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

natorial rdered ene ssembly in (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 (Combi-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 Combi-OGAB. GS plasmids, originally generated using conventional Combi-OGAB, were successfully evolved using Combi-OGAB. carrying the evolved plasmid with unpredictable mutations showed a 1.5-fold improvement in the GS productivity. We thus expect that Combi-OGAB can be applied to various BGCs for useful products, such as antibiotics, to improve their productivity.