Technologies

UW–Madison Technologies Developed Through the Great Lakes Bioenergy Research Center

The Great Lakes Bioenergy Research Center (GLBRC) is led by the University of Wisconsin–Madison, with Michigan State University as a major partner, and is one of three bioenergy research centers established in 2007 by the U.S. Department of Energy (DOE). Member institutions include a DOE National Laboratory, universities and a biotechnology company.

GLBRC is working to meet the nation’s need for a comprehensive suite of clean energy technologies, including next generation and drop-in fuels that can be used in today’s engines. The GLBRC's research supports the development of a robust pipeline from biomass production through pretreatment and final conversion to fuel, with sustainability providing a unifying theme.

UW–Madison has a rich history of developing and commercializing innovative technologies that benefit the world, including sustainable technologies. From engines that emit fewer pollutants to "smart" grids that enable more efficient use of power to solar heating and cooling systems, for decades UW–Madison scientists have been inventing solutions to the energy challenge, including several biofuels technologies developed through the GLBRC.

Technologies

Zip-Lignin™ Assay: An Analysis and Validation Tool

The researchers have now developed the most sensitive assay to date for detecting and quantifying Zip-lignin monomers in plants. They modified an existing lignin assay known as DFRC (Derivatization Followed by Reductive Cleavage) that has been in use for almost a decade. They incorporated several new features to improve the sensitivity of the assay, including extended incubation periods and an additional purification step.

The modified DFRC assay is currently the only known technique capable of determining levels of monolignol ester conjugates in plant lignin.
P150118US02

Natural Antibiotic to Treat Clostridium Infection and More

UW–Madison researchers have identified a potential new antibiotic to treat infections caused by C. difficile, Staphylococcus and other drug-resistant strains. The compound is a natural product called ecteinamycin. It was discovered and isolated from a sea squirt bacterium (Actinomadura). Preliminary data suggests ecteinamycin is potent, selective and able to protect cells against bacterial toxins.
P140428US02

Modified E. coli for Enhanced Production of Pyruvate, Ethanol

UW–Madison researchers have developed a variety of new E. coli strains capable of producing pyruvate up to 95 percent of the maximum theoretical yield from renewable sources under aerobic conditions. This exceeds the highest previously reported yields of 78 percent.

The researchers used a genome-scale metabolic model of E. coli to identify multiple gene deletion targets that couple growth rate with pyruvate production. Further engineering of these new strains enabled them to produce ethanol at near maximum theoretical yields.
P140301US02

Modified Yeast with Enhanced Tolerance for GVL Biomass Solvent

UW–Madison researchers have developed a genetically modified strain of Saccharomyces cerevisiae that is more resistant to GVL toxicity and grows more than 1.5 times faster than wild yeast in the presence of GVL.

The researchers deleted two genes (Pad1p and Fdc1p) in the yeast that play a role in mediating GVL tolerance. The new strain is the first ethanol-producing yeast specifically tailored for GVL-based techniques.
P140430US02

High-Throughput Genome Editing and Engineering of Industrial Yeast, Other Fungi

UW–Madison researchers have developed expression cassettes that facilitate genome editing and sequence replacement in fungi at an extraordinarily high rate. Their HERP (Haploid Engineering Replacement Protocol) cassettes combine thymidine kinase (TK) enzyme with meganucleases, and permit hundreds of thousands of independent transformations to be obtained in a single experiment.

TK (from human Herpes Simplex Virus) serves as both a selectable and counter-selectable marker. Since the common ancestor of all fungi lacked the gene, the marker is likely of nearly pan-fungal utility. Relevant species should include Saccharomyces cerevisiae, Saccharomyces mikatae, Saccharomyces kudriavzevii, Saccharomyces uvarum and Neurospora crassa.
P140240US03

Bio-Based Production of Non-Straight-Chain and Oxygenated Fatty Acids for Fuels and More

UW–Madison researchers have identified several enzymes in the bacterium Rhodobacter sphaeroides that can be purified to produce non-straight-chain fatty acids in vitro or expressed in genetically modified microorganisms including E. coli for synthesis in vivo. Strains may be ‘fine-tuned’ to produce a specific type of non-straight-chain fatty acid (e.g., furan-containing) by expressing, overexpressing or deleting the enzymes in various combinations.
P140318US02

Natural Antimicrobial Agent Derived from Biomass

UW–Madison researchers have identified an antimicrobial agent produced as a byproduct of biomass processing. The agent is a diferulate compound called poacic acid (and sometimes also called ‘8-5-DC’). It has been shown to target and destroy the cell walls of several species of fungus and yeast.
P140248US02

Modified Yeast to Boost Biofuel Yields

A UW–Madison researcher has developed an S. cerevisiae strain that is 80 percent more effective at fermenting xylose. He discovered that knocking out several genes (hog1, isu1, gre3, ira1/2) enables dramatically faster xylose fermentation under the anaerobic conditions favored by industry.
P140199US02

Oxygen-Responsive Bacterial Gene ‘Switch’ for Biofuel Production and More

UW–Madison researchers have taken a known promoter sequence and developed several novel variants capable of modulating downstream expression levels over a 50-fold range.

The new promoters are exquisitely sensitive to changes in O2 concentration as well as other growth conditions applicable to various industrial fermentations. These include changes in the redox state of the cell and also membrane perturbations/stress caused by production or export of hydrophobic compounds, biofuel precursors or recombinant proteins.

Some of the promoters act as tightly regulated on/off gene switches while others offer a more graded or linear response.
P130380US02

High Yield Method to Produce LGO from Biomass

UW–Madison researchers have developed a new method to produce LGO from cellulosic biomass under mild reaction conditions. The biomass material is reacted in a mixture comprising a polar aprotic solvent (e.g., tetrahydrofuran or THF) and an acid in the absence of water. The LGO can be separated out by routine downstream processes such as distillation and evaporation.

Glucose, levoglucosan, furfural and 5-hydroxymethylfurfural also are produced in small quantities.
P150101US01

Microbes Produce High Yields of Fatty Alcohols from Glucose

UW–Madison researchers have developed a method to produce fatty alcohols such as 1-dodecanol and 1-tetradecanol from glucose using genetically engineered microorganisms. The organism, e.g., a modified E. coli strain, overexpresses several genes (including FadD and a recombinant thioesterase gene, acyl-CoA synthetase gene and acyl-CoA reductase gene). Other gene products are functionally deleted to maximize performance.

The strain is cultured in a bioreactor in the presence of glucose.
P140076US02

Grass Modified for Easier Bioprocessing

The researchers have identified another gene of interest in rice, corn/maize and other grasses, called p-coumarate monolignol transferase (PMT). This is the first gene reportedly involved in the acylation of lignin monomers. In essence, interfering with this gene could make plants more amenable to biorefining.
P120040US02

Gene Controls Flowering Time in Corn

The researchers now have found a gene in maize that affects flowering time. By modulating this gene, GRMZM2G171650, the onset of flowering in maize may be delayed or accelerated. Standard vector and transgenic methods can be employed to overexpress or suppress the gene, or introduce it into new crop lines.

The gene was identified by studying more than 500 different maize lines. The researchers mapped single nucleotide polymorphisms (SNPs) correlating to early or late flowering traits. A large concentration of such SNPs was located in GRMZM2G171650, a transcription factor on chromosome 3. The gene was of previously unknown function in corn.
P130256US02

Two-Step Process Converts Lignin into Simple Aromatic Compounds

Building on their work, the researchers have now developed a two-step process for selectively converting lignin and lignin-type material into low molecular weight aromatic compounds.

The lignin is first selectively oxidized via the previously described method, then reacted with an organic carboxylic acid, salt or ester (e.g., formic acid) for a time and temperature sufficient to cleave carbon-carbon or carbon-oxygen bonds. The process results in high yields of simple aromatic compounds.
P140205US01

Concentrated C5 and C6 Sugars from Biomass

UW–Madison researchers have developed a process for producing C5 and C6 sugars from biomass at high yields (70 to 90 percent) in a solvent mixture of water, dilute acid and GVL (gamma-valerolactone). GVL is attractive because it is effective and derived from biomass.

The biomass and solvent system may be reacted at a temperature between 50 and 250oC for less than 24 hours.

The method yields liquid and solid fractions enriched in C5 and C6 sugar, respectively. The fractions are easily separated for post-treatment upgrading. This strategy is well-suited for catalytic upgrading to furans or fermentative upgrading to ethanol at near-theoretical yield.
P140075US01

Powerful New Enzyme for Transforming Biomass

UW–Madison researchers have engineered a multifunctional polypeptide capable of hydrolyzing cellulose, xylan and mannan. It is made of the catalytic core of Clostridium thermocellum Cthe_0797 (also called CelE), a linker region and a cellulose-specific carbohydrate binding module(CBM3).

C. thermocellum is a well-known cellulose-degrading bacterium whose genome has been sequenced, annotated and published.
P120371US02

Biofuel-Producing Lactobacillus Strain

A UW–Madison researcher and others have modified a Lactobacillus casei strain that exhibits the highest ethanol conversion rates yet reported from the genus.

L. casei naturally combines many characteristics of an ideal strain when compared to microorganisms typically considered for biofuel production, like Saccharomyces cerevisiae, Zymomonas mobilis, Escherichia coli and Clostridium sp., which all suffer from various deficiencies. A L. casei strain exhibiting high conversion rates could represent a novel, more efficient path to market for ethanol production.

The modified bacterium is derived from L. casei strain 12A. It is made by (i) inactivating genes that encode a competing lactate enzyme and (ii) introducing genes from another organism (Zymomonas mobilis) that encode a pyruvate decarboxylase and an alcohol dehydrogenase II.
P130023US02

Producing Linear Alpha Olefins from Biomass

UW–Madison researchers have developed a method for producing LAOs cheaply from biomass. In the process, an inexpensive solid acid catalyst is used in a reaction that converts the carboxylic acids and lactones present in the feedstock. The catalyst features Lewis acid catalytic sites and no precious metal components.
P130275US01

Conversion of Biomass Sugars via Fermentation

The researchers have now developed just such an integrated conversion process: using biomass-derived sugars to culture microorganisms that in turn convert biomass into fuels, commodity chemicals and fatty acids.

In the process, biomass is reacted with a lactone like GVL (gamma-valerolactone), water and an acid catalyst. The reaction yields a mixture containing C5 and C6 sugar oligomers and monomers. The lactone is separated out, leaving an aqueous carbohydrate layer that can act as a fermentable substrate for (genetically engineered or wild-type) microorganisms like yeast, E. coli and Lactobacillus casei.
P130272US01

Transgenic Lignin Easier to Break Down for Biofuel

UW–Madison researchers and others have developed methods to genetically alter the structure of plant lignin to be less resistant to chemical (mostly alkaline) degradation.

They have identified and isolated nucleic acids from the Angelica sinensis plant that encode feruloyl-CoA:monolignol transferase. This enzyme produces lignin rich in CAFA and similar chemicals, and thus contains ester bonds that cleave under relatively mild conditions.

Plant cells can be modified to contain the enzyme gene sequence using standard genetic techniques. Whole plants (and their seeds) then can be generated from these cells.
P100281US02

Renewable Plastic from Glucose-Fed Microbes

UW–Madison researchers have developed recombinant E. coli capable of producing high yields of mcl-PHA from non-lipid, carbohydrate sources.

The researchers previously designed and built a bacterial strain that produces high levels of C12 fatty acids (see WARF reference number P09329US02). This strain has been further modified by deleting various fad genes implicated in the breakdown of fatty acids. Also, the bacteria cells incorporate several genes taken from other species to increase conversion efficiency.
P120377US02

Extending Juvenile Stage of Plants for Biofuels and Feedstock

UW–Madison researchers have developed methods for locking plants in a juvenile state by modifying genes related to maturation.

The genes – GRMZM2G362718 or GRMZM2G096016 – have been analyzed by the researchers and shown to influence growth transition in corn. To alter plant development, these genes and their homologs could be knocked out or inhibited by small molecules or biologics. The process could involve additional genes known to affect juvenile to adult growth development.
P120179US02

Modified Microbes Tolerate 50-Fold More Organic Acid

UW–Madison researchers have genetically modified microorganisms to better tolerate organic acids like 3HP, acrylic acid and propionic acid. The modified microorganisms are cyanobacteria such as Synechococcus.

In the modified bacteria, the acsA gene is replaced or deleted. This leads to increased organic acid tolerance.
P120017US02

Selective Conversion of Lignin into Simple Aromatic Compounds

UW–Madison researchers have developed a metal-free, aerobic oxidation method that selectively transforms the benzylic alcohol in lignin to the corresponding ketone. The process uses a nitric acid (HNO3) catalyst combined with another Brønsted acid. The reaction leaves unchanged at least a portion of unprotected primary aliphatic alcohols in the lignin or lignin subunit.

The reaction may be carried out in any suitable polar solvent and in the presence of additional reagents including TEMPO and derivatives.
P130104US01

Better Biomass Conversion with Recyclable GVL Solvent

UW–Madison researchers have developed a method for producing soluble C6 and C5 carbohydrate oligomers and monomers from biomass. These include glucose, xylose and other sugars.

In the process, lignocellulosic material is reacted with water and gamma-valerolactone (GVL) – an organic solvent derived from biomass. This occurs in the presence of an acid catalyst under moderate temperatures, and results in the conversion of water-insoluble to water-soluble carbohydrates. These desired products are partitioned into an aqueous layer, where they can be recovered, concentrated and purified. The GVL separates into another layer to be recycled.
P130123US01

Enhanced Biomass Digestion with Wood Wasp Bacteria

UW–Madison researchers have derived preparations from ActE secretions that highly degrade lignocellulose. The bacteria can be obtained from Sirex noctilio wasps and grown on a substrate containing mostly cellulose, hemicelluloses, xylan, wood or non-wood biomass, and chitin. The substrate may be pretreated for better results. The ActE are grown aerobically to maximize the secretion of both oxidative and hydrolytic enzymes capable of rapid deconstruction of matter. The secretions can be purified and added directly to biomass slurry.
P110314US03

Mild, Nontoxic Production of Fuels and Chemicals from Biomass

UW–Madison researchers have developed methods for producing furans, including HMF and furfural, from biomass carbohydrates using boronic acids that enhance conversion.

Generating furans from cellulose, lignocellulosic biomass or other sugars is catalyzed by a 2-substituted phenylboronic acid alone or in combination with a metal salt. The reactants are mixed and heated at a selected temperature. The reaction may be carried out in a suitable solvent other than an ionic liquid, with water optionally added to increase yield.
P110055US02

More Efficient Ethanol Production from Mixed Sugars Using Spathaspora Yeast

UW–Madison researchers have developed a method for producing ethanol using Spathaspora passalidarum yeast to ferment xylose or cellobiose, even when mixed with glucose.

The ethanol is converted from biomass or other lignocellulosic material from agricultural residues, fast-growing hardwoods and processing byproducts. Sugars, lignin and other components are first extracted from this feedstock using standard methods to form mixtures rich in different sugars. The mixture is contacted with a Spathaspora yeast cell under oxygen-limiting conditions suitable to allow the yeast to ferment a portion of the xylose and/or cellobiose into ethanol.
P110227US02

One- and Two-Phase Conversion of Biomass to Furfural

UW–Madison researchers have developed monophasic and biphasic systems to produce furfural from the C5 sugar fraction of biomass utilizing GVL, itself a product of the reaction, as a solvent. Both methods result in furfural that can be directly distilled out of, or converted into, GVL.

In the monophasic method, a single reaction medium comprises GVL and an acid such as nitric, sulfuric, or solid acid zeolite to minimize the use of water and eliminate the separation step required of mineral acids. Into this solution is introduced pretreated biomass xylose or hemicellulose. The GVL acts as a reaction solvent. By reducing or removing water from the process, less leaching of acid sites occurs and a significantly higher yield of furfural is produced.

The biphasic, or two-layered, method also utilizes GVL, but as an extraction solvent in the form of a suspended organic layer. The lower phase contains an aqueous, acidic solution with a solute such as sodium chloride or fructose. The saturated biomass material dehydrates into furfural, which spontaneously partitions into the upper GVL layer, thereby preventing further degradation via mineral acid catalysis in the aqueous phase.
P120195US01

Genes for Xylose Fermentation, Enhanced Biofuel Production in Yeast

UW–Madison researchers have identified 10 genes in yeast that are involved in xylose fermentation. These genes could be used to create an organism that can ferment both xylose and glucose for enhanced biofuel production.
P100245US03

Ethanol Tolerant Yeast for Improved Production of Ethanol from Biomass

UW–Madison researchers have developed a method of using the Elongase I (ELO1) gene to impart ethanol tolerance to yeast. ELO1 is an enzyme involved in the biosynthesis of unsaturated fatty acids in yeast.  This gene could be incorporated into an industrial yeast strain to increase the amount of ethanol produced from biomass.
P100228US02

Bacteria Modified to Secrete Biologically Active Protein for Large-Scale Production

UW–Madison researchers have discovered E. coli mutations that substantially increase the amount of biologically active, recombinant protein secreted from cells.  The mutations disrupt genes in a YebF-mediated protein secretion pathway.  Bacteria modified to contain these mutations are useful for the production of secreted proteins.  They can be used to produce proteins that might otherwise not be expressed due to toxicity or folding errors.  They also can be used to produce secreted complexes of enzymes such as cellulases and xylanases for the manufacture of cellulosic biofuels.
P100362US02

Lignin from Transgenic Poplar Is Easier to Process

UW–Madison researchers and others have developed genetically modified poplars with lignin that is less resistant to alkaline degradation.

Having previously identified and isolated the gene for FMT, the researchers introduced the nucleic acid sequence into poplar tissue. The enzyme produced lignin rich in monolignol ferulates, including coniferyl ferulate and sinapyl ferulate. The transformed lignin thus contained ester bonds that cleaved under relatively mild ammonia conditions.

The poplar cells were modified using standard genetic techniques.
P100281US03

Fatty Acid-Producing Microbes for Generating Medium- and Long-Chain Hydrocarbons

UW–Madison researchers have developed genetically modified E. coli that are capable of overproducing fatty acid precursors for medium- to long-chain hydrocarbons. The modified bacteria were transformed with exogenous nucleic acids to increase the production of acyl-ACP or acyl-CoA, reduce the catabolism of fatty acid products and intermediates, and/or reduce feedback inhibition at specific points in the biosynthetic pathway.

The modified bacteria can be cultured in the presence of sugars to produce fatty acids. The fatty acid products formed during fermentation then can be separated from the fermentation media via a two-phase separation process or other method. The separated products can be used directly or as feedstock for subsequent reactions, including conversion to medium- and long-chain hydrocarbons.
P09329US02

Translation-Coupling Cassette for Quickly and Reliably Monitoring Protein Translation in Host Cells

UW–Madison researchers have developed a method of using translation coupling to quickly and reliably determine whether a given host is capable of expressing the gene product of any given gene. This method could be used to monitor protein translation efficiency in bacterial cells.

The method involves a cassette that couples the full translation of a desired target gene to that of a detectable response gene. If the target gene is fully translated, so is the response gene. If the target gene is not translated, the response gene product is not detectable.

For example, one embodiment utilizes a DNA plasmid with a cloning site upstream of a DNA hairpin that masks the ribosome binding site required for translation of an antibiotic resistance protein. Scientists clone the protein of interest into the plasmid without a stop codon in frame and with a tag for purification purposes. Expression of the desired protein leads to unwinding of the DNA hairpin, unmasking the ribosome binding site and promoting translation of the antibiotic resistance protein. If the protein of interest is expressed in E. coli, the bacteria will survive in the presence of the antibiotic. If the protein is not expressed, the bacteria will die when exposed to the antibiotic.
P090401US02

High-Yielding Method for Converting Biomass to Fermentable Sugars for Biofuel Production

UW–Madison researchers have developed a new method for degrading lignocellulosic biomass to fermentable sugars.  This simple, high-yielding chemical process, which involves the gradual addition of water to a chloride ionic liquid, enables crude biomass to serve as the sole source of carbon for a scalable biorefinery.

In this method, biomass is mixed with a cellulose-dissolving ionic liquid and heated to form a solution or gel.  Then water and an acid catalyst are added and the resulting mixture is heated, typically to 105°C.  At specified time intervals, more water is added to the mixture until it contains more than 20 percent water by weight.  At this point, the mixture contains free sugars such as xylose and glucose and unhydrolyzed carbohydrate polymers, which often are not dissolved.  The insoluble materials, acid and ionic liquids are separated from the soluble sugars.  The soluble sugars then can serve as the sole carbon source for microorganisms such as E. coli KO11, an ethanologen.
P09305US02

Cell-Free System for Combinatorial Discovery of Enzymes Capable of Transforming Biomass for Biofuels

UW-Madison researchers have developed compositions and methods that expand the ability to make, express and identify target polypeptides, including enzymes capable of enhancing the deconstruction of biomass into fermentable sugars. 

This approach uses a cell-free system to express enzymes and other polypeptides in a combinatorial manner.  Because the system is cell-free, the enzymes can be assayed without intermediate cloning steps or purification of the protein products.  This system also is more reliable than conventional methods for analyzing biomass transformation because it does not utilize living systems, which could rapidly consume soluble sugars.

Specifically, the system compromises a cell-free extract for synthesizing the target polypeptide, a nucleotide sequence that encodes a fusion protein containing a cohesion domain and a biomass binding domain, and a nucleotide sequence that encodes a second fusion protein containing a dockerin domain and a polypeptide capable of catalyzing biomass transformation.  The system also may include additional polypeptides and fusion proteins.  The target polypeptides may be synthesized in the presence of different types of biomass to determine their effects on biomass deconstruction.
P08301US02

Modified Yeast Ferments Biomass Xylose

A UW–Madison researcher and others have developed an S. cerevisiae strain genetically engineered with xylose utilization genes from another yeast, Scheffersomyces stipitis. The new strain (GLBRCY35) has been made to express S. stipitis genes XYL1, XYL2 and XYL3, which are known to improve xylose fermentation.
P120307US01

Modified Yeast Show Improved Xylose Fermentation and Toxin Tolerance

A UW–Madison researcher and others have developed genetically modified S. cerevisiae strains capable of xylose fermentation and better able to tolerate toxins associated with biomass pretreatment. The strains, called GLBRCY73 and GLBRCY87, were evolved in the presence of increasing amounts of p-Coumaric and Ferulic acids. Desirable specimens were selected based on strong growth characteristics.
P130153US01

Wisconsin-Sourced Lager Yeast

The researchers have now found three new strains of S. eubayanus in Wisconsin. They were isolated from an old beech tree stand at Sheboygan Indian Mound Park. The strains were sequenced, and each was found to represent an admixture or mosaic of two Argentinian populations.
P140088US01
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