Paula Alonso A.,Michigan State University |
Paula Alonso A.,Great Lakes Bioenergy Research Center |
Dale V.L.,Monsanto Corporation |
Shachar-Hill Y.,Michigan State University |
Shachar-Hill Y.,Great Lakes Bioenergy Research Center
Metabolic Engineering | Year: 2010
The efficiency with which developing maize embryos convert substrates into seed storage reserves was determined to be 57-71%, by incubating developing maize embryos with uniformly labeled 14C substrates and measuring their conversion to CO2 and biomass products. To map the pattern of metabolic fluxes underlying this efficiency, maize embryos were labeled to isotopic steady state using a combination of labeled 13C-substrates. Intermediary metabolic fluxes were estimated by computer-aided modeling of the central metabolic network using the labeling data collected by NMR and GC-MS and the biomass composition. The resultant flux map reveals that even though 36% of the entering carbon goes through the oxidative pentose-phosphate pathway, this does not fully meet the NADPH demands for fatty acid synthesis. Metabolic flux analysis and enzyme activities highlight the importance of plastidic NADP-dependent malic enzyme, which provides one-third of the carbon and NADPH required for fatty acid synthesis in developing maize embryos. © 2010.
« Dürr acquires Industry 4.0 software specialist iTAC | Main | New type of thermally stable oilseed rape oil could have tremendous impact on global lubricant market » Researchers at the University of Wisconsin-Madison have developed a simple, robust, and efficient method for generating interspecies yeast hybrids. As reported in the journal Fungal Genetics and Biology, this method provides an efficient means for producing novel synthetic hybrids for beverage and biofuel production, as well as for constructing tetraploids to be used for basic research in evolutionary genetics and genome stability. Some 500 years ago, the accidental natural hybridization of Saccharomyces cerevisiae—the yeast responsible for things like ale, wine and bread—and a distant yeast cousin gave rise to lager beer. Today, cold-brewed lager is the world’s most consumed alcoholic beverage, fueling an industry with annual sales of more than $250 billion. The first lagers depended on that serendipitous cross of Saccharomyces species as evolutionarily diverse as humans and chickens. The result, however, yielded a product of enormous economic value, demonstrating the latent potential of interspecies yeast hybrids. In nature, the odds of a similar hybridization event are, conservatively, one in a billion. There are hundreds of known species of yeasts and they occupy almost every ecological niche imaginable worldwide. They are essential to the process of fermentation, where the microbes convert sugars to alcohol and carbon dioxide. Yeasts are used widely to not only make beer, wine and bread, but also cider, whiskey, cheese, yogurt, soy sauce and an array of other fermented foods and beverages. In industry, yeasts are used to produce biofuels and to make enzymes, flavors and pigments and even drugs such as human insulin. An ability to quickly and efficiently churn out new yeast interspecies hybrids means industries that depend on yeasts will have many more organisms to experiment with to make new flavors, enhance production and produce entirely new products, explains Chris Todd Hittinger, a UW-Madison professor of genetics and the senior author of the new study. Hittinger is a world authority on yeast genetics and a co-discoverer of the wild Patagonian yeast that formed the lager beer hybrid. For example, the marriage of Saccharomyces cerevisiae and its distant cousin Saccharomyces eubayanus, a species that inhabits tree galls in nature, permitted the cold-temperature fermentation that made lager beer possible. The new yeast hybridization method uses plasmids, circles of DNA that can be built into an organism to confer a genetic quality. In the lab, plasmids are routinely used to manipulate genes in cells. Genes in the plasmids facilitate yeast hybridization by expressing a naturally occurring yeast protein that allows two distinct species of yeasts to mate. The plasmids used to facilitate the process of hybridization can be removed from the new hybrid yeasts, leaving the genomes of the two fused organisms unchanged. The new study describes four new hybrids, one of which was made using a strain of Saccharomyces eubayanus discovered near Sheboygan, Wis., after Hittinger and his colleagues first found the lager yeast parent in the alpine regions of Patagonia. The new hybrid is being tested in a new beer recipe in the UW-Madison Department of Food Science. The new technique may also help industry overcome a creative bottleneck, as many industrial strains of yeasts are sterile, unable to produce spores. The new study was supported by grants from the National Science Foundation and the Department of Energy through the Great Lakes Bioenergy Research Center.
The molecules that impart strength to paper, bamboo and wood-frame buildings — lignin and cellulose — have long stymied biofuels researchers by locking away more than half of a plant’s energy-yielding sugar. In a study that could point the way to biofuels processes of the future, scientists from Rice University, the Great Lakes Bioenergy Research Center at the University of Wisconsin-Madison and the Joint BioEnergy Institute at Emeryville, Calif., have discovered how two bacterial enzymes work as a team to break apart lignin. “Ultimately, we would like to use enzymatic fermentation — the same process that brewers and winemakers have used for centuries — to convert all the sugar from plants into ethanol and other fuels,” said Rice’s George Phillips, co-author of the study in the Journal of Biological Chemistry. “The big target is cellulose, which is the primary ingredient in wood, grass stems and corn stalks. Cellulose is basically sugar, but it is tightly packed in a crystalline compound that is practically indigestible. There are some fungi and bacteria that have developed enzymes to cut it apart, but it’s a very slow process, which is why it can take years for dead trees to decompose.” Lignin, another major component of plant fibers that accounts for up to one-third of the carbon in biomass, compounds the problem for any microorganism that wants to eat cellulose or any scientist who wishes to turn it into biofuel. Lignin has a gluelike consistency, and it coats and protects cellulose. “The cellulose is tough, but organisms can’t even get to it until they chew through the lignin,” said Phillips, Rice’s Ralph and Dorothy Looney Professor of Biochemistry and Cell Biology and professor of chemistry. For industry, breaking down lignin has often proved an even tougher challenge than cellulose. As a result, the biofuels and paper industries mostly treat lignin as a waste product to be removed, isolated and discarded. Study co-author Timothy Donohue, professor of bacteriology at UW-Madison and director of the Great Lakes Bioenergy Research Center, said, “If we can convert lignin from an undesirable byproduct into a starting material for advanced biofuels and other lucrative chemicals, we would dramatically change the economics of tomorrow’s biorefineries.” The center is one of three funded by the Department of Energy to make transformational breakthroughs in cellulosic biofuels technology. With an eye toward using techniques that nature has evolved to break down lignin, a team of researchers led by Donohue and Phillips began by studying bacterial enzymes that cleave specific chemical bonds inside lignin. The original goal was to design a single new enzyme that could do the job of several found in nature. But that turned out to be an impossible task, in part because lignin molecules are irregular. They’re made of hundreds of components that twist either to the left or the right, but the pattern of twists doesn’t repeat, and an enzyme that’s tailored to break a left-handed bond won’t cleave a right-handed one. “Making a single enzyme would be like trying to make a glove that’s designed for your left hand fit on your right hand,” said Kate Helmich, co-lead author of the study and a recent Ph.D. graduate of UW-Madison’s Biochemistry Department. “Our two hands are different configurations of the same fingers, and lignin is like a chain of many different hands. Degrading that entire chain would require an enzyme, or glove, that can attach to both the left and the right hands within it.” The researchers found that Sphingobium bacteria use two enzymes, known as LigE and LigF, to attack lignin as a team. “The key finding is that we now understand how the left-hand and the right-hand versions are broken,” Phillips said. “It’s not through a single super enzyme but through teamwork where you’ve got one for the left and one for right. “It wasn’t clear how the bacteria did it until Donohue and his team made both the left-handed and right-handed compounds, and then assayed them with purified enzymes. Those experiments proved that one works on left and one works on right.” Helmich and Phillips used X-ray crystallography to analyze the structure of the enzymes and show how each performed its specialized task. Phillips, who moved to Rice from UW-Madison in 2013, said the research suggests that biofuels processors will need a cocktail of specialized enzymes to break lignin into fermentable components. “Now we know that such a cocktail would need to include something like LigE and LigF to get both hands of the lignin broken open,” he said.
Dumesic's group caused a stir in research circles and the media in 2014 by publishing a paper in the journal Science describing a new scheme for breaking down biomass and unlocking its polysaccharides. Those sugars–candy for microbes—can be fermented to ethanol or upgraded into a host of high value chemicals currently made from petroleum. The development of this new scheme was funded by the Great Lakes Bioenergy Research Center, one of three Bioenergy Research Centers supported by the Department of Energy Office of Science. At the crux of their method is a solvent derived from biomass itself, called gamma valerolactone (GVL). It's an elegant process. The GVL created in the reaction is recycled and used to drive it again. The method appears to be faster and cheaper than its competitors. It doesn't rely on pricey enzyme cocktails that take days to work and must be tailored to the reactants. "Our process can work in a matter of hours and on any biomass we have ever used such as corn stover, wood, leftovers from sugar cane and residues from paper mills," says Dumesic, a Vilas Research Professor and Michel Boudart Professor of Chemical and Biological Engineering at the University of Wisconsin-Madison. But he knew that a process that works beautifully in 50 milliliter batches would have little practical value to a biorefinery operating on tens of tons. With support from the Wisconsin Alumni Research Foundation (WARF) Accelerator Program and State of Wisconsin funding available to the Wisconsin Energy Institute, Dumesic and his team have spent the past 18 months proving their method can scale up. Back in his office on campus, he says the funding partnershiphas been "essential to move the process ahead," and shares the exciting results with an engineer's sobriety. And the results are exciting indeed. He reports that the team has bumped up production by 80-fold, with sugar yields topping 75 and 65 percent for xylose and glucose, respectively. Along the way they've learned to streamline steps and optimize factors like reaction temperature and acid concentration. In addition to the sugars, they're also producing strong streams of 'native' lignin that can be used for a variety of products from construction materials to paint. ('Native' means the lignin is not chemically altered by the process and therefore prized by researchers normally restricted to the byproducts of paper mills.) Achieving these milestones took an unexpected collaboration. Colleagues in the engineering department and at the Forest Products Laboratory helped Dumesic 'cobble on' to a reactor already in use, saving significant time and resources. He credits the WARF Accelerator program for bringing the two camps together. "The funding enabled us to work with the Forest Products Laboratory to modify their apparatus. Without that we never could have scaled up to the two liter level. No way," he says. Now, those same colleagues are interested in taking things to the next level by designing an extruder system that operates in a continuous flow mode, like a real refinery. This would make the entire process more commercially viable—a point brought home by the Accelerator Program's expert Catalysts. "They tuned up our vision of where we want to take this," says Dumesic. "That means we need a system that takes biomass, feeds it to a reactor and sends it through in a continuous process. No one has been able to do that yet." Dumesic, a faculty member at UW–Madison since 1976, has no illusions. He is the first to acknowledge the economic headwinds facing the biofuels movement. Cheap gasoline remains the biggest obstacle. "If you only target biofuels you are going to lose," he says. His research takes a broader scope. Industrial chemicals, pharmaceuticals, polymers, even lubricants—many of these products can be made from sustainable resources and compete on price. "Gasoline is very easy to make from petroleum. All you do is distill it. But you can make the case that other things like lubricants are harder to make from petroleum and rather easy to make from renewables," he says. "So there is a market there." And Dumesic is no stranger to the private sector. With support from WARF he has helped found two startup companies in recent years, Virent Inc. and Glucan Biorenewables. He continues to serve as an advisor to the latter. From experience, he knows that moving discoveries from the academic lab to the private sector often requires support from federal, state and local sources like WARF. In both cases Dumesic provided the initial spark before passing the torch to younger researchers in his lab. "It's possible but very challenging to be a professor and stay with a startup," he says. "Oftentimes it's the graduate students and postdocs who want to run with it and get the technology out into the real world." Glucan Bio, with a location in the University Research Park in Madison, is taking the GVL method in several interesting directions. One of these relates to the production of a valuable chemical feedstock called furfural, found in agricultural formulations, herbicides and flavorants. Furfural production has gone almost entirely to China, says Dumesic, because the process of making it is energy intensive and traditionally requires corn cobs. "The way corn is harvested now, no one is saving the cobs," he says. "So there is no domestic source of furfural currently available." Glucan Bio wants to be that source. If the technology they are developing specs out, the main importers of furfural are eager to talk. In the meantime, Dumesic is busy teaching courses on chemical kinetics and keeping up with a dynamic biomass community stretching from Asia to the Iowa cornfields. But he can still take a moment to reflect on the humor of his 40 years at UW-Madison. "As part of my startup package I got an HP-45 calculator," he smiles. "And all the figures in my thesis were drawn with India ink."
« Comet Biorefining awarded C$10.9M SDTC grant for bio-based chemicals plant | Main | UT, Oak Ridge scientists gain new insights into atomic disordering of complex metal oxides; materials for energy applications » Three US Department of Energy-funded research centers—the BioEnergy Science Center (Oak Ridge National Laboratory); the Great Lakes Bioenergy Research Center (University of Wisconsin–Madison and Michigan State University); and the Joint BioEnergy Institute (Lawrence Berkeley National Laboratory) (earlier post)—reported the disclosure of their 500th invention. Created in 2007, the Bioenergy Research Centers (BRCs) work together to address the most significant challenges standing in the way of affordable, sustainable and scalable advanced liquid transportation fuels. In their focus on producing biofuels from cellulosic biomass (i.e., wood, grasses and the inedible parts of plants), the BRCs are developing a portfolio of new bio-based products, methods and tools for use in the biofuels industry. Great Lakes Bioenergy Research Center Director Tim Donohue credits the BRCs’ continued success to its multidisciplinary research model, which brings together a diverse group of experts, including ecologists, economists, engineers, plant biologists, microbiologists, computational scientists and chemists. Enabled by a broad range of genome-driven research methods, the BRCs’ technologies represent a variety of approaches to different bottlenecks in the current biofuel pipeline. Some technologies focus on improved ways of breaking down biomass for conversion into fuel, some on engineering plants with the characteristics most advantageous for biofuels, and still others on creating co-products that can help make advanced biofuels economically viable. That variety, however, does not represent a lack of focus. BioEnergy Science Center Director Paul Gilna points to the 500 invention disclosures as proof of the BRCs’ progress, focusing on the role the BRCs are playing in creating a broad knowledgebase for future biofuels technologies. Joint BioEnergy Institute Chief Executive Officer Jay Keasling praised the pioneering efforts of the BRCs and their role in envisioning a future in which cellulosic biofuels provide transformative advantages. BRC research is supported by the US Department of Energy’s Office of Science. The Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit science.energy.gov.