基于基因组重排技术的多杀菌素高产菌株选育

doi:10.3969/j.issn.0438-1157.2014.09.036

化工学报 ›› 2014, Vol. 65 ›› Issue (9): 3576-3582.DOI: 10.3969/j.issn.0438-1157.2014.09.036

基于基因组重排技术的多杀菌素高产菌株选育

夏燕春^1,2, 王超², 陈园², 吴江磊³, 邹球龙², 张晓琳², 李春¹

1 石河子大学化学化工学院/新疆兵团化工绿色过程重点实验室-省部共建国家重点实验室培育基地, 新疆石河子 832003;
2 国家粮食局科学研究院, 北京 100037;
3 武汉轻工大学生物与制药工程学院, 湖北武汉 430023

收稿日期:2013-12-30 修回日期:2014-03-25 出版日期:2014-09-05 发布日期:2014-09-05
通讯作者: 李春
基金资助:
粮食公益性行业科研专项（201313002）；农业科技成果转化资金项目（SQ2013EC4490009）

Enhanced production of spinosad with strains of Saccharopolyspora spinosa by genome shuffling

XIA Yanchun^1,2, WANG Chao², CHEN Yuan², WU Jianglei³, ZOU Qiulong², ZHANG Xiaolin², LI Chun¹

1 School of Chemistry and Chemical Engineering/Key Laboratory for Green Process of Chemical Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi 832003, Xinjiang, China;
2 Academy of State Administration of Grain, Beijing 100037, China;
3 School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, Hubei, China

Received:2013-12-30 Revised:2014-03-25 Online:2014-09-05 Published:2014-09-05
Supported by:
supported by Food Public Industry Research (201313002) and Agriculture Science and Technology Achievement Transformation Funds (SQ2013EC4490009).

摘要/Abstract

摘要： 利用课题组前期通过不同物理化学诱变和分子生物学方法获得的8株多杀菌素产生菌为出发菌株，采用96孔板发酵培养结合生物检测的高通量筛选方法，探索了原生质体制备、再生、双亲灭活和原生质体融合等条件，并通过多轮基因组重排获得了多杀菌素高产菌株。结果表明：当菌龄为65 h、溶菌酶浓度为4 mg·ml^-1，39℃处理时间20 min时，原生质体的制备率及再生率分别为92.30%和7.66%；60℃恒温水浴90 min以上和紫外照射200 s以上为双亲灭活条件；PEG浓度为50%，在32℃下处理15 min时，原生质体融合率约为1.18%。最终获得1株产量较出发菌株提高了36.07%且遗传稳定的融合株。

关键词: 多杀菌素, 刺糖多孢菌, 原生质体融合, 基因组重排育种

Abstract: In this study, genome shuffling of eight original strains obtained by various methods of chemical and physical mutations was used to achieve rapid improvement of spinosad production. The conditions of preparation, regeneration, parents inactivation and fusion of protoplast of the original strain were investigated. The strain grown for 65 h was treated by 4 mg·ml^-1 lysozyme at 39℃ for 20 min, and preparation rate and regeneration rate were 92.30% and 7.66%, respectively. The protoplasts were inactivated by heating at 60℃ for more than 90 min and UV treatment more than 200 s, respectively. The inactivated protoplasts were fused and regenerated in polyethylene glycol (PEG 6000, 50%) for 15 min, and fusion rate was about 1.18%. After three rounds of genome shuffling, a high yielding strain, designated as S.spinosa 3-652, was isolated. The increased yield was about 36.07% higher than that of the original strain. Subculture experiments indicated that the high producers were stable.

Key words: spinosad, Saccharopolyspora spinosa, protoplast fusion, genome shuffling

中图分类号:

S482.3⁺9

夏燕春, 王超, 陈园, 吴江磊, 邹球龙, 张晓琳, 李春. 基于基因组重排技术的多杀菌素高产菌株选育[J]. 化工学报, 2014, 65(9): 3576-3582.

XIA Yanchun, WANG Chao, CHEN Yuan, WU Jianglei, ZOU Qiulong, ZHANG Xiaolin, LI Chun. Enhanced production of spinosad with strains of Saccharopolyspora spinosa by genome shuffling[J]. CIESC Journal, 2014, 65(9): 3576-3582.

参考文献 20

[1]	Jin Z H, Xu B, Lin S Z, Jin Q C, Cen P L. Enhanced production of spinosad in Saccharopolyspora spinosa by genome shuffling [J]. Applied Biochemistry and Biotechnology, 2009, 159 (3): 655-663
[2]	Susan C V, Pharm D, Robin L B, Pharm D. Head lice and the use of spinosad [J]. Clinical Therapeutics, 2012, 34 (1): 14-23
[3]	Luo Lisi (罗莉斯), Li Nengwei (李能威), Li Li (李丽), Guo Weiqun (郭伟群), Yu Jianping (郁建平), Zhang Xiaolin (张晓琳). Studies on high throughput screening for high spinosad-producing strains with 96-well plates [J]. Journal of Agricultural Science and Technology (中国农业科技导报), 2010, 12 (2): 133-137
[4]	Chen Yuan (陈园), Xiong Jian (熊犍), Wang Chao (王超), Eddie Chio, Zou Qiulong (邹球龙), Zhang Xiaolin (张晓琳). Rapid screening method of high yield strains spinosad [J]. Science and Technology of Cereals, Oils and Foods (粮油食品科技), 2013, 21 (4): 99-102
[5]	Sharma A, Strivastava A, Ram B, Srivastava P C. Dissipation behavior of spinosad insecticide in soil, cabbage and cauliflower under subtropical conditions [J]. Pest Management Science, 2008, 7 (12): 2611-2617
[6]	Chen Yuan (陈园), Xiong Jian (熊犍), Guo Weiqun (郭伟群), Liu Guoli (刘国丽), Zou Qiulong (邹球龙), Zhang Xiaolin (张晓琳). High throughput screening and breeding of high spinosad producing strains [J]. Chinese Journal of Antibiotics (抗生素杂志), 2013, 38 (5): 339-347
[7]	Vayias B J, Athanassiou C G, Milonas D N, Mavrotasb C. Activity of spinosad against three stored-product beelte species on four grain commodities [J]. Crop Protection, 2009, 28 (7): 561-566
[8]	Gao X F, Zhao H, Zhang G H, He K Z, Jin Y L. Genome shuffling of clostridium acetobutylicum CICC 8012 for improved production of acetone-butanol-ethanol (ABE) [J]. Current Microbiology, 2012, 65 (2): 128-132
[9]	Zhang Y X, Perry K, Vinci V A, Powell K, Stemmer W P, del Cardayré S B. Genome shuffling leads to rapid phenotypic improvement in bacteria [J]. Nature, 2002, 415 (6872): 644-646
[10]	Long M, Li P, Zhang W K, Yi Zhang, Guo Y, Yang S H, Wang Z, Liu G W. Genome shuffling of Megasphaera elsdenii for improving acid-tolerance and propionate production [J]. African Journal of Microbiology Research, 2012, 6 (18): 4041-4047
[11]	Wang H K, Zhang J, Wang X J, Qi W, Dai Y J. Genome shuffling improves production of the low-temperature alkalophilic lipase by Acinetobacter johnsonii [J]. Biotechnology Letter, 2012, 34 (1): 145-151
[12]	Patnaik R, Louie S, Gavrilovic V, Perry K, Stemmer W P, Ryan C M, del Cardayré S. Genome shuffling of lactobacillus for improved acid tolerance [J]. Nature Biotechnology, 2002, 20 (7):707-712
[13]	Hiroyuki H, Takashi Y, Yasuhiro Y. Genome shuffling of Streptomyces sp.U121 for improved production of hydroxycitric acid [J]. Appl. Microbiol. Biotechnol., 2007, 73 (6): 1387-1393
[14]	Li Leilei (李雷雷). Improvement of spinosyn producing strain by genome shuffling [D]. Wuhan: Huazhong Agricultural University, 2008
[15]	Yu L, Pei X L, Lei T, Wang Y H, Feng Y. Genome shuffling enhanced L-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus [J]. Journal of Biotechnology, 2008, 134 (1-2):154-159
[16]	Illing G T, Normansell I D, Peberdy J F. Protoplast isolation and regeneration in Streptomyces clavuligerus [J]. Journal of General Microbiology, 1989, 135 (8): 2289-2291
[17]	Gan Qiufeng (甘邱峰), Zhang Xiaolin (张晓琳), Wang Jieying (王洁颖), Wang Yang (汪洋), Huang Biwang (黄必旺), Guan Xiong (关雄). Protoplast formation and regeneration for Saccharopolyspora spinosa [J]. Chinese Journal of Antibiotics (中国抗生素杂志), 2011, 36 (1): 18-24
[18]	Xiong Jian (熊犍), Li Nengwei (李能威), Ye Jun (叶君), Zhang Xiaolin (张晓琳). Determination of spinosad with high-performance liquid chromatography [J]. Modern Food Science and Technology (现代食品科技), 2009, 25 (6): 704-706
[19]	Zhu Jianwei (朱建伟), Liu Yiping (刘颐屏), Zhu Baoquan (朱宝泉), Tong Cun (童村). Protoplast formation, regeneration, and strain improvement of fortimicins-producing Micromonospora sp. SIPI4812 [J]. Chinese Journal of Biotechnology (生物工程学报), 1988, 4 (4): 304
[20]	Wang C, Zhang X L, Chen Z, Wen Y, Song Y. Strain construction for enhanced production of spinosad via intergeneric protoplast fusion [J]. Canadian Journal of Microbiology, 2009, 55 (9): 1070-1075

基于基因组重排技术的多杀菌素高产菌株选育

Enhanced production of spinosad with strains of Saccharopolyspora spinosa by genome shuffling

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献 20

相关文章 2

编辑推荐

Metrics

本文评价

[1]	王靖楠, 庞建, 秦磊, 郭超, 吕波, 李春, 王超. 丁烯基多杀菌素高产菌株的选育和改造策略[J]. 化工学报, 2022, 73(2): 566-576.
[2]	夏燕春, 王超, 陈园, 吴江磊, 邹球龙, 张晓琳, 李春. 基于基因组重排技术的多杀菌素高产菌株选育[J]. 化工学报, 2014, 65(9): 0-0.