Applied Microbiology and Biotechnology

, Volume 100, Issue 5, pp 2257–2266 | Cite as

In vivo investigation to the macrolide-glycosylating enzyme pair DesVII/DesVIII in Saccharopolyspora erythraea

  • Hang Wu
  • Weiwei Li
  • Chen Xin
  • Congming Zhang
  • Yansheng Wang
  • Shaohua Ren
  • Min Ren
  • Wei Zhao
  • Li Yuan
  • Zhongdong Xu
  • Hualing Yuan
  • Ming Geng
  • Lixin Zhang
  • David T. Weaver
  • Buchang Zhang
Applied Genetics and Molecular Biotechnology

Abstract

Glycosyltransferase DesVII and its auxiliary partner DesVIII from Streptomyces venezulae, homologs of EryCIII and EryCII in Saccharopolyspora erythraea, have previously been demonstrated to be flexible on their substrates in vitro. Herein, we investigated their in vivo function by interspecies complementation in the mutant strains of Sac. erythraea A226. As desVII and desVIII were concomitantly expressed in the ΔeryCIII mutant, the erythromycin A (Er-A) production was restored. Interestingly, co-expression of desVII and desVIII in the ΔeryBV mutant exhibited an increased Er-A yield by 15 % in comparison to A226. Hence, DesVII/DesVIII not only replaced EryCIII to upload D-desosamine to C5 position of 3-O-mycarosyl erythronolide B (MEB) but also in vivo attached L-mycarose, not D-desosamine to C3 position of erythronolide B (EB) with a higher activity than EryBV. Furthermore, expression of desVII in ΔeryCIII and ΔeryBV-CIII partially restored the Er-A production; however, no Er-A was detected while desVII was expressed in ΔeryBV. It was implicated that DesVII coupled with EryCII to form the DesVII/EryCII complex for attaching above two deoxysugars in the absence of EryCIII in Sac. erythraea. In addition, when desVII and desVIII were co-expressed in ΔeryBV-CII, Er-A was recovered with a lower yield than ΔeryBV-CIII. Our study presents an opportunity with Sac. erythraea as a cell factory for macrolide glycodiversification.

Keywords

Saccharopolyspora erythraea Glycosyltransferase DesVII/DesVIII EryCII/EryCIII EryBV 

Supplementary material

253_2015_7036_MOESM1_ESM.pdf (269 kb)
ESM 1(PDF 268 kb)

References

  1. Bierman M, Logan R, O’Brien 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(1):43–49Google Scholar
  2. Borisova SA, Liu H-W (2010) Characterization of glycosyltransferase DesVII and its auxiliary partner protein DesVIII in the methymycin/pikromycin biosynthetic pathway. Biochemistry 49(37):8071–8084PubMedCentralCrossRefPubMedGoogle Scholar
  3. Borisova SA, Zhao L, Melançon CE, Kao C-L, H-w L (2004) Characterization of the glycosyltransferase activity of desVII: analysis of and implications for the biosynthesis of macrolide antibiotics. J Am Chem Soc 126(21):6534–6535CrossRefPubMedGoogle Scholar
  4. Borisova SA, Zhang C, Takahashi H, Zhang H, Wong AW, Thorson JS, Liu HW (2006) Substrate specificity of the macrolide-glycosylating enzyme pair DesVII/DesVIII: opportunities, limitations, and mechanistic hypotheses. Angew Chem Int Ed Engl 45(17):2748–2753CrossRefPubMedGoogle Scholar
  5. Borisova SA, Kim HJ, Pu X, Hw L (2008) Glycosylation of acyclic and cyclic aglycone substrates by macrolide glycosyltransferase DesVII/DesVIII: analysis and implications. Chembiochem 9(10):1554–1558PubMedCentralCrossRefPubMedGoogle Scholar
  6. Cane DE (2010) Programming of erythromycin biosynthesis by a modular polyketide synthase. J Biol Chem 285(36):27517–27523PubMedCentralCrossRefPubMedGoogle Scholar
  7. Cortes J, Haydock SF, Roberts GA, Bevitt DJ, Leadlay PF (1990) An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature 348(6297):176–178CrossRefPubMedGoogle Scholar
  8. Donadio S, Staver MJ, McAlpine JB, Swanson SJ, Katz L (1991) Modular organization of genes required for complex polyketide biosynthesis. Science 252(5006):675–679CrossRefPubMedGoogle Scholar
  9. Doumith M, Legrand R, Lang C, Salas JA, Raynal MC (1999) Interspecies complementation in Saccharopolyspora erythraea: elucidation of the function of oleP1, oleG1 and oleG2 from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus and generation of new erythromycin derivatives. Mol Microbiol 34(5):1039–1048CrossRefPubMedGoogle Scholar
  10. Gryczan T, Shivakumar A, Dubnau D (1980) Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis. J Bacteriol 141(1):246–253PubMedCentralPubMedGoogle Scholar
  11. Han AR, Park JW, Lee MK, Ban YH, Yoo YJ, Kim EJ, Kim E, Kim BG, Sohng JK, Yoon YJ (2011a) Development of a Streptomyces venezuelae-based combinatorial biosynthetic system for the production of glycosylated derivatives of doxorubicin and its biosynthetic intermediates. Appl Environ Microbiol 77(14):4912–4923PubMedCentralCrossRefPubMedGoogle Scholar
  12. Han S, Song P, Ren T, Huang X, Cao C, Zhang B (2011b) Identification of SACE_7040, a member of TetR family related to the morphological differentiation of Saccharopolyspora erythraea. Curr Microbiol 63(2):121–125CrossRefPubMedGoogle Scholar
  13. Hong JS, Park SJ, Parajuli N, Park SR, Koh HS, Jung WS, Choi CY, Yoon YJ (2007) Functional analysis of DesVIII homologues involved in glycosylation of macrolide antibiotics by interspecies complementation. Gene 386(1–2):123–130CrossRefPubMedGoogle Scholar
  14. Kao CL, Borisova SA, Kim HJ, Liu HW (2006) Linear aglycones are the substrates for glycosyltransferase DesVII in methymycin biosynthesis: analysis and implications. J Am Chem Soc 128(17):5606–5607PubMedCentralCrossRefPubMedGoogle Scholar
  15. Kieser T, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. The John Innes Foundation, NorwichGoogle Scholar
  16. McDaniel R, Welch M, Hutchinson CR (2005) Genetic approaches to polyketide antibiotics. 1. Chem Rev 105(2):543–558CrossRefPubMedGoogle Scholar
  17. Moncrieffe MC, Fernandez MJ, Spiteller D, Matsumura H, Gay NJ, Luisi BF, Leadlay PF (2012) Structure of the glycosyltransferase EryCIII in complex with its activating P450 homologue EryCII. J Mol Biol 415(1):92–101CrossRefPubMedGoogle Scholar
  18. Park SR, Han AR, Ban Y-H, Yoo YJ, Kim EJ, Yoon YJ (2010) Genetic engineering of macrolide biosynthesis: past advances, current state, and future prospects. Appl Microbiol Biotechnol 85(5):1227–1239CrossRefPubMedGoogle Scholar
  19. Salah-Bey K, Doumith M, Michel JM, Haydock S, Cortes J, Leadlay PF, Raynal MC (1998) Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea. Mol Gen Genet 257(5):542–553CrossRefPubMedGoogle Scholar
  20. Salas JA, Mendez C (2007) Engineering the glycosylation of natural products in actinomycetes. Trends Microbiol 15(5):219–232CrossRefPubMedGoogle Scholar
  21. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  22. Shinde PB, Han AR, Cho J, Lee SR, Ban YH, Yoo YJ, Kim EJ, Kim E, Song MC, Park JW, Lee DG, Yoon YJ (2013) Combinatorial biosynthesis and antibacterial evaluation of glycosylated derivatives of 12-membered macrolide antibiotic YC-17. J Biotechnol 168(2):142–148CrossRefPubMedGoogle Scholar
  23. Staunton J, Weissman KJ (2001) Polyketide biosynthesis: a millennium review. Nat Prod Rep 18(4):380–416CrossRefPubMedGoogle Scholar
  24. Summers RG, Donadio S, Staver MJ, Wendt-Pienkowski E, Hutchinson CR, Katz L (1997) Sequencing and mutagenesis of genes from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in L-mycarose and D-desosamine production. Microbiology 143(Pt 10):3251–3262Google Scholar
  25. Tsuji K, Goetz J (1978) HPLC as a rapid means of monitoring erythromycin and tetracycline fermentation processes. J Antibiot 31(4):302–308CrossRefPubMedGoogle Scholar
  26. Vara J, Lewandowska-Skarbek M, Wang YG, Donadio S, Hutchinson C (1989) Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus). J Bacteriol 171(11):5872–5881Google Scholar
  27. Volchegursky Y, Hu Z, Katz L, McDaniel R (2000) Biosynthesis of the anti-parasitic agent megalomicin: transformation of erythromycin to megalomicin in Saccharopolyspora erythraea. Mol Microbiol 37(4):752–762CrossRefPubMedGoogle Scholar
  28. Weber J, Leung J, Maine G, Potenz R, Paulus T, DeWitt J (1990) Organization of a cluster of erythromycin genes in Saccharopolyspora erythraea. J Bacteriol 172(5):2372–2383PubMedCentralPubMedGoogle Scholar
  29. Wilkinson CJ, Hughes-Thomas ZA, Martin CJ, Bohm I, Mironenko T, Deacon M, Wheatcroft M, Wirtz G, Staunton J, Leadlay PF (2002) Increasing the efficiency of heterologous promoters in actinomycetes. J Mol Microbiol Biotechnol 4(4):417–426PubMedGoogle Scholar
  30. Wu H, Chen M, Mao Y, Li W, Liu J, Huang X, Zhou Y, Ye BC, Zhang L, Weaver DT, Zhang B (2014) Dissecting and engineering of the TetR family regulator SACE_7301 for enhanced erythromycin production in Saccharopolyspora erythraea. Microb Cell Factories 13(1):158CrossRefGoogle Scholar
  31. Xu XJ, Huang XD, Zhao W, Guo JH, Chen HP, Cao C, Zhang BC (2010) Construction of Saccharopolyspora erythraea A226-ΔeryCIII mutant and identification of its product. Bull Acad Mil Med Sci 34(3):247–250Google Scholar
  32. Xue Y, Zhao L, H-w L, Sherman DH (1998) A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: architecture of metabolic diversity. Proc Natl Acad Sci 95(21):12111–12116PubMedCentralCrossRefPubMedGoogle Scholar
  33. Yuan Y, Chung HS, Leimkuhler C, Walsh CT, Kahne D, Walker S (2005) In vitro reconstitution of EryCIII activity for the preparation of unnatural macrolides. J Am Chem Soc 127(41):14128–14129PubMedCentralCrossRefPubMedGoogle Scholar
  34. Zhang C, Fu Q, Albermann C, Li L, Thorson JS (2007) The in vitro characterization of the erythronolide mycarosyltransferase EryBV and its utility in macrolide diversification. Chembiochem 8(4):385–390CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Hang Wu
    • 1
  • Weiwei Li
    • 1
  • Chen Xin
    • 1
  • Congming Zhang
    • 1
  • Yansheng Wang
    • 1
  • Shaohua Ren
    • 1
  • Min Ren
    • 1
  • Wei Zhao
    • 1
  • Li Yuan
    • 1
  • Zhongdong Xu
    • 3
  • Hualing Yuan
    • 3
  • Ming Geng
    • 3
  • Lixin Zhang
    • 1
    • 2
  • David T. Weaver
    • 1
  • Buchang Zhang
    • 1
  1. 1.Institute of Health Sciences, School of Life SciencesAnhui UniversityHefeiChina
  2. 2.CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of MicrobiologyChinese Academy of SciencesBeijingChina
  3. 3.School of Life SciencesHefei Normal UniversityHefeiChina

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