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BioRxiv : the Preprint Server For... Dec 2023A subset of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are encoded in their biosynthetic gene clusters (BGCs) with enzymes annotated as...
A subset of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are encoded in their biosynthetic gene clusters (BGCs) with enzymes annotated as lantibiotic dehydratases. The functions of these putative lantibiotic dehydratases remain unknown. Here, we characterize an NRPS-PKS BGC with a putative lantibiotic dehydratase from the bacterium (). Heterologous expression revealed several metabolites produced by the BGC, and the omission of selected biosynthetic enzymes revealed the biosynthetic sequence towards these compounds. The putative lantibiotic dehydratase catalyzes peptide bond formation that extends the peptide scaffold opposite to the NRPS and PKS biosynthetic direction. The condensation domain of the NRPS catalyzes the formation of a ureido group, and bioinformatics analysis revealed distinct active site residues of ureido-generating condensation (UreaC) domains. This work demonstrates that the annotated lantibiotic dehydratase serves as a separate amide bond-forming machinery in addition to the NRPS, and that the lantibiotic dehydratase enzyme family possesses diverse catalytic activities in the biosynthesis of both ribosomal and non-ribosomal natural products.
PubMed: 38187666
DOI: 10.1101/2023.12.23.573212 -
Nature Communications Jan 2024Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized...
Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized metabolites. The evolutionary origin of the animal FAS and its relationship to the diversity of PKSs remain unclear despite the critical role of lipid synthesis in cellular metabolism. Recently, an animal FAS-like PKS (AFPK) was identified in sacoglossan molluscs. Here, we explore the phylogenetic distribution of AFPKs and other PKS and FAS enzymes across the tree of life. We found AFPKs widely distributed in arthropods and molluscs (>6300 newly described AFPK sequences). The AFPKs form a clade with the animal FAS, providing an evolutionary link bridging the type I PKSs and the animal FAS. We found molluscan AFPK diversification correlated with shell loss, suggesting AFPKs provide a chemical defense. Arthropods have few or no PKSs, but our results indicate AFPKs contributed to their ecological and evolutionary success by facilitating branched hydrocarbon and pheromone biosynthesis. Although animal metabolism is well studied, surprising new metabolic enzyme classes such as AFPKs await discovery.
Topics: Animals; Polyketides; Fatty Acids; Lipid Metabolism; Phylogeny; Polyketide Synthases; Fatty Acid Synthases
PubMed: 38172109
DOI: 10.1038/s41467-023-44497-0 -
International Journal of Molecular... Dec 2023is a class of plant-specific transcription factors that are widely involved in the growth, development and (a)biotic stress response of plants. However, their molecular...
is a class of plant-specific transcription factors that are widely involved in the growth, development and (a)biotic stress response of plants. However, their molecular evolution has not been extensively studied in Malvales, especially in , a commercial and horticultural crop that produces an aromatic resin named agarwood. In this study, 1502 members of the gene family were identified from the genomes of nine species from Malvales and three model plants. The macroevolutionary analysis revealed that whole genome duplication (WGD) and dispersed duplication (DSD) have shaped the current architectural structure of gene families in Malvales plants. Then, 111 genes were systemically characterized in . The phylogenetic analysis suggests that genes in can be classified into 16 known clusters and four new subfamilies, with each subfamily presenting similar gene structures and conserved motifs. RNA-seq analysis showed that presents a broad transcriptional response to the agarwood inducer. The expression patterns of 15 in after injury treatment indicated that and were positively correlated with the expression patterns of four polyketide synthase (PKS) genes. Additionally, and were also found to bind with the promoter and activate its transcription. This comprehensive analysis provides valuable insights into the molecular evolution of the gene family in Malvales plants and highlights the potential mechanisms of for regulating secondary metabolite biosynthesis in , especially for the biosynthesis of 2-(2-phenyl) chromones in agarwood.
Topics: Malvales; Transcription Factors; Phylogeny; Thymelaeaceae; Genes, Plant
PubMed: 38139213
DOI: 10.3390/ijms242417384 -
Genes Dec 2023Peanut () and its wild relatives are among the few species that naturally synthesize resveratrol, a well-known stilbenoid phytoalexin that plays a crucial role in plant...
Peanut () and its wild relatives are among the few species that naturally synthesize resveratrol, a well-known stilbenoid phytoalexin that plays a crucial role in plant defense against biotic and abiotic stresses. Resveratrol has received considerable attention due to its health benefits, such as preventing and treating various human diseases and disorders. Chalcone (CHS) and Stilbene (STS) Synthases are plant-specific type III Polyketide Synthases (PKSs) that share the same substrates and are key branch enzymes in the biosynthesis of flavonoids and stilbenoids, respectively. Although resveratrol accumulation in response to external stimulus has been described in peanut, there are no comprehensive studies of the CHS and STS gene families in the genus . In the present study, we identified and characterized 6 CHS and 46 STS genes in the tetraploid peanut and an average of 4 CHS and 22 STS genes in three diploid wild species ( and ). The CHS and STS gene and protein structures, chromosomal distributions, phylogenetic relationships, conserved amino acid domains, and -acting elements in the promoter regions were described for all species studied. Based on gene expression patterns of wild STS genes in response to different biotic and abiotic stresses, we selected the candidate gene, which is strongly induced by ultraviolet (UV) light exposure, for further functional investigation. The overexpression in peanut hairy roots significantly reduced (47%) root-knot nematode infection, confirming that stilbene synthesis activation in transgenic plants can increase resistance to pathogens. These findings contribute to understanding the role of resveratrol in stress responses in species and provide the basis for genetic engineering for improved production of valuable secondary metabolites in plants.
Topics: Humans; Arachis; Genome-Wide Association Study; Phylogeny; Resveratrol; Fabaceae
PubMed: 38137003
DOI: 10.3390/genes14122181 -
Angewandte Chemie (International Ed. in... Feb 2024Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the...
Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)-bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter-domain interactions after C-C bond formation in a chain-branching module of the rhizoxin PKS. Mechanism-based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C-C bond formation by cryo-electron microscopy (cryo-EM). The possible existence of two ACP binding sites, one of them a potential "parking position" for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C-C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines.
Topics: Polyketide Synthases; Cryoelectron Microscopy; Binding Sites; Catalytic Domain; Acyl Carrier Protein
PubMed: 38134222
DOI: 10.1002/anie.202315850 -
Journal of Fungi (Basel, Switzerland) Dec 2023microsclerotia are fungal aggregates composed of compacted, pigmented hyphae. As they are highly tolerant to desiccation and produce infective conidia, they are...
microsclerotia are fungal aggregates composed of compacted, pigmented hyphae. As they are highly tolerant to desiccation and produce infective conidia, they are promising candidates to be formulated as bioinsecticides. Despite this potential, the nature of the pigments within these structures remains unclear. In this study, routine culture media used for the differentiation of microsclerotia were supplemented with four melanin inhibitors, and the resulting propagules were characterized. Inhibitors of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthetic pathway such as tricyclazole and guaiacol induced significant phenotypic and molecular modifications in the obtained propagules, which exhibited a more spherical shape, reduced size, and increased susceptibility to desiccation, heat, and oxidative stress than microsclerotia obtained without inhibitors. Additionally, genes encoding for a polyketide synthase (Mrpks2) and a putative 1,3,6,8-tetrahydroxynaphthalene reductase (Mrthnr), potentially involved in the DHN-melanin biosynthetic pathway, were upregulated in fungi grown in the inhibitor-added media. In conclusion, microsclerotia contain melanins of type DHN that might play a role in both microsclerotia differentiation and environmental stress tolerance.
PubMed: 38132763
DOI: 10.3390/jof9121162 -
Journal of Agricultural and Food... Jan 2024Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as...
Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as complex mixtures of similar compounds that are difficult to separate. Synthetic biology offers the opportunity to produce various flavonoids in a targeted, bottom-up approach in engineered microbes with high product titers. However, the production of O-methylated flavonoids is currently still highly inefficient. In this study, we investigated and engineered a combination of enzymes that had previously been shown to support homoeriodictyol and hesperetin production in from fed O-methylated hydroxycinnamic acids. We determined the crystal structures of the enzyme catalyzing the first committed step of the pathway, chalcone synthase from , in three ligand-bound states. Based on these structures and a multiple sequence alignment with other chalcone synthases, we constructed mutant variants and assessed their performance in toward producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234 V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than reported previously. Our findings will facilitate further engineering of this enzyme toward higher production of methylated flavonoids.
Topics: Flavonoids; Polyketide Synthases; Escherichia coli; Plants; Sequence Alignment
PubMed: 38109879
DOI: 10.1021/acs.jafc.3c06785 -
Journal of Industrial Microbiology &... Feb 2023Two new macrocyclic secondary metabolites, glycosyl-migrastatin (1) and 5-hydroxy-migrastatin (2), were isolated from a gut bacterium Kitasatospora sp. JL24 in dung...
UNLABELLED
Two new macrocyclic secondary metabolites, glycosyl-migrastatin (1) and 5-hydroxy-migrastatin (2), were isolated from a gut bacterium Kitasatospora sp. JL24 in dung beetle Onthophagus lenzii. Based on a comprehensive analysis of the nuclear magnetic resonance (NMR), MS, and UV spectroscopic data, the planar structures of 1 and 2 were successfully identified as new derivatives of migrastatin. Compound 1 was the first glycosylated member of the migrastatin family. The absolute configuration of the sugar moiety was determined to be d-glucose through the analysis of coupling constants and ROESY correlations, followed by chemical derivatization and chromatographic comparison with authentic d- and l-glucose. Compound 2, identified as 5-hydroxy-migrastatin possessing an additional hydroxy group with a previously unreported chiral center, was assigned using Mosher's method through 19F NMR chemical shifts and confirmed with the modified Mosher's method. Genomic analysis of Kitasatospora sp. strain JL24 revealed a putative biosynthetic pathway involving an acyltransferase-less type I polyketide synthase biosynthetic gene cluster.
ONE-SENTENCE SUMMARY
Two secondary metabolites, glycosyl-migrastatin (1) and 5-hydroxy-migrastatin (2), were discovered from the gut bacterium Kitasatospora sp. JL24 in the dung beetle Onthophagus lenzii.
Topics: Macrolides; Piperidones; Magnetic Resonance Spectroscopy; Bacteria; Molecular Structure
PubMed: 38093455
DOI: 10.1093/jimb/kuad046 -
BioRxiv : the Preprint Server For... Dec 2023Some 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...
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 . Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in frequently results in a myriad of unpredictable issues with 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, such as BGC refactoring, can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as 1) a valuable expression platform for enzymes that are challenging to synthesize , and as 2) a testbed for rapid prototyping that can improve cellular expression. Here, we use a type III polyketide synthase from , RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques We synergistically tune promoter and codon usage to improve flaviolin production from cell-free expressed RppA. We then 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. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs. By showing the coordinators between CFE versus expression, this work advances CFE as a tool for natural product research.
PubMed: 38077034
DOI: 10.1101/2023.11.30.569483 -
Frontiers in Microbiology 2023A novel marine actinomycete, designated strain MCN248, was isolated from the coastal sediment in Songkhla Province, Thailand. Based on the 16S rRNA gene sequences, the...
A novel marine actinomycete, designated strain MCN248, was isolated from the coastal sediment in Songkhla Province, Thailand. Based on the 16S rRNA gene sequences, the new isolate was closely related to DSM45887 (99.2%) and DSM43553 (98.6%). Phylogenetic analyzes based on the 16S rRNA gene sequences showed that strain MCN248 was clustered with DSM45887 and DSM43553. However, the digital DNA-DNA hybridization analyzes presented a low relatedness of 40.2% between strain MCN248 and the above closely related strains. This strain contained meso-diaminopimelic acid. The acyl type of the peptidoglycan was acetyl, and mycolic acids were absent. The major menaquinones were MK-9(H) and MK-9(H). The whole cell sugars consisted of madurose, ribose, mannose, and glucose. Diphosphatidylglycerol, hydroxyl-phosphatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol were detected as the major phospholipids. The predominant cellular fatty acids were -C (40.4%), 10-methyl-C (22.1%), and C8c (10.9%). The DNA G + C content of the genomic DNA was 71.7%. With analyzes, the antiSMASH platform uncovered a diverse 29 secondary metabolite biosynthesis arsenal, including non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) of strain MCN248, with a high prevalence of gene cluster encoding pathways for the production of anticancer and cytotoxic compounds. Consistently, the crude extract could inhibit colorectal HCT-116 cancer cells at a final concentration of 50 μg/mL. Based on the polyphasic approach, strain MCN248 was designated as a novel species of the genus , for which the name sp. nov. is proposed. The type strain of the type species is MCN248 (=NBRC115966 = TBRC17110).
PubMed: 38053561
DOI: 10.3389/fmicb.2023.1226945