Methods for regulating nitrogen metabolism during the production of ethanol from corn by metabolically engineered yeast strains

A method of producing a fermentation product comprising:

• a) providing a recombinant yeast comprising:
• (i) 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;
• (ii) and, at least one engineered genetic modification that leads to the down-regulation of an enzyme in a glycerol-production pathway;
• and,
• (iii) 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;
• b) contacting the composition with a carbon containing feedstock, wherein the recombinant yeast is capable of fermenting the carbon containing feedstock to yield the fermentation product;
• and,
• c) optionally recovering the fermentation production.