Sequential improvement of rimocidin production in Streptomyces rimosus M527 by introduction of cumulative drug-resistance mutations

  • Yanfang Zhao
  • Zhangqing Song
  • Zheng MaEmail author
  • Andreas Bechthold
  • Xiaoping YuEmail author
Biotechnology Methods - Original Paper


Rimocidin is a polyene macrolide that exhibits a strong inhibitory activity against a broad range of plant-pathogenic fungi. In this study, fermentation optimization and ribosome engineering technology were employed to enhance rimocidin production in Streptomyces rimosus M527. After the optimization of fermentation, rimocidin production in S. rimosus M527 increased from 0.11 ± 0.01 to 0.23 ± 0.02 g/L during shake-flask experiments and reached 0.41 ± 0.05 g/L using 5-L fermentor. Fermentation optimization was followed by the generation of mutants of S. rimosus M527 through treatment of the strain with different concentrations of gentamycin (Gen) or rifamycin. One Genr mutant named S. rimosus M527-G37 and one Rifr mutant named S. rimosus M527-R5 showed increased rimocidin production. Double-resistant (Genr and Rifr) mutants were selected using S. rimosus M527-G37 and S. rimosus M527-R5, and subsequently tested. One mutant, S. rimosus M527-GR7, which was derived from M527-G37, achieved the greatest cumulative improvement in rimocidin production. In the 5-L fermentor, the maximum rimocidin production achieved by S. rimosus M527-GR7 was 25.36% and 62.89% greater than those achieved by S. rimosus M527-G37 and the wild-type strain S. rimosus M527, respectively. Moreover, in the mutants S. rimosus M527-G37 and S. rimosus M527-GR7 the transcriptional levels of ten genes (rimAsr to rimKsr) located in the gene cluster involved in rimocidin biosynthesis were all higher than those in the parental strain M527 to varying degrees. In addition, after expression of the single rimocidin biosynthetic genes in S. rimosus M527 a few recombinants showed an increase in rimocidin production. Expression of rimE led to the highest production.


Rimocidin Streptomyces rimosus Resistant mutants Rim gene 



This work was supported by the National Natural Science Foundation of China (31772213), and the excellent youth fund of Zhejiang province, China (LR17C140002).

Supplementary material

10295_2019_2146_MOESM1_ESM.doc (1.4 mb)
Supplementary material 1 (DOC 1395 kb)


  1. 1.
    Brautaset T, Sletta H, Degnes KF, Sekurova ON, Bakke I, Volokhan O, Andreassen T, Ellingsen TE, Zotchev SB (2011) New nystatin-related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces noursei. Appl Environ Microbiol 77(18):6636–6643CrossRefGoogle Scholar
  2. 2.
    Chater KF, Biró S, Lee KJ, Palmer T, Schrempf H (2010) The complex extracellular biology of Streptomyces. FEMS Microbiol Rev 34(2):171–198CrossRefGoogle Scholar
  3. 3.
    Davies C, Bussiere DE, Golden BL, Porter SJ, Ramakrishnan V, White SW (1998) Ribosomal proteins S5 and L6: high-resolution crystal structures and roles in protein synthesis and antibiotic resistance 1. J Mol Biol 279(4):873–888CrossRefGoogle Scholar
  4. 4.
    Escudero L, Al-Refai M, Nieto C, Laatsch H, Malpartida F, Seco EM (2015) New rimocidin/CE-108 derivatives obtained by a crotonyl-CoA carboxylase/reductase gene disruption in Streptomyces diastaticus var. 108: substrates for the polyene carboxamide synthase PcsA. PLoS One 10(8):e0135891CrossRefGoogle Scholar
  5. 5.
    Gao X, He Q, Jiang Y, Huang L (2016) Optimization of nutrient and fermentation parameters for antifungal activity by Streptomyces lavendulae Xjy and its biocontrol efficacies against Fulvia fulva and Botryosphaeria dothidea. J Phytopathol 164(3):155–165CrossRefGoogle Scholar
  6. 6.
    Hu HF, Zhang Q (2008) Enhanced antibiotic production by inducing low level of resistance to gentamicin. Chin J of Nat Med 6(2):146–152 (in Chinese) CrossRefGoogle Scholar
  7. 7.
    Hu H, Zhang Q, Ochi K (2002) Activation of antibiotic biosynthesis by specified mutations in the rpoB gene (encoding the RNA polymerase beta subunit) of Streptomyces lividans. J Bacteriol 184(14):3984–3991CrossRefGoogle Scholar
  8. 8.
    Jeon BJ, Kim JD, Han JW, Kim BS (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):1219–1228CrossRefGoogle Scholar
  9. 9.
    Kemung HM, Tan LT, Khan TM, Chan KG, Pusparajah P, Goh BH, Lee LH (2018) Streptomyces as a prominent resource of future anti-MRSA drugs. Front Microbiol 9:2221CrossRefGoogle Scholar
  10. 10.
    Kitani S, Miyamoto KT, Takamatsu S, Herawati E, Iguchi H, Nishitomi K, Uchida M, Nagamitsu T, Omura S, Ikeda H (2011) Avenolide, a Streptomyces hormone controlling antibiotic production in Streptomyces avermitilis. Proc Natl Acad Sci USA 108(39):16410–16415CrossRefGoogle Scholar
  11. 11.
    Liang W, Chen X, Wu G, Xin Z, Ren X, Shu L, Lei T, Mao Z (2016) Genome shuffling and gentamicin-resistance to improve ε-Poly-l-Lysine productivity of Streptomyces albulus W-156. Appl Biochem Biotechnol 180(8):1601–1617CrossRefGoogle Scholar
  12. 12.
    Ling L, Jian P, Wang Z, Yan X, Dong Y, Zhu X, Shen B, Duan Y, Yong H (2018) Ribosome engineering and fermentation optimization leads to overproduction of tiancimycin A, a new enediyne natural product from Streptomyces sp. CB03234. J Ind Microbiol Biotechnol 45(3):141–151CrossRefGoogle Scholar
  13. 13.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 25(4):402–408CrossRefGoogle Scholar
  14. 14.
    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):929–933CrossRefGoogle Scholar
  15. 15.
    Lu D, Zhao Y, Ma Z, Zhang Y, Wang J, Yu X (2016) Identification of antifungal compound from Streptomyces rimosus M527 and its application in biocontrol of pathogenic fusarium wilt on cucumber. Chin J Biol Control 32(6):783–787 (In Chinese) Google Scholar
  16. 16.
    Ma Z, Luo S, Xu X, Bechthold A, Yu X (2016) Characterization of representative rpoB gene mutations leading to a significant change in toyocamycin production of Streptomyces diastatochromogenes1628. J Ind Microbiol Biotechnol 43(4):463–471CrossRefGoogle Scholar
  17. 17.
    Ma Z, Tao L, Bechthold A, Shentu X, Bian Y, Yu X (2014) Overexpression of ribosome recycling factor is responsible for improvement of nucleotide antibiotic-toyocamycin in Streptomyces diastatochromogenes 1628. Appl Microbiol Biotechnol 98(11):5051–5058CrossRefGoogle Scholar
  18. 18.
    Ochi K (2017) Insights into microbial cryptic gene activation and strain improvement: principle, application and technical aspects. J Antibiot 70(1):25–40CrossRefGoogle Scholar
  19. 19.
    Ochi K, Okamoto S, Tozawa Y, Inaoka T, Hosaka T, Xu J, Kurosawa K (2004) Ribosome engineering and secondary metabolite production. Adv Appl Microbiol 56:155–184CrossRefGoogle Scholar
  20. 20.
    Phornphisutthimas S, Sudtachat N, Bunyoo C, Chotewutmontri P, Panijpan B, Thamchaipenet A (2010) Development of an intergeneric conjugal transfer system for rimocidin-producing Streptomyces rimosus. Lett Appl Microbiol 50(5):530–536CrossRefGoogle Scholar
  21. 21.
    Ren J, Cui Y, Zhang F, Cui H, Ni X, Chen F, Li L, Xia H (2014) Enhancement of nystatin production by redirecting precursor fluxes after disruption of the tetramycin gene from Streptomyces ahygroscopicus. Microbiol Res 169(7–8):602–608CrossRefGoogle Scholar
  22. 22.
    Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  23. 23.
    Seco EM, Pérezzúñiga 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):357–366CrossRefGoogle Scholar
  24. 24.
    Shentu X, Cao Z, Xiao Y, Tang G, Ochi K, Yu X (2018) Substantial improvement of toyocamycin production in Streptomyces diastatochromogenes by cumulative drug-resistance mutations. PLoS One 13(8):e0203006CrossRefGoogle Scholar
  25. 25.
    Shima J, Hesketh A, Okamoto S, Kawamoto S, Ochi K (1996) Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2). J Bacteriol 178(24):7276–7284CrossRefGoogle Scholar
  26. 26.
    Suzuki T, Seta K, Nishikawa C, Hara E, Shigeno T, Nakajimakambe T (2015) Improved ethanol tolerance and ethanol production from glycerol in a streptomycin-resistant Klebsiella variicola mutant obtained by ribosome engineering. Bioresour Technol 176:156–162CrossRefGoogle Scholar
  27. 27.
    Tamehiro N, Hosaka T, Xu J, Hu H, Otake N, Ochi K (2003) Innovative approach for improvement of an antibiotic-overproducing industrial strain of Streptomyces albus. Appl Environ Microb 69(11):6412–6417CrossRefGoogle Scholar
  28. 28.
    Tanaka Y, Izawa M, Hiraga Y, Misaki Y, Watanabe T, Ochi K (2017) Metabolic perturbation to enhance polyketide and nonribosomal peptide antibiotic production using triclosan and ribosome-targeting drugs. Appl Microbiol Biotechnol 101(11):4417–4431CrossRefGoogle Scholar
  29. 29.
    Tong QQ, Zhou YH, Chen XS, Wu JY, Wei P, Yuan LX, Yao JM (2018) Genome shuffling andribosome engineering of Streptomyces virginiae for improved virginiamycin production. Bioprocess Biosyst Eng 41(5):729–738CrossRefGoogle Scholar
  30. 30.
    Viaene T, Langendries S, Beirinckx S, Maes M, Goormachtig S (2016) Streptomyces as a plant’s best friend? FEMS Microbiol Ecol 92(8):fiw119CrossRefGoogle Scholar
  31. 31.
    Wang G, Hosaka T, Ochi K (2008) Dramatic activation of antibiotic production in Streptomyces coelicolor by cumulative drug resistance mutations. Appl Environ Microbiol 74(9):2834–2840CrossRefGoogle Scholar
  32. 32.
    Xu J, Tozawa Y, Lai C, Hayashi H, Ochi K (2002) A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2). Mol Genet Genomics 268(2):179–189CrossRefGoogle Scholar
  33. 33.
    Yao T, Liu Z, Li T, Zhang H, Liu J, Li H, Che Q, Zhu T, Li D, Li W (2018) Characterization of the biosynthetic gene cluster of the polyene macrolide antibiotic reedsmycins from a marine-derived Streptomyces strain. Microb Cell Fact 17(1):98CrossRefGoogle Scholar
  34. 34.
    Liu Z, Zhao X, Bai F (2013) Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering. Appl Microbiol Biotechnol 97(10):4361–4368CrossRefGoogle Scholar
  35. 35.
    Zhang J, An J, Wang JJ, Yan YJ, He HR, Wang XJ, Xiang WS (2013) Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin. Appl Microbiol Biotechnol 97(23):10091–10101CrossRefGoogle Scholar
  36. 36.
    Zhang Y, Huang H, Xu S, Wang B, Ju J, Tan H, Li W (2015) Activation and enhancement of Fredericamycin A production in deepsea-derived Streptomyces somaliensis SCSIO ZH66 by using ribosome engineering and response surface methodology. Microb Cell Fact 14:64CrossRefGoogle Scholar
  37. 37.
    Zhao Y, Lu D, Bechthold A, Ma Z, Yu X (2018) Impact of otrA expression on morphological differentiation, actinorhodin production, and resistance to aminoglycosides in Streptomyces coelicolor M145. J Zhejiang Univ Sci B 19(9):708–717CrossRefGoogle Scholar
  38. 38.
    Zhu X, Kong J, Yang H, Huang R, Huang Y, Yang D, Shen B, Duan Y (2018) Strain improvement by combined UV mutagenesis and ribosome engineering and subsequent fermentation optimization for enhanced 6′-deoxy-bleomycin Z production. Appl Microbiol Biotechnol 102(4):1651–1661CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  1. 1.Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life SciencesChina Jiliang UniversityHangzhouPeople’s Republic of China
  2. 2.Institute for Pharmaceutical Sciences, Pharmaceutical Biology and BiotechnologyUniversity of FreiburgFreiburgGermany

Personalised recommendations