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

## TL;DR

A new method called rmCombi-OGAB improves the productivity of biosynthetic gene clusters by combining random mutagenesis with existing genetic assembly techniques.

## Contribution

The novel rmCombi-OGAB method integrates random mutagenesis with Combi-OGAB to enhance biosynthetic gene cluster productivity.

## Key findings

- rmCombi-OGAB successfully evolved Gramicidin S-producing plasmids in Bacillus subtilis.
- The evolved plasmid improved Gramicidin S productivity by 1.5-fold.
- The method is expected to be applicable to various biosynthetic gene clusters for productivity improvement.

## Abstract

Combinatorial Ordered Gene Assembly in Bacillus 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 random mutagenesis
with Combi-OGAB (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.