Glycerol free ethanol production
The invention relates to a recombinant yeast comprising a recombinant yeast comprising a nucleotide sequence coding for a glycerol dehydrogenase, a nucleotide sequence coding for a ribulose-1,5-biphosphate carboxylase oxygenase (EC 4.1.1.39); a nucleotide sequence coding for a phosphoribulokinasey (EC 2.7.1.19); a nucleotide sequence allowing the expression of a glucoamylase (EC 3.2.1.20 or 3.2.1.3); and optionally a nucleotide sequence 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 yeast comprising:
a nucleotide sequence coding for a glycerol dehydrogenase,
a nucleotide sequence coding for a ribulose-1,5-biphosphate carboxylase oxygenase (EC 4.1.1.39);
a nucleotide sequence coding for a phosphoribulokinase (EC 2.7.1.19);
a nucleotide sequence allowing the expression of a glucoamylase (EC 3.2.1.20 or 3.2.1.3), the glucoamylase comprising the amino acid sequence according to SEQ ID NO: 17 or a functional homologue thereof having a sequence identity of at least 70% to SEQ ID NO: 17; and optionally
a nucleotide sequence coding for a glycerol transporter.
2. The recombinant yeast according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol exporter.
3. The recombinant yeast according to claim 2 , wherein the deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol exporter comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a FPS1 glycerol exporter.
4. The recombinant yeast 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 recombinant yeast which according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol-3-phosphate dehydrogenase (GPD1/2).
6. The recombinant yeast according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol 3-phosphate phosphohydrolase (GPP1/2).
7. The recombinant yeast according to claim 1 which is a Saccharomyces , optionally Saccharomyces cerevisiae.
8. The recombinant yeast according to claim 1 further comprising one or more nucleotide sequences, coding for molecular chaperones from E. coli which are selected from the group consisting of GroEL, GroES, functional homologues of GroEL, and functional homologues of GroES.
9. A product comprising the recombinant yeast according to claim 1 for preparation of ethanol and/or succinic acid.
10. A process for production of ethanol comprising:
fermenting a composition comprising a fermentable carbohydrate under anaerobic conditions in the presence of a recombinant yeast according to claim 1 ; and
recovering the ethanol.
11. The process according to claim 10 , wherein the fermentable carbohydrate is obtained from starch, lignocellulose, and/or pectin.
12. The process according to claim 10 , wherein said composition comprises an amount of undissociated acetic acid of 10 mM or less.
13. The process according to claim 10 wherein said composition comprises an amount of undissociated acetic acid of between 50 μM and 10 mM.
14. The process according to claim 10 , wherein the fermentable carbohydrate is selected from the group of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose, and mannose.