US-12624381-B2 - Acetyl-CoA producing enzymes in yeast

US12624381B2US 12624381 B2US12624381 B2US 12624381B2US-12624381-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.

Inventors

Ulrike Maria MUELLER
Liang Wu
Lourina Madeleine Raamsdonk
Aaron Adriaan Winkler

Assignees

DANISCO US INC.

Dates

Publication Date: 20260512
Application Date: 20231221
Priority Date: 20070723

Claims (4)

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).

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a Continuation of U.S. application Ser. No. 14/045,683, filed Oct. 3, 2013, which is a Continuation of U.S. application Ser. No. 12/670,050, filed May 7, 2010, which is a § 371 National Stage Application of International Application No. PCT/EP2008/059119, filed Jul. 11, 2008, which claims priority to European Application No. 07112956.3, filed Jul. 23, 2007, U.S. Provisional Application No. 60/935,031, filed Jul. 23, 2007, European Application No. 07123976.8, filed Dec. 21, 2007, European Application No. 08101747.7, filed Feb. 19, 2008, and U.S. Provisional Application No. 61/064,120, filed Feb. 19, 2008, the content of all of which are incorporated herein by reference in their entireties. INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as an XML file entitled “IFF26480-US-PCN3_SequenceListing.xml,” created on Dec. 20, 2023, which is 43 KB in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. BACKGROUND OF THE INVENTION The present invention is in the field of metabolites production in yeast using heterologous expression systems. In particular, the present invention relates to the metabolic engineering of yeast strains capable of producing metabolites that require cytosolic acetyl-CoA as a precursor, such as butanol-producing yeast strains. The present invention relates to an assay system for identifying heterologous enzymes capable of converting pyruvate, acetaldehyde or acetate into cytosolic acetyl-CoA when expressed in the cytosol in yeast. Acetyl-coenzyme A (CoA) is an essential intermediate in numerous metabolic pathways, and is a key precursor in the synthesis of many industrial relevant compounds, such as fatty acids, carotenoids, isoprenoids, vitamins, amino acids, lipids, wax esters, (poly)saccharides polyhydroxyalkanoates, statins, polyketides and acetic esters (such as ethyl acetate and isoamyl acetate). In particular, acetyl-CoA is also the precursor of the industrially important bulk chemical 1-butanol. Compared to bacteria, such as E. coli, yeast cells provide a very suitable alternative to produce the above-mentioned acetyl-CoA derived products, in that yeast is not susceptible to phage or other infection since yeast-based processes may be run at low pH. Therefore, the use of yeast does not require a sterile process, thereby lowering the cost price of the product of interest. When natural (wild type) yeast is not able to produce the acetyl-CoA-derived product of interest, the use of metabolic engineering can provide for yeast cells expressing heterologous genes that could support such a process. In such cases, the heterologous gene products are usually targeted to the cytosolic compartment of yeast. As the biosynthesis of acetyl-CoA-derived product will take place completely or partially in the cytosol, the supply of sufficient amounts of the precursor acetyl-CoA in the cytosolic compartment is crucial. In Saccharomyces cerevisiae, biosynthesis of acetyl-CoA takes place in two separate compartments. In mitochondria, acetyl-CoA is synthesized by oxidative decarboxylation of pyruvate catalyzed by the pyruvate dehydrogenase complex (PDH), with the following overall reaction stoichiometry: Pyruvat(Pyr)+CoA+NAD+=acetyl-CoA+CO2+NADH+H+ In cytosol, acetyl-CoA is synthesized via the pyruvate dehydrogenase (PDH) by-pass, involving the enzymes pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD), and acetyl-CoA synthetase (ACS), with the following overall reaction stoichiometry: Pyr+CoA+ATP+NAD(P)+=acetyl-CoA+CO2+NAD(P)H+AMP+Ppi+H+. Pyruvate-decarboxylase-negative (Pdc−) mutant of the yeast S. cerevisiae does not have a functional PDH by-pass, and cannot grow on minimal medium with glucose as the sole carbon source due to inability to supply (sufficient) cytosolic acetyl-CoA for growth (Flikweert et al., (1996) Yeast 12:247-57). The PDH by-pass is therefore essential in providing acetyl-CoA in the cytosolic compartment. However, the PDH bypass in yeast is not optimal with respect to the energy balance, as can be seen from the overall reaction stoichiometry: 2 moles of ATP are needed per acetyl-CoA synthesized via the PDH-bypass since in the acetyl-CoA synthetase reaction ATP is hydrolyzed to AMP. In contrast, the mitochondrial pathway via the PDH requires no ATP. The additional ATP requirement of the PDH by-pass can present a problem for synthesizing the product of interest from cytosolic acetyl-CoA precursor, as more carbon source needs to be diverted for ATP generation, via e.g. oxidative phosphorylation and/or substrate phosphorylation (e.g. glycolysis), thereby lowering the overall yield of the product on carbon. When yeast is metabolically engineered to produce 1-butanol, heterologous biosynthetic genes of 1-butanol can be expressed in the cyt