rmCombi-OGAB for the Directed Evolution of a Biosynthetic Gene Cluster toward Productivity Improvement

natorial rdered ene ssembly in acillus subtilis (Combi-OGAB) enables construction of combinatorial libraries of various genetic elements, such as promoters in a biosynthetic gene cluster (BGC), and screening of highly productive combinations from the library. The combinations are limited by the library design, and the selectable productivity is defined within the combination. To refine the selected BGC using conventional Combi-OGAB with expanded diversity, we devised a directed evolutionary method called as andom utagenesis with (rmCombi-OGAB), which includes random mutagenesis by error-prone PCR and Combi-OGAB. In the present study, Gramicidin S (GS)-producing plasmids were used to examine the utility of rmCombi-OGAB. GS plasmids, originally generated using conventional Combi-OGAB, were successfully evolved using rmCombi-OGAB. B. subtilis carrying the evolved plasmid with unpredictable mutations showed a 1.5-fold improvement in the GS productivity. We thus expect that rmCombi-OGAB can be applied to various BGCs for useful products, such as antibiotics, to improve their productivity.