New Lessons for Combinatorial Biosynthesis from Myxobacteria

Abstract

The biosynthetic mta gene cluster responsible for myxothiazol formation from the fruiting body forming myxobacterium Stigmatella aurantiaca DW4/3-1 was sequenced and analyzed. Myxothiazol, an inhibitor of the electron transport via the bc 1 -complex of the respiratory chain, is biosynthesized by a unique combination of several polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS), which are activated by the 4-phosphopantetheinyl transferase MtaA. Genomic replacement of a fragment of mtaB and insertion of a kanamycin resistance gene into mtaA both impaired myxothiazol synthesis. Genes mtaC and mtaD encode the enzymes for bis-thiazol(ine) formation and chain extension on one pure NRPS (MtaC) and on a unique combination of PKS and NRPS (MtaD). The genes mtaE and mtaF encode PKSs including peptide fragments with homology to methyltransferases. These methyltransferase modules are assumed to be necessary for the formation of the proposed methoxy-and ␤-methoxy-acrylate intermediates of myxothiazol biosynthesis. The last gene of the cluster, mtaG, again resembles a NRPS and provides insight into the mechanism of the formation of the terminal amide of myxothiazol. The carbon backbone of an amino acid added to the myxothiazolacid is assumed to be removed via an unprecedented module with homology to monooxygenases within MtaG.Myxobacteria are Gram-negative soil bacteria that are assigned to the two suborders Cystobacterineae and Sorangineae. Both belong to the ␦-group of the Proteobacteria (1). They are distinguished from most other bacteria by their ability to glide in swarms, to feed cooperatively, and to form fruiting bodies upon starvation (2, 3). In addition, they have been shown to produce a wide variety of secondary metabolites with unique structures and biological activities (for reviews, see Refs. 4 and 5). These include the electron transport inhibitors myxothiazol (6), stigmatellin (7), and myxalamids (5, 8) produced by different strains of Stigmatella aurantiaca (Cystobacterineae) and the epothilones produced by Sorangium cellulosum (Sorangineae) (9) (structures are given in Fig. 1). Due to their antitumor activity, epothilones have attracted great attention (10 -12). Myxothiazol as well as epothilones contain a thiazole ring that is formed by the incorporation of cysteine into the polyketide backbone (13). Thiazoline and thiazolidine structures of bacitracin in Bacillus licheniformis (14) and the bacterial siderophores yersiniabactin and mycobactin have recently been shown to be biosynthesized by a NRPS 1 or a combined PKS/NRPS in Yersinia pestis and Mycobacterium tuberculosis (15)(16)(17) . 22)). No such combinations have been published so far for the formation of a thiazole coupled to a polyketide structure. In addition to the bis-thiazole moiety, myxothiazol has some unique features: the unusual leucine derived starter unit 3-methyl-butyryl-CoA (13) and the linear polyketide backbone, which includes a ␤-methoxy-acrylate and a terminal amide structure.Little is known about the biochemistry o...

Mining the Cinnabaramide Biosynthetic Pathway to Generate Novel Proteasome Inhibitors

“…The PCR fragments generated from cosmid C:A17 showed significant homology to KS and adenylation-domain sequences from the salinosporamide gene cluster. After cosmid end-sequencing from the T3 and the T7 termini of the pOJ436, the cosmid was thus sequenced on both strands, by use of a shotgun library as described previously, [40] and the sequence was introduced into the EMBL nucleotide database (accession number: FR687018).…”

A Unique Mechanism for Methyl Ester Formation via an Amide Intermediate Found in Myxobacteria

“…Bacterial strains and culture conditions: Escherichia coli and Stigmatella aurantiaca DW4/3-1 were cultivated as described previously [7,18].…”

“…For homologous recombination via mtaH, the insert of pESW26 [7] was isolated after digestion with NotI/SpeI and cloned into plasmid pMSW13-2 predigested with NotI/XbaI (Figure 4) to give pMSW20. The latter plasmid was transferred into S. aurantiaca DW4/3-1 by electroporation followed by a homologous recombination process as described previously [8], resulting in mutant strain ESW637.…”

“…Analysis of both biosynthetic gene clusters led to the proposed biosynthetic pathways depicted in Figure 2 [7,8] which are based on PKS/NRPS assembly logic [14]. In this study, the question of the formation of the methyl ester via the hypothetical amide intermediate in melithiazol and myxothiazol biosynthesis is addressed.…”

“…These include the antifungal agents myxothiazol and melithiazol (see Figure 1), which represent very potent inhibitors of the electron transport via the cytochrome bc1-complex of the eukaryotic respiratory chain [4,5]. Similar to the formation of most myxobacterial secondary metabolites that have been studied on the molecular level, melithiazol and myxothiazol are formed by large multifunctional and modular enzymes, termed polyketide synthases (PKSs) and/or nonribosomal peptide synthetases (NRPSs) [6][7][8][9][10]. The enzymology of these megasynthetases is subject to intensive studies and their modular logic has been reviewed recently [11][12][13][14].…”