E.i. Dupont De Nemours and Company

Alliance for Sustainable Energy, LLC

E. I. Du Pont Nemours and Co.

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

Luan Tao

Qiong Cheng

William Dean Hitz

Paul Veikko Viitanen

Perry G Caimi

Natalia Sedkova

Pierre E Rouviere

Min Zhang

Mary Ann Franden

Yat-Chen Chou

Yuying Zhang

Laura McCole

Jean-Francois Tomb

Xiao-Song Tang

Wonchul Suh

Carol M McCutchen

Jianjun Yang

Min Qi

Kyle Knoke

Joanne Y Shyr

Carol Mccutchen

Laura Mccole

Vasantha Nagarajan

Michael G Bramucci

Andrew C Eliot

Michael P Perry

Pamela L Sharpe

Stuart M Thomas

Kelley C Norton

Kristy N Kostichka

Patricia C Brzostowicz

Melissa D Bosak

Henry Yao

Robel Y Kahsay

Pierre E Rouvier

Perry G G Caimi

Paulv Viitanen

Disrupting expression of a protein encoded by the region of thegenome called ZMO0353 in the genomic sequence of the ZM4 strain was found to improve the use of xylose in a recombinant xylose utilizingcell. In addition, utilization of both xylose and arabinose was improved in a xylose and arabinose utilizingcell with this disruption, and increased ethanol production was achieved.

Enhancing D-xylose and L-arabinose utilization in Zymomonas cells

The endogenous pnp gene encoding polynucleotide phosphorylase in thegenome was identified as a target for modification to provide improved xylose utilizing cells for ethanol production. The cells are in addition genetically modified to have increased expression of ribose-5-phosphate isomerase (RPI) activity, as compared to cells without this genetic modification, and are not limited in xylose isomerase activity in the absence of the pnp modification.

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is unable to synthesize pantothenic acid and requires this essential vitamin in growth medium.strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pantothenic acid supplementation in seed culture and fermentation media.

Pantothenic acid biosynthesis in zymomonas

Xylose-utilizingstrains studied were found to accumulate ribulose when grown in xylose-containing media. Engineering these strains to increase ribose-5-phosphate isomerase activity led to reduced ribulose accumulation, improved growth, improved xylose utilization, and increased ethanol production.

Xylose utilization in recombinant zymomonas

expressing xylose isomerase fromwas found to have improved xylose utilization, growth, and ethanol production when grown in media containing xylose. Xylose isomerases related to that ofwere identified structurally through molecular phylogenetic and Profile Hidden Markov Model analyses, providing xylose isomerases that may be used to improve xylose utilization.

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.

Protein engineered CrtO ketolases isolated fromAN12 are provided having increased carotenoid ketolase activity. Methods using the present CrtO ketolases are also provided for increasing ketocarotenoid production in suitable production hosts.

Carotenoid ketolase genes with improved ketocarotenoid yield

CrtW carotenoid ketolases are provided that are useful for the production of astaxanthin and other cyclic ketocarotenoids. The mutant ketolase genes of the present invention encode polypeptides characterized by an improvement in astaxanthin synthesis activity when converting cyclic hydroxylated carotenoid intermediates into astaxanthin. Expression of the mutant carotenoid ketolases in heterologous hosts enabled increased production of astaxanthin relative to theDC18 CrtW.

Mutant carotenoid ketolases

Mutations in chromosomal genes have been identified that affect plasmid copy number in plasmids that are anti-sense RNA regulated such as the pMB1-derived and p15A-derived plasmids.

Mutations affecting plasmid copy number

A carotenogenic biosynthetic gene cluster has been isolated fromstrain DC413, wherein the genetic organization of the cluster is crtE-idi-crtX-crtY-crtI-crtB-crtZ. The genes contained within this cluster encode geranylgeranyl pyrophosphate (GGPP) synthetase (CrtE), isopentenyl pyrophosphate isomerase (Idi), zeaxanthin glucosyl transferase (CrtX), lycopene cyclase (CrtY), phytoene desaturase (CrtI), phytoene synthase (CrtB), and &#x3b2;-carotene hydroxylase (CrtZ). The gene cluster, genes and their products are useful for the conversion of farnesyl pyrophosphate to carotenoids. Vectors containing those DNA segments, host cells containing the vectors and methods for producing those enzymes by recombinant DNA technology in transformed host organisms are disclosed.

Genes of strain DC413 encoding enzymes involved in biosynthesis of carotenoid compounds

Novel CrtW carotenoid ketolase are provided that are useful for the production of ketocarotenoids. The ketolases genes of the present invention exhibit low homology in comparison to other CrtW ketolases previously reported. Expression of the carotenoid ketolases in heterologous hosts enabled production of canthaxanthin and astaxanthin. Coexpression experiments using divergent crtW genes resulted in increased production of the desired ketocarotenoids.

Carotenoid ketolases

The present invention describes a mutant plasmid replication control region having the ability to convey a phenotype of altered plasmid copy number to the plasmid on which it resides. The mutant replication control region is based on a similar region isolated from the pBBR1 plasmid family. Plasmids containing this replication control region cannot be classed as belonging to any known incompatibility group and thus may co-exist with a broad range of other plasmids in a single host.

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Claymont, DE, United States of America

Luan Tao