Development of a Conjugation-Based Genome Editing System in an Undomesticated Bacillus subtilis Strain for...

Poly-γ-glutamic acid (γ-PGA) is a biodegradable polymer produced by microorganisms. Biosynthesizing γ-PGA with diverse molecular masses (M_w) is an urgent industrial technical problem to be solved. Bacillus subtilis KH2, a high-M_w γ-PGA producer, is an ideal candidate for de novo production of γ-PGA with diverse M_w values. However, the inability to transfer DNA to this strain has limited its industrial use. In this study, a conjugation-based genetic operating system was developed in strain KH2. This system enabled us to modify the promoter of γ-PGA hydrolase PgdS in strain KH2 chromosome to de novo biosynthesize γ-PGA with diverse M_ws. The conjugation efficiency was improved to 1.23 × 10^-4 by establishing a plasmid replicon sharing strategy. A further increase to 3.15 × 10^-3 was achieved after knocking out two restriction endonucleases. To demonstrate the potential of our newly established system, the pgdS promoter was replaced by different phase-dependent promoters. A series of strains producing γ-PGA with specific M_ws of 411.73, 1356.80, 2233.30, and 2411.87 kDa, respectively, were obtained. The maximum yield of γ-PGA was 23.28 g/L. Therefore, we have successfully constructed ideal candidate strains for efficient γ-PGA production with a specific M_w value, which provides an important research basis for sustainable production of desirable γ-PGA.