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, wherein the recombinant yeast comprises overexpression of one or more PPP-genes. This cell can be used for the production of ethanol and advantageously produces little or no glycerol.
1. A recombinant 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); and optionally
e) one or more heterologous genes encoding a glycerol transporter, wherein the recombinant cell comprises overexpression of one or more pentose phosphate pathway (PPP)-genes.
2. The cell according to claim 1 , wherein the cell comprises overexpression of one or more of TAL1, TAL2, TKL1, TKL2, RPE1 and RKI1.
3. The cell according to claim 1 , wherein the cell comprises overexpression of TKL1.
4. The cell according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol exporter.
5. 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).
6. 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 a cell which is genetically identical except for the modification causing the increased specific activity of dihydroxyacetone kinase.
7. 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).
8. 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 PDP 1/2, which cell is optionally free of genes encoding NADH-dependent glycerol 3-phosphate dehydrogenase.
9. 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).
10. 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%.
11. 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%.
12. The cell according to claim 1 which is a yeast cell.
13. 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 bailit ; and Brettanomyces , optionally Brettanomyces intermedius, Issatchenkia , optionally Issatchenkia orientalis and Hansenula.
14. 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.
15. The cell according to claim 1 wherein the PRK is under control of a promoter that enables higher expression under anaerobic conditions than under aerobic conditions, with 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.
16. A process for preparing a fermentation product, comprising preparing a fermentation product from a fermentable carbohydrate, optionally selected from the group of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose and mannose, wherein said preparing is carried out by contacting the cell according to claim 1 with the fermentable carbohydrate under anaerobic conditions.
17. The process according to claim 16 wherein the fermentable carbohydrate is obtained from starch, lignocellulose, and/or pectin.
18. 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 cell.
19. The process according to claim 16 , 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.
20. The cell according to claim 15 , wherein the promoter is a PRK promoter.