Novel Hybrid Tetracenomycins through Combinatorial Biosynthesis Using a Glycosyltransferase Encoded by the elm Genes in Cosmid 16F4 and Which Shows a Broad Sugar Substrate Specificity

Abstract

Cosmid 16F4 contains 25 kb of the elloramycin biosynthetic pathway of Streptomyces olivaceus Tü2353. Transformation of this cosmid into a polyketide synthase (PKS)-deleted mutant of the urdamycin producer, Streptomyces fradiae Tü2717/ΔPKS and into the mithramycin producer Streptomyces argillaceus ATCC 12956 resulted in the production of several novel glycosylated tetracenomycins. Four of the structures of these elloramycin analogues (3, 5−7) were elucidated. They carry various deoxysugar moieties (d-olivose, l-rhodinose, d-mycarose, and a disaccharide consisting of two 1,3-linked d-olivoses) attached at C-8-O of the same aglycon, 8-demethyltetracenomycin C (4). The transfer of the sugars is not catalyzed by glycosyltransferases of the S. fradiae or S. argillaceus strains since the novel hybrid tetracenomycins are also produced by a S. argillaceus mutant carrying cosmid 16F4 but lacking all the known mithramycin glycosyltransferases. Furthermore, a Streptomyces lividans strain containing cosmid 16F4 produced the novel tetracenomycins only when a second plasmid containing the cloned mithramycin sugar biosynthetic genes but lacking glycosyltransferase genes was also present. The glycosyl transfer therefore must be catalyzed by an elloramycin glycosyltransferase encoded by cosmid 16F4. Apparently, this glycosyltransferase is able to catalyze the glycosylation of 8-demethyltetracenomycin C (4, = 12a-demethylelloramycinone) using various d- and l-sugars including a disaccharide. Its future use for combinatorial biosynthetic approaches is discussed.

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

“…The two constructs shown in this work (pOLV and pOLE) are the first two plasmids reported capable of synthesizing dTDP-activated sugars, and so they have the potential to be used for modifying different aglycones. 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).…”

Deoxysugar Methylation during Biosynthesis of the Antitumor Polyketide Elloramycin by Streptomyces olivaceus

“…A peak corresponding to RHA-TCMC (as compared with a standard) was observed in all of the experiments, indicating that the L-rhamnose was being synthesized and that the glycosyltransferase ElmGT was linking L-rhamnose to 8DMTC. In addition, some other peaks were found in the experiments in which the three methyltrans- (27). ElmMI, ElmMII, and Elm-MIII are described in this paper.…”

“…In this way, three glycosylated derivatives containing D-sugars were also tested. They were D-olivosyl-, D-mycarosyl-, and D-diolivosyl-tetracenomycin C. These compounds were produced by expressing cosmid 16F4 into the mithramycin producer S. argillaceus (27). When these compounds were individually fed into S. albus clones EPM1, EPM2, and EPM3, no new biotransformation products were found (data not shown), indicating that none of the methyltransferases act on D-sugars.…”

Combinatorial Biosynthesis of Antitumor Deoxysugar Pathways in Streptomyces griseus : Reconstitution of “Unnatural Natural Gene Clusters” for the Biosynthesis of Four 2,6- d -Dideoxyhexoses

“…Moreover, previous experiments also suggested that the glycosyltransferase ElmGT was not able to recognize TDP-D-oliose as a substrate. Thus, it has been shown that by expressing cosmid 16F4 in the mithramycin producer S. argillaceus, no D-oliosyl-tetracenomycin C was obtained, despite the fact that this microorganism synthesizes TDP-D-oliose (44).…”

“…Structure elucidation of D-digitoxosyl-tetracenomycin C and D-boivinosyl-tetracenomycin C. The two novel tetracenomycins were characterized by liquid chromatography-MS (see below) and NMR spectroscopy in comparison with various previously described 8-position glycosylated tetracenomycins and elloramycins (11,31,44).…”