Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin

Abstract

Two genes (mtmD and mtmE) were cloned and sequenced from the mithramycin producer Streptomyces argillaceus. Comparison with proteins in databases and enzymatic assays after expression in Escherichia coli showed that they encode a glucose-1-phosphate:TTP thymidylyl transferase and a TDP-D-glucose 4,6-dehydratase, respectively. The mtmD gene was inactivated by gene replacement, generating a nonproducing mutant that accumulates a tetracyclic compound designated premithramycinone. The identification of premithramycinone reveals new aspects of the mithramycin biosynthetic pathway and suggests that at least some glycosylations occur before breakage of the fourth ring.Mithramycin (also designated aureolic acid, plicamycin, mithracin, LA7017, and A2371) is an antitumor drug synthesized by different actinomycete species and has clinical application in the treatment of several tumors (14). It also possesses antibiotic activity against gram-positive but not gram-negative bacteria. Structurally, it is an aromatic polyketide containing a three-ring chromophoric aglycon derived from the condensation of 10 acetates (11). Genes for a type II polyketide synthase were cloned and sequenced from a mithramycin producer, Streptomyces argillaceus; their involvement in mithramycin biosynthesis has been demonstrated by insertional inactivation (9), and evidence in favor of the biosynthesis of the aglycon through a single polyketide backbone instead of two has been provided elsewhere (3). The mithramycin aglycon (mithramycinone) is glycosylated at positions 6 and 2 with disaccharide and trisaccharide moieties, respectively. These sugars belong to the large family of the 6-deoxyhexoses (6DOHs), which are synthesized through biosynthetic pathways that have early biosynthetic steps in common (8, 12). In 6DOH biosynthesis, first glucose-1-phosphate is converted into TDP-D-glucose by the action of a glucose-1-phosphate:TTP thymidylyl transferase. Then, a TDP-D-glucose 4,6-dehydratase is responsible for the conversion of TDP-D-glucose into TDP-D-4-keto-6-deoxyglucose. Further action of a 3,5-epimerase can direct TDP-D-4-keto-6-deoxyglucose into the L series of 6DOHs; in contrast, if there is no participation of an epimerase, the final sugars will be D-6DOHs. Further modifications in both L-and D-6DOHs via isomerization, methylation, reduction, dehydration, etc., can produce a great variety of 6DOHs, which are part of the structures of different actinomycete metabolites (12, 18).Here we report the cloning, sequencing, and heterologous expression of two genes involved in the biosynthesis of the sugar moieties of the antitumor drug mithramycin. These genes (mtmD and mtmE) encode a glucose-1-phosphate:TTP thymidylyl transferase and a TDP-D-glucose 4,6-dehydratase, respectively. We also report the construction of a non-mithra-FIG. 1. Schematic representation of the region sequenced from cosAR7 and the different deduced ORFs and its location with respect to the mithramycin polyketide synthase (PKS). The bar indicates the region showing homology to t...

Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics The GenBank accession number for the sequence of cosmid K1F2 is AF329398.

“…SuperCos-1 was purchased from Stratagene. pBSKT, an integrative vector carrying carbenicillin and thiostrepton resistances, was described by Lombo et al (1997). Carbenicillin (50 µg ml − ") and thiostrepton (50 µg ml − ") were used in the medium for selection of recombinant plasmids and strains.…”

Identification of the Novobiocin Biosynthetic Gene Cluster of Streptomyces spheroides NCIB 11891

“…A 2.1-kb EcoRI fragment from cosmid 9-6G, which comprises part of the novT gene, was cloned into the EcoRI site of vector pBSKT, a pBluescript SK(ϩ) derivative containing the thiostrepton resistance gene (36), resulting in pAM1. Restriction analysis showed that the novT gene fragment had the same orientation as the thiostrepton and the ampicillin resistance genes of the vector.…”

Purification and Characterization of a Monooxygenase Involved in the Biosynthetic Pathway of the Antitumor Drug Mithramycin

“…Mithramycin has been clinically used for the treatment of certain tumors, such as disseminated embryonic cell carcinoma, as well as for Paget's bone disease (12) and is also used for control of hypercalcemia in patients with malignant disease (1). The biosynthetic gene cluster for mithramycin has been fully characterized (2)(3)(4)(5)(6)9), and it has been found that although mithramycin is a tricyclic aromatic polyketide, its biosynthesis proceeds through tetracyclic intermediates. Many of these intermediates have been isolated from mutants blocked at different steps in the biosynthesis (2)(3)(4)(5)(6).…”

“…The biosynthetic gene cluster for mithramycin has been fully characterized (2)(3)(4)(5)(6)9), and it has been found that although mithramycin is a tricyclic aromatic polyketide, its biosynthesis proceeds through tetracyclic intermediates. Many of these intermediates have been isolated from mutants blocked at different steps in the biosynthesis (2)(3)(4)(5)(6). Insertional inactivation of one of the genes of the mithramycin cluster, mtmOIV, generated a non-mithramycinproducing mutant that accumulated a fully glycosylated tetracyclic intermediate, premithramycin B (6).…”