Yeast with increased butanol tolerance involving a multidrug efflux pump gene
First Claim
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1. A recombinant yeast cell comprising:
- a) an isobutanol biosynthetic pathway, wherein the recombinant yeast cell comprises heterologous genes encoding an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a branched-chain keto acid decarboxylase, and a branched-chain alcohol dehydrogenase that perform the following substrate to product conversions;
i) pyruvate to acetolactate catalyzed by the acetolactate synthase,ii) acetolactate to 2,3-dihydroxyisovalerate catalyzed by the acetohydroxy acid isomeroreductase,iii) 2,3-dihydroxyisovalerate to α
-ketoisovalerate catalyzed by the acetohydroxy acid dehydratase,iv) α
-ketoisovalerate to isobutyraldehyde catalyzed by the branched-chain keto acid decarboxylase,v) isobutyraldehyde to isobutanol catalyzed by the branched-chain alcohol dehydrogenase; and
b) at least one genetic modification in an endogenous gene encoding a multidrug ATP-binding cassette transporter protein, wherein the multidrug ATP-binding cassette transporter protein has at least 95% sequence identity to SEQ ID NO;
50, 52, 54, 56, or 58, wherein the at least one genetic modification decreases activity of the endogenous multidrug resistance ATP-binding cassette transporter protein, and;
wherein the yeast cell has improved tolerance to isobutanol as compared to the corresponding yeast cell that lacks the at least one genetic modification of (b).
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Abstract
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.
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Citations
5 Claims
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1. A recombinant yeast cell comprising:
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a) an isobutanol biosynthetic pathway, wherein the recombinant yeast cell comprises heterologous genes encoding an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a branched-chain keto acid decarboxylase, and a branched-chain alcohol dehydrogenase that perform the following substrate to product conversions; i) pyruvate to acetolactate catalyzed by the acetolactate synthase, ii) acetolactate to 2,3-dihydroxyisovalerate catalyzed by the acetohydroxy acid isomeroreductase, iii) 2,3-dihydroxyisovalerate to α
-ketoisovalerate catalyzed by the acetohydroxy acid dehydratase,iv) α
-ketoisovalerate to isobutyraldehyde catalyzed by the branched-chain keto acid decarboxylase,v) isobutyraldehyde to isobutanol catalyzed by the branched-chain alcohol dehydrogenase; and b) at least one genetic modification in an endogenous gene encoding a multidrug ATP-binding cassette transporter protein, wherein the multidrug ATP-binding cassette transporter protein has at least 95% sequence identity to SEQ ID NO;
50, 52, 54, 56, or 58, wherein the at least one genetic modification decreases activity of the endogenous multidrug resistance ATP-binding cassette transporter protein, and;wherein the yeast cell has improved tolerance to isobutanol as compared to the corresponding yeast cell that lacks the at least one genetic modification of (b). - View Dependent Claims (2, 3, 4)
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5. A method for producing a recombinant yeast cell having increased tolerance to isobutanol comprising:
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a) providing a recombinant yeast cell comprising an isobutanol biosynthetic pathway, wherein the recombinant yeast cell comprises heterologous genes encoding an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a branched-chain keto acid decarboxylase and a branched-chain alcohol dehydrogenase that perform the following substrate to product conversions; i) pyruvate to acetolactate catalyzed by the acetolactate synthase, ii) acetolactate to 2,3-dihydroxyisovalerate catalyzed by the acetohydroxy acid isomeroreductase, iii 2,3-dihydroxyisovalerate to α
-ketoisovalerate catalyzed by the acetohydroxy acid dehydratase,iv α
-ketoisovalerate to isobutyraldehyde catalyzed by the branched-chain keto acid decarboxylase,v isobutyraldehyde to isobutanol catalyzed by the branched-chain alcohol dehydrogenase; and b) engineering the yeast cell of (a) to comprise at least one genetic modification in an endogenous gene encoding a multidrug ATP-binding cassette transporter protein, wherein the multidrug ATP-binding cassette transporter protein has at least 95% sequence identity to SEQ ID NO;
50, 52, 54 56, or 58, wherein the at least one genetic modification decreases activity of the multidrug resistance ATP-binding cassette transporter protein, and wherein the yeast cell has improved tolerance to isobutanol as compared to the corresponding yeast cell that lacks the at least one genetic modification.
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Specification