Genetic Switches for Butanol Production
AssigneeButamax™ Advanced Biofuels LLC
InventorsMichael DAUNER, Arthur Leo Kruckeberg, Robert A. Larossa, Brian James Paul
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Abstract
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.
Claims97
A method of producing butanol or 2-butanone comprising:
- A) contacting a recombinant host cell comprising a heterologous polynucleotide, said heterologous polynucleotide comprising
- i) a promoter nucleic acid sequence;
- and
- ii) a nucleic acid sequence encoding a biocatalyst polypeptide with a carbon substrate under a first set of conditions;
- and
- B) contacting the recombinant host cell with a carbon substrate under a second set of conditions;
- wherein the first set of conditions and second set of conditions differ and wherein the nucleic acid sequence encoding a biocatalyst polypeptide is differentially expressed under the first set of conditions than under the second set of conditions;
- and
- wherein the host cell produces butanol or 2-butanone under at least one of the first set or the second set of conditions.
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An isolated polynucleotide comprising:
- (a) a promoter nucleic acid sequence;
- and
- (b) a nucleic acid sequence encoding a biocatalyst polypeptide;
- wherein the nucleic acid sequence of (b) is coupled to the nucleic acid sequence of (a) such that the biocatalyst polypeptide is differentially expressed during the production phase and the propagation phase of a fermentation process.
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A method for screening candidate promoter sequences that are preferentially expressed during the production phase of fermentation, comprising:
- (a) incubating a microorganism under propagation conditions;
- (b) isolating ribonucleic acid molecules from the microorganism incubated in (a);
- (c) incubating a microorganism under production conditions;
- (d) isolating ribonucleic acid molecules from the microorganism incubated in (c);
- (e) selecting only those isolated ribonucleic acid molecules in (d) that are expressed at a higher level than the corresponding isolated ribonucleic acid molecules in (b);
- and
- (f) determining the polynucleotide sequences of the promoters associated with the expression of the ribonucleic acid molecules selected in (e).
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A recombinant yeast host cell comprising an isobutanol biosynthetic pathway and at least one modified expression construct that differentially expresses a polypeptide under conditions in which propagation of biomass is favored over production of isobutanol whereby isobutanol production under said conditions is substantially reduced as compared to a host cell without said modified expression construct under the same conditions.
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Dependencies are inferred from the claim text.