Cloning And Expression Of The Marine Streptomyces Thioesterase? Gene

Natural microorganisms,especially Streptomyces,can produce a lot of bioactive substances under special conditions,such as fatty acids,anti-tumor,antibacterial and polyketide synthase(PKS).Thioesterase is a widely-used thioester hydrolase.In fatty acid metabolism,acyl coenzyme A thioesterase II can lyse various substrates containing coenzyme A,such as branched fatty acid,short chain and long chain acyl coenzyme A,saturated and unsaturated acyl coenzyme A,can be lysed by acyl coenzyme A thioesterase II.Besides,in the fatty acid synthesis pathway,it has physiological and biochemical functions of signal transduction and energy regulation.In the PKS system,acyl coenzyme A thioesterase II has the functions of hydrolysate and error correction.In this study,the gene of acyl coenzyme A thioesterase II was cloned from Marine Streptomyces,and the structure and function of acyl coenzyme A thioesterase II were studied,which provided a basis for the application and modification of fatty acid metabolism pathway,the formation mechanism of PKS products and the synthesis of emerging drugs.By using the genomic DNA of Streptiomyces sp.L1 preserved in the laboratory as the template,an approximately 800 bp target fragment was amplified.By connecting with p MD19-T,it was transferred to the Escherichia Coli competent cells.The sequencing results of the recombinant plasmid showed that the amplified fragment was 786 bp long and intended to encode 262 amino acids,and its similarity with type II acyl coenzyme A thioesterase(sequence ID:WP 069742521.1)reached100%.BLAST comparison showed that ACOT ? belonged to the Hot Dog superfamily and contained a type II thioesterase domain,which was a unique region of ACOT ? catalyzed reactions.Therefore,it could be concluded that the cloned target gene fragment belonged to acyl coenzyme A thioesterase II.In this study,the expression vector p ET32a-ACOT ? was constructed.The use of IPTG at a concentration of 0.5 mmol / L successfully induced the expression of the target gene.Furthermore,the target protein was expressed by gene induction at this concentration,and SDS-PAGE analysis showed that the molecular weight of protein was about 37.0KDa.The protein induced expression was larger than the theoretical value predicted previously.This might be due to the fusion protein at N-terminal of the expression vector affected the size of the expression protein or the expressed proteins underwent phosphorylation or glycosylation modification to some extent.