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Mycolactone is a diffusible lipid secreted by the human pathogen Mycobacterium ulcerans, which induces the formation of open skin lesions referred to as Buruli ulcers. Here, we show that mycolactone [...]

Meridamycin is a non-immunosuppressant, FKBP-binding macrocyclic polyketide, which has major potential as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke. A biosynthetic cluster predicted to encode biosynthesis of meridamycin was cloned from the prolific secondary-metabolite-producing strain Streptomyces sp. DSM 4137, not previously known to produce this compound, and specific gene deletion was used to confirm the role of this cluster in the biosynthesis of meridamycin. The meridamycin modular polyketide synthase consists of 14 extension modules distributed between three giant multienzyme proteins. The terminal module is flanked by a highly unusual cytochrome P450-like domain. The characterization of the meridamycin biosynthetic locus in this readily manipulated streptomycete species opens the way to the engineering of new, altered meridamycins of potential therapeutic importance. DOI 10.1099/mic.0.29176-0

The macrolide antibiotic concanamycin A has been identified as an exceptionally potent inhibitor of the vacuolar (V-type) ATPase. Such compounds have been mooted as the basis of a potential drug treatment for osteoporosis, since the V-ATPase is involved in the osteoclast-mediated bone resorption that underlies this common condition. To enable combinatorial engineering of altered concanamycins, the biosynthetic gene cluster governing the biosynthesis of concanamycin A has been cloned from Streptomyces neyagawaensis and shown to span a region of over 100 kbp of contiguous DNA. An efficient transformation system has been developed for S. neyagawaensis and used to demonstrate the role of the cloned locus in the formation of concanamycin A. Sequence analysis of the 28 ORFs in the region has revealed key features of the biosynthetic pathway, in particular the biosynthetic origin of portions of the backbone, which arise from the unusual polyketide building blocks ethylmalonyl-CoA and methoxymalonyl-ACP, and the origin of the pendant deoxysugar moiety 4'-O-carbamoyl-2'-deoxyrhamnose, as well as the presence of a modular polyketide synthase (PKS) encoded by six giant ORFs. Examination of the methoxymalonyl-specific acyltransferase (AT) domains has led to recognition of an amino acid sequence motif which can be used to distinguish methylmalonyl-CoA- from methoxymalonyl-ACP-specific AT domains in natural PKSs.

The 174-kb virulence plasmid pMUM001 in Mycobacterium ulcerans epidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation of M. ulcerans Agy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverse M. ulcerans strains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in all M. ulcerans strains. Physical mapping of these plasmids revealed that like M. ulcerans Agy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis that M. ulcerans diverged from the closely related organism Mycobacterium marinum by acquiring pMUM. Out results suggest that pMUM is a defining characteristic of M. ulcerans but that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.

Research has been conducted on the ketoreductases. The investigation of the beta-ketoacyl reductase stereocontrol has been carried out and the results indicate that the stereochemical course of reduction can be monitored via specific substrate analogues and a general substrate analogue modeling substrate for the ketoreductase fatty acid synthase activity.

The His244 residue of adenosylcobalamin-dependent methylmalonyl-CoA mutase appears to be involved in shielding the active site from water to protect the reactive radical intermediates formed during catalysis. The active site is deeply buried inside the enzyme and an His244 mutant created a cavity in the active site containing a water molecule.

An element of aromatic polyketide synthases (PKS), previously called 'chain-length factor' (CLF), has been shown to be a factor needed for polyketide chain initiation. This factor has decarboxylase activity towards malonyl-acyl carrier protein. The discovery of the common function of CLF and KSQ, a domain of previously unknown function which acts like CLF, as chain initiation factors supplies a further significant mechanistic connection between type I and type II PKSs and between PKSs and fatty-acid synthases.

There may be at least three specific roles for methylmalonyl-CoA in the AdoCbl-dependent skeletal rearrangement catalyzed by methylmalonyl-CoA mutase. There is little information about the involvement of active-site amino acids in catalysis of the radical rearrangement. Only a few polar active-site amino acids were found to be in direct contact with the substrate, particularly histidine 244, tyrosine 89 and arginine 207.

Complex polyketides that provide innumerable, significant biological properties are bio-synthesized by polyketide synthases (PKS). PKS-derived compounds are structurally diverse. Keen interest has been expressed for combining the functions of different PKS-derived compounds through genetic engineering to produce a vast amount of innovative compounds. A variety of strategies have been suggested including the use of the beta-ketosynthase (KS) domain of the module 1 found in PKSs using a bimodular derivative of the PKS called 6-deoxyerythronolide B synthase (DEBS).

Research was conducted to examine the use of a (phi)c31 page-mediated gene replacement in the frameshift mutation of a rapL gene lying within the region of the Streptomyces hygroscopicus chromosome. Results show that the wild-type levels of rapamycin production can be restored by growing the rapL mutant on media with L-pipecolate. The synthesis of rapamycin analogs using the rapL mutants were also observed.

The wide-specificity loading module for the avermectin-producing polyketide synthase was grafted onto the first multienzyme component (DEBS1) of the erythromycin-producing polyketide synthase in place of the normal loading module. Expression of this hybrid enzyme in the erythromycin producer Saccharopolyspora erythraea produced several novel antibiotic erythromycins derived from endogenous branched-chain acid starter units typical of natural avermectins. Because the avermectin polyketide synthase is known to accept more than 40 alternative carboxylic acids as starter units, this approach opens the way to facile production of novel analogs of antibiotic macrolides., The emergence of pathogenic bacteria multiply resistant to antibiotics represents a growing threat to human health[1] and has given added impetus to the search for new drugs and for more [...]

Modular type I polyketide synthases (PKSs), including 6-deoxyerythronolide B synthase (DEBS), are responsible for the synthesis of macrolide antibiotic erythromycin. Macrolides were noted by Celmer to have the same configuration at all comparable sterocenters. In vitro studies with DEBS 1-TE, the bimodular derivative of DEBS from Saccharopolyspora erythraea, aimed to elucidate the molecular basis of Celmer's rules. It is shown that condensation of (2S)-methylmalonyl-CoA in module 2 proceeds without cleavage of the C-H bond next tot he methyl group, although the process involves loss of hydrogen in module 1.

The effects of tritium loss from 5'-[3H]adenosylcobalamin during the isomerization of methylmalonyl-CoA and succinyl-CoA and of its subsequent appearance in the substrate and reaction product were investigated by measuring the kinetics of the reaction. The primary kinetic tritium isotope of 4.9 on enzyme-catalyzed hydrogen transfer was found to be lower than the normal range, suggesting that hydrogen transfer is not a rate-limiting step in the reaction.