枯草芽孢杆菌YZ-1突变体库构建与产表面活性素优势菌株筛选.

【Objective】The study aimed to construct a transposable mutant library of highly efficient insecticidal Bacillus subtilis YZ-1 and screen for mutant strains with high yields of surfactant, thereby providing preliminary materials for the analysis and development of the regulation mechanism of surfactant synthesis in YZ-1. 【Method】 The strain YZ-1 was transformed using the shuttle vector pMarA carrying the transposon TnYLB-1 and a temperature-sensitive replicative protein. The transposon was induced to transpose at a high temperature of 50 ℃, and the transposon mutants were subjected to resistance culture rescreening to construct a transposon mutant library for the YZ-1 strain. A combination of initial screening with the blood agar plate method, rescreening with bromothymol blue colorimetry, and quantitative detection with HPLC was used to select mutants with significantly enhanced surfactin synthesis from the mutant library. 【Result】 Through natural transformation, the pMarA plasmid was successfully introduced into B.subtilis YZ-1, resulting in a YZ-1 mutant library containing 3000 transposon mutants. From this library, 11 candidate strains with enhanced surfactin synthesis were screened. HPLC detection revealed that compared to the YZ-1 wild type, there were 5 mutants (M1, M6, M7, M8 and M9) with increased or similar levels of surfactin synthesis, and 6 mutants (M2, M3, M4, M5, M10 and M11) with significantly decreased synthesis. Among them, the M8 mutant, which had the highest surfactin yield at 282.4 mg/L, showed a 13.7% increase compared to the wild type, while the M2 strain, with the lowest yield at 47.53 mg/L, experienced an 80.9% decrease compared to the wild type. 【Conclusion】 The addition of a resistance rescreening cultivation step can improve the accuracy of the YZ-1 mutant library. High-throughput screening based on blood agar culture and bromothymol blue assay can preliminarily select mutant strains with significant changes in lipopeptide products. [ABSTRACT FROM AUTHOR]