Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea
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O R I GI N A L P A P E R
M. Doumith á P. Weingarten á U. F. Wehmeier K. Salah-Bey á B. Benhamou á C. Capdevila J.-M. Michel á W. Piepersberg á M.-C. Raynal
Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea Received: 2 December 1999 / Accepted: 20 July 2000 / Published online: 25 August 2000 Ó Springer-Verlag 2000
Abstract Glycosylation represents an attractive target for protein engineering of novel antibiotics, because speci®c attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-Lmycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein puri®cation and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The Communicated by W. Goebel M. Doumith (&) á K. Salah-Bey á B. Benhamou J.-M. Michel á M.-C. Raynal Infectious Disease Group, Aventis Pharma, Hoechst Marion Roussel, 102 Route de Noisy, 93235 Romainville Cedex, France E-mail: [email protected] Tel.: +33-1-49914375; Fax: +33-1-49916380 P. Weingarten á U. F. Wehmeier á W. Piepersberg Bergische UniversitaÈt-GH, FB 9 Chemische Mikrobiologie, 42097 Wuppertal, Germany C. Capdevila Department of Biotechnology, Aventis Pharma, Hoechst Marion Roussel, 102 Route de Noisy, 93235 Romainville Cedex, France The ®rst two authors contributed equally to the work
substrate 3-a-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly con®rming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases. Key words Polyketide antibiotics á Glycosylation á Pathway engineering á Erythromycin á Saccharopolyspora erythraea
Introduction Erythromycin A is a clinically important antibiotic produced by the gram-positive bacterium Saccharopolyspora erythraea. Structurally, it consists of a 14-membered macrolactone ring to which are attached two deoxysugar residues, L-cladinose and D-desosamine. The biosynthetic pathway to erythromycin A has been extensively studied over the last 10 years because o
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