多杀菌素产生菌基因组重排育种研究
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摘要
多杀菌素(spinosad)是由放线菌刺糖多孢菌Saccharopolyspora spinosa经有氧发酵后的次级代谢产物。多杀菌素具有生物农药的安全性，化学合成农药的速效性，对哺乳动物，昆虫天敌和环境无害，具有独特的作用机理等优点。该产品的开发具有很好的经济效益和社会效益。本论文运用基因组重排技术对多杀菌素产生菌进行选育，并优化了其发酵工艺，最后进行了2.5L发酵罐小试。
首先，本文对发酵培养基进行了优化。采用正交法和单因素法考察了碳源、氮源、前体物质和接种量。优化后的发酵工艺条件如下：发酵培养基为葡萄糖5.5％、玉米粉2.5％、鱼粉1.5％、黄豆饼粉0.2％、淀粉0.5％、豆油0.2％、谷氨酸钠0.2％、酵母粉0.2％、碳酸钙0.5％，pH为7.0，接种量为10％，250 ml摇瓶装量为30 ml。在发酵培养48 h时，同时加入0.3％乙醇和0.3％正丁醇能进一步提高多杀菌素的合成，且比原发酵产量提高了52％。
此外，本文还考察了原生质体制备、再生以及融合的最佳条件。再根据刺糖多孢菌的生物合成途径和代谢调控原理，运用原生质体紫外线复合诱变，原生质体融合，基因组重排等多种方法进行菌种选育，并结合耐前体突变株、耐自身终产物突变株等理性化筛选，使刺糖多孢菌合成多杀菌素的能力不断提高，最后选育出了一株刺糖多孢菌sp．4．d7，其发酵单位为547 mg·l~(-1)，比亲本菌株sp．y．5提高了2.02倍，比原始出发菌株sp107-2提高了4.36倍。同时，本文还对菌株sp．4．d7进行了遗传稳定性试验，结果表明该菌株遗传性能比较稳定。将重排菌株sp．4．d7在2.5 L发酵罐上进行发酵试验，在转速为300 rpm，DO值为60％，温度为28℃下发酵5天，其发酵产量为428 mg·l~(-1)。
Spinosyn, a novel microbial broad-spectrum insecticide, was secondary metabolites from the aerobic fermentation of Saccharopolyspora spinosad. Its favorable mammalian and environmental profile, insect selectivity, unique mode of action and outstanding efficacy lead to high benefit in ecomony and society. In this paper, we are aimed to improve the spinosyn-producing strain by genome shuffling, optimization of fermentation process and scale-up of fermentation processes in 2.5 L fermentor.
Firstly, the medium composition for spinsyn production were investigated by 'one-variable-at-a-time' approach (OVAT) and orthogonal design. The optimal medium composition was as follows: Glucose 5.5%, Fish meal 1.5%, Corn powder 2.5%, Soybean powder 0.2%, Starch 0.5%, Soybean oil 0.2%, Sodium Glutamate 0.2%, Calcium carbona 0.5%, pH 7.0, the inoculum amount 10%, medium volume 30ml in 250ml shaking flask. And the addition of 0.3% alchol and 0.3% n-butyl alcohol can improve the production of spinsyn by 52%.
In this dissertation, the conditions for protoplast preparation, regeneration and fusion were optimized. According to the biosynthesis pathway and the metabolic regulation of spinosad, strain improvement was performed by protoplast mutatation with UV and LiCl, protoplast fusion and genome shuffling. A high spinosyn producing S. spinosad sp.4.d7 was obtained, and its production reached 547 mg·l~(-1), which was 202% more than that of the parent strain sp.y.5, and 436% more than that of the original strain sp 102-7. Subculture experiment indicated that the hereditary character of Sp.4.d7 was stable. Spinosad fermentation of S. spinosad sp.4.d7 was scaled up in a 2.5L fermentor in the condition of 350 rpm, DO 60%, 28°C, and its production was 428 mg·l~(-1).
首先，本文对发酵培养基进行了优化。采用正交法和单因素法考察了碳源、氮源、前体物质和接种量。优化后的发酵工艺条件如下：发酵培养基为葡萄糖5.5％、玉米粉2.5％、鱼粉1.5％、黄豆饼粉0.2％、淀粉0.5％、豆油0.2％、谷氨酸钠0.2％、酵母粉0.2％、碳酸钙0.5％，pH为7.0，接种量为10％，250 ml摇瓶装量为30 ml。在发酵培养48 h时，同时加入0.3％乙醇和0.3％正丁醇能进一步提高多杀菌素的合成，且比原发酵产量提高了52％。
此外，本文还考察了原生质体制备、再生以及融合的最佳条件。再根据刺糖多孢菌的生物合成途径和代谢调控原理，运用原生质体紫外线复合诱变，原生质体融合，基因组重排等多种方法进行菌种选育，并结合耐前体突变株、耐自身终产物突变株等理性化筛选，使刺糖多孢菌合成多杀菌素的能力不断提高，最后选育出了一株刺糖多孢菌sp．4．d7，其发酵单位为547 mg·l~(-1)，比亲本菌株sp．y．5提高了2.02倍，比原始出发菌株sp107-2提高了4.36倍。同时，本文还对菌株sp．4．d7进行了遗传稳定性试验，结果表明该菌株遗传性能比较稳定。将重排菌株sp．4．d7在2.5 L发酵罐上进行发酵试验，在转速为300 rpm，DO值为60％，温度为28℃下发酵5天，其发酵产量为428 mg·l~(-1)。
Spinosyn, a novel microbial broad-spectrum insecticide, was secondary metabolites from the aerobic fermentation of Saccharopolyspora spinosad. Its favorable mammalian and environmental profile, insect selectivity, unique mode of action and outstanding efficacy lead to high benefit in ecomony and society. In this paper, we are aimed to improve the spinosyn-producing strain by genome shuffling, optimization of fermentation process and scale-up of fermentation processes in 2.5 L fermentor.
Firstly, the medium composition for spinsyn production were investigated by 'one-variable-at-a-time' approach (OVAT) and orthogonal design. The optimal medium composition was as follows: Glucose 5.5%, Fish meal 1.5%, Corn powder 2.5%, Soybean powder 0.2%, Starch 0.5%, Soybean oil 0.2%, Sodium Glutamate 0.2%, Calcium carbona 0.5%, pH 7.0, the inoculum amount 10%, medium volume 30ml in 250ml shaking flask. And the addition of 0.3% alchol and 0.3% n-butyl alcohol can improve the production of spinsyn by 52%.
In this dissertation, the conditions for protoplast preparation, regeneration and fusion were optimized. According to the biosynthesis pathway and the metabolic regulation of spinosad, strain improvement was performed by protoplast mutatation with UV and LiCl, protoplast fusion and genome shuffling. A high spinosyn producing S. spinosad sp.4.d7 was obtained, and its production reached 547 mg·l~(-1), which was 202% more than that of the parent strain sp.y.5, and 436% more than that of the original strain sp 102-7. Subculture experiment indicated that the hereditary character of Sp.4.d7 was stable. Spinosad fermentation of S. spinosad sp.4.d7 was scaled up in a 2.5L fermentor in the condition of 350 rpm, DO 60%, 28°C, and its production was 428 mg·l~(-1).
引文
[1] Ying-xin Zhang, Kim Perry, Victor A. Vinci, et al. Genome shuffling leads to rapid phenotypic improvement in bacteria [J]. Nature, 2002, 15:644～646
[2] Patnaik R, Louie S, Gavrilovic V, et al. Genome shuffling of Lactobacillus for improved acid tolerance [J]. Nature Biotechnol, 2002 Jul, 20(7): 707～712
[3] Campo N, Dias M J, Daveran-Mingot ML, Ritzenthaler P. Metabolic engineering by genome shuffling Genome plasticity in Lactococcus lactis [J]. Bourgeois P Antonie Van Leeuwenhoek, 2002, 82:123～132
[4] 张兴，马志卿等．生物农药评述[J]．西北农林科技大学学报(自然科学版)，2002，30(2)：142～148
[5] Thompson CD, Dutton R and Sparks TC. Spinosad—a case study: an example from a natural products discovery programme [J]. Pest Manag Sci., 2000, 56: 696～702
[6] Mertz E, Yao R. Saccharopolyspors spinosa sp nov isolated from soil coolected in a sugar rum still [J]. Int Sust Bacteriol, 1990, 40(1): 34～39
[7] Aon. Spinosad Technical Guide [M]. DowElaco, 1996:25
[8] Baker PJ. U. S. Patent, 5, 227, 295, 1993
[9] Baker PJ. U. S. Patent, 5, 227, 295, 1994
[10] Vincent L. Salgado. Studies on the mode of action of spinosad: insect symptoms and physiological correlates [J]. Pestic. Biochem. and physiol., 1998, 60:91～102
[11] Gerald B. Watson. Action of insecticidal spinosyns on gaminbutyric acid responses from small diameter cockroach neurons [J]. Pesticide biochemistry and physiology, 2001, 71:20～26
[12] 王彦华，王鸣华．多杀菌素的作用机理及其抗药性的研究进展[J]．农药科学与管理，2006，25(11)：12～15
[13] Saunders D. Fate of spinosad in the environment [J]. Down to Earth, 1996, 52 (1): 21～28
[14] Gary D, Robert D. Spinosad a case study: an example from a nature products discovery programme [J]. Pest Manag Sci, 2000, 56 (8): 696～702
[15] Carson W, Yrumble J. Effect of insecticides for control of leafminers on lima beans [J]. Arthrop Management Tests, 1998, 23 (1): 74～75
[16] 张部昌，赵志虎，马清钧．红霉素生物合成的分子生物学[J]．生物技术通讯，2001，12(2)：151～160
[17] 张惠民．途径工程．第三代基因工程IM]．北京：中国轻工业出版社，2002
[18] Cropp A, Chen S, Liu H, et al. Genetic approaches for controlling ratios of related polyketide products in fermentation processes [J]. Ind Microbiol Biotechnol, 2001, 27(6): 368～377
[19] Baltz R H, Crawford K P, Broughton MC, et al. Biosynthetic genes for spinosyn insecticide production. US Patent 6274350, 2001
[20] Waldron C, Madduri K, Crawford K. A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa [J]. Antonie Van Leeuwenhock, 2000, 78(3-4): 385～390
[21] Waldron C, Matsushima P, Rosteck P R Jr, et al. Cloning and analysis of the spinosad biothetic gene cluster of Saccharopolyspora spinosa [J]. Chemistry Biology, 2001, 8(5): 487～499
[22] 柳君科．刺孢小单孢菌和灰色链霉菌原生质体融合的研究[J]．遗传学报，1989，16(1)：49～55
[23] 张苑．多杀菌素高产菌种的推理选育和发酵工艺的优化[D]．硕士论文，浙江大学，岑沛霖，浙江杭州，2004
[24] 肖信法．微生物原生质体融合与菌种选育[J]．抗生素，1983，8(4)：261～265
[25] 辛明秀．微生物的原生质体融合及应用[J]．微生物学通报，1995，22(6)：365～370
[26] 余荔华．克氏固氮菌与枯草芽孢杆菌的原生质体电融合[J]．清华大学学报，1999，39(6)：46～48
[27] 贺莜蓉，黄小倩．链霉菌原生质体的制备[J]．生物学通报，1998，33(1)：40～41
[28] 陈钧鸣，徐玲娣．抗生素工业分析[M]．增订本，北京，中国医药科技出社，1991
[29] 张苑，金志华，林建平，岑沛霖．多杀菌素的高效液相色谱法测定[J]．农药，2003．42(10)：27～28
[30] 王岳五，松林生．电融合技术选育能利用木糖和纤维二糖生产乙醇的菌株[J]．生物工程学报，1992，8(1)：82～86
[31] 朱建伟等．林肯霉菌原生质体的形成、再生及其影响因素[J]．医药工业，1987：18(3)：108～112
[32] 金志华，林建平，梅乐和．工业微生物遗传育种学原理与应用[M]．化学工业出版社，北京，2006．1
[33] 朱林东．普那霉素产生菌基因组重排[D]．硕士论文，浙江大学，金志华，浙江杭州，2006
[34] 赵凯，平文祥，马玺．紫杉醇高产菌株的原生质体诱变选育及其遗传变异初探[J]．微生物学报．2005，45(3)：355～358
[35] 李荣贵，王普，梅建凤．新型生物杀虫剂—刺糖菌素[J]．微生物学通报，2003，30(1)：77～81
[36] 赵凯，周东坡，平文祥，马玺．产紫杉醇菌株原生质体诱变育种的研究[J]．生物工程学报，2005，21(5)：848～851
[37] 徐志南，董悦涵，谢志鹏，岑沛霖．米多霉素产生菌原生质体融合研究[J]．浙江大学学报，2006，40(7)：1262～1267
[38] Dai M, Copley SD, Genome shuffling improves degradation of the anthropogenic pestcide pentachlorophenol by Sphingbium chlorophenolicum ATCC39723 [J]. Appl Environ Microbiol, 2004, 70(4): 2391～2397
[39] 陈涛，王靖宇，周世奇等．基因组改组及代谢通量分析在产核黄素Bacilus subtilis性能改进中的应用[J]．化工学报，2004，55(11)：1842～1848
[40] 徐波，王明蓉，夏永等，应用基因组重排育种新方法筛选替考拉宁高产菌[J]．中国抗生素杂志，2006，31(4)：237～242
[41] Yuhua Wang, Yan Li, Xiaolin Pei, et al. Genome-shuffiing improved acid tolerance and L-lactic acid volumetric productivity in Lactobacillus rhamnosus [J]. Journal of Biotechnology, 2007, 129(3): 510～515
[42] 朱惠，金志华，岑沛霖．纳他霉素产生菌基因组重排育种[J]．中国抗生素，2006 31(12)：739～742
[2] Patnaik R, Louie S, Gavrilovic V, et al. Genome shuffling of Lactobacillus for improved acid tolerance [J]. Nature Biotechnol, 2002 Jul, 20(7): 707～712
[3] Campo N, Dias M J, Daveran-Mingot ML, Ritzenthaler P. Metabolic engineering by genome shuffling Genome plasticity in Lactococcus lactis [J]. Bourgeois P Antonie Van Leeuwenhoek, 2002, 82:123～132
[4] 张兴，马志卿等．生物农药评述[J]．西北农林科技大学学报(自然科学版)，2002，30(2)：142～148
[5] Thompson CD, Dutton R and Sparks TC. Spinosad—a case study: an example from a natural products discovery programme [J]. Pest Manag Sci., 2000, 56: 696～702
[6] Mertz E, Yao R. Saccharopolyspors spinosa sp nov isolated from soil coolected in a sugar rum still [J]. Int Sust Bacteriol, 1990, 40(1): 34～39
[7] Aon. Spinosad Technical Guide [M]. DowElaco, 1996:25
[8] Baker PJ. U. S. Patent, 5, 227, 295, 1993
[9] Baker PJ. U. S. Patent, 5, 227, 295, 1994
[10] Vincent L. Salgado. Studies on the mode of action of spinosad: insect symptoms and physiological correlates [J]. Pestic. Biochem. and physiol., 1998, 60:91～102
[11] Gerald B. Watson. Action of insecticidal spinosyns on gaminbutyric acid responses from small diameter cockroach neurons [J]. Pesticide biochemistry and physiology, 2001, 71:20～26
[12] 王彦华，王鸣华．多杀菌素的作用机理及其抗药性的研究进展[J]．农药科学与管理，2006，25(11)：12～15
[13] Saunders D. Fate of spinosad in the environment [J]. Down to Earth, 1996, 52 (1): 21～28
[14] Gary D, Robert D. Spinosad a case study: an example from a nature products discovery programme [J]. Pest Manag Sci, 2000, 56 (8): 696～702
[15] Carson W, Yrumble J. Effect of insecticides for control of leafminers on lima beans [J]. Arthrop Management Tests, 1998, 23 (1): 74～75
[16] 张部昌，赵志虎，马清钧．红霉素生物合成的分子生物学[J]．生物技术通讯，2001，12(2)：151～160
[17] 张惠民．途径工程．第三代基因工程IM]．北京：中国轻工业出版社，2002
[18] Cropp A, Chen S, Liu H, et al. Genetic approaches for controlling ratios of related polyketide products in fermentation processes [J]. Ind Microbiol Biotechnol, 2001, 27(6): 368～377
[19] Baltz R H, Crawford K P, Broughton MC, et al. Biosynthetic genes for spinosyn insecticide production. US Patent 6274350, 2001
[20] Waldron C, Madduri K, Crawford K. A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa [J]. Antonie Van Leeuwenhock, 2000, 78(3-4): 385～390
[21] Waldron C, Matsushima P, Rosteck P R Jr, et al. Cloning and analysis of the spinosad biothetic gene cluster of Saccharopolyspora spinosa [J]. Chemistry Biology, 2001, 8(5): 487～499
[22] 柳君科．刺孢小单孢菌和灰色链霉菌原生质体融合的研究[J]．遗传学报，1989，16(1)：49～55
[23] 张苑．多杀菌素高产菌种的推理选育和发酵工艺的优化[D]．硕士论文，浙江大学，岑沛霖，浙江杭州，2004
[24] 肖信法．微生物原生质体融合与菌种选育[J]．抗生素，1983，8(4)：261～265
[25] 辛明秀．微生物的原生质体融合及应用[J]．微生物学通报，1995，22(6)：365～370
[26] 余荔华．克氏固氮菌与枯草芽孢杆菌的原生质体电融合[J]．清华大学学报，1999，39(6)：46～48
[27] 贺莜蓉，黄小倩．链霉菌原生质体的制备[J]．生物学通报，1998，33(1)：40～41
[28] 陈钧鸣，徐玲娣．抗生素工业分析[M]．增订本，北京，中国医药科技出社，1991
[29] 张苑，金志华，林建平，岑沛霖．多杀菌素的高效液相色谱法测定[J]．农药，2003．42(10)：27～28
[30] 王岳五，松林生．电融合技术选育能利用木糖和纤维二糖生产乙醇的菌株[J]．生物工程学报，1992，8(1)：82～86
[31] 朱建伟等．林肯霉菌原生质体的形成、再生及其影响因素[J]．医药工业，1987：18(3)：108～112
[32] 金志华，林建平，梅乐和．工业微生物遗传育种学原理与应用[M]．化学工业出版社，北京，2006．1
[33] 朱林东．普那霉素产生菌基因组重排[D]．硕士论文，浙江大学，金志华，浙江杭州，2006
[34] 赵凯，平文祥，马玺．紫杉醇高产菌株的原生质体诱变选育及其遗传变异初探[J]．微生物学报．2005，45(3)：355～358
[35] 李荣贵，王普，梅建凤．新型生物杀虫剂—刺糖菌素[J]．微生物学通报，2003，30(1)：77～81
[36] 赵凯，周东坡，平文祥，马玺．产紫杉醇菌株原生质体诱变育种的研究[J]．生物工程学报，2005，21(5)：848～851
[37] 徐志南，董悦涵，谢志鹏，岑沛霖．米多霉素产生菌原生质体融合研究[J]．浙江大学学报，2006，40(7)：1262～1267
[38] Dai M, Copley SD, Genome shuffling improves degradation of the anthropogenic pestcide pentachlorophenol by Sphingbium chlorophenolicum ATCC39723 [J]. Appl Environ Microbiol, 2004, 70(4): 2391～2397
[39] 陈涛，王靖宇，周世奇等．基因组改组及代谢通量分析在产核黄素Bacilus subtilis性能改进中的应用[J]．化工学报，2004，55(11)：1842～1848
[40] 徐波，王明蓉，夏永等，应用基因组重排育种新方法筛选替考拉宁高产菌[J]．中国抗生素杂志，2006，31(4)：237～242
[41] Yuhua Wang, Yan Li, Xiaolin Pei, et al. Genome-shuffiing improved acid tolerance and L-lactic acid volumetric productivity in Lactobacillus rhamnosus [J]. Journal of Biotechnology, 2007, 129(3): 510～515
[42] 朱惠，金志华，岑沛霖．纳他霉素产生菌基因组重排育种[J]．中国抗生素，2006 31(12)：739～742