Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea

Abstract

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific 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-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification 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 substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.

Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase

“…Escherichia coli ET12567 has been described by MacNeil et al (14); other E. coli strains were from commercial sources; S. lividans TK24 was described by Hopwood et al (15). Plasmid pWHM1238, described in Kulowski et al (1999), was kindly provided by Dr. Ben Shen (8); plasmid pUWL201, described in Doumith et al (2000), was kindly provided by Dr. Udo Wehmeier (16). Ultrafiltration centrifugation tubes (Centriplus YM series) were purchased from Millipore.…”

New Aminocoumarin Antibiotics Derived from 4-Hydroxycinnamic Acid are Formed after Heterologous Expression of a Modified Clorobiocin Biosynthetic Gene Cluster

“…The E. coli -Streptomyces shuttle vector pUWL201, containing the ermE* promoter [7] was used for the construction of the couL expression plasmid pMS91. Therefore, couL was amplified by PCR.…”

“…Subsequently, this modified cosmid was heterologously expressed in S. coelicolor M512 [3]. The amide synthetase gene couL of the coumermycin biosynthetic gene cluster was cloned into the Streptomyces expression vector pUWL201 [7] and introduced into the mutant by protoplast transformation as described previously [12]. When the resulting strain was cultivated as described in the Experimental, the formation of three aminocoumarin antibiotics was observed.…”

Biosynthesis of the unusual 5,5-gem-dimethyl-deoxysugar noviose: investigation of the C-methyltransferase gene cloU

“…The purified PCR product was restricted with BamHI and XbaI and ligated into the same sites of pBluescript SK(2) (Stratagene) to give pAF15. The BamHI-XbaI fragment of 1?3 kb from pAF15 was religated into the BamHI and XbaI restriction sites of the replicative vector pUWL201 (Doumith et al, 2000), downstream of the constitutive ermE* promoter, to give pAF16.…”