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Eriksen D.T.,University of Illinois at Urbana - Champaign | Eriksen D.T.,Energy Biosciences Institute | Hsieh P.C.H.,University of Illinois at Urbana - Champaign | Lynn P.,University of Illinois at Urbana - Champaign | And 2 more authors.
Microbial Cell Factories | Year: 2013

Background: The optimization of metabolic pathways is critical for efficient and economical production of biofuels and specialty chemicals. One such significant pathway is the cellobiose utilization pathway, identified as a promising route in biomass utilization. Here we describe the optimization of cellobiose consumption and ethanol productivity by simultaneously engineering both proteins of the pathway, the β-glucosidase (gh1-1) and the cellodextrin transporter (cdt-1), in an example of pathway engineering through directed evolution.Results: The improved pathway was assessed based on the strain specific growth rate on cellobiose, with the final mutant exhibiting a 47% increase over the wild-type pathway. Metabolite analysis of the engineered pathway identified a 49% increase in cellobiose consumption (1.78 to 2.65 g cellobiose/(L · h)) and a 64% increase in ethanol productivity (0.611 to 1.00 g ethanol/(L · h)).Conclusions: By simultaneously engineering multiple proteins in the pathway, cellobiose utilization in S. cerevisiae was improved. This optimization can be generally applied to other metabolic pathways, provided a selection/screening method is available for the desired phenotype. The improved in vivo cellobiose utilization demonstrated here could help to decrease the in vitro enzyme load in biomass pretreatment, ultimately contributing to a reduction in the high cost of biofuel production. © 2013 Eriksen et al.; licensee BioMed Central Ltd.

News Article | January 26, 2016
Site: phys.org

The new study, published in Plant, Cell and Environment, addresses a central challenge of transgenic plant development: how to reliably evaluate whether genetic material has been successfully introduced. Researchers at the University of Illinois, the Polish Academy of Sciences, the University of Nebraska-Lincoln and the University of California, Berkeley compared the traditional method to several new ones that have emerged from advances in genomic technology and identified one that is much faster than the standard approach, yet equally reliable. The study was led by Illinois postdoctoral fellows Kasia Glowacka and Johannes Kromdijk. "For plants with long life cycles, such as our food crops, this will greatly speed the time between genetic transformation or DNA editing, and development of pure breeding lines," said Long, Gutgsell Endowed Professor of Crop Sciences and Plant Biology and the principal investigator for the study. Long is also a member of the Genomic Ecology of Global Change and Biosystems Design research themes and the Energy Biosciences Institute at the Carl R. Woese Institute for Genomic Biology. To meet the food and fuel needs of an ever-growing global population, researchers benefit from transgenic technologies to develop crops with higher yields and greater resiliency to environmental challenges. None of the technologies used to introduce new genetic material into plants work with 100 percent efficiency. Plants and their offspring must be screened to identify those in which gene transfer was successful. Traditionally, this was done in part by testing successive generations of plants to see if the desired traits are present and breed true over time. In addition, plant scientists can use one of several molecular methods to determine if a gene or genes have actually been successfully introduced into the plant genome. The "tried and true" method, the Southern blot, yields precise data but is slow and unwieldy. It requires isolating relatively large amounts of plant DNA, using fluorescent or radioactive dye to detect the gene of interest, and performing a week's worth of lab work for results from just a few samples at a time. The team compared the Southern blot technique with several that use variations of a chemical process called polymerase chain reaction (PCR). This process allows researchers to quantify specific pieces of the introduced DNA sequences by making many additional copies of them, and then estimating the number of copies—somewhat like estimating the amount of bacteria present in a sample by spreading it on a petri dish and letting colonies grow until they are visible. These methods are much faster than Southern blotting, but if the DNA in each sample does not "grow" at exactly the same rate, the resulting data will be imprecise—size won't be a perfect indicator of the starting quantity. One method examined by Long's group, digital drop PCR (ddPCR), is designed to overcome this weakness. Rather than using the PCR process to amplify all the DNA in a sample, this method first separates each individual fragment of DNA into its own tiny reaction, much like giving each bacterium its own tiny petri dish to grow in. PCR then amplifies each fragment until there are enough copies to be easily detected, and the total number of tiny reactions are counted. Because this method, unlike others, separates the growth-like step from the quantification step, it can be very precise even when the reaction isn't perfect. Results can be obtained in less than two days, and many samples can be processed simultaneously. Long hopes that his group's demonstration that ddPCR is a "reliable, fast and high throughput" technique will help it to become the new standard for those developing transgenic crops. "I believe it will become widely adopted," he said. Although ddPCR is currently more expensive than the other methods, Long said the cost would likely drop quickly, as have the costs of other genomic technologies.

Balakrishnan M.,Energy Biosciences Institute | Sacia E.R.,Energy Biosciences Institute | Sacia E.R.,University of California at Berkeley | Sreekumar S.,Energy Biosciences Institute | And 10 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

Decarbonizing the transportation sector is critical to achieving global climate change mitigation. Although biofuels will play an important role in conventional gasoline and diesel applications, bioderived solutions are particularly important in jet fuels and lubricants, for which no other viable renewable alternatives exist. Producing compounds for jet fuel and lubricant base oil applications often requires upgrading fermentation products, such as alcohols and ketones, to reach the appropriate molecular-weight range. Ketones possess both electrophilic and nucleophilic functionality, which allows them to be used as building blocks similar to alkenes and aromatics in a petroleum refining complex. Here, we develop a method for selectively upgrading biomass-derived alkyl methyl ketones with >95% yields into trimer condensates, which can then be hydrodeoxygenated in near-quantitative yields to give a new class of cycloalkane compounds. The basic chemistry developed here can be tailored for aviation fuels as well as lubricants by changing the production strategy. We also demonstrate that a sugarcane biorefinery could use natural synergies between various routes to produce a mixture of lubricant base oils and jet fuels that achieve net life-cycle greenhouse gas savings of up to 80%. © 2015, National Academy of Sciences. All rights reserved.

Kim I.J.,Korea University | Ko H.-J.,Korea University | Kim T.-W.,Energy Biosciences Institute | Nam K.H.,Cornell University | And 2 more authors.
Applied Microbiology and Biotechnology | Year: 2013

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus x giganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose. © 2012 Springer-Verlag Berlin Heidelberg.

Crago C.L.,Energy Biosciences Institute | Khanna M.,301A Mumford Hall | Barton J.,University of British Columbia | Giuliani E.,Partners at Venture | Amaral W.,University of Sao Paulo
Energy Policy | Year: 2010

Corn ethanol produced in the US and sugarcane ethanol produced in Brazil are the world's leading sources of biofuel. Current US biofuel policies create both incentives and constraints for the import of ethanol from Brazil and together with the cost competitiveness and greenhouse gas intensity of sugarcane ethanol compared to corn ethanol will determine the extent of these imports. This study analyzes the supply-side determinants of cost competitiveness and compares the greenhouse gas intensity of corn ethanol and sugarcane ethanol delivered to US ports. We find that while the cost of sugarcane ethanol production in Brazil is lower than that of corn ethanol in the US, the inclusion of transportation costs for the former and co-product credits for the latter changes their relative competitiveness. We also find that the relative cost of ethanol in the US and Brazil is highly sensitive to the prevailing exchange rate and prices of feedstocks. At an exchange rate of US$1=R$2.15 the cost of corn ethanol is 15% lower than the delivered cost of sugarcane ethanol at a US port. Sugarcane ethanol has lower GHG emissions than corn ethanol but a price of over $113perton of CO2 is needed to affect competitiveness. © 2010 Elsevier Ltd.

Enslow K.R.,Energy Biosciences Institute | Enslow K.R.,University of California at Berkeley | Bell A.T.,Energy Biosciences Institute | Bell A.T.,University of California at Berkeley
ChemCatChem | Year: 2015

The dehydration of xylose yields furfural, a product of considerable value as both a commodity chemical and a platform for producing a variety of fuels. When xylose is dehydrated in aqueous solution in the presence of a Brønsted acid catalyst, humins are formed via complex side processes that ultimately result in a loss in the yield of furfural. Such degradative processes can be minimized via the insitu extraction of furfural into an organic solvent. The partitioning of furfural from water into a given extracting solvent can be enhanced by the addition of salt to the aqueous phase, a process that increases the thermodynamic activity of furfural in water. Although the thermodynamics of using salts to improve liquid-liquid extraction are well studied, their impact on the kinetics of xylose dehydration catalyzed by a Brønsted acid are not. The aim of the present study was to understand how metal halide salts affect the mechanism and kinetics of xylose dehydration in aqueous solution. We found that the rate of xylose consumption is affected by both the nature of the salt cation and anion, increasing in the order no salt

Kim I.J.,Korea University | Ko H.-J.,Korea University | Kim T.-W.,Energy Biosciences Institute | Choi I.-G.,Korea University | Kim K.H.,Korea University
Biotechnology and Bioengineering | Year: 2013

Plant expansin proteins induce plant cell wall extension and have the ability to extend and disrupt cellulose. In addition, these proteins show synergistic activity with cellulases during cellulose hydrolysis. BsEXLX1 originating from Bacillus subtilis is a structural homolog of a β-expansin produced by Zea mays (ZmEXPB1). The Langmuir isotherm for binding of BsEXLX1 to microcrystalline cellulose (i.e., Avicel) revealed that the equilibrium binding constant of BsEXLX1 to Avicel was similar to those of other Type A surface-binding carbohydrate-binding modules (CBMs) to microcrystalline cellulose, and the maximum number of binding sites on Avicel for BsEXLX1 was also comparable to those on microcrystalline cellulose for other Type A CBMs. BsEXLX1 did not bind to cellooligosaccharides, which is consistent with the typical binding behavior of Type A CBMs. The preferential binding pattern of a plant expansin, ZmEXPB1, to xylan, compared to cellulose was not exhibited by BsEXLX1. In addition, the binding capacities of cellulose and xylan for BsEXLX1 were much lower than those for CtCBD3. © 2012 Wiley Periodicals, Inc.

Lee M.E.,University of California at Berkeley | Lee M.E.,Energy Biosciences Institute | Aswani A.,University of California at Berkeley | Han A.S.,University of California at Berkeley | And 3 more authors.
Nucleic Acids Research | Year: 2013

Engineered metabolic pathways often suffer from flux imbalances that can overburden the cell and accumulate intermediate metabolites, resulting in reduced product titers. One way to alleviate such imbalances is to adjust the expression levels of the constituent enzymes using a combinatorial expression library. Typically, this approach requires high-throughput assays, which are unfortunately unavailable for the vast majority of desirable target compounds. To address this, we applied regression modeling to enable expression optimization using only a small number of measurements. We characterized a set of constitutive promoters in Saccharomyces cerevisiae that spanned a wide range of expression and maintained their relative strengths irrespective of the coding sequence. We used a standardized assembly strategy to construct a combinatorial library and express for the first time in yeast the five-enzyme violacein biosynthetic pathway. We trained a regression model on a random sample comprising 3% of the total library, and then used that model to predict genotypes that would preferentially produce each of the products in this highly branched pathway. This generalizable method should prove useful in engineering new pathways for the sustainable production of small molecules. © 2013 The Author(s).

Enslow K.R.,Energy Biosciences Institute | Enslow K.R.,University of California at Berkeley | Bell A.T.,Energy Biosciences Institute | Bell A.T.,University of California at Berkeley
Catalysis Science and Technology | Year: 2015

A number of Lewis acid catalysts were screened for their effectiveness in converting both xylose and glucose in aqueous media to furfural and 5-HMF, respectively. While other catalysts were found to be more active, SnCl4 was identified as the most selective Lewis acid. Hydrolysis of SnCl4 was observed at various concentrations and temperatures resulting in the production of Brønsted acidic protons in a 3.5:1 ratio to Sn4+ at all SnCl4 concentrations above 60°C. As a consequence, there was no need to add a Brønsted acid in order to promote the dehydration of either xylose or glucose. SnCl4-promoted isomerization/dehydration of xylose and glucose at 140°C in water resulted in conversions of 55% and 33%, respectively, after 2 h of reaction, and furfural and 5-HMF selectivities of up to 58% and 27%, respectively. Significant conversion of sugars to humins was observed in both cases, and in the case of glucose, degradation of 5-HMF to levulinic and formic acids was also noted. The effects of secondary reactions could be greatly suppressed by extraction of the furanic product as it was produced. Using n-butanol as the extracting agent, xylose and glucose conversions of 90% and 75%, respectively, were observed after 5 h of reaction, and the selectivities to furfural and 5-HMF increased to 85% and 69%, respectively. Small additional increases in the furfural and 5-HMF selectivities were obtained by adding LiCl to the aqueous phase without much effect on the conversion of either sugar. In this case, the selectivities to furfural and 5-HMF were 88% and 72%, respectively, after 5 h of reaction at 140°C. © The Royal Society of Chemistry 2015.

News Article | April 1, 2016
Site: phys.org

"We evaluated germination and plant growth for prairie cordgrass accessions and switchgrass cultivars in a greenhouse study," says crop scientist D.K. Lee. In crop production, too much salt in the soil can interfere with the plant's ability to absorb water. Water moves into plant roots by osmosis, and when solutes inside root cells are more concentrated than in soil, water moves into the root. In salt-affected soil, the difference in solute concentration inside and outside of the root is not as great, meaning that water may not move in. So, even where soil is moist, plants experience drought-like conditions when too much salt is present. Certain mineral salts are also toxic to plants. When they are taken up along with soil water, plant tissue damage can occur. "Saline soils are characterized by high concentrations of soluble salts, such as sodium, chloride, calcium chloride, or magnesium sulfate, whereas sodic soils are solely characterized by their high sodium concentrations," Lee explains. "Many soils are both saline and sodic." The researchers subjected six prairie cordgrass accessions and three switchgrass cultivars to different levels of sodicity and salinity over two years of growth. The team conducted a similar experiment in an earlier study, but only looked at one cordgrass ('Red River') and one switchgrass ('Cave-In-Rock') cultivar, over only one growing season. "In that study, we found that 'Cave-In-Rock' switchgrass was not good at all in terms of salt tolerance. 'Red River' cordgrass was far superior," Lee recalls. The expanded study showed that prairie cordgrass had, on average, much higher germination rates than switchgrass in saline and sodic conditions. Dry biomass production was not as clearly split between the two species in salty conditions, however. Three prairie cordgrasses, pc17-102, pc17-109, and 'Red River', and one switchgrass, EG-1102, produced equivalent amounts of dry biomass when subjected to high-salt conditions. However, they produced approximately 70 to 80 percent less biomass in salty conditions than they did with no added salt. In contrast, the salt-susceptible switchgrass cultivar, EG-2012, produced approximately 99.5 percent less biomass in high-salt treatments than it did without added salt. The next step for the researchers is to bring this work out of the greenhouse, where climate is controlled and water is unlimited, to real-world scenarios. Preliminary field research has shown that prairie cordgrass is very successful in salt-affected areas in Illinois and South Dakota. "Even in highly saline soils, prairie cordgrass can do very well. Unlike switchgrass, it can take up salt dissolved in water without getting sick because it can excrete it out through specialized salt glands. Then, once the plants grow deep roots, they can access less salty water," Lee explains. More research and agronomic improvements are needed before prairie cordgrass can be recommended widely as a biomass crop, but Lee sees a lot of potential in this species. "Prairie cordgrass is an interesting species," he says. "As a warm season grass, I think it is unique in being able to handle low temperatures, and it is also well adapted to poorly drained soils and lands with frequent flooding. And even in high-salt conditions in the field, we're getting pretty good yields: up to 8 or 9 tons per acre." The article, "Determining effects of sodicity and salinity on switchgrass and prairie cordgrass germination and plant growth," is published in Industrial Crops and Products. Lee's co-authors, Eric Anderson, Tom Voigt, and Sumin Kim are also from the U of I. The project was funded by the Energy Biosciences Institute. More information: Eric K. Anderson et al. Determining effects of sodicity and salinity on switchgrass and prairie cordgrass germination and plant growth, Industrial Crops and Products (2015). DOI: 10.1016/j.indcrop.2014.11.016

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