Heterologoues Expression Of Spinosad Biosynthesis Gene Cluster And Efficient Genetic Transformation Of Saccharopolyspora Spinosa

Spinosyns are active component of the high effective insect control product marketed by Dow AgroSciences,with an excellent environmental and mammalian toxicological profile.It is produced by aerobic fermentation of the actinomycete Saccharopolyspora spinosa.Spinosad is considered to have a good application prospect in the domain of pest control in crop and grain storage.Sac.spinosa is genetically intractable,making targeted modification of this strain very difficult.Production of Spinosad by fermentation is limited by insufficient product yield,poor growth and culturability profiles associated with the original producer.Heterologous expression of spinosad biosynthesis gene cluster in Streptomyces hosts that is either more suited to large scale fermentation,or have more genetic information would be advantages for production of spinosad,,which also provide a chance to study the mechanism of regulation and expression of cluster in new hosts.The entire spinosyn biosynthetic gene cluster has been captured in a BAC vector using genomic library.Then,genes for conjugation and rhamnose biosynthesis were incorporated into heterologous expression vector by Red/ET and the resulting vector was introduced into Streptomyces coelicolor and Streptomyces lividans by conjugal transfer.The production of spinosad in heterologous host was achieved,paving the road for exploration of expression of spn cluster in various hosts.In order to perform the genetic modification of Sac.spinosa and study the regulation system,we have established and optimized the method for conjugal transfer from E.coli to Sac.Spinosa.This strain showed low rates of starch utilization which is the main carbon source in fermentation industry.Given the maltose transport system is responsible for starch and maltose utilization,malEFG gene of S.avermitilis and its own promoter was inserted into pSET152 and introduced into Sac.spinosa.Spinosad was increased by 34.8%in resulting transformant compared to that of parent strain.Moreover,the spn cluster transcripts and expression level in Sac.spinosa were also detected.Comparing the expression profile between the wide-type strain and a spinosad over-producing strain No.73 illustrates the potential limiting-rate step for spinosad synthesis.