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Journal of Microbiology and... Jul 2023Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds...
Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds that possess crucial biological functions and significant pharmaceutical activities. Based on our sequence analysis, we have identified a putative type III polyketide synthase from sp. CS682 was named as ThnA. The role of ThnA, in sp. CS682 during the biosynthesis of 1,3,6,8 tetrahydroxynaphthalene (THN), which is the key intermediate of 1-(α-L-(2--methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene (IBR-3) was characterized. ThnA utilized five molecules of malonyl-CoA as a starter substrate to generate the polyketide 1,3,6,8-tetrahydroxynaphthalene, which could spontaneously be oxidized to the red flaviolin compound 2,5,7-trihydroxy-1,4-naphthoquinone. The amino acid sequence alignment of ThnA revealed similarities with a previously identified type III PKS and identified Cys, Phe, His, and Asn as four highly conserved active site amino acid residues, as found in other known polyketide synthases. In this study, we report the heterologous expression of the type III polyketide synthase in TK24 and the identification of THN production in a mutant strain. We also compared the transcription level of in TK24 and pIBR25-thnA and found that was only transcribed in the mutant.
Topics: Nocardia; Amino Acid Sequence; Naphthols; Polyketide Synthases
PubMed: 37254303
DOI: 10.4014/jmb.2303.03008 -
Angewandte Chemie (Weinheim An Der... Nov 2023Mupirocin is a clinically important antibiotic produced by a -AT Type I polyketide synthase (PKS) in . The major bioactive metabolite, pseudomonic acid A (PA-A), is...
Mupirocin is a clinically important antibiotic produced by a -AT Type I polyketide synthase (PKS) in . The major bioactive metabolite, pseudomonic acid A (PA-A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS shows relaxed substrate specificity, suggesting precise spatiotemporal control of MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.
PubMed: 38515435
DOI: 10.1002/ange.202312514 -
Scientific Reports Nov 2023Escherichia coli harboring polyketide synthase (pks E. coli) has been suggested to contribute to colorectal cancer development. Physical activity is strongly associated...
Escherichia coli harboring polyketide synthase (pks E. coli) has been suggested to contribute to colorectal cancer development. Physical activity is strongly associated with lower colorectal cancer risks, but its effects on pks E. coli remain unclear. The aim of this study was to investigate the association between pks E. coli prevalence and physical activity. A cross-sectional study was conducted on 222 Japanese adults (27-79-years-old, 73.9% female). Triaxial accelerometers were used to measure light-intensity physical activity, moderate-to-vigorous intensity physical activity, the physical activity level, step-count, and time spent inactive. Fecal samples collected from participants were used to determine the prevalence of pks E. coli. Multivariate logistic regression analysis and restricted cubic spline curves were used to examine the association between pks E. coli prevalence and physical activity. The prevalence of pks E. coli was 26.6% (59/222 participants). The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for the highest tertile with reference to the lowest tertile of physical activity variables were as follows: light-intensity physical activity (OR 0.63; 95% CI 0.26-1.5), moderate-to-vigorous intensity physical activity (OR 0.85; 95% CI 0.39-1.87), physical activity level (OR 0.69; 95% CI 0.32-1.51), step-count (OR 0.92; 95% CI 0.42-2.00) and time spent inactive (OR 1.30; 95% CI 0.58-2.93). No significant dose-response relationship was found between all physical activity variables and pks E. coli prevalence. Our findings did not suggest that physical activity has beneficial effects on the prevalence of pks E. coli. Longitudinal studies targeting a large population are needed to clarify this association.
Topics: Adult; Aged; Female; Humans; Male; Middle Aged; Colorectal Neoplasms; Cross-Sectional Studies; East Asian People; Escherichia coli; Exercise; Gastrointestinal Microbiome; Prevalence; Polyketide Synthases
PubMed: 38012174
DOI: 10.1038/s41598-023-47442-9 -
Journal of Medicinal Chemistry Nov 2023There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current...
There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 μM) and ADMET profiles.
Topics: Polyketide Synthases; Antitubercular Agents; Mycobacterium tuberculosis; Benzofurans; Microbial Sensitivity Tests
PubMed: 37948640
DOI: 10.1021/acs.jmedchem.3c01514 -
Frontiers in Microbiology 2023Although species have produced a wide variety of structurally diverse and biologically active natural products, the secondary biosynthetic potential of species is...
INTRODUCTION
Although species have produced a wide variety of structurally diverse and biologically active natural products, the secondary biosynthetic potential of species is widely underestimated due to the limited number of biosynthetic gene clusters (BGCs) in this genus. The significant variation in the diversity and novelty of BGCs across different species within the genus presents a major obstacle to the efficient discovery of novel natural products from .
METHODS
In this study, the number of each class of BGCs in all 6,378 high-quality genomes was predicted using antiSMASH, the species-specificity of BGC distribution in was investigated by Principal component analysis. Then the structural diversity and novelty of the predicted secondary metabolites in species with specific BGC distributions were analyzed using molecular networking.
RESULTS
Our results revealed a certain degree of species-specificity in the distribution of BGCs in , which was mainly contributed by siderophore, type III polyketide synthase (T3PKS), and transAT-PKS BGCs. , , and are rich in RiPP-like and siderophore BGCs, but lack T3PKS BGCs, while and are abundant in transAT-PKS BGCs. These species collectively encode 77,541 BGCs, with NRPS and RiPPs being the two most dominant types, which are further categorized into 4,291 GCFs. Remarkably, approximately 54.5% of GCFs and 93.8% of the predicted metabolite scaffolds are found exclusively in a single species. Notably, , , and exhibit the highest potential for producing species-specific NRPS and PKS bioinformatic natural products. Taking two species-specific NRPS gene clusters as examples, the potential of to synthesize novel species-specific natural products is illustrated.
CONCLUSION
This study highlights the species-specificity of the secondary biosynthetic potential in and provides valuable insights for the targeted discovery of novel natural products from this genus.
PubMed: 37937215
DOI: 10.3389/fmicb.2023.1271418 -
ACS Synthetic Biology Aug 2023Bacterial biosynthetic assembly lines, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), play a crucial role in the synthesis of natural...
Bacterial biosynthetic assembly lines, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), play a crucial role in the synthesis of natural products that have significant therapeutic potential. The ability to engineer these biosynthetic assembly lines offers opportunities to produce artificial nonribosomal peptides, polyketides, and their hybrids with improved properties. In this study, we introduced a synthetic NRPS variant, termed type S NRPS, which simplifies the engineering process and enables biocombinatorial approaches for generating nonribosomal peptide libraries in a parallelized high-throughput manner. However, initial generations of type S NRPSs exhibited a bottleneck that led to significantly reduced production yields. To address this challenge, we employed two optimization strategies. First, we truncated SYNZIPs from the N- and/or C-terminus of the NRPS. SYNZIPs comprise a large set of well-characterized synthetic protein interaction reagents. Second, we incorporated a structurally flexible glycine-serine linker between the NRPS protein and the attached SYNZIP, aiming to improve dynamic domain-domain interactions. Through an iterative optimization process, we achieved remarkable improvements in production yields, with titer increases of up to 55-fold compared to the nonoptimized counterparts. These optimizations successfully restored production levels of type S NRPSs to those observed in wild-type NRPSs and even surpassed them. Overall, our findings demonstrate the potential of engineering bacterial biosynthetic assembly lines for the production of artificial nonribosomal peptides. In addition, optimizing the SYNZIP toolbox can have valuable implications for diverse applications in synthetic biology, such as metabolic engineering, cell signaling studies, or engineering of other multienzyme complexes, such as PKSs.
Topics: Polyketide Synthases; Peptide Synthases; Peptides; Polyketides
PubMed: 37523786
DOI: 10.1021/acssynbio.3c00295 -
Fungal Biology and Biotechnology Aug 2023Non-reducing polyketide synthases (NR-PKSs) account for a major share of natural product diversity produced by both Asco- and Basidiomycota. The present evolutionary...
BACKGROUND
Non-reducing polyketide synthases (NR-PKSs) account for a major share of natural product diversity produced by both Asco- and Basidiomycota. The present evolutionary diversification into eleven clades further underscores the relevance of these multi-domain enzymes. Following current knowledge, NR-PKSs initiate polyketide assembly by an N-terminal starter unit:acyl transferase (SAT) domain that catalyzes the transfer of an acetyl starter from the acetyl-CoA thioester onto the acyl carrier protein (ACP).
RESULTS
A comprehensive phylogenetic analysis of NR-PKSs established a twelfth clade from which three representatives, enzymes CrPKS1-3 of the webcap mushroom Cortinarius rufoolivaceus, were biochemically characterized. These basidiomycete synthases lack a SAT domain yet are fully functional hepta- and octaketide synthases in vivo. Three members of the other clade of basidiomycete NR-PKSs (clade VIII) were produced as SAT-domainless versions and analyzed in vivo and in vitro. They retained full activity, thus corroborating the notion that the SAT domain is dispensable for many basidiomycete NR-PKSs. For comparison, the ascomycete octaketide synthase atrochrysone carboxylic acid synthase (ACAS) was produced as a SAT-domainless enzyme as well, but turned out completely inactive. However, a literature survey revealed that some NR-PKSs of ascomycetes carry mutations within the catalytic motif of the SAT domain. In these cases, the role of the domain and the origin of the formal acetate unit remains open.
CONCLUSIONS
The role of SAT domains differs between asco- and basidiomycete NR-PKSs. For the latter, it is not part of the minimal set of NR-PKS domains and not required for function. This knowledge may help engineer compact NR-PKSs for more resource-efficient routes. From the genomic standpoint, seemingly incomplete or corrupted genes encoding SAT-domainless NR-PKSs should not automatically be dismissed as non-functional pseudogenes, but considered during genome analysis to decipher the potential arsenal of natural products of a given fungus.
PubMed: 37542286
DOI: 10.1186/s40694-023-00164-z -
Molecules (Basel, Switzerland) Apr 2024Chalkophomycin is a novel chalkophore with antibiotic activities isolated from sp. CB00271, while its potential in studying cellular copper homeostasis makes it an...
Chalkophomycin is a novel chalkophore with antibiotic activities isolated from sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of -hydroxylpyrrole, 2-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the biosynthetic gene cluster from . sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 -type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.
Topics: Multigene Family; Streptomyces; Polyketide Synthases; Peptide Synthases; Bacterial Proteins
PubMed: 38731473
DOI: 10.3390/molecules29091982 -
Applied Microbiology and Biotechnology Oct 2023The aim of this study was the whole-genome analysis and assessment of the antimicrobial potential of bacterial isolates from honey harvested in one geographical...
The aim of this study was the whole-genome analysis and assessment of the antimicrobial potential of bacterial isolates from honey harvested in one geographical location-the north of Poland. In total, 132 strains were derived from three honey samples, and the antimicrobial activity of CFAM (cell-free after-culture medium) was used as a criterion for strain selection and detailed genomic investigation. Two of the tested isolates (SZA14 and SZA16) were classified as Bacillus paralicheniformis, and one isolate (SZB3) as Bacillus subtilis based on their ANI and phylogenetic analysis relatedness. The isolates SZA14 and SZA16 were harvested from the same honey sample with a nucleotide identity of 98.96%. All three isolates have been found to be potential producers of different antimicrobial compounds. The secondary metabolite genome mining pipeline (antiSMASH) identified 14 gene cluster coding for non-ribosomal peptide synthetases (NRPs), polyketide synthases (PKSs), and ribosomally synthesized and post-translationally modified peptides (RiPPs) that are potential sources of novel antibacterials. The BAGEL4 analysis revealed the presence of nine putative gene clusters of interest in the isolates SZA14 and SZA16 (including the presence of six similar clusters present in both isolates, coding for the production of enterocin Nkr-5-3B, haloduracin-alpha, sonorensin, bottromycin, comX2, and lasso peptide), and four in B. subtilis isolate SZB3 (competence factor, sporulation-killing factor, subtilosin A, and sactipeptides). The outcomes of this study confirm that honey-derived Bacillus spp. strains can be considered potential producers of a broad spectrum of antimicrobial agents. KEY POINTS: • Bacteria of the genus Bacillus are an important component of honey microbiota. • Honey-derived Bacillus spp. strains are potential producers of new antimicrobials.
PubMed: 37665371
DOI: 10.1007/s00253-023-12732-9 -
Applied and Environmental Microbiology Jun 2023Natural microbial communities produce a diverse array of secondary metabolites with ecologically and biotechnologically relevant activities. Some of them have been used...
Natural microbial communities produce a diverse array of secondary metabolites with ecologically and biotechnologically relevant activities. Some of them have been used clinically as drugs, and their production pathways have been identified in a few culturable microorganisms. However, since the vast majority of microorganisms in nature have not been cultured, identifying the synthetic pathways of these metabolites and tracking their hosts remain a challenge. The microbial biosynthetic potential of mangrove swamps remains largely unknown. Here, we examined the diversity and novelty of biosynthetic gene clusters in dominant microbial populations in mangrove wetlands by mining 809 newly reconstructed draft genomes and probing the activities and products of these clusters by using metatranscriptomic and metabolomic techniques. A total of 3,740 biosynthetic gene clusters were identified from these genomes, including 1,065 polyketide and nonribosomal peptide gene clusters, 86% of which showed no similarity to known clusters in the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) repository. Of these gene clusters, 59% were harbored by new species or lineages of -related phyla and , whose members are highly abundant in mangrove wetlands and for which few synthetic natural products have been reported. Metatranscriptomics revealed that most of the identified gene clusters were active in field and microcosm samples. Untargeted metabolomics was also used to identify metabolites from the sediment enrichments, and 98% of the mass spectra generated were unrecognizable, further supporting the novelty of these biosynthetic gene clusters. Our study taps into a corner of the microbial metabolite reservoir in mangrove swamps, providing clues for the discovery of new compounds with valuable activities. At present, the majority of known clinical drugs originated from cultivated species of a few bacterial lineages. It is vital for the development of new pharmaceuticals to explore the biosynthetic potential of naturally uncultivable microorganisms using new techniques. Based on the large numbers of genomes reconstructed from mangrove wetlands, we identified abundant and diverse biosynthetic gene clusters in previously unsuspected phylogenetic groups. These gene clusters exhibited a variety of organizational architectures, especially for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), implying the presence of new compounds with valuable activities in the mangrove swamp microbiome.
Topics: Wetlands; Multigene Family; Biosynthetic Pathways; Metagenome; Bacteria; Metabolomics; China; Biodiversity
PubMed: 37191511
DOI: 10.1128/aem.00102-23