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

Abstract

SummaryTwo glycosyltransferase genes, oleG1 and oleG2, and a putative isomerase gene, oleP1, have previously been identified in the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus. In order to identify which of these two glycosyltransferases encodes the desosaminyltransferase and which the oleandrosyltransferase, interspecies complementation has been carried out, using two mutant strains of Saccharopolyspora erythraea, one strain carrying an internal deletion in the eryCIII (desosaminyltransferase) gene and the other an internal deletion in the eryBV (mycarosyltransferase) gene. Expression of the oleG1 gene in the eryCIII deletion mutant restored the production of erythromycin A (although at a low level), demonstrating that oleG1 encodes the desosaminyltransferase required for the biosynthesis of oleandomycin and indicating that, as in erythromycin biosynthesis, the neutral sugar is transferred before the aminosugar onto the macrocyclic ring. Significantly, when an intact oleG2 gene (presumed to encode the oleandrosyltransferase) was expressed in the eryBV deletion mutant, antibiotic activity was also restored and, in addition to erythromycin A, new bioactive compounds were produced with a good yield. The neutral sugar residue present in these compounds was identified as L-rhamnose attached at position C-3 of an erythronolide B or a 6-deoxyerythronolide B lactone ring, thus indicating a relaxed specificity of the oleandrosyltransferase, OleG2, for both the activated sugar and the macrolactone substrate. The oleP1 gene located immediately upstream of oleG1 was likewise introduced into an eryCII deletion mutant of Sac. erythraea, and production of erythromycin A was again restored, demonstrating that the function of OleP1 is identical to that of EryCII in the biosynthesis of dTDP-D-desosamine, which we have previously proposed to be a dTDP-4-keto-6-deoxy-D-glucose 3,4-isomerase.

Identification and Expression of Genes Involved in Biosynthesis of l -Oleandrose and Its Intermediate l -Olivose in the Oleandomycin Producer Streptomyces antibioticus

“…This will require the use of "flexible" glycosyltransferases, and in this context, a few examples of glycosyltransferase substrate flexibility have been reported (8,46,61). The S. antibioticus OleG2 glycosyltransferase has been shown to transfer L-oleandrose and L-olivose (this work) and also L-rhamnose (12). This "substrate flexibility" of the OleG2 glycosyltransferase opens up the possibility of using this sugar transfer enzyme as a genetic tool for further studies leading to the generation of novel glycosylated macrolides.…”

“…The other two glycosyltransferases are involved in the transfer of D-desosamine and L-oleandrose to the oleandolide, as was demonstrated by gene disruption (35). Recently, it has been shown that oleG1 complements an S. erythraea mutant lacking eryCIII and that oleG2, when transformed into a mutant lacking eryBV, produces a new compound that contains a rhamnosyl residue attached to the C-3 position of the erythronolide B (12). Therefore, it has been proposed that OleG1 and OleG2 act as the D-desosaminyl-and L-oleandrosyl-glycosyltransferases, respectively (12).…”

“…Recently, it has been shown that oleG1 complements an S. erythraea mutant lacking eryCIII and that oleG2, when transformed into a mutant lacking eryBV, produces a new compound that contains a rhamnosyl residue attached to the C-3 position of the erythronolide B (12). Therefore, it has been proposed that OleG1 and OleG2 act as the D-desosaminyl-and L-oleandrosyl-glycosyltransferases, respectively (12). In order to test the capability of the putative oleandrose-biosynthetic genes to make dTDP-L-oleandrose, and the substrate flexibility of the OleG2 glycosyltransferase, we generated an appropriate S. albus strain expressing the OleG2 oleandrosyl glycosyltransferase.…”

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

“…For example, expression of the gene encoding the desosaminyltransferase (OleG1) from the oleandomyicn pathway in an S. erythraea mutant lacking the endogenous desosaminyltransferase (EryCIII) restored erythromycin A (206) production, [261] establishing the proposed functions for both EryCIII and OleG1. When the gene encoding the oleandrosyltransferase OleG2 from the oleandomyicn pathway was expressed in an S. erythraea mutant Scheme 16.…”

Functional Analysis of OleY l -Oleandrosyl 3- O -Methyltransferase of the Oleandomycin Biosynthetic Pathway in Streptomyces antibioticus

“…Insertional inactivation of oleG1 produced a mutant that accumulates the macrolactone 8,8a-deoxyoleandolide, because both OleG1 and OleG2 glycosyltransferases were affected in this mutant by a polar effect (16). Complementation experiments using eryBV-and eryCIII-minus mutants of the erythromycin producer Saccharopolyspora erythraea showed that oleG1 complemented an eryCIII-minus mutant and oleG2 complemented an eryBV-minus mutant (6). Therefore, it was concluded that D-desosamine and L-oleandrose were the substrates for the OleG1 and OleG2 glycosyltransferases, respectively.…”