Cloning and Characterization of the Ravidomycin and Chrysomycin Biosynthetic Gene Clusters

Abstract

The gene clusters responsible for the biosynthesis of two anti-tumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1-1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D-ravidosamine and D-virenose biosynthetic genes, post-PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross-complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino-and neutral sugars, exemplified through the structurally distinct 6-membered D-ravidosamine and 5-membered D-fucofuranose, to the coumarin-based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.

The Crystal Structure and Mechanism of an Unusual Oxidoreductase, GilR, Involved in Gilvocarcin V Biosynthesis

“…Our laboratory studies the biosynthetic pathways of selected members of the gilvocarcin group, and three closely related biosynthetic gene clusters (of gilvocarcin V 1, of ravidomycin V 4, and of chrysomycin A 5, supplemental Fig. S1) were cloned and characterized (14). Key steps of this pathway include the oxidative rearrangement cascade and the final dehydrogenation step catalyzed by GilR, which converts a hemiacetal into the central lactone moiety of the polyketide-derived tetracyclic ring skeleton of the gilvocarcins ( Fig.…”

“…S1) were cloned and characterized (13,14). Key steps of this pathway are the oxidative rearrangement, which converts an angucyclinone intermediate into the final benzo [d]naphtho [1,2-b]pyran-6-one frame and is initiated by an oxidative 5,6-bond cleavage (15,16).…”

Engineered Biosynthesis of Gilvocarcin Analogues with Altered Deoxyhexopyranose Moieties

“…Regenerated protoplasts were transferred to solid M2 agar supplemented with appropriate antibiotics. Initially, each strain was grown in 250-ml baffled Erlenmeyer flasks containing 100 ml of liquid SG medium (20 g/liter glucose, 10 g/liter soy peptone, 2 g/liter CaCO 3 , 0.001 g/liter cobalt-II chloride, pH 7.2) with appropriate antibiotics and screened for gilvocarcin analogue production using high-performance liquid chromatographymass spectrometry (HPLC-MS) as described previously (11). Escherichia coli XL1-Blue (Stratagene) was used as the subcloning host and was grown at 37°C in lysogeny broth medium, while E. coli ET12567/pUZ8002 (21, 29) was used for conjugation.…”

“…Fractions containing gilvocarcin analogues were dried and lyophilized. Further purification was obtained through high-performance liquid chromatography (HPLC) according to previously described procedures (11). NMR analysis.…”

Synthetic Biology in Streptomyces Bacteria

“…Cosmids: pOJ446, can be used in both E. coli and Streptomyces (Bierman et al, 1992). There are many examples of cloning large fragments of 40-70 kb (Kharel et al, 2010). pKC505, a useful cosmid-based shuttle vector for cloning of large constructs (Richardson et al, 1987), which can be conjugally transferred between Streptomyces strains.…”