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Chemical and Biomolecular Engineering

Microorganisms possess natural product biosynthetic gene clusters (BGCs) that may harbor unique bioactivities for use in drug development and agricultural applications. However, many uncharacterized microbial BGCs remain inaccessible. Researchers at Illinois previously demonstrated a technique using transcription factor decoys to activate large, silent BGCs in bacteria to aid in natural product discovery.

Now, they have developed a direct cloning method that aims to accelerate large-scale discovery of novel natural products. Their findings are reported in the journal Nature Communications.

Named Cas12a assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE), the method allows for direct cloning of large genomic fragments, including those with high-GC content or sequence repeats. Where existing direct cloning methods fail to effectively clone natural product BGCs of this nature, CAPTURE excels.

“Using CAPTURE, microbial natural product BGCs can be directly cloned and heterologously expressed at an unprecedented rate,” said study leader and Steven L. Miller Chair professor of chemical and biomolecular engineering Huimin Zhao (BSD leader/CABBI/GSE/MMG). “As a result, CAPTURE allows large-scale cloning of natural product BGCs from various organisms, which can lead to discovery of numerous novel natural products.”

Researchers first characterized the efficiency and robustness of CAPTURE by cloning 47 natural product BGCs from both Actinomycetes and Bacilli. After demonstrating nearly 100% efficiency of CAPTURE, 43 uncharacterized natural product BGCs from 14 Streptomyces and three Bacillus species were cloned and heterologously expressed by researchers. The produced compounds were purified and determined as 15 novel natural products, including six unprecedented compounds designated as bipentaromycins. Four of the bipentaromycins exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Bacillus anthracis.

“Addressing the current antimicrobial resistance crisis requires discovery of novel molecules capable of treating drug-resistant infections,” said Zhao. “Discovery of bipentaromycins not only demonstrates the possibility of discovering novel antimicrobials, but it also provides an example on how this strategy can be applied for discovery of unique bioactive compounds for use in drug development and agricultural applications.”

The researchers plan next to characterize these compounds for other bioactivities such as anticancer, antiparasitic and anticancer properties. Preliminary results are already showing anticancer properties for some of the compounds.

“Due to its exceptional robustness and efficiency, CAPTURE will likely become the method of choice for direct cloning of large DNA molecules such as natural product BGCs from genomic or metagenomic DNA for various basic and applied biological applications,” said Zhao.

News Source

By: Alisa King-Klemperer | Carl R. Woese Institute for Genomic Biology


The research team included, from left, postdoctoral researcher Saurabh Shukla, graduate student Che Yang, chemical and biomolecular engineering professor Huimin Zhao, physics professor Paul Selvin, graduate student Meng Zhang, postdoctoral researcher Zia Fatma, graduate student Xiong Xiong, and Surbhi Jain, a former doctoral student at the U. of I. who is now a group lead in cancer biology at Northwestern University. Composite image from separate photos, in compliance with COVID-19 safety protocols. Composite photo by L. Brian Stauffer

Researchers used single-molecule imaging to compare the genome-editing tools CRISPR-Cas9 and TALEN. Their experiments revealed that TALEN is up to five times more efficient than CRISPR-Cas9 in parts of the genome, called heterochromatin, that are densely packed. Fragile X syndrome, sickle cell anemia, beta-thalassemia and other diseases are the result of genetic defects in the heterochromatin.

The researchers report their findings in the journal Nature Communications.

The study adds to the evidence that a broader selection of genome-editing tools is needed to target all parts of the genome, said Huimin Zhao, a professor of chemical and biomolecular engineering at the University of Illinois Urbana-Champaign who led the new research.

“CRISPR is a very powerful tool that led to a revolution in genetic engineering,” Zhao said. “But it still has some limitations.”

CRISPR is a bacterial molecule that detects invading viruses. It can carry one of several enzymes, such as Cas-9, that allow it to cut viral genomes at specific sites. TALEN also scans DNA to find and target specific genes. Both CRISPR and TALEN can be engineered to target specific genes to fight disease, improve crop plant characteristics or for other applications.

Zhao and his colleagues used single-molecule fluorescence microscopy to directly observe how the two genome-editing tools performed in living mammalian cells. Fluorescent-labeled tags enabled the researchers to measure how long it took CRISPR and TALEN to move along the DNA and to detect and cut target sites.

“We found that CRISPR works better in the less-tightly wound regions of the genome, but TALEN can access those genes in the heterochromatin region better than CRISPR,” Zhao said. “We also saw that TALEN can have higher editing efficiency than CRISPR. It can cut the DNA and then make changes more efficiently than CRISPR.”

TALEN was as much as five times more efficient than CRISPR in multiple experiments.

The findings will lead to improved approaches for targeting various parts of the genome, Zhao said.

“Either we can use TALEN for certain applications, or we could try to make CRISPR work better in the heterochromatin,” he said.

The National Institutes of Health and National Science Foundation support this work.

Zhao is a member of the Carl R. Woese Institute for Genomic Biology at the U. of I.


News Source

Diana Yates  | Life Sciences Editor, U. of I. News Bureau

The paper “TALEN outperforms Cas9 in editing heterochromatin target sites” is available from the U. of I. News Bureau.

DOI: 10.1038/s41467-020-20672-5

Check out the original story.

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).

Xiaoqiang Huang, post doc in Huimin Zhao lab

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.

photo credit Jordan Goebig

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.

Huimin Zhao, Steven L. Miller Chair in Chemical Engineering

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.

MUSICAL FAMILY
Ultrasound imaging is used extensively in diagnostic medicine. When tiny, stable bubbles like the ones shown here are introduced to the bloodstream as a contrast agent, the difference between blood vessels and the surrounding tissue in echogenicity (the ability to reflect the ultrasound waves) is enhanced. The resulting image quality leads to improved diagnosis of cancers and vascular diseases. Zeiss Stereolumar v12 microscope, Jinrong Chen, Hyunjoon Kong Laboratory 6.0 Exhibition, Funded by the NIH.
LANISPHERE

Genome editing holds immense promise in revolutionizing all aspects of medicine and many other industries. By understanding how gene editing proteins behave inside the cell at the single molecule level, researchers can gain insights into designing highly-specific technologies. CRISPR has been deservedly recognized as an efficient way to edit DNA, but scientists have other strategies to choose from. TALE molecular pathways are more accurate than CRISPR in their identification of DNA sites to be edited. This image evokes the precise pathways taken by the TALE molecules along strands of DNA in living cells. Nikon Ti Eclipse Microscope, Surbhi Jain, Huimin Zhao Laboratory. Funded by the NIH.
THE MEASURE OF EVERY PART
In this image, a few dark points stand out against a softer, textured background. These points represent individual copies of a single gene within human cancer cells. The researchers
who created this image developed a new method that allows them to track, in 3D, the location of individual genes within cells. This tool will allow researchers to visualize fundamental biological processes and reveal how these processes are disrupted in diseases such as cancer. Zeiss Elyra S1 Super Resolution Structured Illumination Microscope. Ipek Tasan, Huimin Zhao Laboratory, Funded by the NIH.
FIRST IMPRESSIONS
Many processes within the body are changed by the presence of cancer. These images show the response of microglia, immune defense cells in the brain, to cancer cells. When microglia encounter glioblastoma multiforme, one of the most aggressive brain cancers, they shift from a relaxed, elongated shape to a rounded, ready-for-combat conformation. These images echo the work of Anna Atkins, a British botanist and photographer who used a contact printing technique called cyanotyping to capture the form of plants and algae. Emily Chen’s work similarly seeks to explore biological function, in this case the immune response to brain cancer, by capturing and comparing biological forms. Zeiss LSM 710 Confocal Microscope, Jee-Wei Emily Chen, Brendan Harley Laboratory. Funded by the NIH.

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.

From left: Scott Weisberg, former undergraduate researcher; Saurabh Sinha, professor of computer science; Mohammad (Sam) Hamedi Rad, former graduate student; and Huimin Zhao, professor of chemical and bimolecular engineering, stand near iBioFAB2.0.

“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 zhao5@illinois.edu.

The paper “Towards a fully automated algorithm driven platform for biosystems design” is available online and from the U. of I. News Bureau. DOI: 10.1038/s41467-019-13189-z

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.

Huimin Zhao, the Steven L. Miller Chair of Chemical and Biomolecular Engineering

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.

From left: Fang Guo, postdoctoral fellow; Huimin Zhao, the Steven L. Miller Chair in Chemical Engineering; and Bin Wang, postdoctoral fellow. Photo by L. Brian Stauffer.

“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: zhao5@illinois.edu.

The paper “Activation of silent biosynthetic gene clusters using transcription factor decoys” is available online.


DOI: 10.1038/s41589-018-0187-0

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.

Nobel Laureate Frances Arnold and Prof. Huimin Zhao at the ceremony.

“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.

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