Combinatorial radiation-induced mutagenesis of Bacillus licheniformis and comparative whole genome sequencing to elucidate mutagenic effects on protease production

Abstract

This study demonstrates that sequential UV, pulse, and laser treatments are effective for inducing mutations in membrane proteins and enhancing the protease activity of Bacillus licheniformis. Of the six different treatment combinations tested, the sequential application of Laser, Pulse, and UV (LPU-2) provided the highest lethality rate (96.44 ± 1.97%) and was selected as the optimal combination for subsequent mutagenesis. Through high-throughput screening of the mutant library, 25 strains exhibiting more than 30% relative enzyme activity were selected for second-step screening. Among the strains, LPU-2.1 exhibited the highest protease activity (57.90 ± 0.45 U/mL) in shaking flask fermentation and maximum hydrolysis zone (3.24 cm). Finally, the whole genome resequencing of LPU-2.1 revealed significant genetic alterations, including 20.3 bp insertions in gene ctg_04128, which encodes a membrane protein (MprF). Compared to the wild strain, LPU-2.1 demonstrated a significant increase in protease activity, which was 68%. The genome of LPU-2.1 encoded a total of 4470 proteins, with 3174 sequences (70.91%) being annotated in the GO database. Further analysis in the KEGG platform indicated the active biosynthesis of lysine via L-Lysyl-tRNA in LPU-2.1 produced by enzyme 6.1.16 involved in the aminoacyl-tRNA biosynthesis pathway. These results disclose that the combinatorial irradiation of B. licheniformis caused a modification in the ABC transporter and the two-component systems, which might have impacted the protease production of the mutant.