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

Patent metadata

Field	Value
Publication number	US-10604772-B2
Application number	US-201414771831-A
Country	US
Kind code	B2
Filing date	Mar 13, 2014
Priority date	Mar 15, 2013
Publication date	Mar 31, 2020
Grant date	Mar 31, 2020

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.

First claim

What is claimed is:

1. 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.

2. The recombinant yeast of claim 1 , wherein the up-regulated enzyme in the nitrogen-assimilation pathway is a Gdh7 isolated from an organism from a genus selected from the group consisting of Saccharomyces and Neurospora (Gdh2); a Glt1 isolated from an organism from the genus Saccharomyces (Glt1); a Gln1 isolated from an organism from the genus Saccharomyces (Gln1′); a MEP protein selected from the group consisting of; Mep1, Mep2, and Mep3 and isolated from an organism from a genus selected from the genus Saccharomyces ; a urea-amido lyase isolated from an organism from the germs Saccharomyces ; a Dur3 or a Dur4 isolated from an organism from the genus Saccharomyces ; or a Gln 3 isolated from the genus Saccharomyces.

3. The recombinant yeast of claim 1 , wherein the down-regulated enzyme in the nitrogen-assimilation pathway is encoded by a polypeptide sequence at least 80%, 90%, 95%, or 100% identical to a polypeptide sequence selected from the group consisting of: SEQ ID NOs: 25 and 31 ( S. cerevisiae Gdh1 and Gdh3).

4. The recombinant yeast of claim 1 , wherein the up-regulated enzyme in the nitrogen-assimilation pathway is encoded by a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence selected from the group consisting of: SEQ ID NOs: 27 and 29 (Gdh2); a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by SEQ ID NO: 33 (Glt1); a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by SEQ ID NO: 35 (Gln1); a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by SEQ ID NO: 19 (Mep1); a polypeptide sequence at least 80%, 90%, 95% or 100% A identical to a polypeptide sequence encoded by SEQ ID NO: 21 (Mep2); a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by SEQ ID NO: 23 (Mep3); a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by SEQ ID NO: 37 (Dur1/2); a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by: SEQ ID NO: 39 (Dur3); or a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by SEQ ID NO: 156 (Gln3).

5. The recombinant yeast of claim 1 , wherein the enzyme in the glycerol production pathway is encoded by a polypeptide sequence at least 80%, 90%, 95% or 100% identical to the polypeptide sequence encoded by: SEQ ID NO: 5 (Gpd1); a polypeptide sequence at least 80%, 90%, 95% or 100% identical to the polypeptide sequence encoded by: SEQ ID NO: 7 (Gpd2); a polypeptide sequence at least 80%, 90%, 95% or 100% identical to the polypeptide sequence encoded by: SEQ ID NO: 159 (Gpp1); or a polypeptide sequence at least 80%, 90%, 95% or 100% identical to the polypeptide sequence encoded by: SEQ ID NO: 161 (Gpp2).

6. The recombinant yeast of claim 1 , wherein the enzyme that acts in the ethanol production pathway is encoded by a polypeptide sequence at least 80%, 90%, 95% or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 9 ( B. adolescentis Pfl); encoded by a polypeptide sequence at least 80%, 90%, 95% or 100% identical, to the polypeptide sequence encoded by SEQ ID NO: 11 ( B. adolescentis Pfl-activating enzyme); encoded by a polypeptide sequence at least 80%, 90%, 95% or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 13 ( B. adolescentis AdhE); or encoded by a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence selected from a group consisting of SEQ ID NOs: 15 and 17.

7. The recombinant yeast of claim 1 , wherein the microorganism further comprises a down-regulation in one or more native enzymes encoded by a formate dehydrogenase enzyme selected from the group consisting of: EC 1.2.1.43 and EC 1.2.1.2;
and/or a heterologous GPD1 polynucleotide operably linked to a native GPD2 promoter;
and/or a heterologous GPD2 polynucleotide operably linked to a native GPD1 promoter;
and/or an up-regulation or down-regulation of a regulatory element.

8. The recombinant yeast of claim 7 wherein the down-regulated enzyme is encoded by a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by SEQ ID NO: 2 (Fdh1); or a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 3 (Fdh2).

9. The recombinant yeast of claim 7 , wherein the regulatory element is selected from the group consisting of Ure2 and Aual.

10. The recombinant yeast of claim 1 , wherein the microorganism further comprises at least one additional up-regulated enzyme, wherein the at least one additional up-regulated enzyme is a glucoamylase enzyme with an EC number 3.2.1.3; a permease; or a protease with EC number: 3.4.23.41.

11. The recombinant yeast of claim 10 , wherein the up-regulated enzyme is encoded by a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by SEQ ID NO: 163 (glucoamylase); a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence encoded by SEQ ID NO: 53 (Gap); or a polypeptide sequence at least 80%, 90%, 95% or 100% identical to a polypeptide sequence selected from a group consisting of SEQ ID NOs: 41, 43, 45, 47, 49, and 51 (protease).

12. The recombinant yeast of claim 1 , wherei

Assignees

• Lallemand Hungary Liquidity Man Llc

Inventors

• Argyros Aaron
• Barrett Trisha

Classifications

• C12N9/50

  Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)} · CPC title

• C12Y104/01004

  Glutamate dehydrogenase (NADP+) (1.4.1.4) · CPC title

• C12P7/10

  substrate containing cellulosic material · CPC title

• C12N9/60

  from yeast · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title