Cloning and expression of the acyl thioesterase 2 (ALT2) gene in Escherichia coli for producing short- and medium-chain 2-methylketones

Abstract

2-Methylketones are organic compounds that are more widely used in the food industry and cosmetics. Some of methylketones found in plants act as pheromones and natural insecticides. Recently, methylketones have been recognized as strong candidates for the production of renewable energy as they possess not only favourable cetane numbers but also lower hydrophilicity and melting points than fatty acids which have been used in biodiesel production. In addition, short chain methylketones have much lower melting points than long chain methylketones. In this study, we cloned and expressed in Escherichia coli C41(DE3) cells a gene for acyl thioesterase 2 (ALT2) isolated from Arabidopsis thaliana. The ALT2 enzyme could hydrolyze 3-ketoacyl-ACP (also called β-ketoacyl-ACP) intermediates in the fatty acid biosynthetic pathway into the corresponding 3-ketoacid. The resulting 3-ketoacids are unstable compounds that will be decarboxylated to produce n-1 methylketones. Methylketones released in the growth medium of E. coli expressing ALT2 were extracted by hexane and analyzed by gas chromatography with the flame ionization detector (GC-FID). The results showed that the E. coli C41(DE3) cells expressing ALT2 recombinantly produced 2-nonanone (9C), 2-undecanone (11C) and 2-tridecanone (13C) in the spent medium when were induced with 0.25 mM IPTG at 37 oC for 3.5 hours.