E.i. Dupont De Nemours and Company

Butamax Advanced Biofuels LLC

Butamax (tm) Advanced Biofuels LLC

Wonchul Suh

Pierre E Rouviere

Brian James Paul

Qiong Cheng

Vasantha Nagarajan

Rick Weizhang Ye

Luan Tao

Dennis Flint

Arthur Leo Kruckeberg

Jean-Francois Tomb

Steven Cary Rothman

Ranjan Patnaik

Anthony John Kinney

Lori Ann Maggio-Hall

Larry Cameron Anthony

Charles Edwin Nakamura

Gail K Donaldson

Tina K Van Dyk

Fateme Sima Sariaslani

Michael Dauner

Anthony Arthur Gatenby

Lixuan Lisa Huang

Robert A Larossa

Ritu Bhalla

Hao Ni

Bhaskar Bhadra

Joseph Frederich Tuminello

Anthony A Gatenby

The invention relates to the fields of industrial microbiology and alcohol production. The invention also relates to the development of a microorganism capable of producing fermentation products via an engineered pathway, and uses of the microorganism. The invention also relates to the methods to improve cell viability and productivity and the use of recycling and acid washing to increase the yield of fermentation products.

Fermentative production of alcohols

Methods for the fermentive production of four carbon alcohols is provided. Specifically, butanol, preferably 1-butanol is produced by the fermentive growth of a recombinant bacterium expressing a 1-butanol biosynthetic pathway.

Fermentive production of four carbon alcohols

The invention relates to suitable screening strategies for evaluating various candidate promoters for differential gene expression during the propagation and production phases of a fermentation process. The invention also relates to recombinant host cells that comprise identified promoter nucleic acid sequences and methods for producing fermentation products employing the same.

Genetic switches for butanol production

An engineering method was developed to allow genetic modification and isolation of lactic acid bacteria cells that lack lactate dehydrogenase and acetolactate decarboxylase activities. In cells with these modifications and an isobutanol biosynthetic pathway, improved production of isobutanol was observed.

Flux to acetolactate-derived products in lactic acid bacteria

A group of bacterial dihydroxy-acid dehydratases having a [2Fe-2S] cluster was discovered. Bacterial [2Fe-2S] DHADs were expressed as heterologous proteins in bacteria and yeast cells, providing DHAD activity for conversion of 2,3-dihydroxyisovalerate to &#x3b1;-ketoisovalerate or 2,3-dihydroxymethylvalerate to &#x3b1;-ketomethylvalerate. Isobutanol and other compounds may be synthesized in pathways that include bacterial [2Fe-2S] DHAD activity.

Identification and use of bacterial [2Fe-2S] dihydroxy-acid dehydratases

Lactic acid bacterial (LAB) cells were modified such that they have a specific activity of dihydroxy-acid dehydratase enzyme activity that is increased to about 0.1 &#x3bc;mol minmg. LAB cells with even higher activities of 0.2 to 0.6 &#x3bc;mol minmgof DHAD activity were obtained. These modified cells may be used to produce isobutanol when additional isobutanol biosynthetic pathway enzymes are expressed.

Enhanced dihydroxy-acid dehydratase activity in lactic acid bacteria

Yeast strains were engineered that have increased activity of heterologous proteins that require binding of an Fe&#x2014;S cluster for their activity. The yeast strains have reduced activity of an endogenous Fe&#x2014;S protein. Activities of heterologous fungal or plant 2Fe-2S dihydroxy-acid dehydratases and Fe&#x2014;S propanediol dehydratase reactivase were increased for increased production of products made using biosynthetic pathways including these enzymes, such as valine, isoleucine, leucine, pantothenic acid (vitamin B5), isobutanol, 2-butanone and 2-butanol.

Recombinant yeast host cell with Fe-S cluster proteins and methods of using thereof

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

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

Expression of at least one plant oleosin gene in a microbial cell engineered to produce hydrophobic/lipophilic compounds significantly increases the overall titer of the compound. The hydrophobic nature of the oleosin core is believed to increase the microbial host cell's internal storage capacity for any hydrophobic/lipophilic compound. The method is exemplified using a bacterial host cell engineered to produce significant amount of various carotenoids.

Method to increase hydrophobic compound titer in a recombinant microorganism

The invention describes a method for the stacking of traits in a recombination proficient host using a phage transduction system. The method makes use of a nucleic acid integration cassette that has homology to a specific site on a host chromosome for the insertion of genetic elements and the stacking of traits. Repetition of the method results in the stacking of traits on a single genetic element.

Parallel chromosomal stacking of traits in bacteria

A method for the in vivo bioconversion of cyclic carotenes having a &#x3b2;-ionone ring to the corresponding aryl carotene is provided. Gram negative host cells expressing a heterologous, codon-optimized gene encoding a carotene desaturase are grown in the presence of a suitable cyclic carotene substrate to effect the production of aromatic carotenoids.

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Hockessin, DE, United States of America

Wonchul Suh