, Zhao’s group looked at 13 different ways to modify the inserted DNA. They found that small changes to the very end of the DNA increased both the speed and efficiency of insertion.
Then, the researchers tested inserting end-modified DNA fragments of varying sizes at multiple points in the genome, using CRISPR-Cas9 to precisely target specific sites for insertion. They found efficiency improved two to five times, even when inserting larger DNA fragments – the most difficult insertion to make.
“We speculate that the efficiency improved so much because the chemical modification to the end stabilizes the DNA we are inserting,” Zhao said. “Normally, when you try to transfer DNA into the cell, it gets degraded by enzymes that eat away at it from the ends. We think our chemical addition protects the ends. More DNA is getting into the nucleus, and that DNA is more stable, so that’s why I think it has a higher chance to be integrated into the chromosome.”
Zhao’s group already is using the method to tag essential genes in gene function studies. They purposely used off-the-shelf chemicals to modify the DNA fragments so that other research teams could use the same method for their own genetic studies.
“We’ve developed quite a few knock-in methods in the past, but we never thought about just using chemicals to increase the stability of the DNA we want to insert,” Zhao said. “It’s a simple strategy, but it works.”
The National Institutes of Health supported this work. Zhao also is affiliated with the Carle Illinois Carle of Medicine.
The paper “An efficient gene knock-in strategy using 5′-modified double-stranded DNA donors with short homology arms” is available online. DOI: 10.1038/s41589-019-0432-1
Several research images from Chemical and Biomolecular Engineering faculty are currently on display at the National Institutes of Health.
The exhibit was organized by the Carl R. Woese Institute for Genomic Biology and includes images from the labs of Professors Hyunjoon Kong, Brendan Harley, and Huimin Zhao. All three are researchers affiliated with IGB.
The IGB’s Art of Science program is a celebration of common ground between science and art. Each exhibit comprises images from IGB’s research portfolio, captured in its core facilities, and enhanced to highlight the beauty and fascination encountered daily in scientific endeavors. Art of Science images have been displayed at O’Hare International Airport in Chicago, at the Illinois State Capitol in Springfield, The Rayburn House Office Building in Washington, DC, the German Center for Innovation in NYC, and Abbott Diagnostics, among other locations.
The images can be found in the hallway just outside the library in the NIH Clinical Center (Building 10), down the hall from the Masur Auditorium in Betheseda, MD.
CHAMPAIGN, Ill. — A new proof-of-concept study details how an automated system driven by artificial intelligence can design, build, test and learn complex biochemical pathways to efficiently produce lycopene, a red pigment found in tomatoes and commonly used as a food coloring, opening the door to a wide range of biosynthetic applications, researchers report.
The results of the study, which combined a fully automated robotic platform called the Illinois Biological Foundry for Advanced Biomanufacturing with AI to achieve biomanufacturing, are published in the journal Nature Communications.
“Biofoundries are factories that mimic the foundries that build semiconductors, but are designed for biological systems instead of electrical systems,” said Huimin Zhao. He is the Steven L. Miller Chair in Chemical Engineering at the University of Illinois who led the research.
However, because biology offers many pathways to chemical production, the researchers assert that a system driven by AI and capable of choosing from thousands of experimental iterations is required for true automation.
Previous biofoundry efforts have produced a wide variety of products such as chemicals, fuels, and engineered cells and proteins, the researchers said, but those studies were not performed in a fully automated manner.
“Past studies in biofoundry development mainly focused on only one of the design, build, test and learn elements,” Zhao said. “A researcher was still required to perform data analysis and to plan for the next experiment. Our system, dubbed BioAutomata, closes the design, build, test and learn loop and leaves humans out of the process.”
BioAutomata completed two rounds of fully automated construction and optimization of the lycopene-production pathway, which includes the design and construction of the lycopene pathways, transfer of the DNA-encoding pathways into host cells, growth of the cells, and extraction and measurement of the lycopene production.
“BioAutomata was able to reduce the number of possible lycopene-production pathways constructed from over 10,000 down to about 100 and create an optimized quantity of lycopene-overproducing cells within weeks – greatly reducing time and cost,” Zhao said.
Zhao envisions fully automated biofoundries being a future revolution in smart manufacturing, not unlike what automation did for the automobile industry.
“A hundred years ago, people built cars by hand,” he said. “Now, that process is much more economical and efficient thanks to automation, and we imagine the same for biomanufacturing of chemicals and materials.”
Zhao also is affiliated with the departments of chemistry, biochemistry and bioengineering, and is a theme leader at the Carl R. Woese Institute for Genomic Biology and at the Center for Advanced Bioenergy and Bioproducts Innovation at the U. of I.
The U.S. Department of Energy’s Center for Advanced Bioenergy and Bioproducts Innovation and the IGB supported this research.
Written by Lois Yoksoulian, Physical Sciences Editor, U of I News Bureau
To reach Huimin Zhao, call 217-333-2631; email firstname.lastname@example.org.
Congratulations to Huimin Zhao, who was recently selected to receive the 2019 Enzyme Engineering Award for his pioneering contributions in the development of directed evolution for enzyme engineering.
The award, given by Engineering Conferences International, recognizes outstanding achievement in the field of enzyme engineering, through basic or applied research in academia or industry. It will be presented to Zhao at the 25th Enzyme Engineering Conference in Whistler, British Columbia, Canada. Previous winners include Frances Arnold, Zhao’s PhD advisor, as well as researchers Alex Klibanov, Harvey Blanch, and Chi Huey Wong.
Zhao is the Steven L. Miller Chair of Chemical and Biomolecular Engineering in the University of Illinois Department of Chemical and Biomolecular Engineering. His primary research interests are in the development and applications of synthetic biology tools to address society’s most daunting challenges in health, energy, and sustainability, and in the fundamental aspects of enzyme catalysis, cell metabolism, and gene regulation. He leads the conversion reserach theme at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a U.S. Department of Energy-funded Bioenergy Research Center, a collaboration between the University of Illinois Institute for Sustainability, Energy, and Environment (iSEE) and the Carl R. Woese Institute for Genomic Biology (IGB). Zhao is also leader of the Biosystems Design research theme at IGB.
By enticing away the repressors dampening unexpressed, silent genes in Streptomyces bacteria, researchers at the University of Illinois have unlocked several large gene clusters for new natural products, according to a study published in the journal Nature Chemical Biology.
Since many antibiotics, anti-cancer agents and other drugs have been derived from genes readily expressed in Streptomyces, the researchers hope that unsilencing genes that have not previously been expressed in the lab will yield additional candidates in the search for new antimicrobial drugs, says study leader and chemical and biomolecular engineering professor Huimin Zhao.
“There are so many undiscovered natural products lying unexpressed in genomes. We think of them as the dark matter of the cell,” Zhao said. “Anti-microbial resistance has become a global challenge, so clearly there’s an urgent need for tools to aid the discovery of novel natural products. In this work, we found new compounds by activating silent gene clusters that have not been explored before.”
The researchers previously demonstrated a technique to activate small silent gene clusters using CRISPR technology. However, large silent gene clusters have remained difficult to unmute. Those larger genes are of great interest to Zhao’s group, since a number of them have sequences similar to regions that code for existing classes of antibiotics, such as tetracycline.
To unlock the large gene clusters of greatest interest, Zhao’s group created clones of the DNA fragments they wanted to express and injected them into the bacteria in hopes of luring away the repressor molecules that were preventing gene expression. They called these clones transcription factor decoys.
“Others have used a similar kind of decoy for therapeutic applications in mammalian cells, but we show here for the first time that it can be used for drug discovery by activating silent genes in bacteria,” said Zhao, who is affiliated with the Carle Illinois College of Medicine, the Carl R. Woese Institute for Genomic Biology and the Center for Advanced Bioenergy and Bioproducts Innovation at Illinois.
To prove that the molecules they coded for were being expressed, researchers tested the decoy method first on two known gene clusters that synthesize natural products. Next, they created decoys for eight silent gene clusters that had been previously unexplored. In bacteria injected with the decoys, the targeted silent genes were expressed and the researchers harvested new products.
“We saw that the method works well for these large clusters that are hard to target by other methods,” Zhao said. “It also has the advantage that it does not disturb the genome; it’s just pulling away the repressors. Then the genes are expressed naturally from the native DNA.”
In the search for drug candidates, each product needs to be isolated and then studied to determine what it does. Of the eight new molecules produced, the researchers purified and determined the structure of two molecules, and described one in detail in the study – a novel type of oxazole, a class of molecules often used in drugs.
The researchers plan next to characterize the rest of the eight compounds and run various assays to find out whether they have any anti-microbial, anti-fungal, anti-cancer or other biological activities.
Zhao’s group also plans to apply the decoy technique to explore more silent biosynthetic gene clusters of interest in Streptomyces and in other bacteria and fungi to find more undiscovered natural products. Other research groups are welcome to use the technique for gene clusters they are exploring, Zhao said.
“The principle is the same, assuming that gene expression is repressed by transcription factors and we just need to release that expression by using decoy DNA fragments,” Zhao said.
The National Institutes of Health supported this work.
By Liz Ahlberg Touchstone, U of I News Bureau
Editor’s notes: To reach Huimin Zhao, call (217) 333-2631; email: email@example.com.
The paper “Activation of silent biosynthetic gene clusters using transcription factor decoys” is available online.
On Monday, December 10, the Royal Swedish Academy of Sciences will award one half of the Nobel Prize in Chemistry to Dr. Frances H. Arnold, Professor of Chemical Engineering, Bioengineering, and Biochemistry at the California Institute of Technology for her work in the directed evolution of enzymes. She shares the prize with the team of George P. Smith of the University of Missouri, Columbia, and Sir Gregory P. Winter of the MRC Laboratory of Molecular Biology, Cambridge, UK, who collaborated on the phage display of peptides and antibodies.
Among those celebrating with Arnold will be Dr. Huimin Zhao, the Steven L. Miller Chair in the Department of Chemical and Biomolecular Engineering at the University of Illinois.
“Frances had a huge impact on my career development and my personal life,” Zhao said. “She has always been my inspiration, dear mentor, and dear friend. She helped me grow my own career tremendously.”
As a PhD student at Caltech, Zhao was Arnold’s first graduate student to work in the directed evolution field.
“Frances pioneered the most powerful approach to enzyme engineering, directed evolution, and has used directed evolution to generate a broad array of new and useful enzymes. Together with Dr. Willem “Pim” Stemmer (who unfortunately passed away a few years ago), she created a new research field on directed evolution in the early 1990s,” he said.
Since then, thousands of papers have been published in this area by researchers across the world, and directed evolution is widely used in academic and industrial laboratories to optimize enzymes, antibodies and other therapeutic proteins, metabolic pathways, and even whole organisms, Zhao said. Directed evolution technologies form the basis of some specialized service companies and are practiced in small and major companies worldwide. These companies use enzymes that have been optimized by directed evolution to make fuels, such as ethanol; chemicals, such as 1,4 butanediol; plastics; consumer products like laundry detergents; research reagents; diagnostics, such as glucose sensors; and valuable pharmaceuticals.
Zhao joined Frances Arnold’s laboratory at Caltech in November 1992 and graduated in March 1998. As a student of Arnold’s, he developed a number of directed evolution technologies and used them to engineer enzymes as biocatalysts. Five of his papers were cited in the scientific background document prepared by the Nobel Prize committee and a Nature Biotechnology paper published in 1998 remains Arnold’s most cited paper related to directed evolution, according to Google Scholar.
After graduation from Caltech, Zhao worked at the Dow Chemical Company for two years. He joined the Department of Chemical and Biomolecular Engineering at University of Illinois in July 2000. His research program at Illinois is focused on protein engineering, metabolic engineering, natural product biosynthesis, and synthetic biology.
“We are interested in using directed evolution to create enzymes as biocatalysts for synthesis of valuable chemicals and drugs in an environmental-friendly manner,” Zhao said. “We are also interested in engineering microorganisms to produce chemicals and biofuels from lignocellulosic materials. In addition, we are interested in developing new tools to discover novel natural products from soil bacteria that can potentially be used as antibiotics and anti-cancer drugs. Finally, we are interested in developing genome editing tools for the treatment of human genetic diseases and fundamental studies of cell biology. As an underlying theme among these four application areas, we are developing a biofoundry, which is an integrated robotic system, to accelerate our bioengineering efforts.”
Earlier this year, Zhao won the Marvin Johnson Award from the American Chemical Society for “his pioneering contributions in the areas of directed evolution and synthetic biology for industrial and medical applications.” He has authored and co-authored more than 290 research articles and over 20 issued and pending patent applications with several being licensed by industry. He also has given plenary, keynote or invited lectures in over 350 international meetings, universities, industries, and research institutes. Twenty-two of his former graduate students and postdocs became professors in the United States (8), China (Mainland 9, Taiwan 1), Korea (2), Singapore (1), and Egypt (1).
Zhao is the theme leader for the Biosystems Design Theme at the Carl R. Woese Institute for Genomic Biology and he holds faculty affiliations in the departments of Chemistry, Biochemistry, and Bioengineering, as well as visiting investigatorship at the Agency for Science, Technology, and Research in Singapore. He is also Conversion Theme Leader at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a U.S. Department of Energy-funded Bioenergy Research Center at the University of Illinois.
University of Illinois researchers have developed a new method that aids in the process of making valuable compounds by using a unique combination of catalysts.
A study published this month in Nature reported a new method that combines enzymatic catalysts with photocatalysts.
The team that created the method was led by Professor Huimin Zhao, the Steven L. Miller Chair of Chemical and Biomolecular Engineering. Zhao is also Conversion Theme Leader at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a U.S. Department of Energy-funded Bioenergy Research Center. Zhao worked alongside John Hartwig, Professor of Chemistry at the University of California at Berkeley, who was previously a professor at Illinois and is a longtime collaborator of Zhao’s. Coauthors include Yajie Wang, a Ph.D. Candidate in Chemical and Biomolecular Engineering at Illinois, and Zachary Litman, a Postdoctoral Research Fellow at the University of Michigan.
All organisms rely on chemical reactions in order to make various natural products. These reactions can be caused by a number of enzymatic or chemical catalysts.
Scientists often use combinations of enzymatic and chemical catalysts to cause reactions that result in higher yields than what can be achieved with enzymes alone. Higher yields are beneficial when scientists want to use these reactions to make useful products such as biofuels and pharmaceuticals.
But combining enzymatic and chemical catalysts is difficult to do — the two catalysts aren’t naturally compatible; they work best under different conditions and temperatures.
Seeing the need for new approaches, Wang and Litman came up with an idea: to combine enzymatic catalysis and photocatalysis.
Photocatalysis, which uses light to spur on a chemical reaction, is currently a popular research area.
“Lots of researchers have tried to develop new photocatalysts for different reactions,” Zhao said. “And enzyme catalysis is a relatively old field.”
While photocatalysis and enzyme catalysis have been studied separately, few researchers have put these two catalysts together.
The team studied several enzymes and photocatalysts and found a pair that works together.
“If you use a light-driven process instead of one driven by heat, then that will be compatible with the enzymes,” Hartwig said.
The scientists not only showed that enzymatic catalysts and photocatalysts can be combined, but that this combination can also be productive. The method proved useful in creating a few important active pharmaceutical intermediates (APIs) for producing pharmaceutical drugs.
“(This research) would be quite useful for synthetic chemistry,” Hartwig said.
Zhao said the team will pursue further research in this area as a part of CABBI, a collaboration between the Carl R. Woese Institute for Genomic Biology (IGB) and the Institute for Sustainability, Energy, and Environment (iSEE) along with 17 partner institutions.
One of CABBI’s goals is to engineer biological systems that produce non-natural compounds, which can be used for biofuels, jet fuels, lubricants, and more.
The researchers hope to find more combinations of photocatalysts and enzymatic catalysts for more chemical reactions.
“This is just a first example,” Zhao said. “We want to find more chemoenzymatic systems for different types of reactions.”
Zhao expects this research will also inspire new discoveries.
“There are many new photocatalysts for different types of reactions, and there are many enzymes,” he said. “So I can see that this will stimulate researchers to develop new systems that can combine these two types of catalysts for making a wide variety of useful compounds.”
The CRISPR-Cas9 system has given researchers the power to precisely edit selected genes. Now, researchers have used it to develop a technology that can target any gene in the yeast Saccharomyces cerevisiae and turn it off by deleting single letters from its DNA sequence.
Such genome-scale engineering – in contrast to traditional strategies that only target a single gene or a limited number of genes – allows researchers to study the role of each gene individually, as well as in combination with other genes. It also could be useful for industry, where S. cerevisiae is widely used to produce ethanol, industrial chemicals, lubricants, pharmaceuticals and more.
Understanding and optimizing the genome could create yeast strains with increased productivity, said study leader Huimin Zhao, the Steven L. Miller Chair of Chemical Engineering and a member of the Carl R. Woese Institute for Genomic Biology at the U. of I. Zhao’s group published the new findings in the journal Nature Biotechnology.
“We want to use microorganisms as cellular factories to make valuable chemicals and biofuels,” Zhao said. “The scale we have demonstrated in this study is unprecedented. CRISPR has been used to introduce point mutations – for example, to address genetic diseases – but Saccharomyces yeast has about 6,000 genes, and we want to be able to knock out each of these genes iteratively and find out how they affect the production of a target compound.”
Researchers produce “knockout” yeast – where one gene has been deleted, or “knocked out” – to study how each gene contributes to the function of the cell. When a beneficial mutation is found, they can selectively breed yeast with that characteristic. Leading methods to produce knockout yeast excise the entirety of the targeted gene. This creates unintended problems, Zhao said, because many genes overlap each other. Deleting one gene also deletes portions of others, affecting multiple functions and making it difficult for researchers to truly isolate the effects of a single gene.
Each letter in a DNA sequence corresponds to a base, the building blocks that make up DNA chains. Zhao’s group harnessed the precision of the CRISPR-Cas9 system to create a technique that allows them to delete just one base in a gene’s DNA sequence. Since a cell “reads” DNA three bases at a time, this shifts the reading frame and knocks out the gene. Genes that overlap with the edited one remain unchanged and functional.
“We can introduce just one single base change on the entire chromosome. That makes a minimal disturbance in the function of the neighboring genes, so we can study how important the gene is in its cellular context. That kind of precision has not been achieved before,” Zhao said.
Their technique, named CRISPR/Cas9 and homology-directed-repair assisted genome-scale engineering or CHAnGE, has the advantages of being quick, efficient and low-cost, in addition to its precision. Zhao’s group developed a library of knockout yeast, one for each gene in the S. cerevisiae genome, and are making it available to other researchers for a $50 handling fee.
“In the past, teams of people would spend several years trying to knock out every gene in a yeast. With CHAnGE, one person can generate a library of yeast mutants covering the entire genome in about a month,” Zhao said.
Zhao’s group is working to develop libraries for other types of yeast, including ones that produce lipids used in lubricants, biofuels and other industrial applications.
The U.S. Department of Energy and the Carl R. Woese Institute for Genomic Biology at the U. of I. supported this work. Zhao is also a professor in the Carle Illinois College of Medicine at Illinois.
By Liz Ahlberg Touchstone, University of Illinois News Bureau