RÉSUMÉ
Polyketides represent an important class of natural products, renowned for their intricate structures and diverse biological activities. In contrast to common fatty acids, polyketides possess relatively more rigid carbon skeletons, more complex ring systems, and chiral centers. These structural features are primarily achieved through distinctive enzymatic cyclizations and oxidations as tailoring steps. In this opinion, we discuss the recent progress in deciphering the mechanisms of cyclization and oxidation within polyketide biosynthesis. By shedding light on these enzymatic processes, this article seeks to motivate the community to unravel the remaining mysteries surrounding cyclase and oxidase functionalities and to explore novel polyketide natural products through genome mining.
Sujet(s)
Oxydoréduction , Polycétides , Polycétides/métabolisme , Polycétides/composition chimique , Cyclisation , Produits biologiques/métabolisme , Produits biologiques/composition chimique , Polyketide synthases/métabolismeRÉSUMÉ
The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. The recently updated module boundary has been successful for engineering short synthases, yet larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases to test every module combination of the pikromycin synthase. Anticipated products are detected from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. We determine ketosynthase gatekeeping and module-skipping are the principal impediments to obtaining functional synthases. The platform is also employed to construct active hybrid synthases by incorporating modules from the erythromycin, spinosyn, and rapamycin assembly lines. The relaxed gatekeeping of a ketosynthase in the rapamycin synthase is especially encouraging in the quest to produce designer polyketides.
Sujet(s)
Macrolides , Polyketide synthases , Polyketide synthases/métabolisme , Polyketide synthases/génétique , Macrolides/métabolisme , Ingénierie des protéines/méthodes , Érythromycine , Polycétides/métabolisme , Polycétides/composition chimique , Streptomyces/enzymologie , Streptomyces/génétique , Sirolimus , Protéines bactériennes/métabolisme , Protéines bactériennes/génétiqueRÉSUMÉ
Prymnesium parvum are harmful haptophyte algae that cause massive environmental fish kills. Their polyketide polyether toxins, the prymnesins, are among the largest nonpolymeric compounds in nature and have biosynthetic origins that have remained enigmatic for more than 40 years. In this work, we report the "PKZILLAs," massive P. parvum polyketide synthase (PKS) genes that have evaded previous detection. PKZILLA-1 and -2 encode giant protein products of 4.7 and 3.2 megadaltons that have 140 and 99 enzyme domains. Their predicted polyene product matches the proposed pre-prymnesin precursor of the 90-carbon-backbone A-type prymnesins. We further characterize the variant PKZILLA-B1, which is responsible for the shorter B-type analog prymnesin-B1, from P. parvum RCC3426 and thus establish a general model of haptophyte polyether biosynthetic logic. This work expands expectations of genetic and enzymatic size limits in biology.
Sujet(s)
Haptophyta , , Polyketide synthases , Haptophyta/enzymologie , Haptophyta/génétique , Polyènes/métabolisme , Polyènes/composition chimique , /biosynthèse , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Polycétides/métabolisme , Domaines protéiquesRÉSUMÉ
The natural compound (R)-(-)-mellein exhibits antiseptic and fungicidal activities. We investigated its biosynthesis using the polyketide synthase encoded by SACE_5532 (pks8) from Saccharopolyspora erythraea heterologously expressed in Streptomyces albus B4, a chassis chosen for its fast growth, genetic manipulability, and ample large short-chain acyl-CoA precursor supply. High-level heterologous (R)-(-)-mellein yield was achieved by pks8 overexpression and duplication. The precursor supply pathways were strengthened by overexpression of SACE_0028 (encoding acetyl-CoA carboxylase) and four genes involved in ß-oxidation (fadD, fadE, fadB, and fadA). Cell growth inhibition by (R)-(-)-mellein production at high concentration was relieved by in situ adsorption using Amberlite XAD16 resin. The final strain, B4mel12, produced (R)-(-)-mellein at 6395.2 mg/L in shake-flask fermentation. Overall, this is the first report of heterologous (R)-(-)-mellein synthesis in microorganism with a high titer. (R)-(-)-mellein prototype in this study opens a possibility for the overproduction of valuable melleins in S. albus B4.
Sujet(s)
Protéines bactériennes , Génie métabolique , Polyketide synthases , Streptomyces , Streptomyces/génétique , Streptomyces/métabolisme , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Fermentation , Saccharopolyspora/génétique , Saccharopolyspora/métabolisme , Acetyl-coA carboxylase/génétique , Acetyl-coA carboxylase/métabolismeRÉSUMÉ
Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSCBE3-NG-Hypa, tailored with both high efficiency and -fidelity for Streptomyces. Of note, eSCBE3-NG-Hypa recognizes NG PAM and exhibits high activity at challenging sites with high GC content or GC motifs, while displaying minimal off-target effects. To illustrate its practicability, we employ eSCBE3-NG-Hypa to achieve precise key amino acid conversion of the dehydratase (DH) domains within the modular polyketide synthase (PKS) responsible for the insecticide avermectins biosynthesis, achieving domains inactivation. The resulting DH-inactivated mutants, while ceasing avermectins production, produce a high yield of oligomycin, indicating competitive relationships among multiple biosynthetic gene clusters (BGCs) in Streptomyces avermitilis. Leveraging this insight, we use eSCBE3-NG-Hypa to introduce premature stop codons into competitor gene cluster of ave in an industrial S. avermitilis, with the mutant Δolm exhibiting the highest 4.45-fold increase in avermectin B1a compared to the control. This work provides a potent tool for modifying biosynthetic pathways and advancing metabolic engineering in Streptomyces.
Sujet(s)
Systèmes CRISPR-Cas , Cytosine , Édition de gène , Polyketide synthases , Streptomyces , Streptomyces/génétique , Streptomyces/métabolisme , Édition de gène/méthodes , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Cytosine/métabolisme , Ivermectine/analogues et dérivés , Ivermectine/métabolisme , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , OligomycinesRÉSUMÉ
Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. KEY POINTS: Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans.
Sujet(s)
Systèmes CRISPR-Cas , Penicillium , Métabolisme secondaire , Penicillium/génétique , Penicillium/métabolisme , Métabolisme secondaire/génétique , Aspergillus nidulans/génétique , Aspergillus nidulans/métabolisme , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Famille multigénique , Ciblage de gène/méthodes , Régulation de l'expression des gènes fongiques , Protéines fongiques/génétique , Protéines fongiques/métabolisme , Voies de biosynthèse/génétique , Génie métabolique/méthodes , Expression des gènesRÉSUMÉ
The catastrophic loss of aquatic life in the Central European Oder River in 2022, caused by a toxic bloom of the haptophyte microalga Prymnesium parvum (in a wide sense, s.l.), underscores the need to improve our understanding of the genomic basis of the toxin. Previous morphological, phylogenetic, and genomic studies have revealed cryptic diversity within P. parvum s.l. and uncovered three clade-specific (types A, B, and C) prymnesin toxins. Here, we used state-of-the-art long-read sequencing and assembled the first haplotype-resolved diploid genome of a P. parvum type B from the strain responsible for the Oder disaster. Comparative analyses with type A genomes uncovered a genome-size expansion driven by repetitive elements in type B. We also found conserved synteny but divergent evolution in several polyketide synthase (PKS) genes, which are known to underlie toxin production in combination with environmental cues. We identified an approximately 20-kbp deletion in the largest PKS gene of type B that we link to differences in the chemical structure of types A and B prymnesins. Flow cytometry and electron microscopy analyses confirmed diploidy in the Oder River strain and revealed differences to closely related strains in both ploidy and morphology. Our results provide unprecedented resolution of strain diversity in P. parvum s.l. and a better understanding of the genomic basis of toxin variability in haptophytes. The reference-quality genome will enable us to better understand changes in microbial diversity in the face of increasing environmental pressures and provides a basis for strain-level monitoring of invasive Prymnesium in the future.
Sujet(s)
Haptophyta , Haptophyta/génétique , Haplotypes , Microalgues/génétique , Toxines de la flore et de la faune marines/génétique , Animaux , Phylogenèse , Poissons/génétique , Polyketide synthases/génétique , Polyketide synthases/métabolismeRÉSUMÉ
BACKGROUND: Microbial genome sequencing and analysis revealed the presence of abundant silent secondary metabolites biosynthetic gene clusters (BGCs) in streptomycetes. Activating these BGCs has great significance for discovering new compounds and novel biosynthetic pathways. RESULTS: In this study, we found that ovmZ and ovmW homologs, a pair of interdependent transcriptional regulators coding genes, are widespread in actinobacteria and closely associated with the biosynthesis of secondary metabolites. Through co-overexpression of native ovmZ and ovmW in Streptomyces neyagawaensis NRRL B-3092, a silent type II polyketide synthase (PKS) gene cluster was activated to produce gephyromycin A, tetrangomycin and fridamycin E with the yields of 22.3 ± 8.0 mg/L, 4.8 ± 0.5 mg/L and 20.3 ± 4.1 mg/L respectively in the recombinant strain of S.ne/pZnWn. However, expression of either ovmZ or ovmW failed to activate this gene cluster. Interestingly, overexpression of the heterologous ovmZ and ovmW pair from oviedomycin BGC of S. ansochromogenes 7100 also led to awakening of this silent angucyclinone BGC in S. neyagawaensis. CONCLUSION: A silent angucyclinone BGC was activated by overexpressing both ovmZ and ovmW in S. neyagawaensis. Due to the wide distribution of ovmZ and ovmW in the BGCs of actinobacteria, co-overexpression of ovmZ and ovmW could be a strategy for activating silent BGCs, thus stimulating the biosynthesis of secondary metabolites.
Sujet(s)
Anthraquinones , Antibactériens , Famille multigénique , Streptomyces , Streptomyces/génétique , Streptomyces/métabolisme , Antibactériens/biosynthèse , Anthraquinones/métabolisme , Régulation de l'expression des gènes bactériens , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Voies de biosynthèse/génétique , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Métabolisme secondaire/génétique ,RÉSUMÉ
A VMYH motif was determined to help ketosynthases in polyketide assembly lines select α,ß-unsaturated intermediates from an equilibrium mediated by an upstream dehydratase. Alterations of this motif decreased ketosynthase selectivity within a model tetraketide synthase, most significantly when replaced by the TNGQ motif of ketosynthases that accept D-ß-hydroxy intermediates.
Sujet(s)
Hydro-lyases , Polyketide synthases , Polyketide synthases/métabolisme , Polyketide synthases/composition chimique , Hydro-lyases/métabolisme , Hydro-lyases/composition chimiqueRÉSUMÉ
Escherichia coli that harbor the polyketide synthase (pks) genomic island produce colibactin and are associated with sporadic colorectal cancer development. Given the considerable prevalence of pks+ bacteria in healthy individuals, we sought to identify strategies to limit the growth and expansion of pks+ E. coli. We found that culture supernatants of the probiotic strain E. coli Nissle 1917 were able to inhibit the growth of the murine pathogenic strain pks+ E. coli NC101 (EcNC101). We performed a nontargeted analysis of the metabolome in supernatants from several E. coli strains and identified putrescine as a potential postbiotic capable of suppressing EcNC101 growth in vitro. The effect of putrescine supplementation was then evaluated in the azoxymethane/dextran sulfate sodium mouse model of colorectal cancer in mice colonized with EcNC101. Putrescine supplementation inhibited the growth of pks+ E. coli, reduced the number and size of colonic tumors, and downmodulated the release of inflammatory cytokines in the colonic lumen. Additionally, putrescine supplementation led to shifts in the composition and function of gut microbiota, characterized by an increase in the Firmicutes/Bacteroidetes ratio and enhanced acetate production. The effect of putrescine was further confirmed in vitro using a pks+ E. coli strain isolated from a patient with colorectal cancer. These results suggest that probiotic-derived metabolites can be used as an alternative to live bacteria in individuals at risk of developing colorectal cancer due to the presence of pks+ bacteria in their colon. SIGNIFICANCE: Putrescine supplementation inhibits the growth of cancer-promoting bacteria in the gut, lowers inflammation, and reduces colon cancer development. The consumption of healthy foods rich in putrescine may be a potential prophylactic approach for individuals at risk of developing colorectal cancer due to the presence of pks+ bacteria in their colon.
Sujet(s)
Escherichia coli , Microbiome gastro-intestinal , Polyketide synthases , Putrescine , Putrescine/pharmacologie , Putrescine/métabolisme , Animaux , Escherichia coli/effets des médicaments et des substances chimiques , Souris , Microbiome gastro-intestinal/effets des médicaments et des substances chimiques , Polyketide synthases/métabolisme , Polyketide synthases/génétique , Tumeurs du côlon/microbiologie , Tumeurs du côlon/anatomopathologie , Humains , Probiotiques/pharmacologie , Probiotiques/administration et posologie , Probiotiques/usage thérapeutique , Tumeurs colorectales/microbiologie , Tumeurs colorectales/anatomopathologie , Tumeurs colorectales/métabolisme , Compléments alimentaires , Polycétides/pharmacologie , Polycétides/métabolisme , Modèles animaux de maladie humaine , Ilots génomiques , Côlon/microbiologie , Côlon/anatomopathologie , Côlon/métabolisme , Côlon/effets des médicaments et des substances chimiques , Oxyde de diméthyl-diazène , PeptidesRÉSUMÉ
Hitachimycin is a bicyclic macrolactam antibiotic with (S)-ß-phenylalanine (ß-Phe) at the starter position of the polyketide skeleton. While the enzymes that recognize ß-amino acids, modify the aminoacyl groups, and transfer the resultant dipeptide groups to the acyl carrier protein domains of polyketide synthases (PKSs) have been studied extensively, the post-PKS modification mechanism responsible for constructing the unique bicyclic structure of hitachimycin remains elusive. In this study, we first inactivated six genes encoding putative post-PKS modification enzymes, namely hitM1 to hitM6, in Streptomyces scabrisporus to determine their involvement in hitachimycin biosynthesis. The ΔhitM4 strain accumulated an all-trans-2,4,6,8,18-pentaene macrolactam, which was confirmed as a true intermediate in hitachimycin biosynthesis by cellular feeding experiments, and appears to be the initial intermediate in the post-PKS modification pathway. The ΔhitM1 strain accumulated 10-O-demethyl-10-oxohitachimycin (M1-A). In enzymatic experiments, M1-A was reduced by the NAD(P)H-dependent reductase HitM1 in the presence of NADPH. The product of the reaction catalyzed by HitM1 was converted to hitachimycin by the methyltransferase HitM6. We thus propose a plausible post-PKS modification mechanism for the biosynthesis of hitachimycin.
Sujet(s)
Polyketide synthases , Streptomyces , Streptomyces/enzymologie , Streptomyces/métabolisme , Streptomyces/génétique , Polyketide synthases/métabolisme , Polyketide synthases/génétique , Antibactériens/biosynthèse , Antibactériens/composition chimique , Antibactériens/métabolisme , Lactames macrocycliques/métabolisme , Lactames macrocycliques/composition chimique , Structure moléculaireRÉSUMÉ
Enediyne natural products are renowned for their potent cytotoxicities but the biosynthesis of their defining 1,5-diyne-3-ene core moiety remains largely enigmatic. Since the discovery of the enediyne polyketide synthase cassette in 2002, genome sequencing has revealed thousands of distinct enediyne biosynthetic gene clusters, each harboring the conserved enediyne polyketide synthase cassette. Here we report that (1) the products of this cassette are an iodoheptaene, a diiodotetrayne and two pentaynes; (2) the diiodotetrayne represents a common biosynthetic intermediate for all known enediynes; and (3) cryptic iodination can be exploited to increase enediyne titers. These findings establish a unified biosynthetic pathway for the enediynes, set the stage to further advance enediyne core biosynthesis and enable fundamental breakthroughs in chemistry, enzymology and translational applications of enediyne natural products.
Sujet(s)
Produits biologiques , Ènediynes , Ènediynes/composition chimique , Ènediynes/métabolisme , Produits biologiques/composition chimique , Produits biologiques/métabolisme , Famille multigénique , Polyketide synthases/métabolisme , Polyketide synthases/génétique , Voies de biosynthèse , Streptomyces/génétique , Streptomyces/métabolismeRÉSUMÉ
Potato common scab (PCS) is a widespread plant disease that lacks effective control measures. Using a small molecule elicitor, we activate the production of a novel class of polyketide antibiotics, streptolateritic acids A-D, in Streptomyces sp. FXJ1.172. These compounds show a promising control efficacy against PCS and an unusual acyclic pentacarboxylic acid structure. A gene cluster encoding a type I modular polyketide synthase is identified to be responsible for the biosynthesis of these metabolites. A cytochrome P450 (CYP) and an aldehyde dehydrogenase (ADH) encoded by two genes in the cluster are proposed to catalyze iterative oxidation of the starter-unit-derived methyl group and three of six branching methyl groups to carboxylic acids during chain assembly. Our findings highlight how activation of silent biosynthetic gene clusters can be employed to discover completely new natural product classes able to combat PCS and new types of modular polyketide synthase-based biosynthetic machinery.
Sujet(s)
Protéines bactériennes , Famille multigénique , Maladies des plantes , Polyketide synthases , Solanum tuberosum , Streptomyces , Streptomyces/génétique , Streptomyces/métabolisme , Streptomyces/composition chimique , Maladies des plantes/microbiologie , Solanum tuberosum/métabolisme , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Protéines bactériennes/composition chimique , Antibactériens/pharmacologie , Antibactériens/composition chimique , Antibactériens/biosynthèse , Voies de biosynthèse , Acides carboxyliques/composition chimique , Acides carboxyliques/métabolismeRÉSUMÉ
Filamentous Actinobacteria, recently renamed Actinomycetia, are the most prolific source of microbial bioactive natural products. Studies on biosynthetic gene clusters benefit from or require chromosome-level assemblies. Here, we provide DNA sequences from >1000 isolates: 881 complete genomes and 153 near-complete genomes, representing 28 genera and 389 species, including 244 likely novel species. All genomes are from filamentous isolates of the class Actinomycetia from the NBC culture collection. The largest genus is Streptomyces with 886 genomes including 742 complete assemblies. We use this data to show that analysis of complete genomes can bring biological understanding not previously derived from more fragmented sequences or less systematic datasets. We document the central and structured location of core genes and distal location of specialized metabolite biosynthetic gene clusters and duplicate core genes on the linear Streptomyces chromosome, and analyze the content and length of the terminal inverted repeats which are characteristic for Streptomyces. We then analyze the diversity of trans-AT polyketide synthase biosynthetic gene clusters, which encodes the machinery of a biotechnologically highly interesting compound class. These insights have both ecological and biotechnological implications in understanding the importance of high quality genomic resources and the complex role synteny plays in Actinomycetia biology.
Sujet(s)
Actinobacteria , Génome bactérien , Famille multigénique , Polyketide synthases , Génome bactérien/génétique , Actinobacteria/génétique , Actinobacteria/classification , Actinobacteria/métabolisme , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Streptomyces/génétique , Streptomyces/classification , Streptomyces/métabolisme , Phylogenèse , Génomique/méthodesRÉSUMÉ
Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of public microbial genomes leads to the discovery of over sixty thousand type 1 polyketide gene clusters. However, the molecular products of only about a hundred of these clusters are characterized, leaving most metabolites unknown. Characterizing polyketides relies on bioactivity-guided purification, which is expensive and time-consuming. To address this, we present Seq2PKS, a machine learning algorithm that predicts chemical structures derived from Type 1 polyketide synthases. Seq2PKS predicts numerous putative structures for each gene cluster to enhance accuracy. The correct structure is identified using a variable mass spectral database search. Benchmarks show that Seq2PKS outperforms existing methods. Applying Seq2PKS to Actinobacteria datasets, we discover biosynthetic gene clusters for monazomycin, oasomycin A, and 2-aminobenzamide-actiphenol.
Sujet(s)
Spectrométrie de masse , Famille multigénique , Polyketide synthases , Polycétides , Polycétides/métabolisme , Polycétides/composition chimique , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Spectrométrie de masse/méthodes , Fouille de données/méthodes , Apprentissage machine , Actinobacteria/génétique , Actinobacteria/métabolisme , Génome bactérien , Algorithmes , Produits biologiques/composition chimique , Produits biologiques/métabolismeRÉSUMÉ
Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.
Sujet(s)
Génome fongique , Famille multigénique , Mycotoxines , Penicillium , Phylogenèse , Métabolisme secondaire , Penicillium/génétique , Penicillium/métabolisme , Mycotoxines/métabolisme , Mycotoxines/génétique , Contamination des aliments/analyse , Patuline/métabolisme , Protéines fongiques/génétique , Protéines fongiques/métabolisme , Noix/microbiologie , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Microbiologie alimentaire , Corylus/microbiologie , Composés hétérocycliques avec 4 noyaux ou plus , Indoles , PipérazinesRÉSUMÉ
BACKGROUND: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied. RESULTS: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin. CONCLUSIONS: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.
Sujet(s)
Antibactériens , Génome bactérien , Famille multigénique , Paenibacillus , Paenibacillus/génétique , Paenibacillus/métabolisme , Antibactériens/pharmacologie , Antibactériens/biosynthèse , Amino-acid ligases/génétique , Polyketide synthases/génétique , Bactériocines/génétique , Bactériocines/pharmacologie , Bactériocines/biosynthèse , Voies de biosynthèse/génétique , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Découverte de médicament/méthodesRÉSUMÉ
Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.
Sujet(s)
Système acellulaire , Régions promotrices (génétique) , Streptomyces griseus/enzymologie , Streptomyces griseus/génétique , Streptomyces griseus/métabolisme , Escherichia coli/génétique , Escherichia coli/métabolisme , Famille multigénique , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Codon/génétique , AcyltransferasesRÉSUMÉ
Utilizing bioinformatics tools, this study expands our understanding of secondary metabolism in Botrytis cinerea, identifying novel genes within polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), sesquiterpene cyclase (STC), diterpene cyclase (DTC), and dimethylallyltryptophan synthase (DMATS) families. These findings enrich the genetic framework associated with B. cinerea's pathogenicity and ecological adaptation, offering insights into uncharted metabolic pathways. Significantly, the discovery of previously unannotated genes provides new molecular targets for developing targeted antifungal strategies, promising to enhance crop protection and advance our understanding of fungal biochemistry. This research not only broadens the scope of known secondary metabolites but also opens avenues for future exploration into B. cinerea's biosynthetic capabilities, potentially leading to novel antifungal compounds. Our work underscores the importance of integrating bioinformatics and genomics for fungal research, paving the way for sustainable agricultural practices by pinpointing precise molecular interventions against B. cinerea. This study sets a foundation for further investigations into the fungus's secondary metabolism, with implications for biotechnology and crop disease management.
Sujet(s)
Botrytis , Amino-acid ligases , Polyketide synthases , Métabolisme secondaire , Botrytis/génétique , Botrytis/pathogénicité , Métabolisme secondaire/génétique , Amino-acid ligases/génétique , Amino-acid ligases/métabolisme , Polyketide synthases/génétique , Polyketide synthases/métabolisme , Protéines fongiques/génétique , Protéines fongiques/métabolisme , Biologie informatique/méthodes , Famille multigénique , Gènes fongiquesRÉSUMÉ
BACKGROUND: The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized. RESULTS: The aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein). CONCLUSIONS: The results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.