Abstract
The transposon mutagenesis strategy has been employed to generate random insertion mutants and analyze the correlation between genes and secondary metabolites in the genus Streptomyces. In this study, our primary objective was to identify an unknown gene involved in rimocidin biosynthesis and elucidate its role in rimocidin production in Streptomyces rimosus M527. To achieve this, we established a random mutant library of S. rimosus M527 using a Tn5 transposon-mediated random mutagenesis strategy. Among the 137 isolated mutants, M527-G10 and M527-W5 exhibited the most significant variations in antagonistic activity against the plant pathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Specifically, M527-G10 displayed a 72.93% reduction, while M527-W5 showed a 49.8% increase in rimocidin production compared to the wild-type (WT) strain S. rimosus M527. Subsequently, we employed a plasmid rescue strategy to identify the insertion loci of the transposon in the genomes of mutants M527-G10 and M527-W5, revealing a response regulator transcription factor (rrt) and a hypothetical protein (hyp), respectively. The roles of rrt and hyp in rimocidin biosynthesis were determined through gene deletion, overexpression in the WT strain, and complemented expression in the transposon mutants. Notably, the gene-deletion mutants M527-ΔRRT and M527-ΔHYP exhibited similar behavior in rimocidin production compared to the corresponding transposon mutants M527-G10 and M527-W5, suggesting that transposon insertions in genes rrt and hyp led to alterations in rimocidin production. Furthermore, both gene deletion and overexpression of rrt and hyp had no discernible effects on cell growth. These results reveal that genes rrt and hyp have positive and negative impacts on rimocidin production in S. rimosus M527, respectively.
Similar content being viewed by others
Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Ahmed Y, Rebets Y, Tokovenko B, Brötz E, Luzhetskyy A (2017) Identification of butenolide regulatory system controlling secondary metabolism in Streptomyces albus J1074. Sci Rep 7(1):9784. https://doi.org/10.1038/s41598-017-10316-y
Bilyk B, Weber S, Myronovskyi M, Bilyk O, Petzke L, Luzhetskyy A (2013) In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1. Appl Microbiol Biotechnol 97(1):351–359. https://doi.org/10.1007/s00253-012-4550-x
Cain AK, Barquist L, Goodman AL, Paulsen IT, Parkhill J, van Opijnen T (2020) A decade of advances in transposon-insertion sequencing. Nat Rev Genet 21(9):526–540. https://doi.org/10.1038/s41576-020-0244-x
Chen Y, Smanski MJ, Shen B (2010) Improvement of secondary metabolite production in Streptomyces by manipulating pathway regulation. Appl Microbiol Biotechnol 86(1):19–25. https://doi.org/10.1007/s00253-009-2428-3
Chen J, Wu Q, Hawas UW, Wang H (2016) Genetic regulation and manipulation for natural product discovery. Appl Microbiol Biotechnol 100(7):2953–2965. https://doi.org/10.1007/s00253-016-7357-3
Cobb RE, Wang Y, Zhao H (2015) High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth Biol 4(6):723–728. https://doi.org/10.1021/sb500351f
Elleuch L, Shaaban M, Smaoui S, Mellouli L, Karray-Rebai I, Fourati-Ben Fguira L, Shaaban KA, Laatsch H (2010) Bioactive secondary metabolites from a new terrestrial Streptomyces sp. TN262. Appl Biochem Biotechnol 162(2):579–593. https://doi.org/10.1007/s12010-009-8808-4
Feng BQ, Feng J, Zhang M, Liu Y, Cao R, Yi HZ, Qi FX, Li ZL, Yi SL (2021) Screening of high avermectin-producing strains via Tn5 transposon mediated mutagenesis. China Biotechnol 41(07):32–41. https://doi.org/10.13523/j.cb.2102033(In Chinese)
Gehring AM, Nodwell JR, Beverley SM, Losick R (2000) Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation. Proc Natl Acad Sci U S A 97(17):9642–9647. https://doi.org/10.1073/pnas.170059797
Gongerowska-Jac M, Szafran MJ, Jakimowicz D (2021) Combining transposon mutagenesis and reporter genes to identify novel regulators of the topA promoter in Streptomyces. Microb Cell Fact 20(1):99. https://doi.org/10.1186/s12934-021-01590-7
Goryshin IY, Jendrisak J, Hoffman LM, Meis R, Reznikoff WS (2000) Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat Biotechnol 18(1):97–100. https://doi.org/10.1038/72017
He H, Williamson RT, Shen B, Graziani EI, Yang HY, Sakya SM, Petersen PJ, Carter GT (2002) Mannopeptimycins, novel antibacterial glycopeptides from Streptomyces hygroscopicus, LL-AC98. J Am Chem Soc 124(33):9729–9736. https://doi.org/10.1021/ja020257s
Higginbotham SJ, Murphy CD (2010) Identification and characterisation of a Streptomyces sp. isolate exhibiting activity against methicillin-resistant Staphylococcus aureus. Microbiol Res 165(1):82–86. https://doi.org/10.1016/j.micres.2008.12.004
Holban AM, Gregoire CM, Gestal MC (2022) Conquering the host: Bordetella spp. and Pseudomonas aeruginosa molecular regulators in lung Infection. Front Microbiol 13:983149. https://doi.org/10.3389/fmicb.2022.983149
Hong SW, Kim DR, Kwon YS, Kwak YS (2019) Genome-wide screening antifungal genes in Streptomyces griseus S4-7, a Fusarium wilt Disease suppressive Microbial Agent. FEMS Microbiol Lett 366(12):fnz133. https://doi.org/10.1093/femsle/fnz133
Hu Y, Wang J, Xu J, Ma Z, Bechthold A, Yu X (2021) Effects of S-adenosylmethionine on production of secondary metabolites in Streptomyces diastatochromogenes 1628. J Zhejiang Univ Sci B 22(9):767–773. https://doi.org/10.1631/jzus.B2100115
Ibrahim WM, Olama ZA, Abou-Elela GM, Ramadan HS, Hegazy GE, El Badan DES (2023) Exploring the antimicrobial, antiviral, antioxidant, and antitumor potentials of marine Streptomyces tunisiensis W4MT573222 pigment isolated from Abu-Qir sediments, Egypt. Microb Cell Fact 194. https://doi.org/10.1186/s12934-023-02106-1
Jiang Y, Zhang J, Huang X, Ma Z, Zhang Y, Bechthold A, Yu X (2023) Improvement of rimocidin production in Streptomyces rimosus M527 by reporter-guided mutation selection. J Ind Microbiol Biotechnol 49(6):kuac030. https://doi.org/10.1093/jimb/kuac030
Kemppainen M, Duplessis S, Martin F, Pardo AG (2008) T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor. Microb Biotechnol 1(3):258–269. https://doi.org/10.1111/j.1751-7915.2008.00029.x
Ko K, Lee SH, Kim SH, Kim EH, Oh KB, Shin J, Oh DC (2014) Lajollamycins, nitro group-bearing spiro-β-lactone-γ-lactams obtained from a marine-derived Streptomyces Sp. J Nat Prod 77(9):2099–2104. https://doi.org/10.1021/np500500t
Li S, Li Z, Pang S, Xiang W, Wang W (2021) Coordinating precursor supply for pharmaceutical polyketide production in Streptomyces. Curr Opin Biotechnol 69:26–34. https://doi.org/10.1016/j.copbio.2020.11.006
Li H, Hu Y, Zhang Y, Ma Z, Bechthold A, Yu X (2023) Identification of RimR2 as a positive pathway-specific regulator of rimocidin biosynthesis in Streptomyces rimosus M527. Microb Cell Fact 22(1):32. https://doi.org/10.1186/s12934-023-02039-9- DOI
Liao Z, Song Z, Xu J, Ma Z, Bechthold A, Yu X (2020) Identification of a gene from Streptomyces rimosus M527 negatively affecting rimocidin biosynthesis and morphological differentiation. Appl Microbiol Biotechnol 104(23):10191–10202. https://doi.org/10.1007/s00253-020-10955-8
Liu G, Tian Y, Yang H, Tan H (2005) A pathway-specific transcriptional regulatory gene for nikkomycin biosynthesis in Streptomyces ansochromogenes that also influences colony development. Mol Microbiol 55(6):1855–1866. https://doi.org/10.1111/j.1365-2958.2005.04512.x
Lu D, Ma Z, Xu X, Yu X (2016) Isolation and identification of biocontrol agent Streptomyces rimosus M527 against Fusarium oxysporum f. sp. cucumerinum. J Basic Microbiol 56(8):929–933. https://doi.org/10.1002/jobm.201500666
Luo S, Chen XA, Mao XM, Li YQ (2018) Transposon-based identification of a negative regulator for the antibiotic hyper-production in Streptomyces. Appl Microbiol Biotechnol 102(15):6581–6592. https://doi.org/10.1007/s00253-018-9103-5
Nett M, Ikeda H, Moore BS (2009) Genomic basis for natural product biosynthetic diversity in the actinomycetes. Nat Prod Rep 26(11):1362–1384. https://doi.org/10.1039/b817069j
Petzke L, Luzhetskyy A (2009) In vivo Tn5-based transposon mutagenesis of Streptomycetes. Appl Microbiol Biotechnol 83(5):979–986. https://doi.org/10.1007/s00253-009-2047-z- DOI
Pimentel-Elardo SM, Kozytska S, Bugni TS, Ireland CM, Moll H, Hentschel U (2010) Anti-parasitic compounds from Streptomyces sp. strains isolated from Mediterranean sponges. Mar Drugs 8(2):373–380. https://doi.org/10.3390/md8020373
Seco EM, Pérez-Zúñiga FJ, Rolón MS, Malpartida F (2004) Starter unit choice determines the production of two tetraene macrolides, rimocidin and CE-108, in Streptomyces diastaticus var. 108. Chem Biol 11(3):357–366. https://doi.org/10.1016/j.chembiol.2004.02.017 - DOI
Song ZQ, Liao ZJ, Hu YF, Ma Z, Bechthold A, Yu XP (2019) Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527. J Zhejiang Univ Sci B 20(11):891–900. https://doi.org/10.1631/jzus.B1900270
Song Z, Ma Z, Bechthold A, Yu X (2020) Effects of addition of elicitors on rimocidin biosynthesis in Streptomyces rimosus M527. Appl Microbiol Biotechnol 104(10):4445–4455. https://doi.org/10.1007/s00253-020-10565-4
Sowiński P, Pawlak J, Borowski E, Gariboldi P (1995) Stereostructure of rimocidin. J Antibiot (Tokyo) 48(11):1288–1291. https://doi.org/10.7164/antibiotics.48.1288
Tian J, Ye L, Yang Y, Zhang Y, Hu C, Liao G (2020) Transposon-based screen identifies a XRE family regulator crucial for candicidin biosynthesis in Streptomyces albus J1074. Sci China Life Sci 63(9):1421–1424. https://doi.org/10.1007/s11427-019-1582-5
Wang Y, Cobb RE, Zhao H (2016) High-efficiency genome editing of Streptomyces species by an engineered CRISPR/Cas system. Meth Enzymol 575:271–284. https://doi.org/10.1016/bs.mie2016.03.014 - DOI
Xu J, Zhang J, Zhuo J, Li Y, Tian Y, Tan H (2017) Activation and mechanism of a cryptic oviedomycin gene cluster via the disruption of a global regulatory gene, adpA, in Streptomyces ansochromogenes. J Biol Chem 292(48):19708–19720. https://doi.org/10.1074/jbc.M117.809145
Zhang Y, He H, Liu H, Wang H, Wang X, Xiang W (2016) Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces Bingchenggensis. Microb Cell Fact 15(1):152. https://doi.org/10.1186/s12934-016-0552-1
Zhao W, Zhong Y, Yuan H, Wang J, Zheng H, Wang Y, Cen X, Xu F, Bai J, Han X, Lu G, Zhu Y, Shao Z, Yan H, Li C, Peng N, Zhang Z, Zhang Y, Lin W, Fan Y, Qin Z, Hu Y, Zhu B, Wang S, Ding X, Zhao GP (2010) Complete genome sequence of the rifamycin SV-producing Amycolatopsis mediterranei U32 revealed its genetic characteristics in phylogeny and metabolism. Cell Res 20(10):1096–1108. https://doi.org/10.1038/cr.2010.87
Zhao Y, Song Z, Ma Z, Bechthold A, Yu X (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. https://doi.org/10.1007/s10295-019-02146-w
Zheng J, Li Y, Guan H, Zhang J, Tan H (2019) Enhancement of neomycin production by engineering the entire biosynthetic gene cluster and feeding key precursors in Streptomyces fradiae CGMCC 4.576. Appl Microbiol Biotechnol 103(5):2263–2275. https://doi.org/10.1007/s00253-018-09597-8
Acknowledgements
This work was supported by National Natural Science Foundation of China (32272616, 31772213), Key Program of Zhejiang Province Natural Science Foundation (LZ22C140002).
Author information
Authors and Affiliations
Contributions
H Bao, H Li and Y Zhang conducted experiments. Z Ma designed research and wrote this article. A Bechthold revised this article. X Yu checked the final version. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Contributors
H Bao, H Li and Y Zhang conducted experiments. Z Ma designed research and wrote this article. A Bechthold revised this article. X Yu checked the final version. All authors read and approved the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Bao, Hy., Li, Hj., Zhang, Yy. et al. Transposon-based identification of genes involved in the rimocidin biosynthesis in Streptomyces rimosus M527. World J Microbiol Biotechnol 39, 359 (2023). https://doi.org/10.1007/s11274-023-03814-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-023-03814-x