Recombinant yeast expressing rubisco and phosphoribulokinase
The invention relates to a recombinant yeast cell, in particular a transgenic yeast cell, functionally expressing one or more recombinant, in particular heterologous, nucleic acid sequences encoding ribulose-1,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK). The invention further relates to the use of carbon dioxide as an electron acceptor in a recombinant chemotrophic micro-organism, in particular a eukaryotic micro-organism.
1. A recombinant yeast cell comprising one or more recombinant heterologous, nucleic acid sequences encoding ribulose-1,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK), wherein the nucleic acid sequence encoding the Rubisco comprises the sequence of SEQ ID NO: 1, and
wherein the recombinant yeast cell further comprises nucleic acid sequences encoding one or more prokaryotic molecular chaperones selected from the group consisting of GroEL, GroES, a functional homologue of GroEL, and a functional homologue of GroES, wherein the respective GroEL and GroES homologues are functional as molecular chaperones.
2. The recombinant yeast cell according to claim 1 , wherein said encoded Rubisco is a single subunit Rubisco.
3. The recombinant yeast cell according to claim 1 , wherein the genus of said yeast cell is selected from the group consisting of Saccharomyceraceae, Schizosaccharomyces, Torulaspora, Kluyveromyces, Pichia, Zygosaccharomyces, Brettanomyces, Metschnikowia, Issatchenkia, Kloeckera , and Aureobasidium.
4. The recombinant yeast cell according to claim 1 , wherein said yeast cell is a Saccharomyceraceae yeast cell.
5. The recombinant yeast cell according to claim 4 , wherein said Saccharomyceraceae yeast cell is a member of a species selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum , and Saccharomyces bayanus.
6. One or more vectors for the expression of a heterologous polypeptide in a yeast cell, wherein:
said vector or vectors comprise one or more recombinant-heterologous nucleic acid sequences encoding ribulose-1,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK);
said encoded Rubisco exhibits carbon fixation activity; and
the nucleic acid sequence encoding the Rubisco comprises the sequence of SEQ ID NO: 1.
7. A method for preparing an alcohol, organic acid, or amino acid, comprising fermenting a carbon source with the yeast cell according to claim 1 , thereby forming the alcohol, organic acid, or amino acid, wherein the yeast cell is present in a reaction medium.
8. The method according to claim 7 , wherein:
the reaction medium comprises carbon dioxide; and
the carbon dioxide concentration in the reaction medium is at least 5% of the carbon dioxide saturation concentration.
9. The method according to claim 7 , wherein ethanol is formed.
10. The method according to claim 7 , wherein the carbon source is a carbohydrate.
11. The method according to claim 8 , wherein the carbon dioxide concentration in the reaction medium is at least 10% or 20% of the carbon dioxide saturation concentration.
12. The recombinant yeast cell according to claim 4 , wherein said yeast cell is a Saccharomyces cerevisiae yeast cell.
13. The recombinant yeast cell according to claim 1 , wherein the nucleic acid sequence encoding GroEL encodes the polypeptide of SEQ ID NO: 10, and the nucleic acid sequence encoding GroES encodes the polypeptide of SEQ ID NO: 12.
14. The one or more vectors according to claim 6 , wherein the nucleic acid sequence encoding the PRK comprises the sequence of SEQ ID NO: 3.
15. The recombinant yeast cell according to claim 1 , wherein the nucleic acid sequence encoding the PRK will hybridize with the full complement of the nucleic acid of SEQ ID NO: 3, under stringent conditions, wherein said hybridization is conducted in a 1 M salt solution, for at least 10 hours, with washing at 65° C. for at least one hour, with at least two changes of the washing solution, wherein the washing solution comprises a 0.1 M salt solution.
16. The one or more vectors according to claim 6 , wherein the nucleic acid sequence encoding the PRK will hybridize with the full complement of the nucleic acid sequence of SEQ ID NO: 3, under stringent conditions, wherein said hybridization is conducted in a 1 M salt solution, for at least 10 hours, with washing at 65° C. for at least one hour, with at least two changes of the washing solution, wherein the washing solution comprises a 0.1 M salt solution.
17. The recombinant yeast cell according to claim 1 , wherein the nucleic acid sequence encoding the Rubisco consists of SEQ ID NO: 1.
18. The recombinant yeast cell according to claim 1 , wherein the nucleic acid sequence encoding the PRK consists of SEQ ID NO: 3.
19. The method according to claim 7 , wherein the reaction medium is free of acetate.