Identification of two genes from Streptomyces argillaceus encoding glycosyltransferases involved in transfer of a disaccharide during biosynthesis of the antitumor drug mithramycin

Abstract

Mithramycin is an antitumor polyketide drug produced by Streptomyces argillaceus that contains two deoxysugar chains, a disaccharide consisting of two D-olivoses and a trisaccharide consisting of a D-olivose, a D-oliose, and a D-mycarose. From a cosmid clone (cosAR3) which confers resistance to mithramycin in streptomycetes, a 3-kb PstI-XhoI fragment was sequenced, and two divergent genes (mtmGI and mtmGII) were identified. Comparison of the deduced products of both genes with proteins in databases showed similarities with glycosyltransferases and glucuronosyltransferases from different sources, including several glycosyltransferases involved in sugar transfer during antibiotic biosynthesis. Both genes were independently inactivated by gene replacement, and the mutants generated (M3G1 and M3G2) did not produce mithramycin. High-performance liquid chromatography analysis of ethyl acetate extracts of culture supernatants of both mutants showed the presence of several peaks with the characteristic spectra of mithramycin biosynthetic intermediates. Four compounds were isolated from both mutants by preparative high-performance liquid chromatography, and their structures were elucidated by physicochemical methods. The structures of these compounds were identical in both mutants, and the compounds are suggested to be glycosylated intermediates of mithramycin biosynthesis with different numbers of sugar moieties attached to C-12a-O of a tetracyclic mithramycin precursor and to C-2-O of mithramycinone: three tetracyclic intermediates containing one sugar (premithramycin A1), two sugars (premithramycin A2), or three sugars (premithramycin A3) and one tricyclic intermediate containing a trisaccharide chain (premithramycin A4). It is proposed that the glycosyltransferases encoded by mtmGI and mtmGII are responsible for forming and transferring the disaccharide during mithramycin biosynthesis. From the structures of the new metabolites, a new biosynthetic sequence regarding late steps of mithramycin biosynthesis can be suggested, a sequence which includes glycosyl transfer steps prior to the final shaping of the aglycone moiety of mithramycin.

FIG. 1

Structures of mithramycin, premithramycinone, and the new premithramycins.

FIG. 2

(A) Schematic representation of the region sequenced from cosAR3 and location of the mtmGI and mtmGII genes with respect to previously reported genes from the mithramycin gene cluster. Mithramycin sugar biosynthetic genes (22) and mithramycin polyketide synthase genes (21) were designated mtm genes, while mithramycin resistance genes (11) were designated mtr genes. B, BamHI; G, BglII; H, SphI; L, SalI; N, NotI; P, PstI; S, SmaI; T, StuI; X, XhoI. (B) Alignment of the deduced amino acid sequences of different GTFs involved in antibiotic biosynthesis. MtmGI and MtmGII, mithramycin GTFs from S. argillaceus (this work); OleG1 and OleG2, oleandomycin GTFs from S. antibioticus (25); EryCIII and EryBV, desosaminyl and mycarosyl GTFs from Saccharopolyspora erythraea (12, 38); DnrH, baumycin GTF from S. peucetius (36); DnrS, daunorubicin GTF from S. peucetius (26); DauH, daunomycin GTF from Streptomyces sp. strain C5 (GenBank accession no. U43704); Gra-Orf5, granaticin GTF from S. violaceoruber (2); TylM2, tylosin GTF from S. fradiae (14).

FIG. 3

Analysis of gene replacement in the generation of mutants M3G1 and M3G2. (A) Scheme representing the replacement in the chromosome of the wild-type mtmGI and mtmGII genes by the in vitro-mutated ones. In the case of the mtmGI gene, an apramycin resistance cassette (black inverted triangle) was subcloned into the blunt-ended SalI site of mtmGI (generating mutant M3G1). In the case of mtmGII, the apramycin resistance cassette was subcloned into the unique SmaI site of mtmGII (generating mutant M3G2). B, BamHI; L, SalI; N, NotI; P, PstI; S, SmaI; X, XhoI. The asterisks above the two PstI sites indicate the boundaries of the probe used. (B) Southern hybridization with the 2.3-kb PstI fragment as the ³²P-labeled probe. Chromosomal DNA from the wild-type strain and that from mutants M3G1 and M3G2 were digested with SmaI and analyzed by Southern hybridization. Lane 1, SmaI-digested chromosomal DNA from the wild-type strain. Lane 2, SmaI-digested chromosomal DNA from mutant M3G1. Lane 3, SmaI-digested chromosomal DNA from mutant M3G2.

FIG. 4

HPLC analysis of the products accumulated by mutants M3G1 and M3G2. The mobility of mithramycin under the chromatographic conditions used was 17.8 min, a retention time between peaks B and C.

FIG. 5

Proposed pathways for mithramycin biosynthesis. CoA, coenzyme A.