Inactivation of gilGT, Encoding a C‐Glycosyltransferase, and gilOIII, Encoding a P450 Enzyme, Allows the Details of the Late Biosynthetic Pathway to Gilvocarcin V to be Delineated

Abstract

Resequencing of the gilGT gene, which encodes a putative glycosyltransferase (GT) that is 495 amino acids (aa) long, from the Streptomyces griseoflavus Gc3592 gilvocarcin V (GV) gene cluster, revealed that the previously reported gilGT indeed contains two genes. These are the larger gilGT, which encodes the C-glycosyltransferase GilGT (379 aa), and the smaller gilV gene, which encodes an enzyme of unknown function (116 aa). The gene gilV is located immediately upstream of gilGT in the GV gene cluster. In-frame deletion of gilGT created a mutant that accumulated defucogilvocarcin E (defuco-GE). The result proves the function of GilGT as a Cglycosyltransferase. Deletion of gilOIII, which is located immediately downstream of gilGT, led to a mutant that accumulated gilvocarcin E (GE). This confirms that the corresponding P450 enzyme, GilOIII, is involved in the vinyl-group formation of GV. Cross-feeding experiments in which GE, defuco-GE, and defucogilvocarcin V (defuco-GV) were fed to an early blocked mutant of the GV biosynthetic pathway, showed that neither GE nor any of the defuco-compounds was an intermediate of the pathway.

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

“…[233] Several natural products, such as gilvocarcin and enterobactin, contain only para-C-glycosides, which would be difficult to form with an O-glycosylation/rearrangement sequence. [18,234,235] Recent studies of UrdGT2, a C-GT involved in the biosynthesis of urdamycin in Streptomyces fradiae Tü2717, have provided important insights into the mechanism of C-glycosylation (Scheme 15 f). While the C-GT activity (198!…”

“…This strain was transformed with several plasmids encoding the production of different NDP-sugars, and each resulting strain was then fed 8-DMTC (235). [270] In these experiments, ElmGT was shown to attach l-olivose and l-rhamnose (its natural sugar substrate) onto 235 to generate 240 and 241 (Scheme 19 c).…”

Engineered Biosynthesis of Gilvocarcin Analogues with Altered Deoxyhexopyranose Moieties

“…The host strain S. lividans TK24 (cosG9B3-U Ϫ ) as well as the two recombinant strains S. lividans TK24 (cosG9B3-U Ϫ / pLN2) and S. lividans TK24 (cosG9B3-U Ϫ /pRHAM) also produced significant amounts of the previously described (17) sugar-free defucogilvocarcins, defucogilvocarcin M (structure 22), defucogilvocarcin V (structure 23), and defucogilvocarcin E (structure 24) (Fig. 3).…”

“…In addition, GV's activity is also attributed to a unique selective cross-linking of DNA and histone H3, a core component of the histone complex that plays an important role for DNA replication and transcription (2,8,22,23,30). The saccharide moiety, D-fucofuranose, of GV is essential for this activity, as it is believed to facilitate binding of histone H3 (17,18).…”

“…It was possible to shut down the biosynthesis of gilvocarcin's natural sugar donor through the inactivation of gilU, the 4-ketoreductase involved in the biosynthesis of GV's deoxysugar moiety. The inactivation of gilU afforded a more active gilvocarcin analogue, 4Ј-hydroxy gilvocarcin V (structure 5, 4Ј-OH GV), illustrating an inherent substrate flexibility of GilGT, the glycosyltranferase responsible for C-glycosylation in GV biosynthesis (17,18). Additionally, the improved bioactivity of 4Ј-OH GV over that of GV clearly demonstrated the importance of further investigations into modifying the glycosylation pattern of gilvocarcins.…”