Abstract
Deoxysugar, 2′, 3′, 4′-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4′-O-methylation of 2′, 3′-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4′-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4′-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4′-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.
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We thank Dow AgroSciences for finical support, Christine Donahue for technical assistance and Waldron Clegg for discussion.
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Huang, Kx., Zahn, J. & Han, L. SpnH from Saccharopolyspora spinosa encodes a rhamnosyl 4′-O-methyltransferase for biosynthesis of the insecticidal macrolide, spinosyn A. J Ind Microbiol Biotechnol 35, 1669–1676 (2008). https://doi.org/10.1007/s10295-008-0431-9
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DOI: https://doi.org/10.1007/s10295-008-0431-9