Heterologous production of myxobacterial α-pyrone antibiotics in Myxococcus xanthus.

Myxopyronins (MXN) and corallopyronins (COR) are structurally related α-pyrone antibiotics from myxobacteria that represent a highly promising compound class for the development of broad-spectrum antibacterial therapeutic agents. Their ability to inhibit RNA polymerase through interaction with the "switch region", a novel target, distant from previously characterized RNA polymerase inhibitors (e.g. rifampicin), makes them particularly promising candidates for further research. To improve compound supply for further investigation of MXN, COR and novel derivatives of these antibacterial agents, establishment of an efficient and versatile microbial production platform for myxobacterial α-pyrone antibiotics is highly desirable. Here we describe design, construction and expression of a heterologous production and engineering platforms for MXN and COR to facilitate rational structure design and yield improvement approaches in the myxobacterial host strain Myxococcus xanthus DK1622. Optimization of the cultivation medium yielded significantly higher production titers of MXN A at around 41-fold increase and COR A at around 25-fold increase, compared to the standard CTT medium.

AibA/AibB Induces an Intramolecular Decarboxylation in Isovalerate Biosynthesis by Myxococcus xanthus.

Isovaleryl coenzyme A (IV-CoA) is an important precursor for iso-fatty acids and lipids. It acts in the development of myxobacteria, which can produce this compound from acetyl-CoA through alternative IV-CoA biosynthesis (aib). A central reaction of aib is catalyzed by AibA/AibB, which acts as a cofactor-free decarboxylase despite belonging to the family of CoA-transferases. We developed an efficient expression system for AibA/AibB that allowed the determination of high-resolution crystal structures in complex with different ligands. Through mutational studies, we show that an active-site cysteine previously proposed to be involved in decarboxylation is not required for activity. Instead, AibA/AibB seems to induce an intramolecular decarboxylation by binding its substrate in a hydrophobic cavity and forcing it into a bent conformation. Our study opens opportunities for synthetic biology studies, since AibA/AibB may be suitable for the production of isobutene, a precursor of biofuels and chemicals.

Genomics-Guided Exploitation of Lipopeptide Diversity in Myxobacteria.

Analysis of 122 myxobacterial genome sequences suggested 16 strains as producers of the myxochromide lipopeptide family. Detailed sequence comparison of the respective mch biosynthetic gene clusters informed a genome-mining approach, ultimately leading to the discovery and chemical characterization of four novel myxochromide core types. The myxochromide megasynthetase is subject to evolutionary diversification, resulting in considerable structural diversity of biosynthesis products. The observed differences are due to the number, type, sequence, and configuration of the incorporated amino acids. The analysis revealed molecular details on how point mutations and recombination events led to structural diversity. It also gave insights into the evolutionary scenarios that have led to the emergence of mch clusters in different strains and genera of myxobacteria.

Solving the Puzzle of One-Carbon Loss in Ripostatin Biosynthesis.

Ripostatin is a promising antibiotic that inhibits RNA polymerase by binding to a novel binding site. In this study, the characterization of the biosynthetic gene cluster of ripostatin, which is a peculiar polyketide synthase (PKS) hybrid cluster encoding cis- and trans-acyltransferase PKS genes, is reported. Moreover, an unprecedented mechanism for phenyl acetic acid formation and loading as a starter unit was discovered. This phenyl-C2 unit is derived from phenylpyruvate (phenyl-C3) and the mechanism described herein explains the mysterious loss of one carbon atom in ripostatin biosynthesis from the phenyl-C3 precursor. Through in vitro reconstitution of the whole loading process, a pyruvate dehydrogenase like protein complex was revealed that performs thiamine pyrophosphate dependent decarboxylation of phenylpyruvate to form a phenylacetyl-S-acyl carrier protein species, which is supplied to the subsequent biosynthetic assembly line for chain extension to finally yield ripostatin.

Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within E1.

UNLABELLED: To explore mechanisms of hepatitis C viral (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. CONCLUSION: These observations reveal novel details about HCV membrane fusion; moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as a cost-effective component of HCV combination therapies.

Biosynthesis of thuggacins in myxobacteria: comparative cluster analysis reveals basis for natural product structural diversity.

The thuggacins are macrolide antibiotics that are active against Mycobacterium tuberculosis, the causative agent of tuberculosis. Distinct variants of these structures are produced by the myxobacteria Sorangium cellulosum So ce895 and Chondromyces crocatus Cm c5, which differ in side chain structure and modification by hydroxylation. We report here a comparative analysis of the biosynthetic gene clusters in these strains, which reveals the mechanistic basis for this architectural diversity. Although the polyketide-nonribosomal peptide cores of the molecules are highly similar, the underlying biosynthetic machineries exhibit an unexpected degree of divergence. Furthermore, the S. cellulosum gene cluster contains a crotonyl-CoA reductase (CCR) homolog not present in C. crocatus, which likely participates in assembling the unusual hexyl side chain of the So ce895 thuggacins, whereas the distinct hydroxylation pattern may result from variable action of a conserved FMN-dependent monooxygenase. Indeed, inactivation of the monooxygenase gene in C. crocatus resulted in production of both mono- and di-deshydroxy thuggacin derivatives, providing direct evidence for the role of this enzyme in the pathway. Finally, integration of a Tn5-derived npt promotor upstream of the thuggacin cluster in C. crocatus led to a significant increase in thuggacin production.