Butamax Advanced Biofuels LLC

Butamax (tm) Advanced Biofuels LLC

Robert A Larossa

Michael G Bramucci

Dana R Smulski

Vasantha Nagarajan

Rick Weizhang Ye

Dennis Flint

Arthur Leo Kruckeberg

Tina K Van Dyk

Brian James Paul

Wonchul Suh

Michael Dauner

Manjari Singh

Joseph Frederich Tuminello

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

Yeast cells with a reduced general control response to amino acid starvation were found to have increased tolerance to butanol in the growth medium. The reduced response was engineered by genetic modification of a gene involved in the response, a GCN gene, to eliminate activity of the encoded protein. Yeast strains with an engineered butanol biosynthetic pathway and a genetic modification in a gene involved in the general control response to amino acid starvation, which have increased butanol tolerance, are useful for production of butanol.

Yeast strain for production of four carbon alcohols

Increasing tolerance to butanol in yeast has been accomplished by increasing activity of the cell wall integrity pathway. Yeast with increased expression of SLT2p, a mitogen activated protein kinase of the MAPK module of the cell wall integrity pathway had increased tolerance to isobutanol. These yeast may be used for improved butanol production.

Yeast with increased butanol tolerance involving cell wall integrity pathway

Increasing tolerance to butanol in yeast has been accomplished by increasing activity of the filamentous growth response. Yeast with increased expression of MSS11p, a transcriptional activator of the filamentous growth response pathway had increased tolerance to isobutanol. These yeast may be used for improved butanol production.

Yeast with increased butanol tolerance involving filamentous growth response

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

Increasing tolerance to butanol in yeast has been accomplished by increasing activity of the high osmolarity/glycerol response pathway. Yeast with increased expression of PBS2p, a mitogen activated protein kinase kinase of the MAPK module of the high osmolarity/glycerol response pathway may be used for improved butanol production.

Yeast with increased butanol tolerance involving high osmolarity/glycerol response pathway

Using screening of transposon random insertion mutants, genes involved in accumulation of (p)ppGpp were found to be involved in bacterial cell response to butanol. Reduced production of proteins with enzymatic activity for (p)ppGpp biosynthesis confers increased butanol tolerance. Bacterial strains with reduced (p)ppGpp accumulation and having a butanol or 2-butanone biosynthetic pathway are useful for production of butanol or 2-butanone.

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Chadds Ford, PA, United States of America

Robert A Larossa