Production of isoamyl acetate in ackA-pta and/or ldh mutants of Escherichia coli with overexpression of yeast ATF2

• Published in: Applied microbiology and biotechnology Vol. 63; no. 6; pp. 698 - 704
• Main Authors: Vadali, R V, Horton, C E, Rudolph, F B, Bennett, G N, San, K-Y
• Format: Journal Article
• Language: English
• Published: Germany Springer Nature B.V 01.02.2004
• Subjects: Acetate Kinase - genetics; Acetate Kinase - metabolism; Acetyltransferases - genetics; Acetyltransferases - metabolism; Alcohol; Bacteria; Biotransformation; Cloning, Molecular; E coli; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - growth & development; Gene Deletion; Gene Expression; Genes, Bacterial; Genes, Fungal; L-Lactate Dehydrogenase - genetics; L-Lactate Dehydrogenase - metabolism; Microbiology; Mutation; Pentanols - metabolism; Phosphate Acetyltransferase - genetics; Phosphate Acetyltransferase - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-003-1452-y

The gene coding for alcohol acetyltransferase ( ATF2), which catalyzes the esterification of isoamyl alcohol and acetyl coenzyme A (acetyl-CoA), was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli. This genetically engineered strain of E. coli produced the ester isoamyl acetate when isoamyl alcohol was added externally to the cell culture medium. Various competing pathways at the acetyl-CoA node were inactivated to increase the intracellular acetyl-CoA pool and divert more carbon flux to the ester synthesis pathway. Several strains with deletions in the ackA-pta and/or ldh pathways and bearing the ATF2 on a high-copy-number plasmid were constructed and studied. Compared to the wild-type, ackA-pta and nuo mutants produced higher amounts of ester and an ackA-pta-ldh-nuo mutant lower amounts. Isoamyl acetate production correlated well with intracellular coenzyme A (CoA) and acetyl-CoA levels. The ackA-pta-nuo mutant had the highest intracellular CoA/acetyl-CoA level and hence produced the highest amount of ester (1.75 mM) during the growth phase under oxic conditions and during the production phase under anoxic conditions.