DKxanthene biosynthesis--understanding the basis for diversity-oriented synthesis in myxobacterial secondary metabolism.

The DKxanthenes are a family of yellow pigments which play a critical role in myxobacterial development. Thirteen unique structures from Myxococcus xanthus DK1622 differ in the length of their characteristic polyene functionality, as well as the extent of methyl branching. We aimed to understand the mechanistic basis for this "molecular promiscuity" by analyzing the gene cluster in DK1622, and comparing it to the DKxanthene biosynthetic locus in a second myxobacterium, Stigmatella aurantiaca DW4/3-1, which produces a more limited range of compounds. While the core biosynthetic machinery is highly conserved, M. xanthus contains a putative asparagine hydroxylase function which is not present in S. aurantiaca. This observation accounts, in part, for the significantly larger metabolite family in M. xanthus. Detailed analysis of the encoded hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) assembly line provides direct evidence for the mechanism underlying the variable polyene length and the observed pattern of methyl functionalities.

Identification and analysis of the core biosynthetic machinery of tubulysin, a potent cytotoxin with potential anticancer activity.

Myxobacteria are well known for their biosynthetic potential, especially for the production of cytotoxic compounds with potential anticancer activities. The tubulysins are currently in preclinical development. They are produced in very low quantities, and genetic manipulation of producing strains has never been accomplished. We report the development of a mariner-based transposon mutagenesis system for Angiococcus disciformis An d48. Extracts from a library of 1200 mutants were analyzed for the presence of tubulysin by a microscopic cell nucleus fragmentation bioassay. The transposition sites of four tubulysin-negative mutants were identified by vector recovery, which led to the identification and the sequencing of the corresponding core biosynthetic gene locus. Sequence analysis of more than 80,000 bp reveals an unusual multimodular hybrid polyketide synthase/peptide synthetase assembly line with a variety of unprecedented features.

Critical variations of conjugational DNA transfer into secondary metabolite multiproducing Sorangium cellulosum strains So ce12 and So ce56: development of a mariner-based transposon mutagenesis system.

Myxobacteria increasingly gain attention as a source of bioactive natural products. The genus Sorangium produces almost half of the secondary metabolites isolated from these microorganisms. Nevertheless, genetic systems for Sorangium strains are poorly developed, which makes the identification of the genes directing natural product biosynthesis difficult. Using biparental and triparental mating, we have developed methodologies for DNA transfer from Escherichia coli via conjugation for the genome sequencing model strain So ce56 and the secondary metabolite multiproducing strain So ce12. The conjugation protocol developed for strain So ce56 is not applicable to other Sorangium strains. Crucial points for the conjugation are the ratio of E. coli and Sorangium cellulosum cells, the choice of liquid or solid medium, the time used for the conjugation process and antibiotic selection in liquid medium prior to the plating of cells. A mariner-based transposon containing a hygromycin resistance gene was generated and used as the selectable marker for S. cellulosum. The transposon randomly integrates into the chromosome of both strains. As a proof of principle, S. cellulosum So ce12 transposon mutants were screened using an overlay assay to target the chivosazole biosynthetic gene cluster.

Completing the puzzle of aurachin biosynthesis in Stigmatella aurantiaca Sg a15.

The aurachins are a family of secondary metabolites, with the main members aurachin A, B, C, and D, produced by the myxobacterium Stigmatella aurantiaca Sg a15. These isoprenoid quinoline alkaloids are classified as A-type or C-type aurachins according to the position of the farnesyl residue either at C4 or C3 of the quinoline core, respectively. Previous feeding studies revealed that the C-type aurachins are converted to A-type aurachins by late stage tailoring reactions. While the core gene cluster coding for the functionalities required for the biosynthesis of the basic structure aurachin D is known, neither of the genes encoding for the successively acting tailoring enzymes was known up to date, which was assumed to be due to a split cluster organisation. Here we describe the identification of a total of five genes, located upstream of the aurachin core cluster and at additional two loci elsewhere in the genome, encoding for the aforementioned functionalities. The generation and evaluation of respective inactivation mutants of S. aurantiaca Sg a15 allowed for the first time to propose an exhaustive model for aurachin biosynthesis. One of the deduced biosynthetic transformations corresponds to a pinacol rearrangement, an unprecedented tailoring reaction in secondary metabolite biosynthesis.

Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase.

The production of epothilone mixtures is a direct consequence of the substrate tolerance of the module 3 acyltransferase (AT) domain of the epothilone polyketide synthase (PKS) which utilises both malonyl- and methylmalonyl-CoA extender units. Particular amino acid motifs in the active site of AT domains influence substrate selection for methylmalonyl-CoA (YASH) or malonyl-CoA (HAFH). This motif appears in hybrid form (HASH) in epoAT3 and may represent the molecular basis for the relaxed specificity of the domain. To investigate this possibility the AT domains from modules 2 and 3 of the epothilone PKS were examined in the heterologous DEBS1-TE model PKS. Substitution of AT1 of DEBS1-TE by epoAT2 and epoAT3 both resulted in functional PKSs, although lower yields of total products were observed when compared to DEBS1-TE (2% and 11.5% respectively). As expected, epoAT3 was significantly more promiscuous in keeping with its nature during epothilone biosynthesis. When the mixed motif (HASH) of epoAT3 within the hybrid PKS was mutated to HAFH (indicative of malonyl-CoA selection) it resulted in a non-productive PKS. When this mixed motif was converted to YASH (indicative of methylmalonyl-CoA selection) the selectivity of the hybrid PKS for methylmalonyl-CoA showed no statistically significant increase, and was associated with a loss of productivity.

Evolutionary implications of bacterial polyketide synthases.

Polyketide synthases (PKS) perform a stepwise biosynthesis of diverse carbon skeletons from simple activated carboxylic acid units. The products of the complex pathways possess a wide range of pharmaceutical properties, including antibiotic, antitumor, antifungal, and immunosuppressive activities. We have performed a comprehensive phylogenetic analysis of multimodular and iterative PKS of bacteria and fungi and of the distinct types of fatty acid synthases (FAS) from different groups of organisms based on the highly conserved ketoacyl synthase (KS) domains. Apart from enzymes that meet the classification standards we have included enzymes involved in the biosynthesis of mycolic acids, polyunsaturated fatty acids (PUFA), and glycolipids in bacteria. This study has revealed that PKS and FAS have passed through a long joint evolution process, in which modular PKS have a central position. They appear to have derived from bacterial FAS and primary iterative PKS and, in addition, share a common ancestor with animal FAS and secondary iterative PKS. Furthermore, we have carried out a phylogenomic analysis of all modular PKS that are encoded by the complete eubacterial genomes currently available in the database. The phylogenetic distribution of acyltransferase and KS domain sequences revealed that multiple gene duplications, gene losses, as well as horizontal gene transfer (HGT) have contributed to the evolution of PKS I in bacteria. The impact of these factors seems to vary considerably between the bacterial groups. Whereas in actinobacteria and cyanobacteria the majority of PKS I genes may have evolved from a common ancestor, several lines of evidence indicate that HGT has strongly contributed to the evolution of PKS I in proteobacteria. Discovery of new evolutionary links between PKS and FAS and between the different PKS pathways in bacteria may help us in understanding the selective advantage that has led to the evolution of multiple secondary metabolite biosyntheses within individual bacteria.