Production of isoamyl acetate in ackA-pta and/or ldh mutants of Escherichia coli with overexpression of yeast ATF2
- 273 Downloads
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.
KeywordsAnoxic Condition Isoamyl Isoamyl Alcohol Oxic Condition Isoamyl Acetate
This research was supported in part by grants from U.S. Department of Agriculture 2002–35505–11638), National Science Foundation (BES-0118815) and by the Robert A. Welch Foundation C-1268 (GNB) and C-1372 (FBR). Experiments comply with the current laws of the USA.
- Aristidou AA (1994) Application of metabolic and biochemical engineering techniques for the enhancement of recombinant protein production in Escherichia coli. PhD thesis, Rice University, Houston, TexasGoogle Scholar
- Boynton ZL, Bennett GN, Rudolph FB (1994) Intracellular concentrations of coenzyme A and its derivatives from Clostridium acetobutylicum ATCC 824 and their roles in enzyme regulation. Appl Environ Microbiol 60:39–44Google Scholar
- Cauet G, Degryse E, Ledoux C, Spagnoli R, Achstetter T (1999) Pregnenolone esterification in Saccharomyces cerevisiae. A potential detoxification mechanism. Eur J of Biochem. 261:317Google Scholar
- De Graaf AA, Eggeling L, Sahm H (2001) Metabolic engineering for L-lysine production by Corynebacterium glutamicum. Adv in Biochem Eng Biotechnol 73:9-29Google Scholar
- Fukuda K, Kiyokawa Y, Yanagiuchi T,Wakai Y, Kitamoto K, Inoue Y, Kimura A (2000) Purification and characterization of isoamyl acetate-hydrolyzing esterase encoded by the IAH1 gene of Saccharomyces cerevisiae from a recombinant Escherichia coli. Appl Microbiol Biotechnol 53:596–600CrossRefPubMedGoogle Scholar
- Nagasawa N, Bogaki T, Iwamatsu A, Hamachi M, Kumagai C (1998) Cloning and nucleotide sequence of the alcohol acetyltransferase II gene (ATF2) from Saccharomyces cerevisiae Kyokai No. 7. Biosci Bioetchnol and Biochem 62:1852–1857Google Scholar
- Tolentino GJ, Meng SY, Bennett GN, San KY (1992) A pH-regulated promoter for the expression of recombinant proteins in Escherichia coli. Biotechnol Lett 14:157–162Google Scholar