Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa

Background: Spinosad is a mixture of novel macrolide secondary metabolites produced bp Saccharopolyspora spinosa. It is used in agriculture as a poten t insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting ma terials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketid e core or in the attached sugars. Results: We cloned the spinosad biosynthetic genes by molecular probing, co mplementation of blocked mutants, and cosmid walking, and sequenced an 80 k b region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediat es in the spinosyn pathway. The spinosyn gene cluster contains five large o pen reading frames that encode a multifunctional, multisubunit type I polyk etide synthase (PKS). The PKS cluster is flanked on one side by genes invol ved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-b ridging. Genes involved in the early common steps in the biosynthesis of fo rosamine and rhamnose, and genes dedicated to rhamnose biosynthesis. were n ot located in the 80 kb cluster. Conclusions: Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the gene s required for the essential deoxy-sugars. Characterization of the clustere d genes suggests that the spinosyns are synthesized largely by mechanisms s imilar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that coul d encode enzymes that generate the most striking structural feature of thes e compounds, a tetracyclic polyketide aglycone nucleus. (C) 2001 Elsevier S cience Ltd. All rights reserved.