Biosynthesis of Spinosyn in Saccharopolyspora spinosa: Synthesis of Permethylated Rhamnose and Characterization of the Functions of SpnH, SpnI, and SpnK

Abstract

Spinosyn A is a polyketide-derived macrolide produced by Saccharopolyspora spinosa and is an active ingredient in several commercial insecticides. It is glycosylated by a tri-O-methylated rhamnose at C-9 and a forosamine at C-17. Previous studies indicated that the rhamnose methyltransferases are encoded by the spnH, spnI and spnK genes. To verify the functions of these methyltransferases and to study how they are coordinated to achieve the desired level of methylation of rhamnose, we studied the catalytic properties of the spnH, spnI and spnK gene products and validated their roles in the permethylation process of spinosyn A. Our data reported herein firmly established that SpnH, SpnI, and SpnK are the respective rhamnose 4′-, 2′-, and 3′-Omethyltransferase. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK and SpnH in sequence is productive for the permethylation of the rhamnose moiety. Moreover, the completion of rhamnose permethylation is likely achieved by the proper control of the expression levels of the methyltransferase genes involved. These results set the stage for future exploitation of spinosyn biosynthetic pathway to produce targeted spinosyn derivatives and, perhaps, new analogues.Spinosyn A (SPA, 1) is a polyketide-derived macrolide produced by Saccharopolyspora spinosa, that is an active ingredient in several commercial insecticides. 1 The structures of SPA and its many analogues have a characteristic perhydro-as-indacene core, which is glycosylated by a rhamnose (see 2) at C-9 and a forosamine at C-17. Both the aglycone (AGL, 3) and the sugar appendages contribute to the observed activity of the spinosyns, among which SPA is most potent. 2 Alteration of the tetracyclic nucleus or removal of either deoxy sugars significantly diminishes the pesticidal activity. Even subtle structural variations, such as the methylation pattern of the rhamnose moiety in spinosyns, change the LD 50 by as much as >200- 3 the study of how the corresponding methyltransferases are coordinated to achieve the desired level of methylation of rhamnose has been a focus of this research. We report herein the function and substrate specificity of the three methyltransferases involved in the methylation reactions, the preferred reaction sequence of their catalyzed reactions, and the likely regulation of permethylation of rhamnose in 1.The spinosyn biosynthetic gene cluster had been cloned from S. spinosa. 4 Sequence analysis and gene disruption experiments revealed that the spnH, spnI and spnK genes, 4 all of which show high sequence identity to those encoding S-adenosyl-L-methionine (SAM) dependent methyltransferases (MTs), are involved in the O-methylation of rhamnose in 1. 4,5 As illustrated in Scheme 1, methylations may take place before (2 → 4 → 6, route A) or after (3 → 5 → 6, route B) the attachment of rhamnose to the aglycone (AGL, 3). It is also possible that methylations are the final tailoring steps after both sugars have been coupled to the aglycone (...

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O-Methyltransferases.

“…We previously reported crystal structures of MycE in binary and ternary complexes with co-substrate and substrate 20 . All characterized MycF subfamily members methylate at the 3′ or 4′ hydroxyl positions 21, 22 . Within this subfamily, the substrate- and metal-free structure of NovP 23 and the kinetic mechanism of the homolog TylF 24, 25 have been reported, however the structural basis of substrate specificity and regioselectivity is unknown 20, 23 .…”

Structural Studies of the Spinosyn Rhamnosyltransferase, SpnG

“…SpnN, SpnQ, SpnR, and SpnS catalyze the construction of the forosamine moiety (8–9). SpnH, SpnI, and SpnK are responsible for permethylation of the rhamnose unit (10). Attachment of these two sugar appendages to the C9 and C17 positions of the aglycone is catalyzed by the rhamnosyl- and forosaminyltransferases, SpnG and SpnP, respectively.…”

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

“…This was probably due to the overexpression of spnK, which can improve the production of PSA. 16 than in WT during the fermentation process. These results indicate that the increased precursors and cofactors from primary metabolism can improve the production of PSA.…”

“…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.…”