Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Abstract

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis--the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly.

Suitable extracellular oxidoreduction potential inhibit

“…By over-expression rhamnose-synthesizing genes with their own promoter the yield of spinosad was significantly improved [1]. Pan et al [7] made a three-fold improvement by over-expression rhamnose-synthesizing genes under the control of PermE* promoter.…”

“…Spinosyns containing a 21-carbon tetracyclic lactone are produced by Saccharopolyspora spinosa [1]. Besides to the tetracyclic lactone core, spinosyns also contain two deoxysugars, tri-O-methylated rhamnose and forosamine.…”

The spinosyn family of insecticides: realizing the potential of natural products research

“…17 Cloning and analysis of the spinosyn biosynthetic gene cluster in S. spinosa provided a wealth of additional information. 44,46,47 The anticipated PKS genes for assembling the polyketide chain were identified, but the specific mechanism(s) by which intramolecular crossbridging occurred to form the four rings of the aglycone was not definitively established. The proposed pathway involved thioesterase cleavage of the polyketide chain to form a 22-membered monocyclic macrolide whose subsequent intramolecular cyclizations were possibly controlled by the genes spnF, spnJ, spnL or spnM.…”

“…19,42,43 One study found that spinosyn aglycone consumed all available deoxysugar, and hence duplication of genes stimulating deoxysugar biosynthesis was performed and spinosyn production was substantially increased. 44 In addition to increasing titers, the strain development program created other mutant strains that were important for learning biosynthetic pathways. Initial studies showed that the aglycone of spinosyn A is assembled from nine acetate and two propionate units through a type I polyketide synthase (PKS) pathway and the two sugars are sequentially added to the aglycone, but many further details about the exact sequence and nature of these events were unknown or ambiguous.…”

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

“…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.…”