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Chemical and Biomolecular Engineering

New approach combines synthetic biology, genome editing tools, and automation to quickly and effectively produce novel yeast strains

One of humankind’s oldest industrial partners is yeast, a familiar microbe that enabled early societies to brew beer and leaven bread and empowers modern ones to synthesize biofuels and conduct key biomedical research. Yeast remains a vital biological agent, yet our ability to explore and influence its genomic activity has lagged.

In a new article in Nature Communications (DOI: 10.1038/NCOMMS15187), University of Illinois researchers describe how their successful integration of several cutting-edge technologies—creation of standardized genetic components, implementation of customizable genome editing tools, and large-scale automation of molecular biology laboratory tasks—will enhance our ability to work with yeast. The results of their new method demonstrate its potential to produce valuable novel strains of yeast for industrial use, as well as to reveal a more sophisticated understanding of the yeast genome.

Zhao and Si
Dr. Huimin Zhao and IGB Research Fellow Tong Si

“The goal of the work was really to develop a genome-scale engineering tool for yeast . . . traditional metabolic engineering focused on just a few genes and the few existing genome-scale engineering tools are only applicable to bacteria, not eukaryotic organisms like yeast,” said Steven L. Miller Chair of Chemical and Biomolecular Engineering Huimin Zhao, who led the study. “A second innovation is the use of synthetic biology concepts, the modularization of the parts, and integration with a robotic system, so we can do it in high-throughput.”

The team focused on yeast in part because of its important modern-day applications; yeasts are used to convert the sugars of biomass feedstocks into biofuels such as ethanol and industrial chemicals such as lactic acid, or to break down organic pollutants. Because yeast and other fungi, like humans, are eukaryotes, organisms with a compartmentalized cellular structure and complex mechanisms for control of their gene activity, study of yeast genome function is also a key component of biomedical research.

“In basic science, a lot of fundamental eukaryotic biology is studied in yeast,” said Tong Si, a Carl R. Woese Institute for Genomic Biology Research Fellow. “People have a limited understanding of these complicated systems. Although there are approximately 6,000 genes in yeast, people probably know less than 1,000 by their functions; all the others, people do not know.”

The group took the first step toward their goal of a novel engineering strategy for yeast by creating what is known as a cDNA library: a collection of over 90% of the genes from the genome of baker’s yeast (Saccharomyces cerevisiae), arranged within a custom segment of DNA so that each gene will be, in one version, overactive within a yeast cell, and in a second version, reduced in activity.

Zhao and colleagues examined the ability of the CRISPR-Cas system, a set of molecules borrowed from a form of immune system in bacteria (CRISPR stands for clustered regularly interspaced short palindromic repeats, describing a feature of this system in bacterial genomes). This system allowed Zhao to make precise cuts in the yeast genome, into which the standardized genetic parts from their library could insert themselves.

“The first time we did this, in 2013, there was no CRISPR . . . the best we could get was 1% of the cells modified in one run,” said Si. “We struggled a little on that, and when CRISPR came out, that worked. We got it to 70% [cells modified], so that was very important.”

With gene activity-modulating parts integrating into the genome with such high efficiency, the researchers were able to randomly generate many different strains of yeast, each with its own unique set of modifications. These strains were subjected to artificial selection processes to identify those that had desirable traits, such as the ability to survive exposure to reagents used in the biofuel production process.

This selection process was greatly aided by the Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB), a robotic system that performs most of the laboratory work described above in an automated way, including selection of promising yeast strains. Use of iBioFAB greatly accelerated the work, enabling simultaneous creation and testing of many unique strains. The iBioFAB was conceived and developed by the Biosystems Design research theme at the Carl R. Woese Institute for Genomic Biology (IGB), which is led by Zhao.

With support from the High Performance Biological Computing Group at Illinois, Zhao, Si and their colleagues analyzed the modified genomes of their most promising yeast strains.  They identified combinations of genes whose altered activities contributed to desirable traits; the functions of some of these genes were previously unknown, demonstrating the technique’s ability to generate new biological knowledge.

“I think the key difference between this method and the other existing metabolic engineering strategies in yeast is really the scale,” said Zhao. “The current metabolic engineering strategies are all focused on just a few genes, dozens of genes at most . . .  it’s very intuitive.  With this we can explore all the genes, we can identify a lot of targets that cannot be intuited.”

The work, which was funded by the Roy J. Carver Charitable Trust, IGB, Defense Advanced Research Program Agency, and National Academies Keck Futures Initiative on Synthetic Biology, paves the way for similar approaches to broad-scale, automated genome engineering of other eukaryotic species.

By Claudia Lutz, Carl R. Woese Institute for Genomic Biology

By Liz Ahlberg Touchstone, University of Illinois News Bureau

In the fight against disease, many weapons in the medicinal arsenal have been plundered from bacteria themselves. Using CRISPR-Cas9 gene-editing technology, researchers have now uncovered even more potential treasure hidden in silent genes.

A new study from researchers at the University of Illinois and colleagues at the Agency for Science, Technology and Research in Singapore used CRISPR technology to turn on unexpressed, or “silent,” gene clusters in Streptomyces, a common class of bacteria that naturally produce many compounds that have already been used as antibiotics, anti-cancer agents and other drugs. The study, led by chemical and biomolecular engineering professor Huimin Zhao, was published in the journal Nature Chemical Biology.

Clockwise from back middle: Behnam Enghiad, graduate student; Shangwen Luo, postdoc; Tajie Luo, grad student; and Huimin Zhao, professor of chemical and biomolecular engineering
Clockwise from back middle: Behnam Enghiad, graduate student; Shangwen Luo, postdoc; Tajie Luo, grad student; and Huimin Zhao, Steven L. Miller Chair in Chemical Engineering. Photo by L. Brian Stauffer.

“In the past, researchers just screened the natural products that bacteria made in the lab to search for new drugs,” Zhao said. “But once whole bacterial genomes were sequenced, we realized that we have only discovered a small fraction of the natural products coded in the genome.

“The vast majority of biosynthetic gene clusters are not expressed under laboratory conditions, or are expressed at very low levels. That’s why we call them silent. There are a lot of new drugs and new knowledge waiting to be discovered from these silent gene clusters. They are truly hidden treasures.”

To mine for undiscovered genomic treasure, the researchers first used computational tools to identify silent biosynthetic gene clusters – small groups of genes involved in making chemical products. Then they used CRISPR technology to insert a strong promoter sequence before each gene that they wanted to activate, prompting the cell to make the natural products that the genes clusters coded for.

“This is a less-explored direction with the CRISPR technology. Most CRISPR-related research focuses on biomedical applications, like treating genetic diseases, but we are using it for drug discovery,” Zhao said. His lab was the first to adapt the CRISPR system for Streptomyces. “In the past, it was very difficult to turn on or off a specific gene in Streptomyces species. With CRISPR, now we can target almost any gene with high efficiency.”

The Illinois team collaborated with a team from A*STAR in Singapore.

Zhao.CRISPR.AStar
Pictured: Wan Lin Yeo, Huimin Zhao, Ee Lui Ang, Mingzi M. Zhang, Fong Tian Wong, Yee Hwee Lim and Elena Heng. Photo courtesy of Huimin Zhao.

The team succeeded in activating a number of silent biosynthetic gene clusters. To look for drug candidates, each product needs to be isolated and studied to determine what it does. As a demonstration, the researchers isolated and determined the structure of one of the novel compounds produced from a silent biosynthetic gene cluster, and found that it has a fundamentally different structure from other Streptomyces-derived drugs – a potential diamond in the rough.

Zhao said such new compounds could lead to new classes of drugs that elude antibiotic resistance or fight cancer from a different angle.

“Antimicrobial resistance is a global challenge. We want to find new modes of action, new properties, so we can uncover new ways to attack cancer or pathogens. We want to identify new chemical scaffolds leading to new drugs, rather than modifying existing types of drugs,” he said.

The U.S. National Institutes of Health and the National Research Foundation of Singapore supported this work.

To reach Huimin Zhao, call (217)333-2631; email: zhao5@illinois.edu.

The paper “CRISPR–Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters” is available online. doi:10.1038/nchembio.2341

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