贝莱斯芽孢杆菌抗真菌能力及基因组分析

Antifungal activity and genomic analysis of Bacillus velezensis X49

摘要:

蛋白水解酶及脂肽类化合物具有广谱的抑菌活性，这些抑菌活性蛋白及物质拥有巨大的研究和开发潜力。本研究通过蛋白水解酶平板筛选到一株对6种不同病原真菌有较明显抑菌效果的菌株，命名为X49，对该菌株进行理化分析，并由16S rRNA基因测序、API测试和电镜分析鉴定菌种，进一步分析其基因组序列，再利用偶氮酪蛋白方法分析蛋白水解酶活性及提取脂肽类化合物进行抗真菌测试。结果表明，该菌株在10–50℃、pH 4.0–9.0范围内均可以生长，对环境盐浓度的适应性较强，在10% NaCl浓度下仍然能够生长。菌种鉴定发现X49菌株属于贝莱斯芽孢杆菌（Bacillus velezensis）。基因组分析则显示，B. velezensis X49菌株有11个编码的丝氨酸蛋白酶，其基因编号1_894属于S8枯草杆菌蛋白酶家族，与已知具有抗真菌能力的丝氨酸蛋白酶有高达99%的相似度。另外有多达30个编码的非核糖体肽合成酶，其中包含参与合成已知的表面活性素、伊枯草菌素、丰原素、杆菌肽及短杆菌肽脂肽类化合物。胞外蛋白水解酶活性分析发现其在高温下仍具有一定的活性。将B. velezensis X49菌株与中药干姜共发酵后，提取出的脂肽类化合物，其抑菌功效大大提高。由此可见，本研究筛选出的B. velezensis X49菌株对于植物及人类病原菌都有很显著的抑制作用。而与中药共发酵的活性物质可作为未来药物的研究开发。

Abstract:

Proteolytic enzymes and lipopeptides contain broad-spectrum antimicrobial activities, which have great potential for research and development. A microbial strain X49 obtained from protease screening plate showed antifungal activities against six fungi. Biochemical analysis, 16S rRNA sequencing, API identification system, and electron microscope analysis were carried out to identify the bacterium. Azocasein method was used to analyze the protease activity. Lipopeptides were extracted for antifungal analysis. The result indicated that strain X49 grew in the range of 10-50℃ and pH 4.0-9.0. Moreover, it survived in 10% NaCl, showing good halotolerance. Strain X49 was identified as Bacillus velezensis. Genomic analysis of B. velezensis X49 revealed eleven genes encoding serine protease. The ID 1_894 belonging to S8 subtilisin family was 99% similar to the serine protease with known antifungal ability. On the other hand, thirty genes encoding non-ribosomal peptide synthetase involved in the lipopeptide biosynthesis, including surfactin, iturin, fengycin, bacitracin, and gramicidin, were identified. Part of the extracellular proteolytic activity remained under high temperature. After co-fermentation of B. velezensis X49 with Zingiber officinale Rosc., the antifungal activity of the lipopeptide extract from the co-fermentation was greatly improved. In conclusion, B. velezensis X49 showed clear inhibitory effect on both plant and human pathogens. The active substances co-fermented with Chinese herbs and microbes can be utilized for further drug development.