Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach

Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method. Antimicrobial activity screens and LC-MS analyses revealed that the production of the kalimantacin analogues in the mutated strains was abolished. These results support the idea that further insight in the specificity of downstream domains in nonribosomal peptide synthetases and polyketide synthases is required to efficiently engineer these strains in vivo

Thioester reduction and aldehyde transamination are universal steps in actinobacterial polyketide alkaloid biosynthesis

Actinobacteria produce a variety of polyketide alkaloids with unusual structures. Recently, it was shown that a type I modular polyketide synthase (PKS) is involved in the assembly of coelimycin P1, a polyketide alkaloid produced by Streptomyces coelicolor M145. However, the mechanisms for converting the product of the PKS to coelimycin P1 remain to be elucidated. Here we show that the C-terminal thioester reductase (TR) domain of the PKS and an ω-transaminase are responsible for release of the polyketide chain as an aldehyde and its subsequent reductive amination. Bioinformatics analyses identified numerous gene clusters in actinobacterial genomes that encode modular PKSs with a C-terminal TR domain and a homolog of the ω-transaminase. These are predicted to direct the biosynthesis of both known and novel polyketide alkaloids, suggesting that reductive chain release and transamination constitutes a conserved mechanism for the biosynthesis of such metabolites

The zeamine antibiotics affect the integrity of bacterial membranes

The zeamines (zeamine, zeamine I, and zeamine II) constitute an unusual class of cationic polyamine-polyketide-nonribosomal peptide antibiotics produced by Serratia plymuthica RVH1. They exhibit potent bactericidal activity, killing a broad range of Gram-negative and Gram-positive bacteria, including multidrug-resistant pathogens. Examination of their specific mode of action and molecular target revealed that the zeamines affect the integrity of cell membranes. The zeamines provoke rapid release of carboxyfluorescein from unilamellar vesicles with different phospholipid compositions, demonstrating that they can interact directly with the lipid bilayer in the absence of a specific target. DNA, RNA, fatty acid, and protein biosynthetic processes ceased simultaneously at subinhibitory levels of the antibiotics, presumably as a direct consequence of membrane disruption. The zeamine antibiotics also facilitated the uptake of small molecules, such as 1-N-phenylnaphtylamine, indicating their ability to permeabilize the Gram-negative outer membrane (OM). The valine-linked polyketide moiety present in zeamine and zeamine I was found to increase the efficiency of this process. In contrast, translocation of the large hydrophilic fluorescent peptidoglycan binding protein PBDKZ-GFP was not facilitated, suggesting that the zeamines cause subtle perturbation of theOMrather than drastic alterations or defined pore formation. At zeamine concentrations above those required for growth inhibition, membrane lysis occurred as indicated by time-lapse microscopy. Together, these findings show that the bactericidal activity of the zeamines derives from generalized membrane permeabilization, which likely is initiated by electrostatic interactions with negatively charged membrane components

Pentamycin biosynthesis in Philippine Streptomyces sp. S816 : cytochrome P450-catalyzed installation of the C-14 hydroxyl group

Pentamycin is a polyene antibiotic, registered in Switzerland for the treatment of vaginal candidiasis, trichomoniasis, and mixed infections. Chemical instability has hindered its widespread application and development as a drug. Here, we report the identification of Streptomyces sp. S816, isolated from Philippine mangrove soil, as a pentamycin producer. Genome sequence analysis identified the putative pentamycin biosynthetic gene cluster, which shows a high degree of similarity to the gene cluster responsible for filipin III biosynthesis. The ptnJ gene, which is absent from the filipin III biosynthetic gene cluster, was shown to encode a cytochrome P450 capable of converting filipin III to pentamycin. This confirms that the cluster directs pentamycin biosynthesis, paving the way for biosynthetic engineering approaches to the production of pentamycin analogues. Several other Streptomyces genomes were found to contain ptnJ orthologues clustered with genes encoding polyketide synthases that appear to have similar architectures to those responsible for the assembly of filipin III and pentamycin, suggesting pentamycin production may be common in Streptomyces species

Structural basis for chain release from the enacyloxin polyketide synthase

Modular polyketide synthases and nonribosomal peptide synthetases are molecular assembly lines consisting of several multienzyme subunits that undergo dynamic self-assembly to form a functional mega-complex. N- and C-terminal docking domains are usually responsible for mediating interactions between subunits. Here we show that communication between two nonribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a ß-hairpin docking domain. The structures, interactions and dynamics of these subunits are characterised using several complementary biophysical techniques, providing extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/ß-hairpin docking domain pairs mediate subunit interactions in numerous nonribosomal peptide and hybrid polyketide-nonribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and ß-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering

Elucidation of the relative and absolute stereochemistry of the kalimantacin/batumin antibiotics

A multidisciplinary approach combining natural product degradation, fragment synthesis, bioinformatics and NMR spectroscopy was used.</p

Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites

A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis

Type I PKSs often utilise programmed β-branching, via enzymes of an “HMG-CoA synthase (HCS) cassette”, to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules