摘要
为提高具有诱导植物抵御逆境等多种生理功能的冠菌素的产量,基于传统分批发酵法建立丁香假单胞菌大豆致病变种Pseudomonas syringae pv. glycinea Z2-6间歇式流加补料发酵方式,并对补料方法中的起始补料时间、补料体积和补料中的合成前体L-异亮氨酸含量进行了优化。结果表明,利用间歇式流加补料发酵方式,于接种后第8天开始每隔24 h补加新鲜GC培养基(含0.75 g/L的L-异亮氨酸和0.3 g/L的丙酮酸钠)1次,每次补料体积占发酵液总体积的2.0%时,丁香假单胞菌大豆致病变种Z2-6的冠菌素产量最大,达241.5 mg/L,较传统分批发酵方式下的产量(152.5 mg/L)提高了58.4%,合成速率达到20.1 mg·L~(-1)·d~(-1)。表明此种补料发酵方式既可以使细菌高效、持续地利用营养物质,又可解除产物的反馈抑制,大幅提高冠菌素产量,最终达到高产目的。
In order to improve the yield of coronatine(COR), which has many physiological functions such as inducing plants to resist adversity, a batch flow fed-batch fermentation method for Pseudomonas syringae pv. glycinea Z2-6 was established based on the method of microbial fermentation. The initial feeding time, the feed volume, and the L-isoleucine content of synthesized precursor in the feed material were optimized. The results showed that the coronatine yield was 241.5 mg/L with a synthetic rate up to 20.1 mg·L~(-1)·d~(-1), when 2.0% fresh GC broth(containing 0.75 g/L L-isoleucine and 0.3 g/L pyruvate) was fed to the total fermentation broth daily from day 8 post-inoculation, increased by 58.4% compared with traditional batch fermentation(152.5 mg/L). By using this feeding method, the nutrients could be efficiently and continuously used by the bacteria, and the feedback inhibition of the products could also be relieved, which finally achieved the goal of high yield, and increased production of coronatine by 58.4%.
引文
Bender CL,Alarcon-Chaidez F,Gross DC.1999.Pseudomonas syringae phytotoxins:mode of action,regulation,and biosynthesis by peptide and polyketide synthetases.Microbiology and Molecular Biology Reviews,63(2):266-292
Ceylan HA,Turkan I,Sekmen AH.2013.Effect of coronatine on antioxidant enzyme response of chickpea roots to combination of PEG-induced osmotic stress and heat stress.Journal of Plant Growth Regulation,32(1):72-82
Chen XS,Dong N,Mao ZG.2013.Enhancement ofε-poly-L-lysine production through optimization of carbon and nitrogen sources feeding approach.Food and Fermentation Industries,39(8):47-52(in Chinese)[陈旭升,董难,毛忠贵.2013.碳源和氮源流加方式对ε-聚赖氨酸补料-分批发酵过程的影响.食品与发酵工业,39(8):47-52]
Duan LS,Jiang F,Li ZH,Wang CY,Yu S.2013.A fermentation medium and its application in the preparation of coronatine:CN103396965A.2013-11-20(in Chinese)[段留生,姜峰,李召虎,王春燕,于莎.2013.一种发酵培养基及其在制备冠菌素中的应用:CN103396965A.2013-11-20]
Ferguson IB,Mitchell RE.1985.Stimulation of ethylene production in bean leaf discs by the pseudomonad phytotoxin coronatine.Plant Physiology,77(4):969-973
Gou L,Tang CQ,Yang SS.2015.Optimization of Aspergillus fumigatus TMS-26 taxol production fermentation system by precursors,elicitors and fermentation conditions.Mycosystema,34(6):1165-1175(in Chinese)[苟莉,唐春强,杨淑慎.2015.前体及诱导子和发酵条件对烟曲霉TMS-26产紫杉醇发酵体系优化.菌物学报,34(6):1165-1175]
Ichihara A,Shiraishi K,Sato H,Sakamura S,Nishiyama K,Sakai R,Furusaki A,Matsumoto T.1977.The structure of coronatine.Journal of the American Chemical Society,99(2):636-637
Lai YQ,Zeng TF,Xiong R,Guo SQ,Liu DD,Li XR.2017.Effect of intermediate metabolites onε-poly-lysine biosynthesis in Streptomyces albulus.Food and Fermentation Industries,43(2):33-38(in Chinese)[赖永勤,曾天府,熊瑞,郭诗琪,刘丹丹,李学如.2017.中间代谢产物对白色链霉菌ε-聚赖氨酸合成的影响.食品与发酵工业,43(2):33-38]
Lee SY,Wong HH,Choi JI,Lee SH,Lee SC,Han CS.2000.Production of medium-chain-length polyhydroxyalkanoates by highcell-density cultivation of Pseudomonas putida under phosphorus limitation.Biotechnology and Bioengineering,68(4):466-470
Li JA,Lin HM,Niu JG,Zhang HZ,Chen DJ.2012.Application of flow feeding technology to lincomycin fermentation.Chinese Journal of Pharmaceuticals,43(9):739-742(in Chinese)[李继安,林惠敏,牛金刚,张宏周,陈代杰.2012.流加补料技术在林可霉素发酵中的应用.中国医药工业杂志,43(9):739-742]
Parry RJ,Mhaskar SV,Lin MT,Walker AE,Mafoti R.1993.Investigations of the biosynthesis of the phytotoxin coronatine.Canadian Journal of Chemistry,72(1):86-99
Rangaswamy V,Jiralerspong S,Parry R,Bender CL.1998.Biosynthesis of the Pseudomonas polyketide coronafacic acid requires monofunctional and multifunctional polyketide synthase proteins.Proceedings of the National Academy of Sciences of the United States of America,95(26):15469-15474
Shen TF,Wang P.2011.Effect of feeding DL-methionine as precursor on the production of S-adenosyl-L-methionine.Journal of Zhejiang University of Technology,39(4):390-394(in Chinese)[沈天丰,王普.2011.补加前体DL-蛋氨酸对S-腺苷-L-蛋氨酸发酵的影响.浙江工业大学学报,39(4):390-394]
Tan H,Zhou JY,Zhong J,Yang J,Xiao L.2012.A method for efficiently preparing natural abscisic acid:CN102399827A.2012-04-04(in Chinese)[谭红,周金燕,钟娟,杨杰,肖亮.2012.一种高效制备天然脱落酸的方法:CN102399827A.2012-04-04]
Uppalapati SR,Ayoubi P,Weng H,Palmer DA,Mitchell RE,Jones W,Bender CL.2005.The phytotoxin coronatine and methyl jasmonate impact multiple phytohormone pathways in tomato.The Plant Journal,42(2):201-217
Vaillancourt FH,Yeh E,Vosburg DA,O’Connor SE,Walsh CT.2005.Cryptic chlorination by a non-haemiron enzyme during cyclopropyl amino acid biosynthesis.Nature,436(7054):1191-1194
Wang BQ,Li ZH,Zhai ZX,Duan LS.2006.Coronatine and its physiological functions.Plant Physiology Communications,42(3):503-510(in Chinese)[汪宝卿,李召虎,翟志席,段留生.2006.冠菌素及其生理功能.植物生理学通讯,42(3):503-510]
Weiler EW,Kutchan TM,Gorba T,Brodschelm W,Niesel U,Bublitz F.1994.The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants.FEBS Letters,345(1):9-13
Wang L,Chen WJ,Wang Q,Eneji AE,Li ZH,Duan LS.2009.Coronatine enhances chilling tolerance in cucumber(Cucumis sativus L.)seedlings by improving the antioxidative defence system.Journal of Agronomy and Crop Science,195(5):377-383
Wu HL.2009.Construction of high coronatine producing strains and function analysis of dapA and hrcC genes in Pseudomonas syringae.Ph.D Thesis.Beijing:China Agricultural University(in Chinese)[吴慧玲.2009.冠菌素高产工程菌的构建及其合成相关基因dapA和hrcC的调控作用.博士学位论文.北京:中国农业大学]
Xie ZX,Duan LS,Li ZH,Wang XD,Liu XJ.2015.Dose-dependent effects of coronatine on cotton seedling growth under salt stress.Journal of Plant Growth Regulation,34(3):651-664
Yu S,Jiang F,An DJ,Tan WM,Li ZH,Duan LS.2017.Study on the production of coronatine by Pseudomonas syringae Z2-6 using semicontinuous fermentation.Journal of Agricultural Biotechnology,25(12):2066-2071(in Chinese)[于莎,姜峰,安冬捷,谭伟明,李召虎,段留生.2017.丁香假单胞菌Z2-6半连续发酵生产冠菌素的研究.农业生物技术学报,25(12):2066-2071]
Zhang B,Wu XL,Wu HL.2010.Study on fed batch fermentation of producing coronatine with P.syringae pv.glycinea M-18.Scientia Agricultura Sinica,43(12):2533-2540(in Chinese)[张博,吴晓丽,吴慧玲.2010.P.syringae pv.glycinea M-18生产冠菌素补料发酵工艺优化研究.中国农业科学,43(12):2533-2540]
Zhang SJ,Liang Y,Pan F,LüRB,Wang J,Wu FA.2011.Study on fermentation condition for biosynthesis of coronatine by Pseudomonas sringae pv.mori M4-13.Modern Chemical Industry,31(S1):240-243(in Chinese)[张生杰,梁垚,潘飞,吕荣宾,王俊,吴福安.2011.生物合成冠菌素的桑丁香假单孢菌发酵条件.现代化工,31(S1):240-243]
Zhou YY,Shen XF,Zhang MC,Li ZH,Duan LS,Li JM.2011.Preliminary study of physiological base of heat resistance induced by coronatine in wheat seedlings.Journal of Triticeae Crops,31(1):139-142(in Chinese)[周于毅,沈雪峰,张明才,李召虎,段留生,李建民.2011.冠菌素诱导小麦幼苗抗高温胁迫的生理机制.麦类作物学报,31(1):139-142]
Zhou YY,Zhang MC,Li JM,Li ZH,Tian XL,Duan LS.2015.Phytotoxin coronatine enhances heat tolerance via maintaining photosynthetic performance in wheat based on electrophoresis and TOF-MS analysis.Scientific Reports,5:13870
