A breeding approach combining genome shuffling with multiple antibiotic-resistance including gentamicin, rifampin and lincomycin, was developed in this research to improve the poly-γ-L-diaminobutanoic acid (γ-PAB) production in Bacillus pumilus LS-1. By this unique strategy, recombinants from the third round of genome shuffling could tolerate higher concentration of compound antibiotics and exhibited higher γ-PAB production as 392.4 mg/L in shake-flask fermentation, tenfold over the parent. In batch fermentation, B. pumilus GS3-M7 could produce γ-PAB as high as 2316.4 mg/L in two days, 5.4-fold higher than the control, which was the highest productivity ever reported. In addition, the optimal pH in B. pumilus for γ-PAB synthesis was decreased after ARTP mutagenesis and protoplast fusion, because the lower pH environment is favorable for accumulation of intracellular ATP. Some key enzymes in GS3-M7 showed higher activities than those in the parent, suggesting a greater flux to TCA circle and DAP pathway, which was a reason for enhanced γ-PAB production.
Keywords: Antibiotic resistance; Fermentation; Genome shuffling; Poly-γ-L-diaminobutanoic acid.