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US 10,604,772 B2Granted
Methods for regulating nitrogen metabolism during the production of ethanol from corn by metabolically engineered yeast strains
AssigneeLallemand Hungary Liquidity Management LLC
InventorsAaron Argyros, Trisha Barrett
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Abstract
The present invention provides for a mechanism to reduce glycerol production and increase nitrogen utilization and ethanol production of recombinant microorganisms. One aspect of this invention relates to strains of S. cerevisiae with reduced glycerol productivity that get a kinetic benefit from higher nitrogen concentration without sacrificing ethanol yield. A second aspect of the invention relates to metabolic modifications resulting in altered transport and/or intracellular metabolism of nitrogen sources present in com mash.
Claims21
A recombinant yeast comprising:
- a) at least one engineered genetic modification that leads to the up-regulation of one or more native and/or heterologous enzymes that function in one or more ethanol production pathways;
- b) at least one engineered genetic modification that leads to the down-regulation of an enzyme in a glycerol-production pathway;
- and,
- c) at least one engineered genetic modification that leads to the up-regulation or down-regulation of an enzyme in a nitrogen-assimilation pathway;
- wherein the upregulated enzyme that acts in the ethanol production pathway is selected from the group consisting of pyruvate formate lyase (EC 2.3.1.54), pyruvate formate lyase activating enzyme (EC 1.91.1.4), bifunctional acetaldehyde-alcohol dehydrogenase selected from a group of enzymes having both of the following Enzyme Commission Numbers:
- EC 1.2.1.10 and 1.111;
- and an NADPH-dependent bifunctional acetaldehyde-alcohol dehydrogenase selected from a group of enzymes having both of the following Enzyme Commission Numbers:
- EC 1.2.1.10 and 1.1.1.2;
- wherein the down-regulated enzyme in the glycerol-production pathway is selected from the group consisting of glycerol-3-phosphate dehydrogenase 1 polynucleotide (GPD1) (EC 1.1.1.8), glycerol-3-phosphate dehydrogenase 1 polypeptide (Gpd1) (EC 1 .1.1.8), glycerol-3-phosphate dehydrogenase 2 polynucleotide (GPD2) (EC 1.1.1.8), glycerol-3-phosphate dehydrogenase 2 polypeptide (Gpd2) (EC 1.1.1.8), glycerol-3-phosphate phosphatase 1 polynucleotide (GPP1) (EC 3.1.3.21), a glycerol-3-phosphate phosphatase polypeptide 1 (Gpp1) (EC 3.1.3.21), a glycerol-3-phosphate phosphatase 2 polynucleotide (GPP2) (EC 3.1.3.21), and glycerol-3-phosphate phosphatase polypeptide 2 (Gpp2) (EC 3.1.3.21);
- and
- wherein the down-regulated enzyme in the nitrogen-assimilation pathway is glutamate dehydrogenase (Gdh) (EC 1.4.1.4);
- or
- wherein the up-regulated enzyme in the nitrogen-assimilation pathway is at least one enzyme selected from the group consisting of glutamate dehydrogenase (Gdh) (EC 1.4.1.2), glutamate synthase (Glt) (EC 1.4.1.14), and glutamine synthase (Gln) (EC 6.3.1.2);
- an ammonium transporter;
- a urea-amido lyase (EC 6.3.4.6);
- and a urea transporter.
13 more elements
A recombinant yeast comprising:
- down-regulated Gpd1, down-regulated Gpd2, down-regulated Fdh1, down-regulated Fdh2, down-regulated Gdh1, up-regulated AdhE, up-regulated pyruvate formate lyase, an up-regulated pyruvate formate lyase-activating enzyme, GPD1 under the control of the GPD2 promoter, GPD2 under the control of the GPD1 promoter, and up-regulated Gdh2;
- or a recombinant yeast comprising:
- down-regulated Gpd1, down-regulated Gpd2, down-regulated Fdh1, down-regulated Fdh2, down-regulated Gdh1, up-regulated AdhE, up-regulated pyruvate formate lyase, an up-regulated pyruvate formate lyase-activating enzyme, GPD1 under the control of the GPD2 promoter, GPD2 under the control of the GPD1 promoter, up-regulated Glt1 and up-regulated Gln1;
- or a recombinant yeast comprising:
- down-regulated Gpd1, down-regulated Fdh1, down-regulated Fdh2, down-regulated Gdh1, up-regulated AdhE, up-regulated pyruvate formate lyase, an up-regulated pyruvate formate lyase-activating enzyme, GPD1 under the control of the GPD2 promoter, GPD2 under the control of the GPD1 promoter, up-regulated Glt1 and up-regulated Gln;
- or a recombinant yeast comprising:
- down-regulated Gpd2, down-regulated Fdh1, down-regulated Fdh2, down-regulated Gdh1, up-regulated AdhE, up-regulated pyruvate formate lyase, and an up-regulated pyruvate formate lyase-activating enzyme;
- or a recombinant yeast comprising:
- down-regulated Gpd1, down-regulated Fdh1, down-regulated Fdh2, down-regulated Gdh1, up-regulated AdhE, up-regulated pyruvate formate lyase, an unregulated pyruvate formate lyase-activating enzyme, and GPD2 under the control of the GPD1 promoter;
- a recombinant yeast comprising:
- down-regulated Gpd1, down-regulated Fdh1, down-regulated Fdh2, down-regulated Gdh1, up-regulated AdhE, up-regulated pyruvate formate lyase, and an up-regulated pyruvate formate lyase-activating enzyme;
- or a recombinant yeast comprising:
- down-regulated Gpd1, down-regulated Fdh1, down-regulated Fdh2, up-regulated AdhE, up-regulated pyruvate formate lyase, an up-regulated pyruvate formate lyase-activating enzyme, upregulated-DUR1/2 , and GPD2 under the control of the GPD1 promoter;
- or a recombinant yeast comprising:
- down-regulated Gpd1, down-regulated Fdh1, down-regulated Fdh2, up-regulated AdhE, up-regulated pyruvate formate lyase, an up-regulated pyruvate formate lyase-activating enzyme, and up-regulated DUR1/2 ;
- or a recombinant yeast comprising:
- down-regulated Gpd1, down-regulated Gpd2, down-regulated Fdh1, down-regulated Fdh2, down-regulated Ure2, up-regulated AdhE, up-regulated pyruvate formate lyase, an up-regulated pyruvate formate lyase-activating enzyme, GPD1 under the control of the GPD2 promoter, and GPD2 under the control of the GPD1 promoter;
- or a recombinant yeast comprising:
- down-regulated Gpd1, down-regulated Gpd2, down-regulated Fdh1, down-regulated Fdh2, up-regulated AdhE, up-regulated pyruvate formate lyase, an up-regulated pyruvate formate lyase-activating enzyme, up-regulated GDH2, GPD1 under the control of the GPD2 promoter, and GPD2 under the control of the GPD1 promoter.
16 more elements
Dependencies are inferred from the claim text.