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On-line Access: 2019-10-09

Received: 2019-05-24

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Journal of Zhejiang University SCIENCE B 2019 Vol.20 No.11 P.891-900


Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527

Author(s):  Zhang-Qing Song, Zhi-Jun Liao, Ye-Feng Hu, Zheng Ma, Andreas Bechthold, Xiao-Ping Yu

Affiliation(s):  Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China; more

Corresponding email(s):   mazheng520@163.com

Key Words:  Streptomyces rimosus M527, Intergeneric conjugation, Promoter, β, -Glucuronidase (GUS)

Zhang-Qing Song, Zhi-Jun Liao, Ye-Feng Hu, Zheng Ma, Andreas Bechthold, Xiao-Ping Yu. Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527[J]. Journal of Zhejiang University Science B, 2019, 20(11): 891-900.

@article{title="Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527",
author="Zhang-Qing Song, Zhi-Jun Liao, Ye-Feng Hu, Zheng Ma, Andreas Bechthold, Xiao-Ping Yu",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527
%A Zhang-Qing Song
%A Zhi-Jun Liao
%A Ye-Feng Hu
%A Zheng Ma
%A Andreas Bechthold
%A Xiao-Ping Yu
%J Journal of Zhejiang University SCIENCE B
%V 20
%N 11
%P 891-900
%@ 1673-1581
%D 2019
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1900270

T1 - Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527
A1 - Zhang-Qing Song
A1 - Zhi-Jun Liao
A1 - Ye-Feng Hu
A1 - Zheng Ma
A1 - Andreas Bechthold
A1 - Xiao-Ping Yu
J0 - Journal of Zhejiang University Science B
VL - 20
IS - 11
SP - 891
EP - 900
%@ 1673-1581
Y1 - 2019
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1900270

An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10−5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in β;-Glucuronidase (GUS) activity compared with the control promoter permE*. promoter SPL-57 showed activity comparable to that of permE*. promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.


目的:建立并优化适用于龟裂链霉菌(Streptomyces rimosus)M527的属间接合转移体系,并在此基础上分析四个启动子的表达活性.
创新点:有效的遗传转化系统和合适的启动子是链霉菌基因表达和基因工程的必要前提.由于链霉菌的遗传背景复杂,接合转移实验参数对菌种具有高度特异性.因此,本研究建立并优化了一套适用于S. rimosus M527的属间接合转移体系,并在此基础上分析比较了四个启动子的表达活性.这将为进一步遗传修饰S. rimosus M527奠定基础.
方法:S. rimosus M527为研究对象,通过对接合转移体系中的一些重要的实验参数(如接合转移培养
结论:建立了一种有效的适用于S. rimosus M527接合转移体系,优化后的接合效率最高达3.05×10−5.分析测试的四个启动子中,合成启动子SPL-21表现出最高表达活性,比常用强组成型启动子permE*活性高出2.2倍,合成启动子SPL-57与permE*的活性无显著差异,内源启动子potrB的活性比permE*降低了50%.


Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


[1]Cao XM, Luo ZS, Zeng WZ, et al., 2018. Enhanced avermectin production by Streptomyces avermitilis ATCC 31267 using high-throughput screening aided by fluorescence-activated cell sorting. Appl Microbiol Biotechnol, 102(2): 703-712.

[2]Du L, Liu RH, Ying L, et al., 2012. An efficient intergeneric conjugation of DNA from Escherichia coli to mycelia of the lincomycin-producer Streptomyces lincolnensis. Int J Mol Sci, 13(4):4797-4806.

[3]Enríquez LL, Mendes MV, Antón N, et al., 2006. An efficient gene transfer system for the pimaricin producer Streptomyces natalensis. FEMS Microbiol Lett, 257(2):312-318.

[4]Jeon BJ, Kim JD, Han JW, et al., 2016. Antifungal activity of rimocidin and a new rimocidin derivative BU16 produced by Streptomyces mauvecolor BU16 and their effects on pepper anthracnose. J Appl Microbiol, 120(5):1219-1228.

[5]Ji CH, Kim JP, Kang HS, 2018. Library of synthetic Streptomyces regulatory sequences for use in promoter engineering of natural product biosynthetic gene clusters. ACS Synth Biol, 7(8):1946-1955.

[6]Kakule TB, Jadulco RC, Koch M, et al., 2015. Native promoter strategy for high-yielding synthesis and engineering of fungal secondary metabolites. ACS Synth Biol, 4(5):625-633.

[7]Kemung HM, Tan LTH, Khan TM, et al., 2018. Streptomyces as a prominent resource of future anti-MRSA drugs. Front Microbiol, 9:2221.

[8]Kieser T, Bibb MJ, Buttner MJ, et al., 2000. Practical Streptomyces Genetics. John Innes Foundation, Norwich, UK.

[9]Li SS, Wang JY, Xiang WS, et al., 2018. An autoregulated fine-tuning strategy for titer improvement of secondary metabolites using native promoters in Streptomyces. ACS Synth Biol, 7(2):522-530.

[10]Luo YZ, Zhang L, Barton KW, et al., 2015. Systematic identification of a panel of strong constitutive promoters from Streptomyces albus. ACS Synth Biol, 4(9):1001-1010.

[11]Ma Z, Liu JX, Bechthold A, et al., 2014. Development of intergeneric conjugal gene transfer system in Streptomyces diastatochromogenes 1628 and its application for improvement of toyocamycin production. Curr Microbiol, 68(2):180-185.

[12]Mazodier P, Petter R, Thompson C, 1989. Intergeneric conjugation between Escherichia coli and Streptomyces species. J Bacteriol, 171(6):3583-3585.

[13]Myronovskyi M, Luzhetskyy A, 2016. Native and engineered promoters in natural product discovery. Nat Prod Rep, 33(8):1006-1019.

[14]Myronovskyi M, Welle E, Fedorenko V, et al., 2011. β-Glucuronidase as a sensitive and versatile reporter in actinomycetes. Appl Environ Microbiol, 77(15):5370-5383.

[15]Noomnual S, Thasana N, Sungkeeree P, et al., 2016. Streptanoate, a new anticancer butanoate from Streptomyces sp. DC3. J Antibiot (Tokyo), 69(2):124-127.

[16]Phornphisutthimas S, Sudtachat N, Bunyoo C, et al., 2010. Development of an intergeneric conjugal transfer system for rimocidin-producing Streptomyces rimosus. Lett Appl Microbiol, 50(5):530-536.

[17]Rey T, Dumas B, 2017. Plenty is no plague: Streptomyces symbiosis with crops. Trends Plant Sci, 22(1):30-37.

[18]Rocha D, Ruiz-Villafán B, Manzo M, et al., 2018. Development of an efficient conjugal DNA transfer system between Escherichia coli and a non-sporulating Streptomyces strain. J Microbiol Methods, 144:60-66.

[19]Schlatter DC, Kinkel LL, 2015. Do tradeoffs structure antibiotic inhibition, resistance, and resource use among soil-borne Streptomyces? BMC Evol Biol, 15:186.

[20]Seghezzi N, Amar P, Koebmann B, et al., 2011. The construction of a library of synthetic promoters revealed some specific features of strong Streptomyces promoters. Appl Microbiol Biotechnol, 90(2):615-623.

[21]Siegl T, Tokovenko B, Myronovskyi M, et al., 2013. Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes. Metab Eng, 19:98-106.

[22]Sohoni SV, Fazio A, Workman CT, et al., 2014. Synthetic promoter library for modulation of actinorhodin production in Streptomyces coelicolor A3(2). PLoS ONE, 9(6): e99701.

[23]Sun FH, Luo D, Shu D, et al., 2014. Development of an intergeneric conjugal transfer system for xinaomycins-producing Streptomyces noursei xinao-4. Int J Mol Sci, 15(7):12217-12230.

[24]Wang HX, 2007. Studies on strain breeding and fermentation process of flavomycin. MS Thesis, Heilongjiang Bayi Agricultural University, Daqing, China (in Chinese).

[25]Wang WD, Zhang NN, Chanda W, et al., 2018. Antibacterial and anti-biofilm activity of the lipid extract from Mantidis ootheca on Pseudomonas aeruginosa. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 19(5):364-371.

[26]Wang WS, Li X, Wang J, et al., 2013. An engineered strong promoter for streptomycetes. Appl Environ Microbiol, 79(14):4484-4492.

[27]Wang XK, Jin JL, 2014. Crucial factor for increasing the conjugation frequency in Streptomyces netropsis SD-07 and other strains. FEMS Microbiol Lett, 357(1):99-103.

[28]Xu XH, Wang J, Bechthold A, et al., 2017. Selection of an efficient promoter and its application in toyocamycin production improvement in Streptomyces diastatochromogenes 1628. World J Microbiol Biotechnol, 33(2):30.

[29]Yi JS, Kim MW, Kim M, et al., 2017. A novel approach for gene expression optimization through native promoter and 5' UTR combinations based on RNA-seq, Ribo-seq, and TSS-seq of Streptomyces coelicolor. ACS Synth Biol, 6(3):555-565.

[30]Yoo YJ, Kim H, Park SR, et al., 2017. An overview of rapamycin: from discovery to future perspectives. J Ind Microbiol Biotechnol, 44(4-5):537-553.

[31]Zhang SM, Chen TS, Jia J, et al., 2018. Establishment of a highly efficient conjugation protocol for Streptomyces kanamyceticus ATCC12853. MicrobiologyOpen, 8(6): e00747.

[32]Zhao YF, Lu DD, Bechthold A, et al., 2018. Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 19(9):708-717.

[33]Zhao YF, Song ZQ, Ma Z, et al., 2019. Sequential improvement of rimocidin production in Streptomyces rimosus M527 by introduction of cumulative drug-resistance mutations. J Ind Microbiol Biotechnol, 46(5):697-708.

[34]Zhou H, Wang YM, Yu YF, et al., 2012. A non-restricting and non-methylating Escherichia coli strain for DNA cloning and high-throughput conjugation to Streptomyces coelicolor. Curr Microbiol, 64(2):185-190.

[35]Zhou YS, Gu YX, Qi BZ, et al., 2017. Porcine circovirus type 2 capsid protein induces unfolded protein response with subsequent activation of apoptosis. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 18(4):316-323.

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