ccpA基因对蜡样芽胞杆菌氨肽酶生产的影响

Effect of ccpA gene on the production of aminopeptidase in Bacillus cereus

摘要:

[目的]构建蜡样芽胞杆菌（Bacillus cereus）ccpA缺失菌株，并初步探索ccpA基因对其碳代谢及氨肽酶生产的影响。[方法]利用温敏型质粒pKSV7构建蜡样芽胞杆菌CZ ccpA基因缺失突变株CZΔccpA，通过回补菌株对敲除株表型进行验证；不同碳源发酵对比菌株碳代谢的变化，进行氨肽酶发酵优化。[结果]成功构建ccpA缺失菌株CZΔccpA与回补菌株CZ1，三株菌在LB培养基中生长无差异；在柠檬酸钠以及甘露低聚糖为碳源时，菌株的代谢产生明显变化；以D-木糖为单一碳源时，氨肽酶的产量提高48.25%。[结论]CZccpA基因对柠檬酸钠、甘露低聚糖、D-木糖为单一碳源时的代谢可能具有调控作用，ccpA基因缺失可以提高蜡样芽胞杆菌CZ的氨肽酶产量。

关键词:蜡样芽胞杆菌;基因敲除;分解代谢物控制蛋白A;氨肽酶

Abstract:

[Objective] We constructed Bacillus cereus CZ ccpA deletion strains to explore its effect on carbon metabolism and aminopeptidase production. [Methods] Through homologous recombination mediated by the temperature-sensitive plasmid pKSV7, we acquired the mutant strain CZΔccpA successfully. We also constructed ccpA gene revertant strain CZ1 for phenotypic validation of knockout strains. We compared the metabolic differences of three strains cultured with different carbon source and optimized aminopeptidase fermentation conditions. [Results] The ccpA deletion strain CZΔccpA and the revertant strain CZ1 were successfully constructed. There was no difference in the growth of the three strains in LB medium. When fermented with sodium citrate or mannan oligosaccharides as a single carbon source, the CZΔccpA showed significant metabolic differences from the CZ and CZ1. Besides, the activity of aminopeptidase increased by 48.25 percent when D-xylose was used as the sole carbon source. [Conclusion] Bacillus cereus CZ ccpA gene affects the production of aminopeptidase by regulating the carbon metabolism process and we can raise aminopeptidase production by ccpA gene knockout.

Key words:Bacillus cereus;gene deletion mutants;catabolite control protein A;aminopeptidase