E.i. Dupont De Nemours and Company

Butamax (tm) Advanced Biofuels LLC

Butamax Advanced Biofuels LLC

Tina K Van Dyk

Dennis Flint

Vasantha Nagarajan

Fateme Sima Sariaslani

Michael G Bramucci

Natalia Sedkova

Edward S Miller, Jr

Manjari Singh

Robert A LaRossa

Robert Alan Larossa

Robert DiCosimo

J Antoni Rafalski

Rick Weizhang Ye

Sharon Loretta Haynie

Jean-Francois Tomb

Ranjan Patnaik

Wonchul Suh

Anthony Arthur Gatenby

Zheng You

Kristin Ruebling-Jass

Lixuan Lisa Huang

Robert A Larossa

Michael K Hanafey

William R Majarian

Robert J Trotman

Gregory E Gonye

Dana Michelle Walters Pollak

Edward S Miller Jr

Anthony A Gatenby

Recombinant bacteria capable of metabolizing sucrose are described. The recombinant bacteria comprise in their genome or on at least one recombinant construct, a novel nucleotide sequence encoding a polypeptide having sucrose transporter activity and a nucleotide sequence encoding a polypeptide having sucrose hydrolase activity. These nucleotide sequences are each operably linked to the same or a different promoter. Recombinant bacteria capable of metabolizing sucrose to produce glycerol and/or glycerol-derived products such as 1,3-propanediol and 3-hydroxypropionic acid are also described.

Recombinant bacteria comprising novel sucrose transporters

Variant sucrose transporter polypeptides that enable bacterial growth over a wide range of gene expression levels and sucrose concentrations are described. Additionally, recombinant bacteria comprising these variant sucrose transporter polypeptides, and methods of utilizing the bacteria to produce products such as glycerol and glycerol-derived products are provided.

Variant sucrose transporter polypeptides

Bacteria that are not natural butanol producers were found to have increased tolerance to butanol when the membrane content of unsaturated trans fatty acids was increased. Feeding cells with unsaturated trans fatty acids increased their concentration in the membrane, which may also be accomplished by expressing a fatty acid cistrans isomerase.

Strain for butanol production with increased membrane unsaturated trans fatty acids

Increasing tolerance to butanol in yeast has been accomplished by decreasing activity of Pdr5p encoded by an endogenous PDR5 gene. A deletion mutation of the PDR5 gene led to improved growth yield in the presence of butanol. Yeast cells with reduced Pdr5p activity, or other multidrug resistance ATP-binding cassette transporter protein activity encoded by CDR1 or BFR1, and a butanol biosynthetic pathway may be used for improved butanol production.

Yeast with increased butanol tolerance involving a multidrug efflux pump gene

TAL cell biocatalyst was immobilized in alginate cross-linked beads using low concentrations of glutaraldehyde. The biocatalyst beads have highly stable TAL activity and mechanical strength such that they withstand prolonged recycling in production of pHCA.

Immobilized TAL biocatalyst for production of para-hydroxycinnamic acid

Screening of fatty acid fed bacteria which are not natural butanol producers identified increased membrane cyclopropane fatty acid as providing improved butanol tolerance. Increasing expression of cyclopropane fatty acid synthase in the presence of the enzyme substrate that is either endogenous to the cell or fed to the cell, increased butanol tolerance. Bacterial strains with increased cyclopropane fatty acid synthase and having a butanol biosynthetic pathway are useful for production of butanol.

Strain comprising increased expression of a CFA coding region for butanol production

A method for the production of 1-butanol by fermentation using a microbial production host is disclosed. The method employs a reduction in temperature during the fermentation process that results in a more robust tolerance of the production host to the butanol product.

Method for the production of 1-butanol

A method for the production of 2-butanol by fermentation using a microbial production host is disclosed. The method employs a reduction in temperature during the fermentation process that results in a more robust tolerance of the production host to the butanol product.

Method for the production of 2-butanol

Recombinant bacteria capable of metabolizing sucrose are described. The recombinant bacteria comprise in their genome or on at least one recombinant construct: a nucleotide sequence fromATCC&#xae; 14580 encoding a polypeptide having sucrose transporter activity and a nucleotide sequence fromATCC&#xae; 14580 encoding a polypeptide having sucrose hydrolase activity. These nucleotide sequences are each operably linked to the same or a different promoter. Recombinant bacteria capable of metabolizing sucrose to produce glycerol and/or glycerol-derived products such as 1,3-propanediol and 3-hydroxypropionic acid are also described.

Recombinant bacteria having the ability to metabolize sucrose

Recombinant bacteria capable of metabolizing sucrose are described. The recombinant bacteria comprise in their genome or on at least one recombinant construct: a nucleotide sequence fromPf5 (ATCC&#xae; BAA-477) encoding a polypeptide having sucrose transporter activity and a nucleotide sequence fromPf5 (ATCC&#xae; BAA-477) encoding a polypeptide having sucrose hydrolase activity. These nucleotide sequences are each operably linked to the same or a different promoter. Recombinant bacteria capable of metabolizing sucrose to produce glycerol and/or glycerol-derived products such as 1,3-propanediol and 3-hydroxypropionic acid are also described.

An enteric bacterial strain was engineered to over-produce L-tyrosine using a one-step method. The pheA-tyrA chromosomal region of the bacterial genome was replaced with an engineered chromosomal segment, resulting in inactivation of the pheA coding region and strong expression of the tyrA coding region, resulting in high levels of L-tyrosine production.

Method for producing an L-tyrosine over-producing bacterial strain

A method for the facile and inexpensive inducible expression of heterologous genes has been discovered. The yhcS regulator gene has been found to be inducible by aromatic carboxylic acids and to alter the expression of operons in the LysR gene family, including the yhcRQP operon, common in enteric bacteria. Heterologous nucleic acid molecules placed under the control of yhcS responsive promoters may be overexpressed in response to the presence of inexpensive aromatic carboxylic acids.

Regulator/promoter for tunable gene expression and metabolite sensing

The invention provides methods of increasing the production of aromatic carboxylic acids from a host cell via manipulation of the yhcRQP operon encoding a family of efflux proteins. Up-regulation of all or a sub-set of the genes in the yhcRQP were additionally found to enhance tolerance to aromatic carboxylic acids toxicity.

Methods for increasing the resistance of a host cell to aromatic carboxylic acids

A method for the facile and inexpensive inducible expression of heterologous genes has been discovered. The yhcS regulator gene has been found to be inducible by aromatic carboxylic acids and to alter the expression of operons in the LysR gene family, including the yhcRQP operon, common in enteric bacteria. Heterologous nucleic acid molecules placed under the control of yhcs responsive promoters may be overexpressed in response to the presence of inexpensive aromatic carboxylic acids.

The present invention relates to the generation and use of a cellular array or a cellular array in combination with other genome-registered arrays (an array of arrays) for the determination of gene function and/or perturbation mode of action. Each cellular array consists of a number of microbial strains. Each strain comprises one reporter gene fusion made up of a gene or gene fragment operably linked to a reporter gene. Each gene or gene fragment has been &ldquo;registered&rdquo; or mapped to a specific location in the genome of the organism. The genome-registered collection of the invention may be used to determine alterations in gene expression under a variety of conditions. Such collections are amenable to rapid assay and may be used to confirm, correct or augment data generated from DNA micro array technology.

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Wilmington, DE, United States of America

Tina K Van Dyk