Genetic replacement of tesB with PTE1 affects chain-length proportions of 3-hydroxyalkanoic acids produced through ß-oxidation of oleic acid in Escherichia coli.

ABSTRACT

Acyl-CoA thioesterase II (TesB), which catalyzes hydrolysis of acyl-CoAs to free fatty acids and CoA, is involved in 3-hydroxyalkanoic acid production in Escherichia coli. Effects of genetic replacement of tesB with Saccharomyces cerevisiae acyl-CoA thioesterase gene PTE1 on 3-hydroxyalkanoic acid production from oleic acid through ß-oxidation were examined. Kinetic analyses using ß-oxidation intermediates showed that hydrolyses of C4-acyl substrates are more efficient by PTE1 than by TesB. Deletion of tesB in E. coli decreased 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, and hexanoic acid in medium after cultivation with oleic acid as a sole carbon source. Hexanoic acid concentration was much lower than those of 3-hydroxyacids. In genetic complementation of tesB deletion, use of PTE1, instead of tesB, affected proportions of the 3-hydroxyalkanoic acids. Proportion of 3-hydroxybutyric acid was higher in a PTE1-complemented strain than in a tesB-complemented strain, while proportions of 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid markedly increased in the tesB-complemented strain. Proportion of 3-hydroxyoctanoic acid did not significantly increase in the PTE1-complemented strain. These data indicate possibilities of 3-hydroxyalkanoic acid production from oleic acid through ß-oxidation and customization of their chain-length proportions by genetic replacement of tesB with a gene encoding acyl-CoA thioesterase with a different kinetic property.