RECOMBINANT MICRO-ORGANISM FOR USE IN METHOD WITH INCREASED PRODUCT YIELD
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, functionally expressing one or more recombinant, heterologous nucleic acid sequences encoding ribulose-1,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK).
2 . The recombinant yeast cell of claim 1 , wherein said yeast cell further comprises one or more prokaryotic molecular chaperones.
3 . The recombinant yeast cell of claim 2 , wherein said chaperones are selected from the group consisting of GroEL, GroES, functional homologues of GroEL and functional homologues of GroES.
4 . The recombinant yeast cell of claim 1 , wherein said Rubisco is a single subunit Rubisco.
5 . The recombinant yeast cell of claim 1 , wherein said Rubisco is a prokaryotic form-II Rubisco.
6 . The recombinant yeast cell of 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.
7 . The recombinant yeast cell of claim 6 , wherein the genus of yeast cell is Saccharomyceraceae.
8 . The recombinant yeast cell of claim 7 , wherein the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus.
9 . The recombinant yeast cell of claim 1 , wherein the PRK is a PRK originating from a eukaryote.
10 . The recombinant yeast cell of claim 9 , wherein the PRK originates from a Caryophyllales plant.
11 . The recombinant yeast cell of claim 1 , wherein the Rubisco has an activity, defined by the rate of ribulose-1,5-bisphosphate-dependent 14 C-bicarbonate incorporation by cell extracts, of at least 1 nmol.min − .(mg protein) (at 30° C.).
12 . One or more vectors for the functional expression of a heterologous polypeptide in a yeast cell, wherein said vector or vectors comprise one or more heterologous nucleic acid sequence encoding Rubisco and PRK, wherein said Rubisco exhibits activity of carbon fixation.
13 . A method for preparing an alcohol, organic acid or amino acid, comprising fermenting a carbon source with the recombinant yeast cell of claim 1 , thereby forming the alcohol, organic acid or amino acid, wherein the recombinant yeast cell is present in a reaction medium.
14 . The method of claim 13 , wherein the reaction medium comprises carbon dioxide wherein the carbon dioxide concentration in the reaction medium is at least 5% of the carbon dioxide saturation concentration.
15 . The method of claim 13 , wherein ethanol is formed.
16 . A recombinant micro-organism, having an enzymatic system comprising one or more recombinant enzymes that allow the micro-organism to use carbon dioxide as an electron acceptor under chemotrophic (non-phototrophic) conditions, said recombinant micro-organism further comprising:
a heterologous nucleic acid sequence encoding a polypeptide from a naturally autotrophic organism, which polypeptide is selected from the group consisting of carbonic anhydrases, carboxylases, oxygenases, hydrogenases, dehydrogenases, isomerases, aldolases, transketolases, transaldolases, phosphatases, epimerases, kinases, carboxykinases, oxidoreductases, aconitases, fumarases, reductases, lactonases, phosphoenolpyruvate (PEP) carboxylases, phosphoglycerate kinases, glyceraldehyde 3-phosphate dehydrogenases, triose phosphate isomerases, fructose-1,6-bisphosphatases, sedoheptulose-1,7-bisphosphatases, phosphopentose isomerases, phosphopentose epimerase, phosphoribulokinases (PRK), glucose 6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, ribulose 5-phosphate isomerases, ribulose 5-phosphate 3-epimerases, Ribulose-1,5-bisphosphate carboxylase oxygenases, lactate dehydrogenases, malate synthases, isocitrate lyases, pyruvate carboxylases, phosphoenolpyruvate carboxykinases, fructose-1,6-bisphosphatases, phosphoglucoisomerases, glucose-6-phosphatases, hexokinases, glucokinases, phosphofructokinases, pyruvate kinases, succinate dehydrogenases, citrate synthases, isocitrate dehydrogenases, α-ketoglutarate dehydrogenases, succinyl-CoA synthetases, malate dehydrogenases, nucleoside-diphosphate kinases, xylose reductases, xylitol dehydrogenases, xylose isomerases, isoprenoid synthases, and xylonate dehydratases; and
a heterologous nucleic acid sequence encoding a first prokaryotic chaperone for said polypeptide;
wherein the recombinant micro-organism produces an organic compound under anaerobic conditions, and/or wherein the carbon dioxide serves as an electron acceptor in a process with NADH as an electron donor.
17 . The recombinant yeast cell of claim 2 , wherein the chaperone originates from a bacterium.
18 . The recombinant yeast cell of claim 17 , wherein the bacterium is Escherichia coli.
19 . The recombinant yeast cell of claim 10 , wherein the Caryophyllales plant is Amaranthaceae or Spinacia.
20 . The method of claim 14 , wherein the carbon dioxide concentration in the medium is at least 10% of the carbon dioxide saturation concentration.