Application of Counterseletable Gene <i>upp</i> in Genetic Manipulation of <i>Streptomyces fungicidicus</i>

doi:10.13523/j.cb.20191109

China Biotechnology

2019, Vol. 39

Issue (11): 78-86 DOI: 10.13523/j.cb.20191109

Application of Counterseletable Gene upp in Genetic Manipulation of Streptomyces fungicidicus

WU Guo-guo¹,SONG Shu-ting¹,YUE Rong¹,ZHANG Jing¹,GUAN Ying¹,WANG Yue¹,LIU Bao-ai²,LV Xue-min²,WEI Jian-jun²,ZHANG Hui-tu^1,^**(

)

1 Laboratory of Enzyme and Applied Microbiology, College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457, China
2 Tianjin Xinxing Veterinary Pharmaceutical Factory, Tianjin 300402, China

Download:

HTML

PDF(1250KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

In order to further simplify the genetic operation process of actinomycetes and shorten the screening cycle of the recombinant strains, a counter selectable marker gene —— uracil phosphoribosyl transferase gene (upp) was introduced into the genetic operation of Streptomyces fungicidicus ATCC 21013. By deletion of the upp gene in the wild type strain and construction of a suicide vector that carries the upp gene expression cassette, a markerless inframe deletion system was developed, and the StrR gene in S. fungicidicus genome was successfully knocked out after vector integration, plasmid excision and counterselection. The introduction of upp gene shortened the average screening cycle of Streptomyces recombinant strains by about 2 weeks, and further reduced the probability of false positive recombinant strains. The simplicity of this system should make it adaptable for continuous markerless gene deletion in other actinomycetes, so it is worth further promotion and application.

Key words： Streptomyces fungicidicus Counterselectable marker Uracil phosphoribosyl transferase Gene knockout

Received: 03 April 2019 Published: 17 December 2019

ZTFLH:

Q78

Corresponding Authors: Hui-tu ZHANG E-mail: hzhang@tust.edu.cn

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Guo-guo WU
	Shu-ting SONG
	Rong YUE
	Jing ZHANG
	Ying GUAN
	Yue WANG
	Bao-ai LIU
	Xue-min LV
	Jian-jun WEI
	Hui-tu ZHANG

Cite this article:

WU Guo-guo,SONG Shu-ting,YUE Rong,ZHANG Jing,GUAN Ying,WANG Yue,LIU Bao-ai,LV Xue-min,WEI Jian-jun,ZHANG Hui-tu. Application of Counterseletable Gene upp in Genetic Manipulation of Streptomyces fungicidicus. China Biotechnology, 2019, 39(11): 78-86.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20191109 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I11/78

Table 1 Strains and plasmids used in this research

Table 2 Primers used in this study

Fig.1 Construction of the recombinant plasmid

Table 3 Plate culture of S. fun ATCC 21013 in the different 5-fluorouracil concentration

Fig.2 Culture of transconjugant in 5-FU and non- antibiotics plates (a) 5-Fluorouracil plates(5μg/ml) (b) Non- antibiotics plates

Fig.3 Screening of S. fun Δupp in 5-FU plate (a) S.fun Δupp (b) S. fun ATCC 21013

Fig.4 Identification of S.fun Δupp strain by PCR 1,2: S.fun ATCC 21013;3,4:S.fun Δupp;M:Marker

Fig.5 Homologous crossing-over in this study A,B:Homologous arm of upp

Fig.6 Effect of upp gene deletion on S.fun Δupp (a) 1:S.fun ATCC 21013;2:S.fun Δupp (b)1,2,3:S.fun ATCC 21013;4,5,6:S.fun Δupp (c)A: S.fun ATCC 21013;B:S.fun Δupp (d) A: S.fun ATCC 21013;B:S.fun Δupp

Fig.7 Identification of S.fun Δupp/upp strain by PCR 1,2,3:S.fun Δupp/upp;4:S.fun Δupp;M:Marker

Fig.8 Culture of S.fun Δupp/upp in 5-FU plates (a) S.fun Δupp (b) S.fun Δupp/upp

Fig. 9 Schematic of gene knockout based on S.fun Δupp A,B:Homologous arm of StrR

Fig.10 Identification of S. fun Δupp ΔStrR strain by PCR 1,2:S. fun Δupp ΔStrR;3:S.fun Δupp;M:Marker

Fig. 11 Measuring plate 1:S. fun ATCC 21013;2:S.fun Δupp;3:S. fun Δupp ΔStrR;4:S.fun Δupp/upp


[1]	Komatsu M, Uchiyama T, Omura S , et al. Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proceedings of the National Academy of Sciences, 2010,107(6):2646-2651.

[2]	张红岩, 申乃坤, 周兴 . 基因敲除技术及其在微生物育种中的应用. 酿酒科技, 2010,190(4):21-25.

[2]	Zhang H Y, Shen N K, Zhou X . Gene knockout technology and its application in microbial breeding. Liquor-Making Sci Technol, 2013,190(4):21-25.

[3]	Steinmetz M, Coq D L, Djemia H B , et al. Genetic analysis of sacB, the structural gene of a secreted enzyme, levansucrase of Bacillus subtilis Marburg. Molecular & General Genetics Mgg, 1983,191(1):138-44. doi: 10.4014/jmb.1910.10055 pmid: 31838832

[4]	Lee D J, Bingle L E, Heurlier K , et al. Gene doctoring: A method for recombineering in laboratory and pathogenic Escherichia coli strains. BMC Microbiol, 2009,9(12):252-266. doi: 10.1186/1471-2180-9-252 pmid: 20003185

[5]	Hasegawa N, Abei M, Yokoyama K K , et al. Cyclophosphamide enhances antitumor efficacy of oncolytic adenovirus expressing uracil phosphoribosyltransferase (UPRT) in immunocompetent Syrian hamsters. International Journal of Cancer, 2013,133(6):1479-1488. doi: 10.1002/ijc.28132 pmid: 23444104

[6]	Andersen P S, Smith J M, Mygind B . Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12. Febs Journal, 2010,204(1):51-56. doi: 10.1111/j.1432-1033.1992.tb16604.x pmid: 1371255

[7]	Siegl T, Luzhetskyy A . Actinomycetes genome engineering approaches. Antonie Van Leeuwenhoek, 2012,102(3):503-516. doi: 10.1007/s10482-012-9795-y pmid: 22926833

[8]	Paget M S B, Chamberlin L, Atrih A , et al. Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2). Journal of Bacteriology, 1999,181(1):204-211. pmid: 9864331

[9]	Kieser T, Bibb M J, Buttner M J , et al. Practical Streptomyces Genetics: a Laboratory Manual. Norwich: John Innes Foundation, 2000. doi: 10.1016/bs.acc.2018.12.004 pmid: 30797468

[10]	方志锴, 洪文荣, 严凌斌 , 等. 金色链霉菌接合转移体系的构建. 福建农林大学学报, 2011,40(5):521-524.

[10]	Fang Z K, Hong W R, Yan L B , et al. Construction of the conjugal transfer system of Streptomyces aureofaciens. Journal of Fujian Agriculture and Forestry University, 2011,40(5):521-524.

[11]	许铭玉, 宋淑婷, 张莹 , 等. 恩拉霉素生物检测方法的改进. 中国畜牧兽医, 2017,44(2):384-390.

[11]	Xu M Y, Song S T, Zhang Y , et al. Improvement of the biological assay method for enramycin. Chinese Animal Husbandry and Veterinary Medicine, 2017,44(2):384-390.

[12]	Li C, Liu X, Lei C , et al. RifZ (AMED_0655) is a pathway-specific regulator for rifamycin biosynjournal in Amycolatopsis mediterranei. Applied and Environmental Microbiology, 2017,83(8):3201-3216. doi: 10.1128/AEM.03201-16 pmid: 28159794

[13]	Lewinson O, Livnat-Levanon N . Mechanism of action of ABC importers: conservation, divergence, and physiological adaptations. Journal of Molecular Biology, 2017,429(5):606-619. doi: 10.1016/j.jmb.2017.01.010 pmid: 28104364

[14]	Braibant M, Gilot P, Content J . The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis. Fems Microbiology Reviews, 2000,24(4):449-467. doi: 10.1111/j.1574-6976.2000.tb00550.x pmid: 10978546

[15]	Komatsu M, Uchiyama T, Omura S , et al. Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proceedings of the National Academy of Sciences, 2010,107(6):2646-2651.

[16]	Gust B, Challis G L, Fowler K , et al. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynjournal of the sesquiterpene soil odor geosmin. Proceedings of the National Academy of Sciences of the United States of America, 2003,100(4):1541-1546.

[17]	Herrmann S, Siegl T, Luzhetska M , et al. Site-specific recombination strategies for engineering actinomycete genomes. Applied and Environmental Microbiology, 2012,78(6):1804-1812. doi: 10.1128/AEM.06054-11

[1]	GUO Sheng-nan, LI Xin-xiao, WANG Feng, LIU Kun-mei, DING Na, HU Qi-kuan, SUN Tao. *Establishment and Identification of the Neocortex and Hippocampus GABRG2* Knockout Mice and Its Preliminary Study in Generalized Epilepsy with Febrile Seizures Plus**[J]. China Biotechnology, 2020, 40(3): 9-20.

[2]	WAN Ying-han,CI Lei,WANG Jue,GONG Hui,LI Jun,DONG Ru,SUN Rui-lin,FEI Jian,SHEN Ru-ling. Construction and Preliminary Phenotypic Verification of PD-L1 Knockout Mice[J]. China Biotechnology, 2019, 39(12): 42-49.

[3]	LU Hai-yan,LI Jia-man,SUN Si-fan,ZHANG Xiao-mao,DING Juan-juan,ZOU Shao-lan. *Construction of an Auxotrophic Mutant from an Industrial Saccharomyces cerevisiae* Strain by CRISPR-Cas9 System**[J]. China Biotechnology, 2019, 39(10): 67-74.

[4]	Chun-xiao SU,Xiao-yu ZHANG,Han ZENG,Ya-xi CHEN,Xiong-zhong RUAN,Ping YANG. Establishment and Identification of Liver-Specific CD36 Knockout Mice[J]. China Biotechnology, 2018, 38(8): 26-33.

[5]	Yu-rui SHENG,Bin LI,Bin WANG,Di ZUO,Lin MA,Xiao-fan REN,Le GUO,Kun-mei LIU. The Construction of AEG-1-Knockout U251 Cell Line by CRISPR/Cas9 Technology and Study of The Effect of AEG-1 on the Metastasis in U251 Cells[J]. China Biotechnology, 2018, 38(10): 38-47.

[6]	ZHANG Zhen-yang, YANG Yan-kun, ZHAN Chun-jun, LI Xiang, LIU Xiu-xia, BAI Zhong-hu. Pichia pastoris X-33 ΔGT2 Release the Glycerol Repression on AOX1 and Ef-ficiently Express Heterologous Proteins[J]. China Biotechnology, 2017, 37(1): 38-45.

[7]	DU Hong-yan, LI Tian-ming, LIU Jin-lei, FENG Hui-yong. *Construct the Uracil Phosphoribosyl Transferase Gene Mutant Strain in Gluconobacter suboxydans* for Seamless Genome Editing**[J]. China Biotechnology, 2016, 36(7): 64-71.

[8]	HAN Hai hong, WANG Jun qing, WANG Teng fei, XIAO Jing, HAN Deng lan, WANG Rui ming. *Method and Application of Gene Knockout Based Single Cross in Bacillus licheniformis* 20085**[J]. China Biotechnology, 2016, 36(11): 63-69.

[9]	CHANG Yu-mei, HOU Zhan-ming . Research on Gene Knockout and Function of FgPDE1 in Fusarium graminearum[J]. China Biotechnology, 2015, 35(8): 59-67.

[10]	SHEN Dong-ling, SHANG Shu-mei, LI Wei-na, YAN Jin-ping, HANGAN Ir-bis. *Characterization of the Disrupted ack* Genes on Fermentation by Thermoanaerobacterium calidifontis Rx1**[J]. China Biotechnology, 2015, 35(7): 37-44.

[11]	TAO Si-mei, ZHENG Wei, ZHAO Peng-chao, ZHOU Wei, QUAN Chun-shan, FAN Sheng-di. *Effects of bmy* Gene knockout on Hemolysis and Antifungal activity of Bacillus amyloliquefaciens Q-426**[J]. China Biotechnology, 2014, 34(3): 56-60.

[12]	GE Gao-shun, ZHANG Li-chao, ZHAO Xin, HU Xue-jun, LI Ya-jie. *Optimization of the Method for Scarless Gene Knockout in Escherichia coli* Genome**[J]. China Biotechnology, 2014, 34(06): 68-74.

[13]	YE Xiang-li, LI Da-li. Rapid Construction of GPR126 Conditional Gene-targeting Vector[J]. China Biotechnology, 2013, 33(4): 106-113.

[14]	JIANG Na, WANG Yan-Chun, MA Zhi-Hong, LUO Lin, LIU Chun-Jie. A Novel Temperatrue Sensitive Plasmid-based Method for Deletion of Chromosomal Genes[J]. China Biotechnology, 2010, 30(03): 85-89.

[15]	YAN Ji-Ai, ZHANG Xue, ZHANG Yun, ZUO Dian-Guang, CHEN Ning, WEN Ting-Yi. Construction of Genetic Engineering Strains for L-threonine Production by Red Recombination[J]. China Biotechnology, 2010, 30(03): 79-84.

Viewed

Full text

Abstract

Cited

Shared

Discussed