Patent
A Saccharomyces yeast cell, preferably a Saccharomyces cerevisae yeast cell, comprising one or more nucleic acid sequences encoding an enzyme having NADH-dependent nitrate reductase activity and/or one or more nucleic acid sequences encoding an enzyme having NADH-dependent nitrite reductase activity. An anaerobic fermentation process for the production of a fermentation product using such Saccharomyces yeast cell and a fermentation co-product produced.
1 . A Saccharomyces yeast cell, comprising one or more nucleic acid sequences encoding an enzyme having NADH-dependent nitrate reductase activity and one or more nucleic acid sequences encoding an enzyme having NADH-dependent nitrite reductase activity;
wherein the one or more nucleic acid sequences encoding an enzyme having NADH-dependent nitrate reductase activity encode the amino acid sequence set forth by SEQ ID NO: 1 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1; and
wherein the one or more nucleic acid sequences encoding an enzyme having NADH-dependent nitrite reductase activity encode the amino acid sequence set forth by SEQ ID NO: 3 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3 and the amino acid sequence set forth by SEQ ID NO: 4 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4.
2 . The Saccharomyces yeast cell according to claim 1 , wherein the Saccharomyces yeast cell further comprises one or more nucleic acid sequences encoding a nitrate/nitrite transporter.
3 . The Saccharomyces yeast cell according to claim 1 , further comprising flavin adenine dinucleotide (FAD), one or more heme prosthetic groups, a molybdenum cofactor (MoCo), or any combination thereof.
4 . The Saccharomyces yeast cell according to claim 1 , further comprising one or more nucleic acid sequences encoding enzymes for the synthesis of one or more of a flavin adenine dinucleotide (FAD), one or more heme prosthetic groups, or a molybdenum cofactor (MoCo).
5 . The Saccharomyces yeast cell according to claim 1 , further comprising one or more of:
a nucleic acid sequence encoding a GTP 3′,8-cyclase;
a nucleic acid sequence encoding a Cyclic pyranopterin monophosphate synthase;
a nucleic acid sequence encoding a Molybdopterin synthase catalytic subunit;
a nucleic acid sequence encoding a Molybdopterin synthase sulfur carrier subunit;
a nucleic acid sequence encoding a Molybdopterin adenylyltransferase;
a nucleic acid sequence encoding a Molybdopterin-synthase adenylyltransferase; or
a nucleic acid sequence encoding a Molybdopterin molybdenumtransferase.
6 . The Saccharomyces yeast cell according to claim 1 , further comprising one or more of:
a polypeptide selected from the group consisting of SEQ ID NO:6 and a functional homologue having at least 80% amino acid sequence identity to SEQ ID NO:6; or
a polypeptide selected from the group consisting of SEQ ID NO: 22 and a functional homologue having at least 80% amino acid sequence identity to SEQ ID NO: 22.
7 . The Saccharomyces yeast cell according to claim 1 , wherein the Saccharomyces yeast cells are either free of glycerol phosphate phosphatase activity or have reduced glycerol phosphate phosphatase activity compared to corresponding wild-type cells.
8 . The Saccharomyces yeast cell according to claim 1 , wherein the Saccharomyces yeast cells are either free of glycerol 3-phosphate dehydrogenase activity or have reduced glycerol 3-phosphate dehydrogenase activity compared to corresponding wild-type cells.
9 . The Saccharomyces yeast cell according to claim 1 , wherein the Saccharomyces yeast cell further comprises one or more of:
one or more nucleic acid sequences encoding a glycerol dehydrogenase;
one or more nucleic acid sequences encoding a dihydroxyacetone kinase; or
one or more nucleic acid sequences encoding a glycerol transporter.
10 . An anaerobic fermentation process for production of a fermentation product, comprising fermenting a feed, which feed comprises an oxidized nitrogen source and a fermentable carbon source, using a Saccharomyces yeast cell as claimed in claim 1 .
11 . The anaerobic fermentation process according to claim 10 wherein the Saccharomyces yeast cell produces less glycerol compared to corresponding wild-type cells.
12 . The anaerobic fermentation process according to claim 10 , wherein the Saccharomyces yeast cell produces less ethyl carbamate compared to corresponding wild-type cells.
13 . The anaerobic fermentation process according to claim 10 , wherein the fermentable carbon source comprises or consists of one or more monosaccharides, disaccharides or polysaccharides.
14 . The anaerobic fermentation process according to claim 10 , wherein the oxidized nitrogen source comprises or consists of one or more of:
an acid, salt or ion of nitrate; or
an acid, salt or ion of nitrite.
15 . The anaerobic fermentation process according to claim 10 , wherein the oxidized nitrogen source comprises one or more of nitrate or nitrite in combination with one or more other nitrogen sources selected from the group consisting of ammonium, ammonia and urea.
16 . The anaerobic fermentation process according to claim 10 , wherein the fermentation product is selected from the group consisting of ethanol, n-butanol, isobutanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, fumaric acid, malic acid, itaconic acid, maleic acid, citric acid, and adipic acid.
17 . The anaerobic fermentation process according to claim 10 , wherein further a fermentation co-product is produced.
18 . The anaerobic fermentation process according to claim 17 , wherein the fermentation co-product comprises equal to or less than 17 ng/g of ethyl carbamate.
19 . The Saccharomyces yeast cell according to claim 1 , wherein the Saccharomyces yeast cell is a Saccharomyces cerevisiae yeast cell.
20 . The Saccharomyces yeast cell according to claim 19 , wherein the Saccharomyces cerevisiae yeast cell comprises a genomic mutation in at least one gene selected from the group consisting of glycerol 3-phosphate dehydrogenase 1 (GPD1), glycerol 3-phosphate dehydrogenase 2 (GPD2), glycerol phosphate phosphatase 1 (GPP1) and glycerol phosphate phosphatase 2 (GPP2).