University of Florida Research Foundation, Incorporated

Lonnie O Ingram

Brent E Wood

Keelnatham T Shanmugam

Kazuyoshi Ohta

Sean W York

Lorraine P Yomano

Tyrrell Conway

Maria D Barbosa-Alleyne

James L Peterson

Shengde Zhou

Julie A Maupin-Furlow

Claudia Geddes

Qingzhao Wang

Xiaokuang Lai

David P Clark

Youngnyun Kim

Flavio Alterthum

Gerhard F Burchhardt

David S Beall

Michael T Mullinnix

Walter V Guimaraes

Lee Ann Talarico

Krishnan Chandra Raj

Mohammed Moniruzzaman

Lai Xiaokuang

The invention provides methods for treatment of feedstock to reduce the relative viscosity and promote release of fermentable sugars.

Methods of using cellulase for reducing the viscosity of feedstock

Genetically modified microorganisms having the ability to produce D(&#x2212;)-lactic acid at temperatures between 30&#xb0; C. and 55&#xb0; C. are provided. In various embodiments, the microorganisms may have the chromosomal lactate dehydrogenase (ldh) gene and/or the chromosomal acetolactate synthase (alsS) gene inactivated. Exemplary microorganisms for use in the disclosed methods arespp., such as

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The invention provides recombinant bacteria, which comprise a full complement of heterologous ethanol production genes. Expression of the full complement of heterologous ethanol production genes causes the recombinant bacteria to produce ethanol as the primary fermentation product when grown in mineral salts medium, without the addition of complex nutrients. Methods for producing the recombinant bacteria and methods for producing ethanol using the recombinant bacteria are also disclosed.

Re-engineering bacteria for ethanol production

Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.

Recombinant host cells and media for ethanol production

Non-recombinant bacteria that produce ethanol as the primary fermentation product, associated nucleic acids and polypeptides, methods for producing ethanol using the bacteria, and kits are disclosed.

Ethanol production in non-recombinant hosts

The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like, recombinantand other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous gene

This invention presents a method of improving enzymatic degradation of lignocellulose, as in the production of ethanol from lignocellulosic material, through the use of ultrasonic treatment. The invention shows that ultrasonic treatment reduces cellulase requirements by &frac13; to &frac12;. With the cost of enzymes being a major problem in the cost-effective production of ethanol from lignocellulosic material, this invention presents a significant improvement over presently available methods.

Ethanol production from lignocellulose

This invention presents a method for the production of ethanol that utilizes a soy hydrolysate-based nutrient medium or a yeast autolysate-based medium nutrient medium in conjunction with ethanologenic bacteria and a fermentable sugar for the cost-effective production of ethanol from lignocellulosic biomass. The invention offers several advantages over presently available media for use in ethanol production, including consistent quality, lack of toxins and wide availability.

Ethanol production using a soy hydrolysate-based medium or a yeast

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated

This invention relates to a recombinant microorganism which expresses pyruvate decarboxylase, alcohol dehydrogenase, Klebsiella phospho-.beta.-glucosidase and Klebsiella (phosphoenolpyruvate-dependent phosphotransferase system) cellobiose-utilizing Enzyme II, wherein said phospho-.beta.-glucosidase and said (phosphoenolpyruvate-dependent phosphotransferase) cellobiose-utilizing Enzyme II are heterologous to said microorganism and wherein said microorganism is capable of utilizing both hemicellulose and cellulose, including cellobiose, in the production of ethanol.

Recombinant organisms capable of fermenting cellobiose

The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

Ethanol production in gram-positive microbes

Ethanol production in Gram-positive microbes

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

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Gainesville, FL, United States of America

Lonnie O Ingram