Development of a Conjugation-Based Genome Editing System in an Undomesticated Bacillus subtilis Strain for Poly-?-glutamic Acid Production with Diverse Molecular Masses

Poly-?-glutamic acid (?-PGA) is a biodegradable polymer produced by microorganisms. Biosynthesizing ?-PGA with diverse molecular masses (M (w)) is an urgent industrial technical problem to be solved. Bacillus subtilis KH2, a high-M (w) ?-PGA producer, is an ideal candidate for de novo production of ?-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 ?-PGA hydrolase PgdS in strain KH2 chromosome to de novo biosynthesize ?-PGA with diverse M (w)s. The conjugation efficiency was improved to 1.23 x 10(-4) by establishing a plasmid replicon sharing strategy. A further increase to 3.15 x 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 ?-PGA with specific M (w)s of 411.73, 1356.80, 2233.30, and 2411.87 kDa, respectively, were obtained. The maximum yield of ?-PGA was 23.28 g/L. Therefore, we have successfully constructed ideal candidate strains for efficient ?-PGA production with a specific M (w) value, which provides an important research basis for sustainable production of desirable ?-PGA.