Produce Dihydoramectin By Combinatorial Biosynthesis

Avermectins are 16-membered macrolide compounds with insecticidal,acaricidal and nematocidal activity produced by Streptomyces avermitilis.Due to the C5,C22-C23 and C25 functional group varitation,avermectins are divided into eight components,the main active component is avermectin B1a.And commercially available Ivermectin is obtained by chemical hydrogenation of the C22-C23 position of avermectin.In this study,the avermectins biosynthetic gene cluster was loaded into a Bacterial Artificial Chromosome(BAC)vector.The biosynthetic gene was successfully expressed in Streptomyces lividans.Meilingmycin polyketide synthase Mei A1 module 2 DH-ER-KR was employed to substitute the Avermectin polyketide synthase Ave A1 module 2 DH-KR to give a hybride polyketide synthase gene.And the aveD gene was knocked out further.The modified gene cluster synthesized Ivermectin B component successfully.The starter group 2-methylbutyryl-CoA and isobutyryl-CoA were produced from the catabolism of isoleucine and valine catalyzed by branched-chain amino acid degradation pathway.Isoleucine and valine were catalyzed by branched-chain amino acid transaminase to produce ?-keto acid,and then dehydrogenation catalyzed by branched chain amino acid dehydrogenase(BCDH).The starter group 2-methyl butyryl-CoA and isobutyryl-CoA are synthesized by the same pathway.In order to obtain a single Ivermectin component,the Ave start module was replaced by lovF,unfortunately,the anticipated compounds have not been detected yet.The branched chain amino acid dehydrogenase of S.avermitilis contains three subunits of E1?,E1? and E2.Meanwhile,in some eukaryotic organisms,there are two BCDH regulatory proteins,which are branched chain amino acid dehydrogenase kinase(BCDH Kinase)and branched chain amino acid dehydrogenase phosphatase(BCDH phosphatase),BCDH Kinase phosphatased E1? to inactive it and BCDH phosphatase dephosphatased E1? to reactive it.In order to regulate the branched chain amino acid degradation pathway in prokaryotic S.avermitilis,we have screened branched chain amino acid dehydrogenase kinase from Saccharomyces cerevisiae,the BCDH Kinase blocked branched amino acid metabolic pathway in S.avermitilis successfully.Dihydoramectin was produced by the recombinant S.avermitilis 319,when cyclohexanoyl-SNAC was fed.In this study,we found out a BCDH Kinase which works well in prokaryotic organisms.It provided a practical case for the expression of eukaryotic genes in prokary-otic cells.Based on BCDH Kinase activity,we have detected a new compound Dihydoramectin.The biosynthetic starter group or extender group of many natural drugs are derived from the branched amino acid degradation pathway.So,BCDH Kinase can be employed to regulate the branched amino acid degradation pathway in a wide range.A variety of related derivatives could be achieved by specifically feeding the substrate or converting the biosynthesis gene cluster of certain substrate.It paved the way for discovering more active new drugs.