SpnH from Saccharopolyspora spinosa encodes a rhamnosyl 4′- O -methyltransferase for biosynthesis of the insecticidal macrolide, spinosyn A

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.