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ACS Infectious Diseases May 2024DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein...
DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.
Topics: Animals; Humans; Mice; Antitubercular Agents; Bacterial Proteins; Crystallography, X-Ray; Disease Models, Animal; Drug Discovery; Drug Evaluation, Preclinical; Enzyme Inhibitors; Mycobacterium tuberculosis; Polyketide Synthases; Small Molecule Libraries; Thiolester Hydrolases; Tuberculosis
PubMed: 38577994
DOI: 10.1021/acsinfecdis.3c00592 -
FEMS Microbes 2024Microorganisms inhabiting hypersaline environments have received significant attention due to their ability to thrive under poly-extreme conditions, including high...
Microorganisms inhabiting hypersaline environments have received significant attention due to their ability to thrive under poly-extreme conditions, including high salinity, elevated temperatures and heavy metal stress. They are believed to possess biosynthetic gene clusters (BGCs) that encode secondary metabolites as survival strategy and offer potential biotechnological applications. In this study, we mined BGCs in shotgun metagenomic sequences generated from Lake Afdera, a hypersaline lake in the Afar Depression, Ethiopia. The microbiome of Lake Afdera is predominantly bacterial, with (18.6%) and (11.8%) being ubiquitously detected. A total of 94 distinct BGCs were identified in the metagenomic data. These BGCs are found to encode secondary metabolites with two main categories of functions: (i) potential pharmaceutical applications (nonribosomal peptide synthase NRPs, polyketide synthase, others) and (ii) miscellaneous roles conferring adaptation to extreme environment (bacteriocins, ectoine, others). Notably, NRPs (20.6%) and bacteriocins (10.6%) were the most abundant. Furthermore, our metagenomic analysis predicted gene clusters that enable microbes to defend against a wide range of toxic metals, oxidative stress and osmotic stress. These findings suggest that Lake Afdera is a rich biological reservoir, with the predicted BGCs playing critical role in the survival and adaptation of extremophiles.
PubMed: 38560625
DOI: 10.1093/femsmc/xtae008 -
Mycology 2024Chromoblastomycosis is a chronic granulomatous subcutaneous fungal disease caused mainly by in southern China. Melanin is an important virulence factor in wild strain...
Chromoblastomycosis is a chronic granulomatous subcutaneous fungal disease caused mainly by in southern China. Melanin is an important virulence factor in wild strain (Mel+), and the strains lack of the polyketide synthase gene is a melanin-deficient mutant strain (Mel-). We investigated the effect of melanin in on Dectin-1 receptor-mediated immune responses in macrophages. Conidia and tiny hyphae of Mel+ and Mel- were co-cultured with THP-1 macrophages expressing normal or low levels of Dectin-1. Compare the killing rate, phagocytosis rate, and expression levels of the inflammatory cytokines tumour necrosis factor-α, interleukin-1β, interleukin-6, and nitric oxide in each group. The results showed that the killing rate, phagocytosis rate, and pro-inflammatory factor levels of Mel+ infected macrophages with normal expression of Dectin-1 were lower than those of Mel-. And the knockdown of Dectin-1 inhibited the phagocytic rate, killing rate, and proinflammatory factor expression in macrophages infected with Mel+ and Mel-. And there was no significant difference in the above indexes between Mel+ and Mel- groups in Dectin-1 knockdown macrophages. In summary, the study reveals that melanin of inhibits the immune response effect of the host by hindering its binding to Dectin-1 on the surface of macrophage, which may lead to persistent fungal infections.
PubMed: 38558842
DOI: 10.1080/21501203.2023.2249010 -
The Journal of Pathology Jun 2024Environmental factors like the pathogenicity island polyketide synthase positive (pks+) Escherichia coli (E. coli) could have potential for risk stratification in...
Environmental factors like the pathogenicity island polyketide synthase positive (pks+) Escherichia coli (E. coli) could have potential for risk stratification in colorectal cancer (CRC) screening. The association between pks+ E. coli measured in fecal immunochemical test (FIT) samples and the detection of advanced neoplasia (AN) at colonoscopy was investigated. Biobanked FIT samples were analyzed for both presence of E. coli and pks+ E. coli and correlated with colonoscopy findings; 5020 CRC screening participants were included. Controls were participants in which no relevant lesion was detected because of FIT-negative results (cut-off ≥15 μg Hb/g feces), a negative colonoscopy, or a colonoscopy during which only a nonadvanced polyp was detected. Cases were participants with AN [CRC, advanced adenoma (AA), or advanced serrated polyp (ASP)]. Existing DNA isolation and quantitative polymerase chain reaction (qPCR) procedures were used for the detection of E. coli and pks+ E. coli in stool. A total of 4542 (90.2%) individuals were E. coli positive, and 1322 (26.2%) were pks+ E. coli positive. The prevalence of E. coli in FIT samples from individuals with AN was 92.9% compared to 89.7% in FIT samples of controls (p = 0.010). The prevalence of pks+ E. coli in FIT samples from individuals with AN (28.6%) and controls (25.9%) was not significantly different (p = 0.13). The prevalences of pks+ E. coli in FIT samples from individuals with CRC, AA, or ASP were 29.6%, 28.3%, and 32.1%, respectively. In conclusion, the prevalence of pks+ E. coli in a screening population was 26.2% and did not differ significantly between individuals with AN and controls. These findings disqualify the straightforward option of using a snapshot measurement of pks+ E. coli in FIT samples as a stratification biomarker for CRC risk. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Topics: Humans; Colorectal Neoplasms; Feces; Escherichia coli; Male; Early Detection of Cancer; Female; Middle Aged; Aged; Polyketide Synthases; Colonoscopy; Risk Factors; Adenoma; Risk Assessment; Biomarkers, Tumor; Case-Control Studies
PubMed: 38551073
DOI: 10.1002/path.6276 -
Molecules (Basel, Switzerland) Mar 2024Glycosylated polyene macrolides are important antifungal agents that are produced by many actinomycete species. Development of new polyenes may deliver improved...
Glycosylated polyene macrolides are important antifungal agents that are produced by many actinomycete species. Development of new polyenes may deliver improved antibiotics. Here, was genetically re-programmed to synthesise pentaene analogues of the heptaene amphotericin B. These pentaenes are of interest as surrogate substrates for enzymes catalysing unusual, late-stage biosynthetic modifications. The previous deletion of amphotericin polyketide synthase modules 5 and 6 generated M57, which produces an inactive pentaene. Here, the chain-terminating thioesterase was fused to module 16 to generate strain M57-16TE, in which cycles 5, 6, 17 and 18 are eliminated from the biosynthetic pathway. Another variant of M57 was obtained by replacing modules 15, 16 and 17 with a single 15-17 hybrid module. This gave strain M57-1517, in which cycles 5, 6, 15 and 16 are deleted. M57-16TE and M57-1517 gave reduced pentaene yields. Only M57-1517 delivered its predicted full-length pentaene macrolactone in low amounts. For both mutants, the major pentaenes were intermediates released from modules 10, 11 and 12. Longer pentaene chains were unstable. The novel pentaenes were not glycosylated and were not active against . However, random mutagenesis and screening may yet deliver new antifungal producers from the M57-16TE and M57-1517 strains.
Topics: Amphotericin B; Polyketide Synthases; Polyenes; Antifungal Agents; Macrolides; Anti-Bacterial Agents
PubMed: 38543033
DOI: 10.3390/molecules29061396 -
Journal of Fungi (Basel, Switzerland) Mar 2024The phytopathogenic fungus has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as...
The phytopathogenic fungus has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as light or nitrogen. Here, we focused our attention on fusarins, a class of mycotoxins whose synthesis is downregulated by nitrogen starvation. Previous data showed that mutants of genes involved in carotenoid regulation (, encoding a RING finger protein repressor), light detection (, White Collar photoreceptor), and cAMP signaling (AcyA, adenylate cyclase) affect the synthesis of different metabolites. We studied the effect of these mutations on fusarin production and the expression of the gene, which encodes the key polyketide synthase of the pathway. We found that the three proteins are positive regulators of fusarin synthesis, especially WcoA and AcyA, linking light regulation to cAMP signaling. Genes for two other photoreceptors, the cryptochrome CryD and the Vivid flavoprotein VvdA, were not involved in fusarin regulation. In most cases, there was a correspondence between fusarin production and mRNA, indicating that regulation is mainly exerted at the transcriptional level. We conclude that fusarin synthesis is subject to a complex control involving regulators from different signaling pathways.
PubMed: 38535211
DOI: 10.3390/jof10030203 -
PNAS Nexus Mar 2024Parrots have remarkable plumage coloration that result in part from a unique ability to produce pigments called psittacofulvins that yield yellow to red feather colors....
Parrots have remarkable plumage coloration that result in part from a unique ability to produce pigments called psittacofulvins that yield yellow to red feather colors. Little is known about the evolution of psittacofulvin-based pigmentation. Widespread color mutations of captive-bred parrots provide perfect opportunities to study the genetic basis of this trait. An earlier study on budgerigars, which do not possess psittacofulvins, reveals the involvement of an uncharacterized polyketide synthase (MuPKS) in yellow psittacofulvin synthesis. The phenotype had repeatedly appeared in different parrot species, similar to independent experimental replications allowing the study of convergent evolution and molecular mechanism of psittacofulvin-based pigmentation. Here, we investigated the genetic basis of the phenotypes in two species of parrots, Fischer's lovebird () and Yellow-collared lovebird (). Using whole-genome data, we identified a single genomic region with size <2 Mb to be strongly associated with the color difference between and wild-type (WT) birds in both species. Surprisingly, we discovered that the mutation associated with the phenotype was identical to the previously described substitution causing the functional change of MuPKS in budgerigars. Together with the evidence of shared -associated haplotypes and signatures of a selective sweep in this genomic region in both species, we demonstrated both mutation and interspecific introgression play a role in the evolution of this trait in different species. The convergent substitution in the same gene in both lovebirds and budgerigars also indicates a strong evolutionary constraint on psittacofulvin-based coloration.
PubMed: 38528953
DOI: 10.1093/pnasnexus/pgae107 -
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 -
Microbial Cell Factories Mar 2024Natural tetramates are a family of hybrid polyketides bearing tetramic acid (pyrrolidine-2,4-dione) moiety exhibiting a broad range of bioactivities. Biosynthesis of...
BACKGROUND
Natural tetramates are a family of hybrid polyketides bearing tetramic acid (pyrrolidine-2,4-dione) moiety exhibiting a broad range of bioactivities. Biosynthesis of tetramates in microorganisms is normally directed by hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) machineries, which form the tetramic acid ring by recruiting trans- or cis-acting thioesterase-like Dieckmann cyclase in bacteria. There are a group of tetramates with unique skeleton of 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione, which remain to be investigated for their biosynthetic logics.
RESULTS
Herein, the tetramate type compounds bripiodionen (BPD) and its new analog, featuring the rare skeleton of 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione, were discovered from the sponge symbiotic bacterial Streptomyces reniochalinae LHW50302. Gene deletion and mutant complementation revealed the production of BPDs being correlated with a PKS-NRPS biosynthetic gene cluster (BGC), in which a Dieckmann cyclase gene bpdE was identified by sit-directed mutations. According to bioinformatic analysis, the tetramic acid moiety of BPDs should be formed on an atypical NRPS module constituted by two discrete proteins, including the C (condensation)-A (adenylation)-T (thiolation) domains of BpdC and the A-T domains of BpdD. Further site-directed mutagenetic analysis confirmed the natural silence of the A domain in BpdC and the functional necessities of the two T domains, therefore suggesting that an unusual aminoacyl transthiolation should occur between the T domains of two NRPS subunits. Additionally, characterization of a LuxR type regulator gene led to seven- to eight-fold increasement of BPDs production. The study presents the first biosynthesis case of the natural molecule with 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione skeleton. Genomic mining using BpdD as probe reveals that the aminoacyl transthiolation between separate NRPS subunits should occur in a certain population of NRPSs in nature.
Topics: Biosynthetic Pathways; Polyketide Synthases; Bacteria; Pyrans; Skeleton; Peptide Synthases; Pyrrolidinones
PubMed: 38515152
DOI: 10.1186/s12934-024-02364-7 -
Science (New York, N.Y.) Mar 2024Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural...
Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook -acyltransferase (-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, -AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered -AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.
Topics: Acyltransferases; Polyketide Synthases; Polyketides; Directed Molecular Evolution; Bacterial Proteins; Serratia; Amino Acid Motifs; Recombinant Fusion Proteins
PubMed: 38513027
DOI: 10.1126/science.adj7621