Alliance for Sustainable Energy, LLC

E.i. Dupont De Nemours and Company

Midwest Research Institute

E.i. Du Pont De Nemours and Company Alliance for Sustainable Energy LLC

Midwest Research Institute National Renewable Energy Laboratory Div.

Yat-Chen Chou

Min Zhang

Paul Veikko Viitanen

Perry G Caimi

Mary Ann Franden

Michael E Himmel

Luan Tao

Mark Emptage

Yuying Zhang

Carol M McCutchen

William S Adney

Xu Li

John O Baker

Stephen Robert Decker

Shi-You Ding

Laura McCole

Kyle Knoke

Stephen K Picataggio

Kent Evans

Mark Finkelstein

Ali Mohagheghi

Steven R Thomas

Christine Eddy

William Howe

Carol M Mc Cutchen

Carol M Mccutchen

Carol Mccutchen

Laura Mccole

Non-naturally occurringstrains useful for the production of 2,3-butanediol are provided.

Engineered zymomonas for the production of 2,3-butanediol

Strains of xylose utilizingwith improved xylose utilization and ethanol production during fermentation in stress conditions were obtained using an adaptation method. The adaptation involved continuously growing xylose utilizingin media containing high sugars, acetic acid, ammonia, and ethanol.

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Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A)) enzymes fromdemonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase fromand tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-nativehost. In a specific example, the specific performance of the formulation using purified recombinant Cel7A fromexpressed inis increased by more than 200% when compared to a formulation using purified Cel7A from

Strains ofwere engineered by introducing a chimeric xylose isomerase gene that contains a mutant promoter of theglyceraldehyde-3-phosphate dehydrogenase gene. The promoter directs increased expression of xylose isomerase, and when the strain is in addition engineered for expression of xylulokinase, transaldolase and transketolase, improved utilization of xylose is obtained.

Zymomonas with improved xylose utilization

Identified are mutants of the promoter of theglyceraldehyde-3-phosphate dehydrogenase gene, which direct improved expression levels of operably linked heterologous nucleic acids. These are high expression promoters useful for expression of chimeric genes in, and other related bacteria.

Through screening of amutant library the himA gene was found to be involved in the inhibitory effect of acetate onperformance. Xylose-utilizingstrains further engineered to reduce activity of the himA gene were found to have increased ethanol production in comparison to a parental strain, when cultured in mixed-sugars medium comprising xylose, and, in particular, in the presence of acetate.

Through screening of amutant library the himA gene was found to be involved in the inhibitory effect of acetate onperformance. Xylose-utilizingfurther engineered to reduce activity of the himA gene were found to have increased ethanol production in comparison to a parental strain, when cultured in medium comprising xylose and acetate.

Production of ethanol using a strain of xylose-utilizingwith a genetic modification of the glucose-fructose oxidoreductase gene was found to be improved due to greatly reduced production of xylitol, a detrimental by-product of xylose metabolism synthesized during fermentation.

Ethanol production using xylitol synthesis mutant of xylose-utilizing zymomonas

A strain of xylose-utilizingwas engineered with a genetic modification to the glucose-fructose oxidoreductase gene resulting in reduced expression of GFOR enzyme activity. The engineered strain exhibits reduced production of xylitol, a detrimental by-product of xylose metabolism. It also consumes more xylose and produces more ethanol during mixed sugar fermentation under process-relevant conditions.

Xylitol synthesis mutant of xylose-utilizing zymomonas for ethanol production

Xylose-utilizingstrains were found to have improved ethanol production when grown in medium containing mixed sugars including xylose if sorbitol or mannitol was included in the medium. The effect was seen in concentrations of mixed sugars where no growth lag period occurs, as well as in higher sugars concentrations.

Ethanol production in fermentation of mixed sugars containing xylose

Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A) enzymes fromdemonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase fromand tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-nativehost. In a specific example, the specific performance of the formulation using purified recombinant Cel7A fromexpressed inis increased by more than 200% when compared to a formulation using purified Cel7A from

The present invention briefly provides a method of site-specific insertion in, comprising, providing agene fragment, interrupting a DNA sequence the fragment, and transforming thethrough homologous recombination with the interrupted fragment.

The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided. The transposon and shuttle vectors are useful in constructing significantly differentstrains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.

Stable zymomonas mobilis xylose and arabinose fermenting strains

Disclosed in the present invention is aintegrant and derivatives of these integrants that posses the ability to ferment pentose into ethanol. The genetic sequences encoding for the pentose-fermenting enzymes are integrated into thein a two-integration event of homologous recombination and transposition. Each operon includes more than one pentose-reducing enzyme encoding sequence. The integrant in some embodiments includes enzyme sequences encoding xylose isomerase, xylulokinase, transketolase and transketolase. Theintegrants are highly stable, and retain activity for producing the pentose-fermenting enzyme for between 80 to 160 generations. The integrants are also resistant to acetate inhibition, as the integrants demonstrate efficient ethanol production even in the presence of 8 up to 16 grams acetate per liter media. These stably integrated sequences provide a uniquethat may then be used for the efficient conversion of pentose sugars (xylose, arabinose) to ethanol. Method of using theintegrants and derivatives thereof in production of ethanol from cellulosic feedstock is also disclosed. The invention also provides a method for preparing aintegrant as part of the present invention. The hoststrain found particularly useful in the creation of these compositions and methods is31821.

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Lakewood, CO, United States of America

Yat-Chen Chou