贝莱斯芽孢杆菌PJP10的抑菌物质稳定性及活性代谢产物挖掘

Stability evaluation and genome mining of antimicrobial metabolites of Bacillus velezensis PJP10

摘要:

【背景】食源性致病菌在食品中易生长繁殖且致病性强，严重威胁着公共卫生和食品安全。【目的】通过多个指标分析贝莱斯芽孢杆菌(Bacillus velezensis) PJP10的抑菌物质稳定性，利用基因组测序预测编码抑菌活性相关酶和代谢产物合成蛋白的基因并检测酶和代谢产物的离体活性，为该菌工农业开发和应用提供参考数据。【方法】通过抑菌试验确定pH、温度、蛋白酶、金属离子、紫外线照射和盐浓度等因素对抑菌物质稳定性的影响。利用全基因组测序了解菌株PJP10潜在的产酶基因，并通过酶活试验检测其产酶能力。使用在线软件antiSMASH预测代谢产物合成基因簇，并利用酸沉淀法和乙酸乙酯萃取法获得无菌发酵液粗提物，通过高效液相色谱和抑菌试验初步探究菌株PJP10的胞外活性代谢产物成分。【结果】菌株PJP10无菌发酵液能够耐受强酸、强碱、高温、高盐和紫外线照射等环境，对蛋白酶K和胃蛋白酶不敏感，但对胰蛋白酶和金属离子敏感。全基因组中存在编码淀粉酶、氨肽酶、蛋白酶、几丁质酶、纤维素酶、酯酶、果胶酶、葡聚糖酶和酰胺酶等抑菌酶的基因，酶活测定结果表明该菌株具有蛋白酶、淀粉酶、纤维素酶和酯酶活性。antiSMASH预测发现菌株PJP10基因组中包含杆菌素(bacillaene)、地非西丁(difficidin)、大环内酰亚胺(macrolactin) H、表面活性素(surfactin)、杆菌霉素(bacillomycin) D、泛革素(fengycin)、溶杆菌素(bacilysin)、儿茶酚型铁载体(bacillibactin)，以及解淀粉芽孢杆菌素(amylocyclicin)和植物唑霉素(plantazolicin)等多种活性代谢产物合成基因簇。此外，抑菌试验发现酸沉淀粗提物对沙门氏菌和大肠杆菌抑菌活性显著，对辣椒青枯菌无抑菌活性，乙酸乙酯粗提物对金黄色葡萄球菌和辣椒青枯菌抑菌活性显著，对沙门氏菌和大肠杆菌抑菌活性明显减弱，根据HPLC结果推测，菌株PJP10无菌发酵液中的抑菌物质可能是iturin、fengycin和surfactin这3个家族的成员。【结论】菌株PJP10抑菌物质稳定性良好，基因组中含有丰富的活性酶基因和活性代谢产物合成基因簇，在工农业生产领域具有较好的应用潜力。

Abstract:

[Background] Food-borne pathogens are easy to grow and reproduce in food and have high pathogenicity, posing critical threats to food safety and public health.[Objective] To analyze the antimicrobial stability of Bacillus velezensis PJP10 from multiple perspectives, discover the genes encoding antimicrobial enzymes and involved in the biosynthesis of antimicrobial substances by genome sequencing, and measure the in vitro activities of enzymes and active metabolites, so as to provide data for the application of this strain in industry and agriculture. [Methods] The microbial inhibition tests were carried out to examine the effects of pH, temperature, protease, metal ions, ultraviolet (UV), and salt concentration on the antimicrobial stability of the strain. The potential genes encoding antimicrobial enzymes of PJP10 were investigated by whole genome sequencing and the enzyme-producing abilities assessed by enzyme activity assays. The secondary metabolites of PJP10 were predicted by antiSMASH. HPLC and microbial inhibition tests were employed to analyze the components of the crude extract obtained by the acid precipitation and ethyl acetate extraction. [Results] The cell-free fermentation supernatant exhibited excellent stability at high temperatures, strong acid, strong alkali, high salt and UV, and was sensitive to trypsin and metal ions, but not proteinase K or pepsin. The genome of PJP10 carried abundant genes encoding amylase, aminopeptidase, protease, chitinase, cellulase, esterase, pectinase, glucanase, and amidase, and the strain could produce protease, amylase, cellulase, and esterase. The antiSMASH predicted that strain PJP10 had a variety of gene clusters for the synthesis of secondary metabolites, including bacillaene, difficidin, macrolactin H, surfactin, bacillomycin D, fengycin, bacilysin, bacillibactin, amylocyclicin, and plantazolicin. In addition, the extract obtained by acidification exhibited strong inhibitory activities against Salmonella enteritidis and Escherichia coli but no activity against Ralstonia solanacearum. The ethyl acetate extract showed strong inhibitory effects on Staphylococcus aureus and R. solanacearum and weak inhibitory effects on S. enteritidis and E. coli. According to HPLC results, we hypothesized that the antimicrobial substances in the cell-free supernatant of PJP10 were iturin, fengycin, and surfactin. [Conclusion] The strain PJP10 shows high antimicrobial stability and carries rich genes encoding active enzyme and abundant gene clusters for the synthesis of antimicrobial substances, demonstrating a promising prospect of application in industry and agriculture.