In order to supply a large amount of RM-A for the in vivo experiments, we investigated the molecular basis of biosynthetic machinery of RM-A. The RM-A gene cluster consists of 21 open reading frames spanning 91 kb. To understand the post-PKS modification pathway, all of the genes found in the RM-A cluster were disrupted. Then, the metabolites accumulated in each mutant strain were analyzed.
We revealed the responsible genes involved in each biosynthesis process, such as polyketide chain truncation (revH, revN), spiroacetal formation (revG, revJ), hydroxylation and succinylation (revI, revK, revL, revM) etc. To clarify the enzymatic mechanism of biosynthesis, we performed the heterologous expression of the enzymes, which were purified for biochemical characterization. Finally we succeeded in the in vitro reconstitution of enzymes and obtaining RM-A (final product) from RM-A1a (post PKS product).