A novel method developed by University of Illinois researchers could change the way metabolic engineering is done.
Researchers from the Carl R. Woese Institute for Genomic Biology’s Biosystems Design theme, including Huimin Zhao, the Steven L. Miller Chair of Chemical and Biomolecular Engineering, recently published a paper in Nature Communications outlining their new method, which could make the metabolic engineering process more efficient.
Metabolic engineering involves engineering microorganisms to produce value-added products such as biofuels and chemicals. This is achieved by changing or deleting the expression of genes to modify the microorganism’s genome. In this process, several targets in the genome are modified in order to achieve specific goals.
“We can easily find several metabolic engineering targets to improve the desired phenotype,” said Jiazhang Lian, a visiting research associate at the IGB who is a co-author of the paper. “How to combine these beneficial genetic modifications is one of the biggest challenges in metabolic engineering.”
Traditionally, researchers test these targets individually in a series of time-consuming steps. These steps limit productivity and the yield of the final product — two crucial components in the metabolic engineering process.
The researchers decided to create a method that combines all of these steps and executes them simultaneously, making the process faster and easier.
They based this method on the CRISPR system, a method of genetic manipulation that uses a set of DNA sequences to modify genes within a cell.
This system uses three genetic manipulations that are frequently used in metabolic engineering: transcriptional activation, transcriptional interference, and gene deletion.
By using these manipulations simultaneously, scientists can explore different combinations of manipulations and discover which combination is best.
“We can now work with 20 targets,” Zhao said. “We can implement all of these (manipulations) for each target in a combinatorial manner to find out which combination actually will give us higher productivity or yield of the final product.”
The researchers tested the method in a species of yeast that is used in winemaking, baking, and the production of biofuels. They showed that using this method could improve the production of a specific product.
Their system, called CRISPR-AID, will allow researchers to easily explore all the possible target combinations. But the key is to find the optimal combination.
“If we compare metabolic engineering to a basketball team, we cannot build a strong team by simply putting the best players together,” Lian said. “Instead, we should try to find those who can collaborate and work synergistically.”
Their new system opens up thousands — even millions — of possibilities, which presents another logistical challenge.
They plan to find the best combinations by developing a high throughput screening method or using a robotic system such as the iBioFAB, a system located in the IGB that automatically produces synthetic biosystems.
“I believe the combination of CRISPR-AID with high throughput screening and iBioFAB will significantly advance the metabolic engineering field in the near future,” Lian said.
Zhao hopes to test their method on other organisms, using the same engineering principles but modifying the protocol for different organisms.
Eventually, they hope to extend to the genome scale — to be able to test all the genes in an organism at once — which would be a considerable leap in the field of metabolic engineering.
“If we can do that, we can make it truly modularized and also standardize the procedure,” Zhao said. “Then we really increase the throughput and the speed of metabolic engineering.”
Several research efforts aim to engineer microorganisms for the production of biofuels and chemicals, so any tools that can speed up the process are significant. Zhao believes this is true for their method.
“It’s not just an incremental improvement,” he said. “It’s a new way to do metabolic engineering.”
Chemical and Biomolecular Engineering faculty member Brendan Harley and a team of researchers from the Carl R. Woese Institute for Genomic Biology’s Regenerative Biology and Tissue Engineering theme were awarded a National Science Foundation grant that will provide funding for a new 3D-bioprinting instrument.
The Major Research Instrumentation grant will fund the purchase of an EnvisionTEC 3D-Bioplotter, a bioprinting system that is essentially a 3D printer for tissues.
Much of the research in the Regenerative Biology and Tissue Engineering (RBTE) theme involves developing approaches to regenerate tissues in order to address problems of human health. But this is not a simple task, considering human tissues are all uniquely complex.
Bioprinting, which uses 3D printing technology to create cell patterns and fabricate biomaterials, will allow researchers to print materials that closely resemble human tissues.
Unlike many 3D printers, which print metals or other hard materials, this bioplotter will let researchers print a wide variety of materials — including materials that are similar in consistency to toothpaste, as well as materials that are the size of human hair or smaller.
“This bioplotter really allows us to do all that,” said Brendan Harley, Associate Professor of Chemical and Biomolecular Engineering and theme leader. “It really lets us open up the toolbox of the materials we can use.”
The bioplotter will be housed within IGB as a shared-use facility that will be accessible to researchers across campus.
Several research efforts at the IGB and the University of Illinois are dedicated to developing tissue engineering solutions that could impact a range of important healthcare issues such as cancer, stem cell behavior and more.
Harley hopes researchers will look to the IGB and the RBTE theme as a hub for tissue engineering on campus.
He said 3D bioplotters have finally become capable of reliably printing at the scale needed to produce complex tissues, and with the acquisition of this printer, IGB will be at the forefront of tissue engineering research.
“We’re going to be able to do some really exciting new work,” he said. “Everything’s in place to really attack big, challenging problems.”
By Emily Scott, IGB Science Writer and Outreach Specialist
A new, multimillion dollar bioenergy research center at the University of Illinois that promises to be a catalyst for the development of sustainable, cost-effective biofuels and bioproducts will involve several faculty from Chemical and Biomolecular Engineering.
The Department of Energy announced earlier this summer it has awarded the University of Illinois $104 million for the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI). The center is a collaboration between Illinois’ Institute for Sustainability, Energy, and Environment (iSEE) and the Carl R. Woese Institute for Genomic Biology (IGB), and will include 17 partner institutions.
“As the United States seeks energy independence, we need to look at the most efficient ways to grow, transform, and market biofuels,” said Evan H. DeLucia, the G. William Arends Professor of Plant Biology and Baum Family Director of iSEE. DeLucia will serve as CABBI Director. “This grant is a game-changer, and CABBI will be at the forefront as we press toward a new bio-based economy. Our center’s holistic approach will generate new products directly from biomass, reducing our nation’s dependence on fossil fuels and making us more secure.”
CABBI is one of four Department of Energy Bioenergy Research Centers, joining the Great Lakes Bioenergy Research Center led by the University of Wisconsin, the Center for Bioenergy Innovation led by the DOE’s Oak Ridge National Laboratory, and the Joint Bioenergy Institute led by the DOE’s Lawrence Berkeley National Lab.
At Illinois, researchers will develop fuels and products by integrating three interconnected priority areas: Growing the Right Crops (feedstock development), Turning Plants into Fuel (conversion), and Determining the Environmental and Economic Bottom Line (sustainability). Crop Sciences professor Stephen Moose will lead the feedstock development theme, in which scientists will integrate recent advances in genomics, synthetic biology, and computational biology to increase the value of biomass crops. Madhu Khanna, ACES Distinguished Professor in Environmental Economics in the Department of Agricultural and Consumer Economics, will lead the sustainability theme in which researchers will provide an overarching framework for viewing outcomes from the feedstocks and conversion themes through an environmental and economic lens.
Dr. Huimin Zhao, the Steven L. Miller Chair in Chemical Engineering at Illinois, will lead the conversion area. His team of about 19 principal investigators, which include two other ChBE faculty at Illinois—Professor Chris Rao and Assistant Professor David Flaherty—will further develop a versatile, automated “biofoundry” for rapidly engineering microbial strains that can efficiently produce diverse, high-value molecules such as biodiesel, organic acids, jet fuels, lubricants, and alcohols. Using the design-build-test-learn framework, research in the conversion theme will overcome the challenges associated with driving biological systems to produce non-natural compounds.
“It’s a unique vision. We want to use the plants as the factories to produce lipid-based chemicals and then we’ll couple that with the microbial conversion platform to make more high-value added products. Furthermore, we will use the lignocellulose from those plants as the feedstocks to make a wide variety of chemicals,” Zhao said.
This approach is different from the other three existing bioenergy centers funded by the Department of Energy, he said. The other centers focus their work on developing technologies to deconstruct the lignocellulose to generate fermentable sugars like glucose and xylose and using those sugars to make a variety of fuels and chemicals.
He and his team of researchers also want to understand what constrains the production of those biofuels or chemicals in the microorganisms. And they want to produce more chemicals and biofuels as well. That’s why Zhao has brought in experts in catalysis like Dr. David Flaherty who will develop chemical catalysts to upgrade the fuels and chemicals produced by the microorganisms.
Flaherty’s group will be collaborating with others in CABBI to develop catalysts to convert advantaged molecules produced from microorganisms, such as butanol and unsaturated fatty acids, into clean burning fuels. They’ll also be coproducing high value chemicals to help increase the economic viability of the overall process.
“The networks of catalytic reactions in these systems are incredibly complex, and to be successful, we will need to develop maps of the reaction pathways that exist and use that information to identify opportunities to control the selectivity to specific desired products,” Flaherty said.
Dr. Chris Rao will focus on the engineering of oleaginous yeast to produce biofuels and chemicals and to understand what constrains the production of those products in the yeast.
“If you think from the organisms’ point of view, they don’t want to produce the product we want at large amounts because it will not benefit their survival and growth. … So we have to hijack the native metabolism to make the organisms themselves produce the product we want,” Zhao said.
The goal of metabolic engineering is to essentially engineer microorganisms to produce useful chemicals, but if they produce at low levels, that would not be economical, Zhao said. How does one make the organism produce a lot of product in a very short period of time? That’s another challenge researchers will address.
“In the traditional chemical industry, what a chemist or chemical engineer often does is to develop chemical catalysts and use them to convert non-renewable petroleum oils into fuels and chemicals. Now we want to develop biological catalysts such as microorganisms and enzymes and use them to convert renewable plant biomass into fuels and chemicals, which represents a paradigm shift in the chemical industry,” Zhao said.
Zhao, Rao and Flaherty have collaborated before. All three were involved with the Energy Biosciences Institute, which was established in 2007 as a partnership between Illinois and the University of California at Berkeley and Lawrence Berkeley National Lab, with initial funding from BP. Because of their work with EBI, Illinois had a strong track record of collaboration and accomplishments in bioenergy research and an infrastructure in place, Zhao said.
At the Carl R. Woese Institute for Genomic Biology, Zhao leads the Biosystems Design theme and he has been involved in development of the Illinois Biological Foundry for Advanced Biomanufacturing, or iBioFAB. Housed at IGB, iBioFAB is a computational and physical infrastructure that supports rapid design, fabrication, validation/quality control, and analysis of genetic constructs and organisms.
IGB will oversee and integrate CABBI’s core science team under one roof.
“The IGB, now with over a decade of experience in successfully addressing grand challenges by transdisciplinary integration of the life sciences, physical sciences, and engineering, will provide an outstanding environment for the talented CABBI team,” said director Gene Robinson.
The Institute for Sustainability, Energy, and Environment will coordinate and integrate field work off campus and at the Illinois Energy Farm, a 320-acre site that enables researchers to trial promising biofuel feedstocks at scale, and it will utilize the nearly complete, $32 million Integrated Bioprocessing Research Laboratory.
“We are very excited about this project because this will further build on our strengths in this area. This is definitely the future. I’m pleased the Department of Energy is committed to this direction,” Zhao said.
The center is expected to receive $4 million in fiscal year 2018, then $25 million a year in 2019-22.
Partner institutions include Brookhaven (N.Y.) National Laboratory; the Lawrence Berkeley National Laboratory’s Joint Genome Institute in Berkeley, Calif.; the U.S. Department of Agriculture’s (USDA) Agricultural Research Service (ARS) in Houma, La., the USDA ARS in Peoria, Ill.; Iowa State University; Princeton University; Mississippi State University; the University of California-Berkeley; West Virginia University; Boston University; the University of Wisconsin-Madison; Colorado State University; the University of Idaho; the University of Florida; the University of Nebraska; the Institute for Systems Biology in Seattle; and the HudsonAlpha Institute for Biotechnology in Huntsville, Ala.
The Department of Energy’s Bioenergy Research Program was established in 2007 and has led to 2,630 peer-reviewed publications, 607 invention disclosures, 378 patent applications, 191 licenses or options, 92 patents, and 14 start-up companies.
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.
“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
Researchers at the University of Illinois report they can alter blood cell development through the use of biomaterials designed to mimic characteristics of the bone marrow.
The findings, reported in the journal Science Advances, are a first step toward developing more effective bone marrow treatments for diseases like leukemia and lymphoma.
Blood cells flow throughout the body delivering life-supporting oxygen and nutrients. As these cells are used and recycled they are regenerated by bone marrow, the soft tissue inside the body’s long and hollow bones.
Certain regions of bone marrow contain hematopoietic stem cells, the precursors of all blood and immune cells, said University of Illinois chemical and biomolecular engineering professor Brendan Harley, who led the research with postdoctoral researcher Ji Sun Choi.
“The tissue environment that surrounds these cells in the bone marrow provides a wealth of signals that can alter how these precursor cells behave. This paper looked at the signals provided by the tissue matrix itself,” said Harley, who also is affiliated with the Carl R. Woese Institute for Genomic Biology at the U. of I.
One of the major tools that oncologists use to treat leukemia and lymphoma involves transplanting HSCs. The donor stem cells must locate marrow cavities and start producing blood and immune cells. However, there is a limited quantity of available donor HSCs and the success rate of transplantation is low.
“We’re interested in this problem from an engineering standpoint,” Harley said. “The goal is to create better tools to both expand the number of donor HSCs and improve their capacity to repopulate the bone marrow after transplantation.”
Like cells throughout the body, HSCs are contained in a three-dimensional tissue environment known as the extracellular matrix. Harley and Choi gathered samples of HSCs from mice and then grew them in the laboratory using biomaterials engineered to mimic some of the extracellular matrix properties of the native bone marrow. Their goal was to examine how these engineered systems could alter the HSCs’ capacity to proliferate and differentiate to become blood cells.
The researchers examined two main elements of the matrix that regularly interact with HSCs: collagen and fibronectin. They found that the HSCs that were exposed to collagen proliferated more rapidly but that they had differentiated, meaning they were no longer stem cells. When exposed to fibronectin, the stem cells proliferated less rapidly, but were able to maintain their stem cell-like nature.
“With the collagen substrates, we got more cells but not useful cells,” Harley said. “With the right combination of stiffness in the matrix and the presence of fibronectin, we identified a class of biomaterials that show promise for being able to maintain and eventually expand these stem cells outside of the body. An engineered bone marrow will be of enormous value for treating hematopoietic cancers such as leukemia, but also for understanding the process of bone marrow failure and other hematopoietic diseases.”
This project is only the first step in controlling the signals from the matrix that influence HSCs, Harley said. He and other researchers in his lab are currently investigating other features of the matrix that can be manipulated to increase the number of stem cells and make them more effective in transplantation.
The National Science Foundation, National Institutes of Health and the American Cancer Society of Illinois supported this research.
by Sarah Banducci, University of Illinois News Bureau Intern
To reach Brendan Harley, call 217-244-7112; email email@example.com.
Research by Professor of Chemical and Biomolecular Engineering Huimin Zhao and graduate student Behnam Enghiad is pioneering a new method of genetic engineering for basic and applied biological research and medicine. Their work, reported in ACS Synthetic Biology, has the potential to open new doors in genomic research by improving the precision and adherence of sliced DNA.
“Using our technology, we can create highly active artificial restriction enzymes with virtually any sequence specificity and defined sticky ends of varying length,” said Zhao. “This is a rare example in biotechnology where a desired biological function or reagent can be readily and precisely designed in a rational manner.”
Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research, however have limited sequence specificity and availability. The Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) is capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length.
Restriction enzymes are an important tool in genomic research: by cutting DNA at a specific site, they create a space wherein foreign DNA can be introduced for gene-editing purposes. This process is not only achieved by naturally-occurring restriction enzymes; other artificial restriction enzymes, or AREs, have risen to prominence in recent years. CRISPR-Cas9, a bacterial immune system used for “cut-and-paste” gene editing, and TALENs, modified restriction enzymes, are two popular examples of such techniques.
Though useful in genetic engineering, no AREs generate defined “sticky ends”—an uneven break in the DNA ladder-structure that leaves complementary overhangs, improving adhesion when introducing new DNA. “If you can cleave two different DNA samples with the same restriction enzyme, the sticky ends that are generated are complementary,” explained Enghiad. “They will hybridize with each other, and if you use a ligase, you can stick them together.”
However, restriction enzymes themselves have a critical drawback: the recognition sequence which prompts them to cut is very short—usually only four to eight base pairs. Because the enzymes will cut anywhere that sequence appears, researchers rely on finding a restriction enzyme whose cut site appears only once in the genome of their organism or plasmid—an often difficult proposition when the DNA at hand might be thousands of base pairs long.
This problem has been partially solved simply by the sheer number of restriction enzymes discovered: more than 3600 have been characterized, and over 250 are commercially available. “Just in our freezer, for our other research, we have probably over 100 different restriction enzymes,” said Enghiad. “We look through them all whenever we want to assemble something … the chance of finding the unique restriction site is so low.
“Our new technology unifies all of those restriction enzymes into a single system consisting of one protein and two DNA guides. Not only have you replaced them, but you can now target sites that no available restriction enzymes can.”
Enghiad and Zhao’s new technique creates AREs through the use of an Argonaute protein (PfAgo) taken from Pyrococcus furiosus, an archeal species. Led by two DNA guides, PfAgo is able to recognize much longer sequences when finding its cut site, increasing specificity and removing much of the obstacles posed by restriction enzymes. Further, PfAgo can create longer sticky ends than even restriction enzymes, a substantial benefit as compared to other AREs.
“When we started, I was inspired by a paper about a related protein—TtAgo. It could use DNA guides to cleave DNA, but the protein is only active at temperatures up to 75 degrees,” explained Enghiad. “DNA strands start to separate at temperatures higher than 75 degrees, which could allow Ago proteins to cleave double stranded DNA. If there were a protein that was active at higher temperatures, I reasoned, that protein could be used as an artificial restriction enzyme.
“So I started looking for that, and what I found was PfAgo.”
In addition to replacing restriction enzymes in genetic engineering processes, Enghiad and Zhao believe their technology will have broad applications in the biological research. By creating arbitrary sticky ends, PfAgo could make assembly of large DNA molecules easier, and enables cloning of large DNA molecules such as biochemical pathways and large genes.
The application of these techniques is broad-reaching: ranging from discovery of new small molecule drugs to engineering of microbial cell factories for synthesis of fuels and chemicals to molecular diagnostics of genetic diseases and pathogens, which are the areas Zhao and Enghiad are currently exploring.
“Due to its unprecedented simplicity and programmability (a single protein plus DNA guides for targeting), as well as accessibility … we expect PfAgo-based AREs will become a powerful and indispensable tool in all restriction enzyme or nuclease-enabled biotechnological applications and fundamental biological research,” said Zhao. “It is to molecular biology as the CRISPR technology is to cell biology.”
Written by Kathryne Metcalf of the Carl R. Woese Institute for Genomic Biology.