Cloning And Reconstitution Of The Spinosad Biosynthetic Pathwhay Of Saccharopolyspora Spinosa NRRL18395

Spinosyns, the active ingredients in insects control products, are produced by fermentation of the actinomycete Saccharopolyspora spinosa, have broad prospects in commercial use. However, spinosyns yields in our country are still at a very low level, and spinosyns improvement and gene engineering are with difficulty due to the genetic manipulation of Saccharopolyspora spinosa. It turns to be a rational alternative to move the biosynthetic pathway into the heterologous hosts for expression, combinatorial biosynthesis and stain improvement.The spinosyns biosynthesis gene cluster has been cloned and sequenced, all genes involved in the biosynthesis and the biosynthesis pathway were specified. Tri-O-methylated rhamnose and dimethyl-forosamine are two essential deoxysugars in spinosyns biosynthesis. The 4 genes involved in rhamnose and forosamine biosynthesis were unlinked to the spinosyn gene cluster (80 kb) but located in three scattered regions of the genome. The 4 genes, gtt, gdh, epi and kre were cloned from S.spinosa by means of PCR and construction of genomic cosmid library and constituted into an integrative vector to give rise to a heterologous expressed vector.The spinosad gene cluster is over 80 kb in lenghth and it needs the bacterial artificial chromosome (BAC) procedure to clone the whole gene cluster for heterologous expression. In theory, for very large DNA fragments and small vector DNA, it will produce mainly linear product in a standard ligation reaction. However linear DNA does not transform E. coli efficiently. In this study, the Cre-loxP recombination system from P1 phage was employed to integrate into the conventional BAC host-vector system, circulizing the imcoming linear DNA in the host cell to yield recomibinant BAC clones. This novel system was verified by cloning ramdom genomic fragments from S. spinosa.