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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (7): 32-41    DOI: 10.13523/j.cb.2102033
技术与方法     
利用Tn5型转座突变系统筛选高产阿维菌素菌株*
冯宝琪1,冯娇1,张苗1,刘洋2,曹睿2,尹涵之3,齐凤仙3,李子龙3,**(),尹守亮2,**()
1 华北理工大学基础医学院　唐山　063210
2 华北理工大学生命科学学院　唐山　063210
3 中国科学院微生物研究所 微生物资源前期开发国家重点实验室　北京　100101
Screening of High Avermectin-producing Strains via Tn5 Transposon Mediated Mutagenesis
Bao-qi FENG1,Jiao FENG1,Miao ZHANG1,Yang LIU2,Rui CAO2,Han-zhi YIN3,Feng-xian QI3,Zi-long LI3,**(),Shou-liang YIN2,**()
1 School of Basic Medicine Sciences, North China University of Science and Technology, Tangshan　063210, China
2 School of Life Sciences, North China University of Science and Technology, Tangshan　063210, China
3 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing　100101, China
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摘要：

目的: 转座突变技术是发现新功能基因和获得高产天然产物菌株的一种有效策略。通过理性设计和构建Tn5型转座突变系统,并将其应用于阿维链霉菌,筛选高产阿维菌素的工程菌株。方法: 在转座突变载体pUCTN转座插入片段的上游和下游分别引入链霉菌常用的强启动子kasOp*和P21,强化插入位置上游和下游基因的转录表达;在插入片段两端分别添加双向转录终止子T1和T2,有效终止插入序列两端靶基因的转录,引入强启动子和终止子的目的在于增强对转座突变株生理代谢活动的扰动。结果: 通过优化供体菌和受体菌的比例,转座效率显著提高。随机选择500株转座突变株进行发酵和阿维菌素产量测试,筛选到3株突变株的阿维菌素产量明显高于出发菌株产量的50%以上。结论: Tn5转座突变系统为研究阿维链霉菌的基因功能和生理代谢提供了有效的分子遗传工具。
关键词： 阿维链霉菌;  阿维菌素;  Tn5转座突变;  分子遗传工具    
Abstract:

Objective: Transposition is an effective strategy for discovering new functional genes and developing high-titer production strains. The Tn5-based transposon mutagenesis system was rationally designed and constructed, and applied to Streptomyces avermitilis to screen the engineered strains with high avermectin production.Methods: In pUCTN, two strong promoters' sequence of kasOp* and P21, commonly used in Streptomyces, were introduced into the upstream and downstream of the transposable elements, respectively, which can enhance the transcription and expression level of the genes located at the insertion position. Two transcriptional terminators T1 and T2 were also added into the upstream and downstream of the transposable elements to terminate the transcription of genes adjacent to the insertion site. The purpose of introducing promoters and terminators is to enhance the disturbance to the physiological metabolism of the mutants.Results: To improve the transposition efficiency, the ratio of donor and recipient bacteria was optimized, and 500 transposon mutants were selected and tested for avermectin production, 3 high avermectin-producing strains were screened, and the yield is significantly increased 50% than that of the parent strain.Conclusion: This Tn5 transposon mutation system established in this paper provides an effective molecular genetic tool for exploring the gene function and physiological metabolism of Streptomyces avermitilis.
Key words: Streptomyces avermitilis    Avermectin    Tn5 transposon mediated mutagenesis    Molecular genetic tool
收稿日期: 2021-02-25 出版日期: 2021-08-03
ZTFLH:  Q819  
基金资助: * 河北省科技计划农业关键共性技术攻关专项(18222916);河北省自然科学基金资助项目(C2019209399)
通讯作者: 李子龙,尹守亮     E-mail: lizl@im.ac.cn;yinsl@ncst.edu.cn
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引用本文:

冯宝琪,冯娇,张苗,刘洋,曹睿,尹涵之,齐凤仙,李子龙,尹守亮. 利用Tn5型转座突变系统筛选高产阿维菌素菌株*[J]. 中国生物工程杂志, 2021, 41(7): 32-41.

Bao-qi FENG,Jiao FENG,Miao ZHANG,Yang LIU,Rui CAO,Han-zhi YIN,Feng-xian QI,Zi-long LI,Shou-liang YIN. Screening of High Avermectin-producing Strains via Tn5 Transposon Mediated Mutagenesis. China Biotechnology, 2021, 41(7): 32-41.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2102033    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I7/32

Name Description Source
Streptomyces
S. avermitilis MA-4680
(ATCC 31267)		 Used for production of avermectin American Type
Culture Collection
E. coli
DH5α General cloning host for plasmid manipulation Invitrogen
ET12567 (pUZ8002) Donor strain for conjugation between E. coli and Streptomycetes [17]
Plasmids
pUC19 Commonly used cloning vector for E. coli, ampR (ampicillin resistance gene), lacZ, oripMB1 (origin of replication in E. coli) [18]
pTNM Carrying the synthetic tnp (a) gene (Tn5 transposase), oriT origin of plasmid transfer, ME mosaic end recognition sequence for transposase, aac (3)IV (apramycin resistance gene) [15]
permE*-otcR Carrying the synthetic terminator sequence [19]
pIJ8660: BsaI-sfgfp Carrying the major transcription terminator of phage tfd [20]
pKasO*-otcR Carrying the promoter of kasOp* [19]
pUCTN Carrying the synthetic tnp (a) gene (Tn5 transposase), oriT origin of plasmid transfer, ME mosaic end recognition sequence for transposase, aac (3)IV (apramycin resistance gene), strong and constitutive promoter kasOp* and P21 This study
表1  菌株和质粒
Name Sequence (5' to 3')
Eto-F ACAGGGTACCGTTGTGGGCTGGACAATCGTGCCGGTTGGTAGGATCCAGCGGTAGCAACGGAGGTACGGAC (Kpn Ⅰ)
Eto-R CCAGTGAATTCGAGCTCGGTGTATCCAAC (EcoR Ⅰ)
P21ME-F CTAGAGTCGACCTGCAGCCCTGTGCGGGCTCTAACACGTCCTAGTATGGTAGGA (Pst Ⅰ)
P21ME-R AACGGTACCCTGTCTCTTATACACATCTTTGCTCATCCTACCATACTAGGA (Kpn Ⅰ)
Termi-F CAACAAGAGCTTCAGGGTTGACAGTGATAAGCATTACCCTG
Termi-R GGCTGCAGGTCGACTCTAGTAAAAAAGAAAG (Pst Ⅰ)
R6K-F GAACCAAGCTTTAAAAGCCTTATATATTCTTTTTTTTCT (Hind Ⅲ)
R6K-R CAGGGTAATGCTTATCACTGTCAACCCTGAAGCTCTTGTTG
Aac3-F AACCTCATACAGAAAATTCATCAACCATCATCGATGAATTTTC
Aac3-R TTTTAAAGCTTGGTTCATGTGCAGCTCCATAAGC (Hind Ⅲ)
Tfd-F GAGACCGTTCGAATGTGAACAGATCCCGCAAAAGCGGCCTTTG
Tfd-R GAAAATTCATCGATGATGGTTGATGAATTTTCTGTATGAGGTT
PkasO-F TACGCCAAGCTTGCATGCCTGTCTCTTATACACATCTAAC (Hind Ⅲ)
PkasO-R CAAAGGCCGCTTTTGCGGGATCTGTTCACATTCGAACGGTCTC
Test-F GGCAATGGATCAGAGATGATCT
Test-R GCAACTTAAATAGCCTCTAAGG
表2  引物序列
图1  阿维链霉菌MA-4680对不同种类不同浓度抗生素的敏感性
图2  转座载体pUCTN的物理图谱及转座插入片段对基因表达的影响
图3  转座条件的优化
图4  转座突变株的鉴定
图5  转座突变株合成阿维菌素产量
[1] 陈金松, 刘梅, 张立新. 从阿维菌素获得诺贝尔奖到中国创造. 微生物学报, 2016, 56(3):543-558.
Chen J S, Liu M, Zhang L X. Avermectin, from winning the Nobel Prize to “innovation in China”. Acta Microbiologica Sinica, 2016, 56(3):543-558.
[2] Ōmura S. A splendid gift from the earth: the origins and impact of the avermectins (Nobel lecture). Angewandte Chemie (International Ed in English), 2016, 55(35):10190-10209.
doi: 10.1002/anie.201602164
[3] Thuan N H, Pandey R P, Sohng J K. Recent advances in biochemistry and biotechnological synthesis of avermectins and their derivatives. Applied Microbiology and Biotechnology, 2014, 98(18):7747-7759.
doi: 10.1007/s00253-014-5926-x
[4] Kaur H, Shekhar N, Sharma S, et al. Ivermectin as a potential drug for treatment of COVID-19: an in-sync review with clinical and computational attributes. Pharmacological Reports, 2021, 73(3):736-749.
doi: 10.1007/s43440-020-00195-y
[5] Mittal N, Mittal R. Inhaled route and anti-inflammatory action of ivermectin: Do they hold promise in fighting against COVID-19. Medical Hypotheses, 2021, 146:110364.
doi: 10.1016/j.mehy.2020.110364
[6] Chaccour C, Casellas A, Blanco-Di Matteo A, et al. The effect of early treatment with ivermectin on viral load, symptoms and humoral response in patients with non-severe COVID-19: a pilot, double-blind, placebo-controlled, randomized clinical trial. EClinicalMedicine, 2021, 32:100720.
doi: 10.1016/j.eclinm.2020.100720
[7] Rizzo E. Ivermectin, antiviral properties and COVID-19: a possible new mechanism of action. Naunyn-Schmiedeberg’s Archives of Pharmacology, 2020, 393(7):1153-1156.
doi: 10.1007/s00210-020-01902-5
[8] 年洪娟, 陈丽梅, 李昆志. Tn5转座突变技术在革兰氏阴性细菌分子遗传研究中的应用. 中国生物工程杂志, 2009, 29(12):114-118.
Nian H J, Chen L M, Li K Z. Application of Tn5 transposon mutagenesis technology in molecular and genetic researches of gram-negative bacteria. China Biotechnology, 2009, 29(12):114-118.
[9] Weaden J, Dyson P. Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis. Microbiology, 1998, 144(Pt 7):1963-1970.
doi: 10.1099/00221287-144-7-1963
[10] Volff J N, Altenbuchner J. High frequency transposition of the Tn5 derivative Tn5493 in Streptomyces lividans. Gene, 1997, 194(1):81-86.
pmid: 9266676
[11] Widenbrant E M, Kao C M. Introduction of the foreign transposon Tn4560 in Streptomyces coelicolor leads to genetic instability near the native insertion sequence IS1649. Journal of Bacteriology, 2007, 189(24):9108-9116.
pmid: 17951387
[12] Baltz R H, McHenney M A, Cantwell C A, et al. Applications of transposition mutagenesis in antibiotic producing Streptomycetes. Antonie Van Leeuwenhoek, 1997, 71(1-2):179-187.
pmid: 9049029
[13] Solenberg P J, Baltz R H. Transposition of Tn5096 and other IS493 derivatives in Streptomyces griseofuscus. Journal of Bacteriology, 1991, 173(3):1096-1104.
pmid: 1846854
[14] Reznikoff W S. Tn5 as a model for understanding DNA transposition. Molecular Microbiology, 2003, 47(5):1199-1206.
pmid: 12603728
[15] Petzke L, Luzhetskyy A. In vivo Tn5-based transposon mutagenesis of Streptomycetes. Applied Microbiology and Biotechnology, 2009, 83(5):979-986.
doi: 10.1007/s00253-009-2047-z pmid: 19479250
[16] Gao H, Liu M, Liu J T, et al. Medium optimization for the production of avermectin B1a by Streptomyces avermitilis 14-12A using response surface methodology. Bioresource Technology, 2009, 100(17):4012-4016.
doi: 10.1016/j.biortech.2009.03.013
[17] Kieser T, Bibb M J, Buttner M J, et al. Practical Streptomyces Genetics. 2nd ed. Norwich, UK: The John Innes Foundation, 2000: 249-250.
[18] Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene, 1985, 33(1):103-119.
pmid: 2985470
[19] Yin S L, Wang W S, Wang X F, et al. Identification of a cluster-situated activator of oxytetracycline biosynthesis and manipulation of its expression for improved oxytetracycline production in Streptomyces rimosus. Microbial Cell Factories, 2015, 14:46.
doi: 10.1186/s12934-015-0231-7
[20] Yang T J, Yang K Q, Chen Y H, et al. Characterization of a Bi-directional promoter OtrRp involved in oxytetracycline biosynthesis. Current Microbiology, 2019, 76(11):1264-1269.
doi: 10.1007/s00284-019-01753-1
[21] Wang W S, Li X, Wang J, et al. An engineered strong promoter for Streptomycetes. Applied and Environmental Microbiology, 2013, 79(14):4484-4492.
doi: 10.1128/AEM.00985-13
[22] Siegl T, Tokovenko B, Myronovskyi M, et al. Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes. Metabolic Engineering, 2013, 19:98-106.
doi: 10.1016/j.ymben.2013.07.006
[23] 周华艳, 周长林, 窦洁, 等. 阿维链霉菌QL0827接合转移体系的建立. 生物技术通讯, 2016, 27(2):188-191.
Zhou H Y, Zhou C L, Dou J, et al. Construction of conjugal transfer system of Streptomyces avermitilis QL0827. Letters in Biotechnology, 2016, 27(2):188-191.
[24] Wang X K, Jin J L. Crucial factor for increasing the conjugation frequency in Streptomyces netropsis SD-07 and other strains. FEMS Microbiology Letters, 2014, 357(1):99-103.
doi: 10.1111/fml.2014.357.issue-1
[25] 孟令卓, 王勇, 储炬. 阿维链霉菌转化系统的优化及其不同PKS敲除菌株的构建. 中国医药工业杂志, 2018, 49(8):1112-1120.
Meng L Z, Wang Y, Chu J. Construction of 7 PKS-deleted mutants for Streptomyces avermitilis and improvement of conjugal transformation system. Chinese Journal of Pharmaceuticals, 2018, 49(8):1112-1120.
[26] 杜茜, 张红丹, 张正坤, 等. 链霉菌769接合转移体系的建立. 吉林农业科学, 2012, 37(2):30-33.
Du Q, Zhang H D, Zhang Z K, et al. Construction of conjugal transfer system of Streptomyces ahygroscopicus gongzhulingensis. Journal of Jilin Agricultural Sciences, 2012, 37(2):30-33.
[27] 孟令卓, 王勇, 储炬. 阿维链霉菌中一个双组分调控系统SAV931/932功能初探. 华东理工大学学报(自然科学版), 2015, 41(4):443-448.
Meng L Z, Wang Y, Chu J. Characterization of two-component system SAV931/932 for the biosynthesis of avermectin in Streptomyces avermitilis. Journal of East China University of Science and Technology (Natural Science Edition), 2015, 41(4):443-448.
[28] 文莹, 张立新. 阿维菌素的中国“智”造. 遗传, 2018, 40(10):888-899.
Wen Y, Zhang L X. Avermectins, intelligently made in China. Hereditas, 2018, 40(10):888-899.
[29] Chen J S, Liu M, Liu X T, et al. Interrogation of Streptomyces avermitilis for efficient production of avermectins. Synthetic and Systems Biotechnology, 2016, 1(1):7-16.
doi: 10.1016/j.synbio.2016.03.002
[30] Ikeda H, Ishikawa J, Hanamoto A, et al. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nature Biotechnology, 2003, 21(5):526-531.
doi: 10.1038/nbt820
[31] 曹晓梅. 阿维菌素高产菌株的定向选育. 无锡: 江南大学, 2018.
Cao X M. Directive breeding of avermectin-high-producing strains. Wuxi, China: Jiangnan University, 2018.
[32] 宋渊, 曹贵明, 陈芝, 等. 阿维菌素高产菌株的选育及阿维菌素B1的鉴定. 生物工程学报, 2000, 16(1):31-35.
pmid: 10883272
Song Y, Cao G M, Chen Z, et al. Selection of high avermectins producing strain and identification of avermectin B1. Chinese Journal of Biotechnology, 2000, 16(1):31-35.
pmid: 10883272
[33] 薄永恒. 阿维链霉菌高产菌株诱变选育及其基因与蛋白差异效果研究. 兰州: 甘肃农业大学, 2012.
Bo Y H. Research on the mutation breeding of high avermectin producing strains and the mutagenic effect about genes and proteins of Streptomyces avermitilis. Lanzhou: Gansu Agricultural University, 2012.
[34] Metcalf W W, Jiang W H, Wanner B L. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers. Gene, 1994, 138(1-2):1-7.
pmid: 8125283
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