Enhanced lincomycin production by co-overexpression of metK1 and metK2 in Streptomyces lincolnensis

  • Yurong Xu
  • Guoqing Tan
  • Meilan Ke
  • Jie Li
  • Yaqian Tang
  • Sitong Meng
  • Jingjing Niu
  • Yansheng Wang
  • Ruihua Liu
  • Hang Wu
  • Linquan Bai
  • Lixin Zhang
  • Buchang Zhang
Genetics and Molecular Biology of Industrial Organisms - Original Paper


Streptomyces lincolnensis is generally utilized for the production of lincomycin A (Lin-A), a clinically useful antibiotic to treat Gram-positive bacterial infections. Three methylation steps, catalyzed by three different S-adenosylmethionine (SAM)-dependent methyltransferases, are required in the biosynthesis of Lin-A, and thus highlight the significance of methyl group supply in lincomycin production. In this study, we demonstrate that externally supplemented SAM cannot be taken in by cells and therefore does not enhance Lin-A production. Furthermore, bioinformatics and in vitro enzymatic assays revealed there exist two SAM synthetase homologs, MetK1 (SLCG_1651) and MetK2 (SLCG_3830) in S. lincolnensis that could convert l-methionine into SAM in the presence of ATP. Even though we attempted to inactivate metK1 and metK2, only metK2 was deleted in S. lincolnensis LCGL, named as ΔmetK2. Following a reduction of the intracellular SAM concentration, ΔmetK2 mutant exhibited a significant decrease of Lin-A in comparison to its parental strain. Individual overexpression of metK1 or metK2 in S. lincolnensis LCGL either elevated the amount of intracellular SAM, concomitant with 15% and 22% increase in Lin-A production, respectively. qRT-PCR assays showed that overexpression of either metK1 or metK2 increased the transcription of lincomycin biosynthetic genes lmbA and lmbR, and regulatory gene lmbU, indicating SAM may also function as a transcriptional activator. When metK1 and metK2 were co-expressed, Lin-A production was increased by 27% in LCGL, while by 17% in a high-yield strain LA219X.


Streptomyces lincolnensis Lincomycin S-adenosylmethionine (SAM) SAM synthetase 



We are grateful to Dr. Sabrina Huber from Department of Biological Engineering at Massachusetts Institute of Technology for critical editing of the manuscript. This work was supported by the National Natural Science Foundation of China (31300081, 31570074, 31600064), the National Program on Key Basic Research Project (973 programs, 2013CB734000), Open Project of State Key Laboratory of Microbial Metabolism from Shanghai Jiao Tong University (MMLKF13-05), the Initial Foundation of Doctoral Scientific Research in Anhui University (01001904, J01001935), and the National Innovation Experiment Program for University Students (J10118516053).

Compliance with ethical standards

conflict of interest

The authors declare that they have no competing interests.

Research involving human and animal participants

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10295_2018_2029_MOESM1_ESM.docx (1.5 mb)
Supplementary material 1 (DOCX 1492 kb)


  1. 1.
    Bierman M, Logan R, Obrien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116:43–49. CrossRefPubMedGoogle Scholar
  2. 2.
    Chiang PK, Gordon RK, Tal J, Zeng GC, Doctor BP, Pardhasaradhi K, Mccann PP (1996) S-adenosylmethionine and methylation. FASEB J 10:471–480CrossRefPubMedGoogle Scholar
  3. 3.
    Choi D, Cho K (2004) Effect of carbon source consumption rate on lincomycin production from Streptomyces lincolnensis. J Microbiol Biot 14:532–539Google Scholar
  4. 4.
    Chu J, Qian J, Zhuang Y, Zhang S, Li Y (2013) Progress in the research of S-adenosyl-l-methionine production. Appl Microbiol Biot 97:41–49. CrossRefGoogle Scholar
  5. 5.
    Copeland RA (2000) Enzymes: a practical introduction to structure, mechanism and data analysis, 2nd edn. Wiley, HobokenCrossRefGoogle Scholar
  6. 6.
    Du L, Liu R, Ying L, Zhao G (2012) An efficient intergeneric conjugation of DNA from Escherichia coli to mycelia of the lincomycin-producer Streptomyces lincolnensis. Int J Mol Sci 13:4797–4806. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Han S, Song P, Ren T, Huang X, Cao C, Zhang B (2011) Identification of SACE_7040, a member of TetR family related to the morphological differentiation of Saccharopolyspora erythraea. Curr Microbiol 63:121–125. CrossRefPubMedGoogle Scholar
  8. 8.
    Hou B, Wu H, Guo M, Petkovic H, Tao L, Zhu X, Ye J, Zhang H (2018) The novel transcriptional regulator LmbU promotes lincomycin biosynthesis through regulating expression of its target genes in Streptomyces lincolnensis. J Bacteriol 200:e00447–e00417. PubMedGoogle Scholar
  9. 9.
    Janata J, Kadlcik S, Koberska M, Ulanova D, Kamenik Z, Novak P, Kopecky J, Novotna J, Radojevic B, Plhackova K (2015) Lincosamide synthetase—a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism. PLoS ONE 10:e0118850. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kim DY, Hwang YI, Choi SU (2011) Cloning of metK from Actinoplanes teichomyceticus ATCC31121 and effect of its high expression on antibiotic production. J Microbiol Biot 21:1294–1298. CrossRefGoogle Scholar
  11. 11.
    Koběrska M, Kopecký J, Olsovska J, Jelinkova M, Ulanova D, Man P, Flieger M, Janata J (2008) Sequence analysis and heterologous expression of the lincomycin biosynthetic cluster of the type strain Streptomyces lincolnensis ATCC 25466. Folia Microbiol 53:395–401. CrossRefGoogle Scholar
  12. 12.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT. Methods 25:402–408. CrossRefPubMedGoogle Scholar
  13. 13.
    Meng S, Wu H, Wang L, Zhang B, Bai L (2017) Enhancement of antibiotic productions by engineered nitrate utilization in actinomycetes. Appl Microbiol Biot 101:5341–5352. CrossRefGoogle Scholar
  14. 14.
    Najmanova L, Kutejova E, Kadlec J, Polan M, Olsovska J, Benada O, Novotna J, Kamenik Z, Halada P, Bauer J (2013) Characterization of N-demethyllincosamide methyltransferases LmbJ and CcbJ. ChemBioChem 14:2259–2262. CrossRefPubMedGoogle Scholar
  15. 15.
    Oh T, Niraula NP, Liou K, Sohng J (2010) Identification of the duplicated genes for S-adenosyl-l-methionine synthetase (metK1-sp and metK2-sp) in Streptomyces peucetius var. caesius ATCC 27952. J Appl Microbiol 109:398–407. PubMedGoogle Scholar
  16. 16.
    Okamoto S, Lezhava A, Hosaka T, Okamotohosoya Y, Ochi K (2003) Enhanced expression of S-adenosylmethionine synthetase causes overproduction of actinorhodin in Streptomyces coelicolor A3(2). J Bacteriol 185:601–609. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Ostash B, Makitrinskyy R, Walker S, Fedorenko V (2009) Identification and characterization of Streptomyces ghanaensis ATCC14672 integration sites for three actinophage-based plasmids. Plasmid 61:171–175. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Pang AP, Du L, Lin C, Qiao J, Zhao G (2015) Co-overexpression of lmbW and metK led to increased lincomycin A production and decreased byproduct lincomycin B content in an industrial strain of Streptomyces lincolnensis. J Appl Microbiol 119:1064–1074. CrossRefPubMedGoogle Scholar
  19. 19.
    Peschke U, Schmidt H, Zhang H, Piepersberg W (1995) Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78-11. Mol Microbiol 16:1137–1156. CrossRefPubMedGoogle Scholar
  20. 20.
    Sambrook JRD (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  21. 21.
    Sekurova ON, Brautaset T, Sletta H, Borgos SEF, Jakobsen OM, Ellingsen TE, Strom AR, Valla S, Zotchev SB (2004) In vivo analysis of the regulatory genes in the Nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis. J Bacteriol 186:1345–1354. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Shin S, Xu D, Kwon H, Suh J (2006) S-adenosylmethionine activates adpA transcription and promotes streptomycin biosynthesis in Streptomyces griseus. FEMS Microbiol Lett 259:53–59. CrossRefPubMedGoogle Scholar
  23. 23.
    Smith MCM, Brown W, Mcewan AR, Rowley PA (2010) Site-specific recombination by φC31 integrase and other large serine recombinases. Biochem Soc T 38:388–394. CrossRefGoogle Scholar
  24. 24.
    Smokvina T, Mazodier P, Boccard F, Thompson CJ, Guerineau M (1990) Construction of a series of pSAM2-based integrative vectors for use in actinomycetes. Gene 94:53–59. CrossRefPubMedGoogle Scholar
  25. 25.
    Spizek J, Rezanka T (2017) Lincosamides: chemical structure, biosynthesis, mechanism of action, resistance, and applications. Biochem Pharmacol 133:20–28. CrossRefPubMedGoogle Scholar
  26. 26.
    Spižek J, Řezanka T (2004) Lincomycin, clindamycin and their applications. Appl Microbiol Biot 64:455–464CrossRefGoogle Scholar
  27. 27.
    Te Poele EM, Dijkhuizen L (2008) Actinomycete integrative and conjugative elements. Antonie Van Leeuwenhoek 94:127–143. CrossRefGoogle Scholar
  28. 28.
    Te Poele EM, Oliynyk M, Leadlay PF, Bolhuis H, Dijkhuizen L (2008) Actinomycete integrative and conjugative pMEA-like elements of Amycolatopsis and Saccharopolyspora decoded. Plasmid 59:202–216. CrossRefGoogle Scholar
  29. 29.
    Wu H, Chen M, Mao Y, Li W, Liu J, Huang X, Zhou Y, Ye B, Zhang L, Weaver DT (2014) Dissecting and engineering of the TetR family regulator SACE_7301 for enhanced erythromycin production in Saccharopolyspora erythraea. Microbial Cell Fact 13:158. CrossRefGoogle Scholar
  30. 30.
    Wu J, Zhang Q, Deng W, Qian J, Zhang S, Liu W (2011) Toward improvement of erythromycin A production in an industrial Saccharopolyspora erythraea strain via facilitation of genetic manipulation with an artificial attB site for specific recombination. Appl Environ Microbiol 77:7508–7516. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Wu P, Pan H, Zhang C, Wu H, Yuan L, Huang X, Zhou Y, Ye B, Weaver DT, Zhang L (2014) SACE_3986, a TetR family transcriptional regulator, negatively controls erythromycin biosynthesis in Saccharopolyspora erythraea. J Ind Microbiol Biot 41:1159–1167. CrossRefGoogle Scholar
  32. 32.
    Xu D, Kwon H, Suh J (2008) S-adenosylmethionine induces BldH and activates secondary metabolism by involving the TTA-codon control of bldH expression in Streptomyces lividans. Arch Microbiol 189:419–426. CrossRefPubMedGoogle Scholar
  33. 33.
    Ye R, Wang Q, Zhou X (2009) Lincomycin, rational selection of high producing strain and improved fermentation by amino acids supplementation. Bioprocess Biosyst Eng 32:521–529CrossRefPubMedGoogle Scholar
  34. 34.
    Zhao Q, Wang M, Xu D, Zhang Q, Liu W (2015) Metabolic coupling of two small-molecule thiols programs the biosynthesis of lincomycin A. Nature 518:115–119. CrossRefPubMedGoogle Scholar
  35. 35.
    Zhao X, Wang Q, Guo W, Cai Y, Wang C, Wang S, Xiang S, Song Y (2013) Overexpression of metK shows different effects on avermectin production in various Streptomyces avermitilis strains. World J Microbiol Biotechnol 29:1869–1875. CrossRefPubMedGoogle Scholar
  36. 36.
    Zhao XQ, Gust B, Heide L (2010) S-adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces. Arch Microbiol 192:289–297. CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  • Yurong Xu
    • 1
  • Guoqing Tan
    • 1
  • Meilan Ke
    • 1
  • Jie Li
    • 1
  • Yaqian Tang
    • 1
  • Sitong Meng
    • 3
  • Jingjing Niu
    • 1
  • Yansheng Wang
    • 1
  • Ruihua Liu
    • 4
  • Hang Wu
    • 1
  • Linquan Bai
    • 3
  • Lixin Zhang
    • 1
    • 2
  • Buchang Zhang
    • 1
  1. 1.School of Life Sciences, School of Chemistry and Chemical Engineering, Institute of Physical Science and Information TechnologyAnhui UniversityHefeiChina
  2. 2.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiChina
  3. 3.State Key Laboratory of Microbial MetabolismShanghai Jiao Tong UniversityShanghaiChina
  4. 4.Xinyu Pharmaceutical Co. Ltd.SuzhouChina

Personalised recommendations