IP Library › Granted Patent US 12,624,381
Granted Patent B2
US 12,624,381 · App. 18/392,655 · Granted May 12, 2026

Acetyl-CoA producing enzymes in yeast

Inventors: Ulrike Maria Mueller (Echt, NL); Liang Wu (Echt, NL); Lourina Madeleine Raamsdonk (Echt, NL); Aaron Adriaan Winkler (The Hague, NL)
Assignee: DANISCO US INC.
C12Q1/32C07K14/33C12N9/001C12P7/16Y02E50/10
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Quick Facts
Patent No.
US 12,624,381
App. No.
18/392,655
Granted
May 12, 2026
Kind
B2
Abstract

A method of identifying a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in cytosol of a yeast cell comprising: a) providing a mutated yeast cell comprising a deletion of at least one gene of the by-pass, selected from the genes encoding the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase, and acetylCoA synthetase; b) transforming said mutated yeast cell with an expression vector comprising a heterologous nucleotide sequence encoding a candidate polypeptide having potential enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA; c) testing said recombinant mutated yeast cell for its ability to grown on minimal medium containing glucose as sole carbon source, and d) identifying said candidate polypeptide as a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in the cytosol of said yeast cell when growth of said cell is observed.

Claims (8)

1 . A method of identifying a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in a cytosol of a yeast cell comprising:

providing a mutated yeast cell, wherein said mutation comprises an inactivation of at least one gene of a pyruvate dehydrogenase (PDH) by-pass, selected from the genes encoding the enzymes pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD), and acetyl-CoA synthetase (ACS);

transforming said mutated yeast cell with an expression vector to produce a recombinant mutated yeast cell, wherein the expression vector comprises comprising at least one heterologous nucleotide sequence operably linked to a promoter functional in yeast and said heterologous nucleotide sequence encodes encoding a candidate polypeptide having potential enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA acetyl CoA;

testing said recombinant mutated yeast cell for its ability to grow on minimal medium containing glucose as sole carbon source and wherein the minimal medium does not contain xylose as a carbon source, and

identifying said candidate polypeptide as a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA acetyl CoA in the cytosol of said yeast cell when growth of said cell is observed.

2 . The method according to claim 1 , wherein said yeast cell is a cell of Saccharomyces cerevisiae and wherein said heterologous nucleotide sequence is codon pair optimized for expression in Saccharomyces cerevisiae.

3 . The method according to claim 2 , wherein said mutation comprises an inactivation of the gene for acetyl-CoA synthetase isoform 2 (acs2).

4 . The method-according to claim 1 , wherein said candidate polypeptide having enzymatic activity for converting acetaldehyde into acetyl-CoA is an (putative) acetylating acetaldehyde dehydrogenase (acdh).

Priority Claims (3)
EP 07112956 · Jul 23, 2007 · regional
EP 07123976 · Dec 21, 2007 · regional
EP 08101747 · Feb 19, 2008 · regional
Continuity (6)
Continuation 17081187 · Oct 27, 2020
Continuation 14045683 · Oct 3, 2013
Continuation 12670050
Provisional Application 61064120 · Feb 19, 2008
Provisional Application 60935031 · Jul 23, 2007
Related Publication 20240191277A1 · Jun 13, 2024
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