-
Microbiology Resource Announcements Apr 2024F29-3, a Gram-positive bacterium, is recognized for its synthesis of the antifungal substance fengycin. This announcement introduces the complete genome sequence and...
F29-3, a Gram-positive bacterium, is recognized for its synthesis of the antifungal substance fengycin. This announcement introduces the complete genome sequence and provides insights into the genetic products related to antibiotic secondary metabolites, including non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and NRPS/PKS combination.
PubMed: 38451104
DOI: 10.1128/mra.01246-23 -
Chemical Science Feb 2024Luteodienoside A is a novel glycosylated polyketide produced by the Australian fungus MST-FP2246, consisting of an unusual 1--β-d-glucopyranosyl--inositol (glucinol)...
Discovery and heterologous biosynthesis of glycosylated polyketide luteodienoside A reveals unprecedented glucinol-mediated product offloading by a fungal carnitine -acyltransferase domain.
Luteodienoside A is a novel glycosylated polyketide produced by the Australian fungus MST-FP2246, consisting of an unusual 1--β-d-glucopyranosyl--inositol (glucinol) ester of 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid. Mining the genome of identified a putative gene cluster for luteodienoside A biosynthesis (), harbouring a highly reducing polyketide synthase (HR-PKS, LtbA) fused at its C-terminus to a carnitine -acyltransferase (cAT) domain. Heterologous pathway reconstitution in , substrate feeding assays and gene truncation confirmed the identity of the cluster and demonstrated that the cAT domain is essential for offloading luteodienoside A from the upstream HR-PKS. Unlike previously characterised cAT domains, the LtbA cAT domain uses glucinol as an offloading substrate to release the product from the HR-PKS. Furthermore, the PKS methyltransferase (MT) domain is capable of catalysing -dimethylation of the 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid intermediate, without requiring reversible product release and recapture by the cAT domain. This study expands the repertoire of polyketide modifications known to be catalysed by cAT domains and highlights the potential of mining fungal genomes for this subclass of fungal PKSs to discover new structurally diverse secondary metabolites.
PubMed: 38425541
DOI: 10.1039/d3sc05008d -
Applied Microbiology and Biotechnology Feb 2024Members of the bacterial phylum Planctomycetota have recently emerged as promising and for the most part untapped sources of novel bioactive compounds. The...
Members of the bacterial phylum Planctomycetota have recently emerged as promising and for the most part untapped sources of novel bioactive compounds. The characterization of more than 100 novel species in the last decade stimulated recent bioprospection studies that start to unveil the chemical repertoire of the phylum. In this study, we performed systematic bioinformatic analyses based on the genomes of all 131 described members of the current phylum focusing on the identification of type III polyketide synthase (PKS) genes. Type III PKSs are versatile enzymes involved in the biosynthesis of a wide array of structurally diverse natural products with potent biological activities. We identified 96 putative type III PKS genes of which 58 are encoded in an operon with genes encoding a putative oxidoreductase and a methyltransferase. Sequence similarities on protein level and the genetic organization of the operon point towards a functional link to the structurally related hierridins recently discovered in picocyanobacteria. The heterologous expression of planctomycetal type III PKS genes from strains belonging to different families in an engineered Corynebacterium glutamicum strain led to the biosynthesis of pentadecyl- and heptadecylresorcinols. Phenotypic assays performed with the heterologous producer strains and a constructed type III PKS gene deletion mutant suggest that the natural function of the identified compounds differs from that confirmed in other bacterial alkylresorcinol producers. KEY POINTS: • Planctomycetal type III polyketide synthases synthesize long-chain alkylresorcinols. • Phylogenetic analyses suggest an ecological link to picocyanobacterial hierridins. • Engineered C. glutamicum is suitable for an expression of planctomycete-derived genes.
Topics: Humans; Planctomycetes; Phylogeny; Acyltransferases; Operon
PubMed: 38407604
DOI: 10.1007/s00253-024-13065-x -
Chemical Science Feb 2024Genome mining has facilitated the efficient discovery of untapped natural products. We performed global genome mining in fungi and discovered a series of biosynthetic...
Genome mining has facilitated the efficient discovery of untapped natural products. We performed global genome mining in fungi and discovered a series of biosynthetic gene clusters (BGCs) that appeared to afford polyketide-terpenoid hybrids a distinct biosynthetic mechanism from those adopted by known pathways. Characterization of one of the BGCs revealed that it yields the drimane-phthalide hybrid 1. During the biosynthesis of 1, the farnesyl group is unusually introduced by the dimethylallyltryptophan synthase-type prenyltransferase MfmD and is then cyclized by the Pyr4-family terpene cyclase MfmH. The replacement of MfmH with its homologue OcdTC gave another hybrid molecule with a monocyclic terpenoid moiety. Moreover, PsetPT, an MfmD homologue, was found to perform dimethylallylation and was then engineered to install a geranyl group. Our study unraveled an unusual biosynthetic mechanism for fungal phthalide-terpenoid hybrids and provided insights into how their structural diversification could be achieved.
PubMed: 38404388
DOI: 10.1039/d3sc06001b -
International Journal of Molecular... Feb 2024Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in...
Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house "Carlos Sialer (CS)" strain collection consists of seventy-one strains isolated from the cuticle of leaf-cutting ants of the tribe . Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC in sp. CS113, which encodes the biosynthesis of the hybrid polyketide-depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene and the piperazate synthase gene . The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis.
Topics: Streptomyces; Antimicrobial Cationic Peptides; Polyketide Synthases; Multigene Family; Depsipeptides; Amino Acids; Pyridazines
PubMed: 38397022
DOI: 10.3390/ijms25042347 -
Journal of Fungi (Basel, Switzerland) Feb 2024Fungi belonging to the genus have garnered increasing attention in recent years. One of the members of the genus, , has been identified as the causal agent of a severe...
Expanding the Toolbox for Genetic Manipulation in : RNAi-Mediated Silencing and CRISPR/Cas9-Mediated Disruption of a Polyketide Synthase Gene Involved in Red Pigment Production in .
Fungi belonging to the genus have garnered increasing attention in recent years. One of the members of the genus, , has been identified as the causal agent of a severe bat disease. Simultaneously, the knowledge of species has expanded, in parallel with the increased availability of genome sequences. Moreover, exhibits great potential as a producer of specialized metabolites, displaying a diverse array of biological activities. Despite these significant advancements, the genetic landscape of remains largely unexplored due to the scarcity of suitable molecular tools for genetic manipulation. In this study, we successfully implemented RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption in , using an Antarctic strain of as a model. Both methods were applied to target , a gene involved in red pigment biosynthesis. Silencing of the gene to levels of 90% or higher eliminated red pigment production, resulting in transformants exhibiting a white phenotype. On the other hand, the CRISPR/Cas9 system led to a high percentage (73%) of transformants with a one-nucleotide insertion, thereby inactivating and abolishing red pigment production, resulting in a white phenotype. The successful application of RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption represents a significant advancement in research, opening avenues for comprehensive functional genetic investigations within this underexplored fungal genus.
PubMed: 38392828
DOI: 10.3390/jof10020157 -
Journal of Fungi (Basel, Switzerland) Jan 2024Wild-type asexual spores (conidia) are green due to a pigment that protects the spores against ultraviolet light. The pigment is produced by a biosynthetic pathway, the...
Wild-type asexual spores (conidia) are green due to a pigment that protects the spores against ultraviolet light. The pigment is produced by a biosynthetic pathway, the genes of which are dispersed in the genome. The backbone molecule of the pigment is a polyketide synthesized by a polyketide synthase encoded by the gene. If is not functional, the conidia are white. The polyketide is modified by a laccase encoded by the gene and inactivation of in an otherwise wild-type background results in yellow spores. Additional spore color mutations have been isolated and mapped to a locus genetically, but the genes that correspond to these loci have not been determined. Spore color markers have been useful historically, and they remain valuable in the molecular genetics era. One can determine if a transforming fragment has been successfully integrated at the or locus by simply looking at the color of transformant conidia. The genes of the potentially useful color loci (chartreuse conidia) and (fawn conidia) have not been identified previously. We chose a set of candidate genes for each locus by comparing the assembled genome with the genetic map. By systematically deleting these candidate genes, we identified a cytochrome P450 gene (AN10028) corresponding to . Deletions of this gene result in chartreuse conidia and chartreuse mutations can be complemented in trans by a functional copy of this gene. With , we found that the existing fawn mutation, , is a deletion of 2241 base pairs that inactivates three genes. By deleting each of these genes, we determined that is AN1088, an EthD domain protein. Deletion of AN1088 results in fawn conidia as expected. Neither deletion of nor restricts growth and both should be valuable target loci for transformations. Combinations of deletions have allowed us to investigate the epistasis relationships of , , and .
PubMed: 38392776
DOI: 10.3390/jof10020104 -
Journal of the American Chemical Society Mar 2024Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite...
Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the "blind watchmaker" of evolution creates skeletal diversity is a key question in natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here, we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from with promising activity against multidrug-resistant , and etnangien, a structurally related antibiotic produced by . Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the -acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all the structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a -acting enoylreductase (ER) domain in module 1 and in recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons.
Topics: Polyketide Synthases; Acyltransferases; Anti-Bacterial Agents; Polyketides; Biological Products; Thiophenes; Sulfonamides; Macrolides; Polyenes; Imidazoles
PubMed: 38389455
DOI: 10.1021/jacs.3c13667 -
BioRxiv : the Preprint Server For... Jan 2024are harmful haptophyte algae that cause massive environmental fish-kills. Their polyketide polyether toxins, the , are amongst the largest nonpolymeric compounds in...
are harmful haptophyte algae that cause massive environmental fish-kills. Their polyketide polyether toxins, the , are amongst the largest nonpolymeric compounds in nature, alongside structurally-related health-impacting "red-tide" polyether toxins whose biosynthetic origins have been an enigma for over 40 years. Here we report the 'PKZILLAs', massive polyketide synthase (PKS) genes, whose existence and challenging genomic structure evaded prior detection. PKZILLA-1 and -2 encode giant protein products of 4.7 and 3.2 MDa with 140 and 99 enzyme domains, exceeding the largest known protein titin and all other known PKS systems. Their predicted polyene product matches the proposed pre-prymnesin precursor of the 90-carbon-backbone A-type prymnesins. This discovery establishes a model system for microalgal polyether biosynthesis and expands expectations of genetic and enzymatic size limits in biology.
PubMed: 38352448
DOI: 10.1101/2024.01.29.577497 -
SLAS Technology Apr 2024Antimicrobial-resistant (AMR) bacterial pathogens are a continually growing threat as our methods for combating these infections continue to be overcome by the evolution... (Review)
Review
Antimicrobial-resistant (AMR) bacterial pathogens are a continually growing threat as our methods for combating these infections continue to be overcome by the evolution of resistance mechanisms. Recent therapeutic methods have not staved off the concern of AMR infections, so continued research focuses on new ways of identifying small molecules to treat AMR pathogens. While chemical modification of existing antibiotics is possible, there has been rapid development of resistance by pathogens that were initially susceptible to these compounds. Synthetic biology is becoming a key strategy in trying to predict and induce novel, natural antibiotics. Advances in cloning and mutagenesis techniques applied through a synthetic biology lens can help characterize the native regulation of antibiotic biosynthetic gene clusters (BGCs) to identify potential modifications leading to more potent antibiotic activity. Additionally, many cryptic antibiotic BGCs are derived from non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) biosynthetic pathways; complex, clustered genetic sequences that give rise to amino acid-derived natural products. Synthetic biology can be applied to modify and metabolically engineer these enzyme-based systems to promote rapid and sustainable production of natural products and their variants. This review will focus on recent advances related to synthetic biology as applied to genetic pathway characterization and identification of antibiotics from naturally occurring BGCs. Specifically, we will summarize recent efforts to characterize BGCs via general genomic mutagenesis, endogenous gene expression, and heterologous gene expression.
Topics: Anti-Bacterial Agents; Synthetic Biology; Bacteria; Biosynthetic Pathways; Biological Products
PubMed: 38340893
DOI: 10.1016/j.slast.2024.100120