前体调控对Bacillus sp. 108菌株发酵合成抑制甘蔗鞭黑粉菌活性物质产量的影响
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摘要
【目的】研究前体调控对Bacillus sp. 108菌株发酵合成抑制甘蔗鞭黑粉菌活性物质24元大环内酯产量的影响,为Bacillus sp.108菌株发酵工艺规模化放大及甘蔗黑穗病的防治提供新途径。【方法】利用单因素优化方法,考察乙酸钠、丙酸钠、正丙醇、异亮氨酸和缬氨酸对Bacillus sp. 108菌株发酵合成24元大环内酯的作用,确定有效前体的最佳添加浓度和添加时间;采用平板对峙法测定Bacillus sp. 108菌株发酵产物24元大环内酯对甘蔗鞭黑粉菌的抑制作用。【结果】乙酸钠是Bacillus sp. 108菌株发酵合成24元大环内酯的最佳前体,在发酵第96 h时添加1.0%乙酸钠,24元大环内酯的产量达104.45μg/mL,比未添加前体发酵的对照组提高58.74%。Bacillus sp. 108菌株发酵粗提物对甘蔗鞭黑粉菌菌丝生长具有较强的抑制作用,半数有效浓度(EC_(50))0为157.94μg/mL。【结论】乙酸钠是Bacillus sp.108菌株发酵合成24元大环内酯较适宜的前体物质,24元大环内酯对甘蔗鞭黑粉菌菌丝生长具有明显的抑制作用,具有开发成微生物农药的潜力。
【Objective】The effects of precursor regulation on the inhibition of the production of active substances of Sporisorium scitamineum by Bacillus sp. 108 fermentation and synthesis was studied,which provided a new way for the scale-up of Bacillus sp. 108 fermentation process and the prevention and control of sugarcane smut.【Method】The effects of sodium acetate,sodium propionate,n-propanol,isoleucine and valine on the fermentation of Bacillus sp. 108 strain to synthesize 24-membered macrolide were investigated by single factor optimization method,and the optimum concentration and adding time of effective precursors were determined. The inhibition effect of Bacillus sp. 108 strain fermentation products 24-membered macrolide on S. scitamineum was determined by plate confrontation method.【Result】Sodium acetate was the optimal precursor for 24-membered macrolide biosynthesis by Bacillus sp. 108 strain. By feeding of 1.0% sodium acetate into the fermentation broth at the 96 thh of cultivation,the 24-membered macrolide production was 104.45μg/mL,58.74% higher than that in the control without any addition of precursor. The mycelium growth experiment of S.scitamineum showed that extract of Bacillus sp. 108 strain fermentation products could inhibit the growth of S. scitamineum mycelium,the median effective concentration(EC_(50))values was 157.94 μg/mL.【Conclusion】Sodium acetate is suitable precursor for 24-membered macrolide biosynthesis by Bacillus sp. 108 strain fermentation. The study confirms that24-membered macrolide has an inhibitory effect on the growth of the mycelium of S. scitamineum,and has the potential to be developed into a microbial pesticide.
【Objective】The effects of precursor regulation on the inhibition of the production of active substances of Sporisorium scitamineum by Bacillus sp. 108 fermentation and synthesis was studied,which provided a new way for the scale-up of Bacillus sp. 108 fermentation process and the prevention and control of sugarcane smut.【Method】The effects of sodium acetate,sodium propionate,n-propanol,isoleucine and valine on the fermentation of Bacillus sp. 108 strain to synthesize 24-membered macrolide were investigated by single factor optimization method,and the optimum concentration and adding time of effective precursors were determined. The inhibition effect of Bacillus sp. 108 strain fermentation products 24-membered macrolide on S. scitamineum was determined by plate confrontation method.【Result】Sodium acetate was the optimal precursor for 24-membered macrolide biosynthesis by Bacillus sp. 108 strain. By feeding of 1.0% sodium acetate into the fermentation broth at the 96 thh of cultivation,the 24-membered macrolide production was 104.45μg/mL,58.74% higher than that in the control without any addition of precursor. The mycelium growth experiment of S.scitamineum showed that extract of Bacillus sp. 108 strain fermentation products could inhibit the growth of S. scitamineum mycelium,the median effective concentration(EC_(50))values was 157.94 μg/mL.【Conclusion】Sodium acetate is suitable precursor for 24-membered macrolide biosynthesis by Bacillus sp. 108 strain fermentation. The study confirms that24-membered macrolide has an inhibitory effect on the growth of the mycelium of S. scitamineum,and has the potential to be developed into a microbial pesticide.
引文
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李菲,黄庶识,王伟,江蕾,米顺利,余炼.2018.海绵Pseudoceratina sp.共栖细菌多样性及其抑制甘蔗鞭黑粉菌活性研究[J].广西科学,25(1):87-93.[Li F,Huang S S,Wang W,Jiang L,Mi S L,Yu L.2018.Diversity of sponge-derived bacteria isolated from Pseudoceratina sp.against Sporisorium scitamineum in vitro[J].Guangxi Sciences,25(1):87-93.]
刘扬,郑华,姚婷婷,田黎,李文利.2014.海洋芽孢杆菌B-9987中macrolactin生物合成基因簇分析及反式酰基转移酶高表达[J].中国海洋药物,33(3):69-75.[Liu Y,Zheng H,Yao T T,Tian L,Li W L.2014.Charaterization of macrolactins gene cluster from Bacillus marinus B-9987and overexpression of trans-acyl transferase[J].Chinese Journal of Marine Drugs,33(3):69-75.]
覃媚,于清武,竺利波,李菲,颜栋美,余炼,高程海.2016.三种江蓠共附生细菌多样性及抑菌活性分析[J].南方农业学报,47(11):1966-1973.[Qin M,Yu Q W,Zhu L B,Li F,Yan D M,Yu L,Gao C H.2016.Diversity of epiphytic bacteria of three species of Gracilaria and their bacteriostatic activities[J].Journal of Southern Agriculture,47(11):1966-1973.]
王波,周涧楠,黄忠勤,丁震乾,常勇,苏在兴,周兴根.2017.一株多粘类芽孢杆菌Paenibacillus polymyxa对甘薯黑斑病的生物防治效果及作用机理初探[J].江西农业学报,29(10):40-43.[Wang B,Zhou J N,Huang Z Q,Ding ZQ,Chang Y,Su Z X,Zhou X G.2017.Biocontrol efficacy of a Paenibacillus polymyxa strain against black rot of sweet potato and its action mechanism[J].Acta Agriculturae Jiangxi,29(10):40-43.]
王竹青.2016.甘蔗对黑穗病菌侵染的生理生化和基因表达响应[D].福州:福建农林大学.[Wang Z Q.2016.Physiological,Biochemical and gene expression response of sugarcane to Sporisorium scitamineum infection[D].Fuzhou:Fujian Agriculture and Forestry University.]
杨世红,李能威,郭伟群,韩伟,陈新,张晓琳.2011.前体对多拉菌素生物合成的影响[J].中国抗生素杂志,36(11):829-832.[Yang S H,Li N W,Guo W Q,Han W,Chen X,Zhang X L.2011.Effect of precursors on doramectin biosynthesis[J].Chinese Journal of Antibiotics,36(11):829-832.]
余炼.2016.柳珊瑚与内生菌次生代谢产物分离及对甘蔗黑穗霉菌抑制研究[D].南宁:广西大学.[Yu L.2016.Secondary metabolites isolayionI of anthogorgia and bacteria and prevention of sugarcane smut[D].Nanning:Guangxi University.]
周剑,张引.2018.达托霉素产生菌前体物耐受选育及其流加补料发酵[J].中国抗生素杂志,43(7):817-823.[Zhou J,Zhang Y.2018.Precursor resistance screening on mutation breeding and fed-batch fermentation for daptomycin production[J].Chinese Journal of Antibiotics,43(7):817-823.]
Mojid Mondol M A,Kim J H,Lee H S,Lee Y J,Shin H J.2011.Macrolactin W,a new antibacterial macrolide from a marine Bacillus sp.[J].Bioorganic and Medicinal Chemistry Letters,21(12):3832-3835.
Schenck S,Pearl H M,Liu Z,Moore P H,Ming R.2005.Genetic variation of Ustilago scitaminea pathotypes in Hawaii evaluated by host range and AFLP markers[J].Sugar Cane International,23(1):15-19.
Schneider K,Chen X H,Vater J,Franke P,Nicholson G,Borriss R,Sussmuth R D.2007.Macrolactin is the polyketide biosynthesis product of the pks2 cluster of Bacillus amyloliquefaciens FZB42[J].Journal of Natural Products,70(9):1417-1423.
Singh N,Somai B M,Pillay D.2005.Molecular profiling demonstrates limited diversity amongst geographically separate strains of Ustilago scitaminea[J].Fems Microbiology Letters,247(1):7-15.
Su Y C,Wang S S,Guo J L,Xue B T,Xu L P,Que Y X.2013.A TaqMan real-time PCR assay for detection and quantification of sporisorium scitamineum in sugarcane[J].The Scientific World Journal,(9):1351-1358.
Wang T,Liang Y F,Wu M B,Chen Z J,Li J P,Yang L R.2015.Natural products from Bacillus subtilis with antimicrobial properties[J].Chinese Journal of Chemical Engineering,23(4):744-754.
Xue C M,Tian L,Xu M J,Deng Z W,Lin W H.2008.Anew 24-membered lactone and a new polyene delta-lactone from the marine bacterium Bacillus marinu[J].The Journal of Antibiotics,61(11):668-674.
陈慎,黄颖颖,陆东和,杨成龙.2017.外加营养源对红曲霉固态发酵产莫纳可林K和洛伐他汀的影响[J].中国食品添加剂,(9):102-113.[Chen S,Huang Y Y,Lu D H,Yang C L.2017.Effects of additional nutrition sources on the production of Monacolin K and Lovastatin by solid state fermentation of Monascus[J].China Food Additives,(9):102-113.]
段海明,程红,余利,黄伟东,余海兵.2017.解淀粉芽孢杆菌gfj-4发酵上清液及其混剂对玉米纹枯病菌的抑制活性[J].江苏农业学报,33(6):1249-1256.[Duan H M,Cheng H,Yu L,Huang W D,Yu H B.2017.Inhibitory activity of Bacillus amyloliquefaciens gfj-4 fermentation supernatant and its association with chemical fungicides against Rhizoctonia solani[J].Jiangsu Journal of Agricultural Sciences,33(6):1249-1256.]
樊乐.2017.阿维菌素生产菌种的选育及发酵工艺的优化[D].呼和浩特:内蒙古大学.[Fan L.2017.Avermectin strain screening and optimization of fermentation process[D].Hohhot:Inner Mongolia University.]
方舟,孟慧云,杨渊,林家富,翟龙飞,褚以文,王欣荣.2018.前体物质对Glarea lozoyensis发酵产生纽莫康定B0的影响(英文)[J].中国抗生素杂志,43(4):437-443.[Fang Z,Meng H Y,Yang Y,Lin J F,Zhai L F,Chu Y W,Wang X R.2018.Precursors supplement effects on the level of pneumocandin B0produced by Glarea lozoyensis[J].Chinese Journal of Antibiotics,43(4):437-443.]
高春霞.2018.克拉维酸发酵培养基及发酵工艺的优化[D].呼和浩特:内蒙古大学.[Gao C X.2018.Optimization of the fermentation medium and fermention process of clavulanic acid[D].Hohhot:Inner Mongolia University.]
何碧珀,郝学政,刘红彦,倪云霞,刘新涛,赵新贝,赵辉.2018.解淀粉芽孢杆菌B10-26对芝麻的促生防病效果及其定殖能力分析[J].河南农业科学,47(12):78-83.[He B B,Hao X Z,Liu H Y,Ni Y X,Liu X T,Zhao X B,Zhao H.2018.Analysis of growth-promotion,disease control effect and colonization capacity of Bacillus amyloliquefaciens B10-26 in sesame[J].Journal of Henan A-gricultural Sciences,47(12):78-83.]
江蕾.2018.抑制甘蔗鞭黑粉菌的活性菌株分离、诱变筛选及其代谢产物作用机理研究[D].南宁:广西民族大学.[Jiang L.2018.Study on separating,mutagenesis screening active strains and mechanism of inhibiting Sporisorium scitamineum of metabolites[D].Nanning:Guangxi University for Nationalities.]
江蕾,李菲,李智,易湘茜,邓家刚,周桂,高程海.2018.海黄瓜共附生细菌多样性及其对甘蔗鞭黑粉菌的抑制作用[J].南方农业学报,49(3):488-494.[Jiang L,Li F,Li Z,Yi X X,Deng J G,Zhou G,Gao C H.2018.Diversity of symbiotic bacteria isolated from Pseudocnus echinatus and its antifungal activity against Sporisorium scitamineum[J].Journal of Southern Agriculture,49(3):488-494.]
李菲,黄庶识,王伟,江蕾,米顺利,余炼.2018.海绵Pseudoceratina sp.共栖细菌多样性及其抑制甘蔗鞭黑粉菌活性研究[J].广西科学,25(1):87-93.[Li F,Huang S S,Wang W,Jiang L,Mi S L,Yu L.2018.Diversity of sponge-derived bacteria isolated from Pseudoceratina sp.against Sporisorium scitamineum in vitro[J].Guangxi Sciences,25(1):87-93.]
刘扬,郑华,姚婷婷,田黎,李文利.2014.海洋芽孢杆菌B-9987中macrolactin生物合成基因簇分析及反式酰基转移酶高表达[J].中国海洋药物,33(3):69-75.[Liu Y,Zheng H,Yao T T,Tian L,Li W L.2014.Charaterization of macrolactins gene cluster from Bacillus marinus B-9987and overexpression of trans-acyl transferase[J].Chinese Journal of Marine Drugs,33(3):69-75.]
覃媚,于清武,竺利波,李菲,颜栋美,余炼,高程海.2016.三种江蓠共附生细菌多样性及抑菌活性分析[J].南方农业学报,47(11):1966-1973.[Qin M,Yu Q W,Zhu L B,Li F,Yan D M,Yu L,Gao C H.2016.Diversity of epiphytic bacteria of three species of Gracilaria and their bacteriostatic activities[J].Journal of Southern Agriculture,47(11):1966-1973.]
王波,周涧楠,黄忠勤,丁震乾,常勇,苏在兴,周兴根.2017.一株多粘类芽孢杆菌Paenibacillus polymyxa对甘薯黑斑病的生物防治效果及作用机理初探[J].江西农业学报,29(10):40-43.[Wang B,Zhou J N,Huang Z Q,Ding ZQ,Chang Y,Su Z X,Zhou X G.2017.Biocontrol efficacy of a Paenibacillus polymyxa strain against black rot of sweet potato and its action mechanism[J].Acta Agriculturae Jiangxi,29(10):40-43.]
王竹青.2016.甘蔗对黑穗病菌侵染的生理生化和基因表达响应[D].福州:福建农林大学.[Wang Z Q.2016.Physiological,Biochemical and gene expression response of sugarcane to Sporisorium scitamineum infection[D].Fuzhou:Fujian Agriculture and Forestry University.]
杨世红,李能威,郭伟群,韩伟,陈新,张晓琳.2011.前体对多拉菌素生物合成的影响[J].中国抗生素杂志,36(11):829-832.[Yang S H,Li N W,Guo W Q,Han W,Chen X,Zhang X L.2011.Effect of precursors on doramectin biosynthesis[J].Chinese Journal of Antibiotics,36(11):829-832.]
余炼.2016.柳珊瑚与内生菌次生代谢产物分离及对甘蔗黑穗霉菌抑制研究[D].南宁:广西大学.[Yu L.2016.Secondary metabolites isolayionI of anthogorgia and bacteria and prevention of sugarcane smut[D].Nanning:Guangxi University.]
周剑,张引.2018.达托霉素产生菌前体物耐受选育及其流加补料发酵[J].中国抗生素杂志,43(7):817-823.[Zhou J,Zhang Y.2018.Precursor resistance screening on mutation breeding and fed-batch fermentation for daptomycin production[J].Chinese Journal of Antibiotics,43(7):817-823.]
Mojid Mondol M A,Kim J H,Lee H S,Lee Y J,Shin H J.2011.Macrolactin W,a new antibacterial macrolide from a marine Bacillus sp.[J].Bioorganic and Medicinal Chemistry Letters,21(12):3832-3835.
Schenck S,Pearl H M,Liu Z,Moore P H,Ming R.2005.Genetic variation of Ustilago scitaminea pathotypes in Hawaii evaluated by host range and AFLP markers[J].Sugar Cane International,23(1):15-19.
Schneider K,Chen X H,Vater J,Franke P,Nicholson G,Borriss R,Sussmuth R D.2007.Macrolactin is the polyketide biosynthesis product of the pks2 cluster of Bacillus amyloliquefaciens FZB42[J].Journal of Natural Products,70(9):1417-1423.
Singh N,Somai B M,Pillay D.2005.Molecular profiling demonstrates limited diversity amongst geographically separate strains of Ustilago scitaminea[J].Fems Microbiology Letters,247(1):7-15.
Su Y C,Wang S S,Guo J L,Xue B T,Xu L P,Que Y X.2013.A TaqMan real-time PCR assay for detection and quantification of sporisorium scitamineum in sugarcane[J].The Scientific World Journal,(9):1351-1358.
Wang T,Liang Y F,Wu M B,Chen Z J,Li J P,Yang L R.2015.Natural products from Bacillus subtilis with antimicrobial properties[J].Chinese Journal of Chemical Engineering,23(4):744-754.
Xue C M,Tian L,Xu M J,Deng Z W,Lin W H.2008.Anew 24-membered lactone and a new polyene delta-lactone from the marine bacterium Bacillus marinu[J].The Journal of Antibiotics,61(11):668-674.
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