不同碳氮源对枯草芽胞杆菌BAB-1产抗菌脂肽的影响

doi:10.16409/j.cnki.2095-039x.2021.02.007

摘要/Abstract

摘要： 枯草芽胞杆菌BAB-1是一株对设施蔬菜气传病害具有较好防治效果的生防菌，前期研究表明脂肽类抗生素是其产生的主要抑菌物质之一。为提高菌株BAB-1脂肽类抗菌物质的产量，本研究通过快速蛋白液相层析（Fast protein liquid chromatography，FPLC）分析了5种碳源及5种氮源对菌株BAB-1脂肽类抗菌物质产量的影响，进一步采用溶血圈试验及排油圈法测定粗脂肽的性质。结果表明，熊果苷是BAB-1菌株产生surfactin的最佳碳源，其surfactin产量分别是葡萄糖、D-木糖、D-核糖及L-阿拉伯糖的2.95、7.01、4.26及5.60倍，此时菌株BAB-1粗脂肽的溶血活性与排油能力最强；熊果苷与葡萄糖利于该菌株产生fengycin，二者的fengycin产量没有显著差异，但显著高于D-木糖、D-核糖及L-阿拉伯糖。L-天冬酰胺与L-谷氨酸钠是菌株BAB-1产生抗菌脂肽的良好氮源，二者的surfactin与fengycin产量没有显著差异，但显著高于L-半胱氨酸、L-脯氨酸及L-谷氨酰胺，以这2种物质为氮源时菌株BAB-1粗脂肽的溶血活性与排油能力均较强。通过RT-qPCR技术分析了不同碳源对surfactin合成酶基因srfAA表达的影响结果表明，以熊果苷为碳源时，菌株BAB-1中srfAA基因的表达量相比葡萄糖提高了2.9倍。上述结果为菌株BAB-1产抗菌脂肽类物质发酵工艺的优化及其工业化生产提供了一定的理论支持。

关键词: 枯草芽胞杆菌, 抗菌脂肽, 碳源, 氮源

Abstract: Bacillus subtilis BAB-1 showed strong antifungal activity against the airborne diseases of facility vegetables. Previous studies have shown that lipopeptides were one of the major antifungal compounds produced by B. subtilis BAB-1. In order to increase the yield of lipopeptides produced by strain BAB-1, the effects of five carbon sources and five nitrogen sources on the yield of lipopeptides were analyzed by FPLC. The characteristics of crude lipopeptides were assayed by hemolysis test and oil spreading technique. The results showed that arbutin was the best carbon source for production of surfactin, with the yield of 2.95 times, 7.01 time, 4.26 times and 5.60 times as many as that of glucose, D-xylose, D-ribose and L-arabinose, respectively, and the crude lipopeptides of strain BAB-1 showed strongest hemolytic activity and oil discharge capacity. Arbutin and glucose were beneficial to production of fengycin, without significant difference in fengycin yield between them, but significantly higher than that of D-xylose, D-ribose and L-arabinose. L-asparagine and L-sodium glutamate were good nitrogen sources for production of antifungal lipopeptides, and there were no significant difference in the yield of surfactin and fengycin between them, while the yield of antifungal lipopeptides were significantly higher than that of L-cysteine, L-glutamine and L-proline. When L-asparagine and L-sodium glutamate were used as nitrogen sources, the crude lipopeptide of strain BAB-1 showed stronger hemolytic activity and oil discharge capacity. The effects of five carbon substances on the expression of surfactin synthetase gene-srfAA were analyzed by quantitative reverse transcription PCR (RT-qPCR). The results showed that the gene-srfAA was positively affected by arbutin and the expression of srfAA was improved about 2.9 times compared to that of glucose. The above results provided theoretical support for the optimization of the fermentation of strain BAB-1 for production of lipopeptides and its lagre-scale industrial production.

Key words: Bacillus subtilis, lipopeptides, carbon source, nitrogen source

中图分类号:

S476

张晓云, 郭庆港, 王培培, 苏振贺, 鹿秀云, 赵卫松, 曲远航, 董丽红, 丛蓉, 李社增, 马平. 不同碳氮源对枯草芽胞杆菌BAB-1产抗菌脂肽的影响[J]. 中国生物防治学报, 2021, 37(2): 251-258.

ZHANG Xiaoyun, GUO Qinggang, WANG Peipei, SU Zhenhe, LU Xiuyun, ZHAO Weisong, QU Yuanhang, DONG Lihong, CONG Rong, LI Shezeng, MA Ping. Effects of Different Carbon and Nitrogen Sources on the Production of Antifungal Lipopeptides from Bacillus subtilis BAB-1[J]. Chinese Journal of Biological Control, 2021, 37(2): 251-258.

参考文献

[1] Tang Q Y, Bie X M, Lu Z X, et al. Effects of fengycin from Bacillus subtilis fmbJ on apoptosis and necrosis in Rhizopus stolonifer[J]. Journal of Microbiology, 2014, 52(8):675-680.
[2] Yin H P, Guo C L, Wang Y, et al. Fengycin inhibits the growth of the human lung cancer cell line 95D through reactive oxygen species production and mitochondria-dependent apoptosis[J]. Anti-cancer Drugs, 2013, 24(6):587-598.
[3] Hu L B, Shi Z Q, Zhang T, et al. Fengycin antibiotics isolated from B-FS01 culture inhibit the growth of Fusarium moniliforme Sheldon ATCC 38932[J]. FEMS Microbiology Letters, 2007, 272(1):91-98.
[4] Arrebola E, Jacobs R, Korsten L. Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens[J]. Journal of Applied Microbiology, 2010, 108(2):386-395.
[5] Bais H P, Fall R, Vivanco J M. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringaeis facilitated by biofilm formation and surfactin production[J]. Plant Physiology, 2004, 134(1):307-319.
[6] Desai J D, Banat I M. Microbial production of surfactants and their commercial potential[J]. Microbiology and Molecular Biology Review, 1997, 61(1):47-64.
[7] Ongena M, Jourdan E, Adam A, et al. Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants[J]. Environmental Microbiology, 2007, 9(4):1084-1090.
[8] Steller S, Vollenbroich D, Leenders F, et al. Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3[J]. Chemistry and Biology, 1999, 6(1):31-41.
[9] 赵朋超, 权春善, 金黎明, 等. 氮源和碳源对解淀粉芽胞杆菌Q-426抗菌脂肽合成的影响[J]. 中国生物工程杂志, 2012, 32(10):50-56.
[10] Yi L, Zhang G Y, Zhu Z, et al. Optimization of medium composition for lipopeptide production from Bacillus subtilis N7 using response surface methodology[J]. Korean Journal of Microbiology and Biotechnology, 2013, 41(1):52-59.
[11] Liu X Y, Ren B, Gao H, et al. Optimization for the production of surfactin with a new synergistic antifungal activity[J]. PLoS ONE, 2012, 7(5):e34430.
[12] 刘宁, 郭庆港, 安海, 等. 番茄灰霉病生防细菌BAB-1的鉴定及发酵条件的优化[J]. 中国农业科技导报, 2009, 11(2):56-62.
[13] 张晓云, 马平, 李社增, 等. 枯草芽胞杆菌BAB-1防治瓜类白粉病的应用[P]. 中国发明专利. C10158059.7. 2017.
[14] 李宝庆, 鹿秀云, 郭庆港, 等. 枯草芽胞杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定[J]. 中国农业科学, 2010, 43(17):3547-3554.
[15] 钱常娣, 李宝庆, 郭庆港, 等. 枯草芽胞杆菌菌株BAB-1表面活性素的分离纯化及性质分析[J]. 植物病理学报, 2011, 41(2):196-202.
[16] Stein T. Bacillus subtilis antibiotics:structures, syntheses and specific functions[J]. Molecular Microbiology, 2005, 56(4):845-857.
[17] Kalai-Grami L, Karkouch I, Naili O, et al. Production and identification of iturin A lipopeptide from Bacillus methyltrophicus TEB1 for control of Phoma tracheiphila[J]. Journal of Basic Microbiology, 2016, 56(8):864-871.
[18] Guo Q G, Dong W X, Li S Z, et al. Fengycin produced by Bacillus subtilis NCD-2 plays a major role in biocontrol of cotton seedling damping-off disease[J]. Microbiological Research, 2014, 169(7-8):533-540.
[19] Akpa E, Jacques P, Wathelet B, et al. Influence of culture conditions on lipopeptide production by Bacillus subtilis[J]. Applied Biochemistry and Biotechnology, 2001, 91(1):551-561.
[20] Davis D A, Lynch H C, Varley J. The production of surfactin in batch culture by Bacillus subtilis ATCC 21332 is strongly influenced by the conditions of nitrogen metabolism[J]. Enzyme and Microbial Technology, 1999, 25(3):322-329.
[21] Sen R, Swaminathan T. Application of response-surface methodology to evaluate the optimum environmental conditions for the enhanced production of surfactin[J]. Applied Microbiology and Biotechnology, 1997, 47(4):358-363.
[22] Görke B, Stülke J. Carbon catabolite repression in bacteria:many ways to make the most out of nutrients[J]. Nature Reviews Microbiology, 2008, 6(8):613-624.
[23] Buffing M F, Link H, Christodoulou D, et al. Capacity for instantaneous catabolism of preferred and non-preferred carbon sources in Escherichia coli and Bacillus subtilis[J]. Scientific Reports, 2018, 8(1):1-10.
[24] Sandrin C, Peypoux F, Michel G. Co-production of surfactin and iturin A, lipopeptides with surfactant and antifungal properties by Bacillus subtilis[J]. Applied Biochemistry and Biotechnology, 1990, 12(4):370-376.
[25] Nihorimbere V, Cawoy H, Seyer A, et al. Impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefaciens S499[J]. FEMS Microbiology Ecology, 2012, 79(1):176-191.
[26] Roongawang N, Thaniyavarn J, Thaniyavarn S, et al. Isolation and characterization of a halotolerant Bacillus subtilis BBK-1 which produces three kinds of lipopeptides:bacillomycin L, plipastatin, and surfactin[J]. Extremophiles, 2002, 6(6):499-506.
[27] 张晓云, 郭庆港, 鹿秀云, 等. 利用表型芯片技术筛选利于枯草芽胞杆菌Bacillus subtilis BAB-1生长与芽胞形成的营养物质[J]. 植物保护学报, 2020, 47(2):263-272.
[28] Nakano M M, Zuber P. Cloning and characterization of srfB, a regulatory gene involved in surfactin production and competence in Bacillus subtilis[J]. Journal of Bacteriology, 1989, 171(10):5347-5353.
[29] 别小妹, 吕凤霞, 路兆新, 等. Bacillus subtilis fmbJ脂肽类抗菌物质的分离和鉴定[J]. 生物工程学报, 2006, 22(4):644-649.
[30] Peypoux F, Michel G. Controlled biosynthesis of Val7- and Leu7-surfactins[J]. Applied Microbiology and Biotechnology, 1992, 36(4):515-517.
[31] Doekel S, Marahiel M A. Biosynthesis of natural products on modular peptide synthetases[J]. Metabolic Engineering, 2001, 3(1):64-77.
[32] 孙力军, 路兆新, 别小妹, 等. 培养基对解淀粉芽胞杆菌ES-2菌株产抗菌脂肽的影响[J]. 中国农业科学, 2008(10):3389-3398.
[33] Kinsella K, Schulthess C P, Morris T F, et al. Rapid quantification of Bacillus subtilis antibiotics in the rhizosphere[J]. Soil Biology and Biochemistry, 2009, 41(2):374-379.
[34] 朱震, 罗毅, 张鹏, 等. 产表面活性素和伊枯草菌素A菌株的筛选及其脂肽类产物的特性[J]. 微生物学通报, 2011, 38(10):1488-1498.
[35] 董丽红, 郭庆港, 王培培, 等. PhoR/PhoP双组份对枯草芽胞杆菌NCD-2菌株中surfactin合成的影响[J]. 植物病理学报, 2018, 48(1):119-127.
[36] Li D Q, Nie F Y, Wei L H, et al. Screening of high-yielding biocontrol bacterium Bs-916 mutant by ion implantation[J]. Applied Microbiology and Biotechnology, 2007, 75:1401-1408.