螺旋链霉菌遗传操作系统-接合转移体系的建立

doi:10.13523/j.cb.2010009

中国生物工程杂志

2021, Vol. 41

Issue (2/3): 45-52 DOI: 10.13523/j.cb.2010009

技术与方法

螺旋链霉菌遗传操作系统-接合转移体系的建立

王优蓓¹,郭思妤¹,常碧博²,叶蕊芳¹,花强^1,^*(

)

1 华东理工大学生物工程学院生物反应器工程国家重点实验室上海 200237
2 河南天方药业股份有限公司驻马店 463000

Establishment of Conjugation System for the Spiramycin Producer Streptomyces spiramyceticus

WANG You-bei¹,GUO Si-yu¹,CHANG Bi-bo²,YE Rui-fang¹,HUA Qiang^1,^*(

)

1 State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai 200237, China
2 Topfond Pharmaceutical Co. Ltd, Zhumadian 463000, China

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摘要：

背景:螺旋链霉菌(Streptomyces spiramyceticus)为国内螺旋霉素(spiramycin,SPM)的生产菌,但目前工业生产中螺旋链霉菌利用遗传操作进行基因改造还没有成功报道。目的:建立螺旋链霉菌遗传操作系统,利用遗传改造的方法改变SPM的组分比例,降低SPM的分离成本。方法:以大肠杆菌(Escherichia coli ET12567/pUZ8002)为供体,螺旋链霉菌为受体进行接合转移实验,建立螺旋链霉菌接合转移的遗传操作系统,并对影响接合转移效率的培养基种类、抗生素覆盖时间、供受体比例等条件进行优化。结果:螺旋链霉菌不适合利用孢子进行接合转移,利用菌体进行接合转移时ISP4培养基为接合转移的最适培养基,供受体比例为10³∶1得到的接合子数量最多,接合转移效率为1.93×10^-4,SPM中三种组分百分含量变化显著。结论：实验首次建立了螺旋链霉菌接合转移的方法,利用菌体进行接合转移操作,简化了实验操作过程,并且利用CRISRP/Cas9基因编辑系统成功阻断3-O-酰基转移酶基因,并在该位置导入φC31整合位点attB,为后续螺旋链霉菌的基因改造奠定了基础。

关键词： 螺旋链霉菌; 菌体接合转移; 螺旋霉素; CRISRP/Cas9

Abstract:

Background:Streptomyces spiramyceticus is used to produce spiramycin (SPM) in China. So far, the successful genetic modification has not been reported in S. spiramyceticus.Objective:The appropriate genetic transformation system of S. spiramyceticus was established in order to modify the composition of SPM and reduce the separation costs of SPM based on genetic operation.Methods:The conjugal transfer system was conducted by conjugation with Escherichia coli ET12567/pUZ8002, and the factors that influence the conjugation efficiency, including culture medium, antibiotic coverage time, and donor/recipient ratio, were investigated and optimized.Results:The experimental results showed that S. spiramyceticus spores were not suitable for conjugal transfer and the best medium for conjugal transfer was ISP4. The maximum transconjugants were obtained when the donor/recipient ratio was 10³∶1. The best conjugation efficiency was 1.93×10^-4 and the percentage of SPM changed significantly.Conclusion:The efficient and simple genetic transformation system for producing strain S. spiramyceticus was established. Based on this method, the sspA gene was knocked out and φC31 locus attB was integrated into S. spiramyceticus genome successfully, which laid the foundation for further biosynthetic gene modification in the strain.

Key words: Streptomyces spiramyceticus Mycelium conjugal transfer Spiramycin CRISRP/Cas9

收稿日期: 2020-11-12 出版日期: 2021-04-08

ZTFLH:

Q815

通讯作者: 花强 E-mail: qhua@ecust.edu.cn

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引用本文:

王优蓓,郭思妤,常碧博,叶蕊芳,花强. 螺旋链霉菌遗传操作系统-接合转移体系的建立[J]. 中国生物工程杂志, 2021, 41(2/3): 45-52.

WANG You-bei,GUO Si-yu,CHANG Bi-bo,YE Rui-fang,HUA Qiang. Establishment of Conjugation System for the Spiramycin Producer Streptomyces spiramyceticus. China Biotechnology, 2021, 41(2/3): 45-52.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2010009 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I2/3/45

表1 螺旋链霉菌的产孢情况

图1 孢子培养基28℃培养时的螺旋链霉菌菌落形态

表2 螺旋链霉菌抗生素的耐药性

表3 螺旋链霉菌在不同培养基上的生长情况

图2 pCRISRP/Cas9 sspA+HA质粒图

表4 不同载体对螺旋链霉菌的接合转移效率比较

图3 供受体比例对接合转移效率的影响

图4 工程菌株△sspA ∷φC31 attB 测序结果

图5 SPM三种组分的百分含量

图6 螺旋链霉菌接合转移过程示意图

[1]	司良. 大环内酯类抗生素的作用机制与应用进展. 现代预防医学, 2010,37(22):4397-4398.
	Si L. The action mechanism and application progress of macrolide antibiotics. Modern Preventive Medicine, 2010,37(22):4397-4398.
[2]	孙丽文. 螺旋霉素类抗生素体内过程探讨. 国外医药(抗生素分册), 1998,19(5):358-361, 365.
	Sun L W. Study on the in vivo process of spiramycin antibiotics. World Notes on Antibiotics, 1998,19(5):358-361, 365
[3]	朱峰, 王尔健. 螺旋霉素的再评价. 中国抗生素杂志, 1991,16(3):231-236.
	Zhu F, Wang E J. Spiramycin reassessed. Chinese Journal of Antibiotics, 1991,16(3):231-236.
[4]	姚开亚. 降低螺旋霉素发酵杂质F、A、D、B的工艺优化及影响因素分析. 上海: 华东理工大学, 2015.
	Yao K Y. Reducing of impurities F,A,D,B and analysis of the influence factors in the fermentation of spramycin by Streptomyces ambofaciens. Shanghai: East China University of Science and Technology, 2015.
[5]	曾俊. 螺旋霉素的生物合成及有效组分优化研究. 上海: 华东理工大学, 2015.
	Zeng J. Study on biosynthesis of spiramycin and effective components optimization. Shanghai: East China University of Science and Technology, 2015.
[6]	梁培培, 高淑红, 姚开亚, 等. 丙三醇对螺旋霉素Ⅰ发酵的影响及机理分析. 华东理工大学学报(自然科学版), 2020,46(1):41-47.
	Liang P P, Gao S H, Yao K Y, et al. Effect and mechanism of glycerol addition on spiramycin I fermentation. Journal of East China University of Science and Technology, 2020,46(1):41-47.
[7]	吴胜, 夏焕章. 链霉菌基因转移的方法. 生物工程进展, 2002,22(1):91-94.
	Wu S, Xia H Z. The methods of gene transfer in Streptomyces. Progress in Biotechnology, 2002,22(1):91-94.
[8]	李晓华, 龙慈凡, 周秀芬, 等. 链霉菌质粒pSET152电转化稀有放线菌小单孢菌的研究. 微生物学报, 2007,47(4):718-720.
	Li X H, Long C F, Zhou X F, et al. Study on electroporation of rare Actinomycete Micromonospora sp.40027 with Streptomyces plasmid pSET152. Acta Microbiologica Sinica, 2007,47(4):718-720.
[9]	Ma Z, Liu J X, Shentu X P, et al. Optimization of electroporation conditions for toyocamycin producer Streptomyces diastatochromogenes 1628. Journal of Basic Microbiology, 2014,54(4):278-284. doi: 10.1002/jobm.201200489 pmid: 23775805
[10]	Wang H, Wang W Y, Wu M, et al. Establishment and optimization of a conjugation system for the daptomycin producer Streptomyces roseosporus NRRL11379. Chinese Journal of Pharmaceutical Biotechnology, 2014,21(3):194-198.
[11]	Zhang S M, Chen T S, Jia J, et al. Establishment of a highly efficient conjugation protocol for Streptomyces kanamyceticus ATCC12853. MicrobiologyOpen, 2018,8(6):e00747.
[12]	Song Z Q, Liao Z J, Hu Y F, et al. Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527. Journal of Zhejiang University-Science B(Biomedicine & Biotechnology), 2019,20(11):891-900.
[13]	王露, 朱世扬, 赵春田, 等. 加纳链霉菌接合转移体系的建立与优化. 浙江农业学报, 2018,30(11):1965-1971.
	Wang L, Zhu S Y, Zhao C T, et al. Establishment and optimization of Streptomyces ghanaensis conjugation system. Acta Agriculturae Zhejiangensis, 2018,30(11):1965-1971.
[14]	张晋龙, 马怡茗, 舒伟学, 等. 羽毛降解杆状链霉菌S-28遗传转化系统的建立与优化. 西北农林科技大学学报(自然科学版), 2018,46(5):94-100.
	Zhang J L, Ma Y M, Shu W X, et al. Establishment and optimization of genetic transformation system for feather-degrading strain Streptomyces bacillaris S-28. Journal of Northwest A& F University(Natural Science Edition), 2018,46(5):94-100.
[15]	郑雷, 颜望明, 刘振盈. 细菌接合转移及其调控机制的研究进展. 微生物学通报, 1997,24(4):244-246.
	Zheng L, Yan W M, Liu Z Y. Advances in the study of bacterial conjugal transfer and its regulatory mechanism. Microbiology China, 1997,24(4):244-246.
[16]	Arber W, Linn S. DNA modification and restriction. Annual review of Biochemistry, 1969,38(1):467-500.
[17]	Arber W, Wauters-Willems D. Host specificity of DNA produced by Escherichia coli. Molecular and General Genetics MGG, 1970,108(3):203-217. doi: 10.1007/BF00283350 pmid: 4920152
[18]	邓会群. 链霉菌AM-7161遗传操作系统的建立和糖基转移酶基因med-ORF8原核表达的研究. 武汉: 华中师范大学, 2009.
	Deng H Q. Establishment of the genetic manipulation system of AM-7161 and prokaryotic expression study on the C-glycosyltransferase gene med-ORF8. Wuhan: Central China Normal University, 2009.
[19]	戴剑漉, 卢智黎, 林灵, 等. 利用异源正调控基因acyB2构建埃莎霉素Ⅰ高产菌株. 中国生物工程杂志, 2017,37(3):65-72.
	Dai J L, Lu Z L, Lin L, et al. Construction of the isomycin Ⅰ high-yield strain by introducing a heterologous positive regulatory gene acyB2. China Biotechnology, 2017,37(3):65-72.
[20]	郭燕华, 安邦. 基于CRISPR-Cas9技术构建橡胶树胶孢炭疽菌的基因敲除系统. 微生物学通报, 2020,47(1):109-117.
	Guo Y H, An B. CRISPR-Cas9 ribonucleoprotein-mediated gene knock-out in Colletotrichum gloeosporioides from Hevea brasiliensis. Microbiology China, 2020,47(1):109-117.
[21]	Tong Y J, Charusanti P, Zhang L X, et al. CRISPR-Cas9 based engineering of actinomycetal genomes. Acs Synthetic Biology, 2015,4(9):1020-1029.

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