Studies On Heterologous Expression Of The Avermectin Biosynthetic Gene Cluster

Avermectins,produced by Streptomyces avermitilis,are widely used in the field of agriculture with good potency,broad-spectrum and low toxicity.Avermectins are a series of 16-membered macrocyclic lactons(A1a,A1b,A2a,A2b,B1a,B1b,B2a,and B2b),among which the B1a component shows the most effective antiparasite activity and the lowest level of side effects against host organisms.Avermectins have good application value and broad market prospects in medicine,agriculture and animal husbandry.Even with the elucidation of avermectin biosynthesis and the sequencing of the S.avermitilis genome,the regulatory mechanisms of avermectin production are still not well studied.In this study,the aveAl gene(11,919 bp)encoding AVES1,one of the polyketide synthases of the avermectin biosynthetic gene cluster,was obtained by Red/ET and subsequently expressed in E.coli.The whole avermectin biosynthetic gene cluster was cloned into several Streptomyces hosts for heterologous expression of avermectin production.Biosynthetic elements obtained in this study will provide good materials for the compatibility between heterologous modules and chassis as well as the synthesis of new compounds.The main conclusions in this dessertation are as follows:The aveAl gene was successfully obtained by Red/ET technology from a cosmid containing aveAl and aveA2.Heterologous expression of AVES1 along with the thioesterase domain TE in E.coli was verified with two different methods,including Western blot and the biarsenical-tetracysteine(TC)tagged approach.The result shows that the AveA1+TE protein was successfully expressed in E.coli.According to the Red/ET recombination system,the integrative plasmid pSET152 from the bacteriophage cpC31 containing aveA1+TE gene under the control of the erythromycin promoter(ermE*P1)was constructed,and then transformed into three different Streptomyces hosts by intergeneric conjugation.This will lay the foundation for proving aveA1 genes' function.By using Red/ET recombination technology,the native chloramphenicol-resistant gene Cmr in BAC vector carrying the whole biosynthetic gene cluster of avermectin was replaced by integrase gene int,apramycin-resistant gene aac(3)? and other features necessary for the conjugal transfer of DNA from E.coli to Streptomyces spp.,then the whole biosynthetic gene cluster of avermectin was transformed into Streptomyces coelicolor M1146 by intergeneric conjugation.The recombinant strain M1146/BAC was obtained.After fermentation and optimization of detecting conditions,six peaks in strain M1146/BAC were detected by HPLC.These peaks were further identified respectively to be avermectin A2b,B2a,A2a,B1a,A1b,A1a by LC-MS analysis.