In vivo investigation to the macrolide-glycosylating enzyme pair DesVII/DesVIII in Saccharopolyspora erythraea
- 592 Downloads
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.
KeywordsSaccharopolyspora erythraea Glycosyltransferase DesVII/DesVIII EryCII/EryCIII EryBV
We are grateful to Prof. Xingyuan Yang from Anhui University for critical editing of the manuscript. This work was supported by the National Program on Key Basic Research Project (973 program, 2013CB734000), The National Natural Science Foundation of China (31300081, 31570074), and The Initial Foundation of Doctoral Scientific Research in Anhui University (01001904). LZ is an awardee for National Distinguished Young Scholar Program in China.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no competing interests.
- 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
- 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
- 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
- Kieser T, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. The John Innes Foundation, NorwichGoogle Scholar
- Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
- 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
- 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
- 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