Glycerol free ethanol production
The invention relates to a recombinant cell, preferably a yeast cell comprising: a) one or more heterologous genes encoding a glycerol dehydrogenase activity; b) one or more genes encoding a dihydroxyacetone kinase (E.C. 2.7.1.28 and/or E.C. 2.7.1.29); c) one or more heterologous genes encoding a ribulose-1,5-biphosphate carboxylase oxygenase (EC 4.1.1.39, RuBisCO); and d) one or more heterologous genes encoding a phosphoribulokinase (EC 2.7.1.19, PRK); and optionally e) one or more heterologous genes encoding 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 recombinant yeast cell comprising:
a) one or more heterologous genes encoding a glycerol dehydrogenase;
b) one or more genes encoding a dihydroxyacetone kinase (E.C. 2.7.1.28 and/or E.C. 2.7.1.29);
c) one or more heterologous genes encoding a ribulose-1,5-biphosphate carboxylase oxygenase (EC 4.1.1.39, RuBisCO); and
d) one or more heterologous genes encoding a phosphoribulokinase (EC 2.7.1.19, PRK);
e) optionally, one or more heterologous genes encoding a glycerol transporter; and
f) a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol exporter, wherein the deleted or disrupted endogenous nucleotide sequence encoding a glycerol exporter is a nucleotide sequence encoding FPS1.
2. The cell according to claim 1 wherein the glycerol dehydrogenase is a NAD+ linked glycerol dehydrogenase (EC 1.1.1.6) or a NADP + linked glycerol dehydrogenase (EC 1.1.1.72).
3. The cell according to claim 1 which comprises a genetic modification that increases the specific activity of dihydroxyacetone kinase in the cell, as compared to the wild-type cell.
4. The cell according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol kinase (EC 2.7.1.30).
5. The cell according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol-3-phosphate dehydrogenase, which glycerol-3-phosphate dehydrogenase optionally belongs to EC 1.1.5.3, optionally gut2, or to EC 1.1.1.8, optionally PDP1/2, which cell is optionally free of genes encoding NADH-dependent glycerol 3-phosphate dehydrogenase.
6. The cell according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol 3-phosphate phosphohydrolase (GPP 1/2).
7. The cell according to claim 1 which comprises one or more nucleic acid sequences encoding a heterologous glycerol transporter represented by amino acid sequence SEQ ID NO: 7 or a functional homologue thereof having sequence identity of at least 50%.
8. The cell according to claim 1 which comprises one or more nucleic acid sequences encoding a heterologous glycerol transporter represented by amino acid sequence SEQ ID NO: 8 or a functional homologue thereof having sequence identity of at least 50%.
9. The cell according to claim 1 which is a yeast cell.
10. The cell according to claim 1 which is selected from Saccharomycetaceae, optionally from the group 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.
11. The cell according to claim 1 further comprising one or more genes, optionally a heterologous genes, encoding molecular chaperones, said chaperones optionally originating from a prokaryote, optionally a bacterium, optionally E coli ; optionally said chaperones are selected from the group consisting of GroEL, GroES, functional homologues of GroEL, and functional homologues of GroES.
12. The cell according to claim 1 wherein the PRK is under control of a promoter (the “PRK promoter”) that enables higher expression under anaerobic conditions than under aerobic conditions, which a PRK expression ratio anaerobic/aerobic of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more or 50 or more.
13. A cell according to claim 1 for preparation of ethanol and/or succinic acid.
14. A process for preparing a fermentation product, comprising culturing the cell of claim 1 under anaerobic conditions with a fermentable carbohydrate, optionally selected from the group of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose and mannose.
15. A process according to claim 14 wherein the fermentable carbohydrate is obtained from starch, lignocellulose, and/or pectin.
16. A process according to claim 15 , 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 cell.
17. A process according to claim 14 , 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.