Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa11The DNA sequence reported here was deposited in GenBank under the accession number AY007564.

Abstract

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 genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.

Functional Characterization and Substrate Specificity of Spinosyn Rhamnosyltransferase by in Vitro Reconstitution of Spinosyn Biosynthetic Enzymes

“…Structurally, spinosyns are novel glycosylated macrolides consisting of a 21-carbon tetracyclic lactone decorated with tri-O-methyl-Lrhamnose and D-forosamine (see Scheme 1). The tetracyclic aglycone scaffold is delicately constructed first by type I polyketide (PK-I) 2 synthases, followed by proposed postmodification events, including FAD-oxidation at C-15, Michael addition between C-3 and C-14, ␥-dehydration at C-11, and Diels-Alder cyclization (4,5). The spinosyn biosynthesis genes recently cloned and sequenced are located in an ϳ80-kb cluster of the S. spinosa genome, except that the four genes involved in L-rhamnose (L-Rha) biosynthesis, gtt (NDP-glucose synthase), gdh (NDP-glucose 4,6-dehydratase), epi (NDP-4-keto-6-deoxyglucose epimerase), and kre (NDP-4-ketorhamnose reductase), are located in three different regions of the genome (6).…”

“…Thus far, only relatively few Gtfs have been characterized in vitro and at the mechanistic level, considering the vast number of Gtf sequences revealed in the data base (9,(23)(24)(25)(27)(28)(29)(30)(31)(32)(33)(34). Especially, SpnG is involved in the biosynthetic construction of unique multicyclic or highly modified PK-I glycoside (4,5). Unlike general PK-I macrolides (e.g.…”

A Comparative Metabolomics Analysis ofSaccharopolyspora spinosaWT, WH124, and LU104 Revealed Metabolic Mechanisms Correlated with Increases in Spinosad Yield

“…11) PSA was converted to spinosad completely when the genes involved in the early steps of deoxysugar biosynthesis were duplicated. 12) At the same time, the yield of spinosad also improved. In this study, despite the overexpression of the genes for D-forosamine synthesis and the enhancement of spinosad, an accumulation of PSA was still obvious in LU104 (Fig.…”

“…Finally D-forosamine is added to the hydroxyl group at C-17 to yield the spinosad molecule. 11,12) Though little is known about the spinosad synthesis, many research efforts have managed to improve the yield of spinosad by mutagenesis and metabolic engineering approaches. Liang et al 13) enhanced spinosad production by 2.85-fold using ultraviolet (UV) irradiation.…”

“…The identification of these genetic targets was achieved mainly by genetic perturbation and fermentation experiment. 12,16) The metabolic engineering method can introduce extra imbalance into the spinosad biosynthetic pathway because there is no systematic and accurate quantitation analysis, such as metabolic flux analysis and metabolic control analysis. So, to clarity the enhanced mechanism of the mutant strains and to provide useful information for metabolic engineering, metabolomics analysis using LC-MS is necessary.…”