IMPROVED GLYCEROL FREE ETHANOL PRODUCTION
The invention relates to a recombinant cell, preferably a yeast cell comprising one or more genes coding for an enzyme having glycerol dehydrogenase activity, one or more genes coding dihydroxyacetone kinase (E.C. 2.7.1.28 and/or E.C. 2.7.1.29); one or more genes coding for an enzyme in an acetyl-CoA-production pathway and one or more genes coding for an enzyme having at least NAD + dependent acetylating acetaldehyde dehydrogenase activity (EC 1.2.1.10 or EC 1.1.1.2), and optionally one or more genes coding for a glycerol transporter. This cell can be used for the production of ethanol and advantageously produces little or no glycerol.
1 . A recombinant cell, optionally a yeast cell, said recombinant cell comprising:
one or more genes coding for an enzyme having glycerol dehydrogenase activity;
one or more genes coding dihydroxyacetone kinase (E.C. 2.7.1.28 and/or E.C. 2.7.1.29);
one or more genes coding for an enzyme in an acetyl-CoA-production pathway; and
one or more genes coding for an enzyme having at least NAD + dependent acetylating acetaldehyde dehydrogenase activity (EC 1.2.1.10 or EC 1.1.1.2); and optionally
one or more genes coding for a glycerol transporter.
2 . The Cell according to claim 1 wherein the enzyme having glycerol dehydrogenase activity is a NAD + linked glycerol dehydrogenase (EC 1.1.1.6).
3 . The Cell according to claim 1 wherein the enzyme having glycerol dehydrogenase activity is a NADP + linked glycerol dehydrogenase (EC 1.1.1.72).
4 . The recombinant cell according to claim 1 wherein the one or more genes coding for an enzyme in an acetyl-CoA-production pathway comprises:
one or more genes coding for an enzyme having phosphoketolase (PKL) activity (EC 4.1.2.9 or EC 4.1.2.22) or an enzyme having an amino acid sequence according SEQ ID NO: 5, 6, 7, or 8, or functional homologues thereof having a sequence identity of at least 50%, and/or
one or more genes coding for an enzyme having phosphotransacetylase (PTA) activity (EC 2.3.1.8) or an enzyme having an amino acid sequence according SEQ ID NO: 9, 10, 11, or 12, or functional homologues thereof having a sequence identity of at least 50%; and/or
one or more genes coding for an enzyme having acetate kinase (ACK) activity (EC 2.7.2.12), or an enzyme having an amino acid sequence according SEQ ID NO: 1 or 2, or functional homologues thereof having a sequence identity of at least 50%.
5 . The recombinant cell according to claim 1 which either lacks enzymatic activity needed for the production of acetic acid from acetaldehyde or has reduced enzymatic activity needed for production of acetic acid from acetaldehyde compared to a corresponding wild type cell thereof, optionally said cell comprises a deletion or disruption of one or more endogenous genes encoding an enzyme having NAD(P)H dependent aldehyde reductase activity (EC 1.2.1.4).
6 . The recombinant cell according to claim 1 wherein the one or more genes encoding an enzyme having at least NAD + dependent acetylating acetaldehyde dehydrogenase activity encodes an enzyme having an amino acid sequence according to SEQ ID NO: 3, 22, 23, 24, or 25 or a functional homologue thereof having a sequence identity of at least 50%.
7 . The recombinant cell according to claim 1 wherein the enzyme having at least NAD + dependent acetylating acetaldehyde dehydrogenase activity catalyses reversible conversion of acetyl-Coenzyme-A to acetaldehyde and subsequent reversible conversion of acetaldehyde to ethanol.
8 . The recombinant cell according to claim 7 wherein the enzyme comprises both NAD + dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10 or EC 1.1.1.2) activity and NAD + dependent alcohol dehydrogenase activity (EC 1.1.1.1).
9 . The recombinant cell according to claim 1 which comprises a deletion or disruption of one or more endogenous genes encoding a glycerol exporter.
10 . The Cell according to claim 1 which either lacks enzymatic activity needed for production of glycerol 3-phosphate or has reduced enzymatic activity needed for production of glycerol 3-phosphate compared to a corresponding wild type (yeast) cell thereof, optionally said cell comprises a deletion or disruption of one or more endogenous genes encoding a glycerol kinase (EC 2.7.1.30).
11 . The recombinant cell according to claim 1 wherein said cell either lacks enzymatic activity needed for NADH-dependent glycerol synthesis or wherein said cell has reduced enzymatic activity needed for NADH-dependent glycerol synthesis compared to a corresponding wild type (yeast) cell thereof.
12 . The Cell according to any of the preceding claim 1 which comprises a deletion or disruption of one or more endogenous genes encoding a glycerol-3-phosphate dehydrogenase optionally S. cerevisiae GPD1 and GPD2which cell is optionally free of genes encoding NADH-dependent glycerol 3-phosphate dehydrogenase.
13 . The recombinant cell according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol 3-phosphate phosphohydrolase, optionally S. cerevisiae GPP1 or GPP2.
14 . The recombinant cell according to claim 1 which comprises one or more genes encoding a heterologous glycerol transporter represented by SEQ ID NO: 13 or 14 or a functional homologue thereof having a sequence identity of at least 60% thereof.
15 . The recombinant cell according to claim 1 which is selected from the group consisting of Saccharomycetaceae, optionally from the group consisting of Saccharomyces, optionally Saccharomyces cerevisiae; Kluyveromyces, optionally Kluyveromyces marxianus; Pichia, optionally Pichia stipitis or Pichia angusta; Zygosaccharomyces, optionally Zygosaccharomyces bailii; and Brettanomyces, optionally Brettanomyces intermedius, Issatchenkia, optionally Issatchenkia orientalis and Hansenula.
16 . A product comprising a cell according to claim 1 for preparation of ethanol and/or succinic acid.
17 . Process for production of a fermentation product comprising:
fermenting a composition comprising a fermentable carbohydrate, optionally selected from the group of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose and mannose under anaerobic conditions in the presence of a recombinant cell according to claim 1 ; and
recovering the fermentation product.
18 . The Process according to claim 17 wherein the fermentable carbohydrate is obtained from starch, lignocellulose, and/or pectin.
19 . The Process according to claim 17 , wherein the starch, lignocellulose, and/or pectin is contacted with an enzyme composition, wherein one or more sugar is produced, and wherein the produced sugar is fermented to give a fermentation product, wherein the fermentation is conducted with said recombinant cell.
20 . Process according to any of claim 19 , wherein the fermentation product is one or more of ethanol, butanol, lactic acid, succinic acid, a plastic, an organic acid, a solvent, an animal feed supplement, a pharmaceutical, a vitamin, an amino acid, an enzyme or a chemical feedstock.
21 . Process according to claim 17 wherein said composition comprises an amount of undissociated acetic acid of 10 mM or less.
22 . Process according to claim 17 wherein said composition comprises an amount of undissociated acetic acid of between 50 μM and 10 mM.