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Journal of Fungi (Basel, Switzerland) May 2024pigments (MPs), a class of secondary metabolites produced by spp., can be classified into yellow, orange, and red MPs according to their differences in the wavelength...
pigments (MPs), a class of secondary metabolites produced by spp., can be classified into yellow, orange, and red MPs according to their differences in the wavelength of the maximum absorption. However, the biosynthetic sequence and cellular biosynthesis mechanism of different MPs components are still not yet completely clear in spp. In this study, the subcellular localization of five MPs synthases was investigated using fluorescent protein fusion expression. The results revealed that the proteins encoded by the MPs biosynthetic gene cluster were compartmentalized in various subcellular locations, including the mitochondrial polyketide synthase MrPigA, cytosolic enzymes consisting of the ketoreductase MrPigC, the oxidoreductase MrPigE, and the monooxygenase MrPigN, and the cell-wall-bound oxidoreductase MrPigF. Moreover, the correct localization of MrPigF to the cell wall was crucial for the synthesis of orange MPs. Lastly, we discussed the compartmentalized biosynthetic pathway of MPs. This study will not only be helpful in clarifying the biosynthetic sequence and biosynthesis mechanism of different MPs but also provides new insights into the cellular biosynthesis of secondary metabolites in filamentous fungi.
PubMed: 38921362
DOI: 10.3390/jof10060375 -
Nature Communications Jun 2024Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of...
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.
Topics: Polyketides; Multigene Family; Polyketide Synthases; Mass Spectrometry; Data Mining; Machine Learning; Actinobacteria; Genome, Bacterial; Algorithms; Biological Products
PubMed: 38918378
DOI: 10.1038/s41467-024-49587-1 -
Phytopathology Jun 2024, the causal agent of pear canker disease, typically induces cankers on the bark of infected trees and even leads to tree mortality. Secondary metabolites (SMs) produced...
, the causal agent of pear canker disease, typically induces cankers on the bark of infected trees and even leads to tree mortality. Secondary metabolites (SMs) produced by pathogenic fungi play a crucial role in the pathogenic process. In this study, secondary metabolic regulator VpLaeA was identified in . was found to strongly affect the pathogenicity, fruiting body formation and toxicity of SMs of . Additionally, was also found to be required for the response of to some abiotic stresses. Transcriptome data analysis revealed that many of differentially expressed genes were involved in the secondary metabolite biosynthesis (SMB). Among them, about one third of SMB core genes were regulated by at different periods. Seven differentially expressed SMB core genes (, , , , , , and ) were selected for knockout. Two modular polyketide synthase (PKS) genes ( and ), which were closely related to the virulence of from the above seven genes were identified. Notably, and also affected the production of fruiting body of , but didn't participate in the resistance of to abiotic stresses. Overall, this study demonstrates the multifaceted biological functions of in , and identifies two toxicity-associated PKS genes in species fungi for the first time.
PubMed: 38916927
DOI: 10.1094/PHYTO-12-23-0498-R -
Structure (London, England : 1993) Jun 2024Docking domains (DDs) located at the C- and N-termini of polypeptides play a crucial role in directing the assembly of polyketide synthases (PKSs), which are multienzyme...
Docking domains (DDs) located at the C- and N-termini of polypeptides play a crucial role in directing the assembly of polyketide synthases (PKSs), which are multienzyme complexes. Here, we determined the crystal structure of a complex comprising the C-terminal DD (DD) and N-terminal DD (DD) of macrolactin trans-acyltransferase (AT) PKS that were fused to a functional enzyme, AmpC EC2 β-lactamase. Interface analyses of the DD/DD complex revealed the molecular intricacies in the core section underpinning the precise DD assembly. Additionally, circular dichroism and steady-state kinetics demonstrated that the formation of the DD/DD complex had no influence on the structural and functional fidelity of the fusion partner, AmpC EC2. This inspired us to apply the DD/DD assembly to metabolon engineering. Indeed, DD assembly induced the formation of a complex between 4-coumarate-CoA ligase and chalcone synthase both involved in flavonoid biosynthesis, leading to a remarkable increase in naringenin production in vitro.
PubMed: 38908377
DOI: 10.1016/j.str.2024.05.017 -
Nucleic Acids Research Jun 2024Filamentous Actinobacteria, recently renamed Actinomycetia, are the most prolific source of microbial bioactive natural products. Studies on biosynthetic gene clusters...
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.
PubMed: 38908028
DOI: 10.1093/nar/gkae523 -
Journal of Agricultural and Food... Jun 2024Potato 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...
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 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.
PubMed: 38899439
DOI: 10.1021/acs.jafc.4c02572 -
International Journal of Molecular... May 2024Utilizing bioinformatics tools, this study expands our understanding of secondary metabolism in , identifying novel genes within polyketide synthase (PKS), non-ribosomal...
Utilizing bioinformatics tools, this study expands our understanding of secondary metabolism in , 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 '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 '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 . This study sets a foundation for further investigations into the fungus's secondary metabolism, with implications for biotechnology and crop disease management.
Topics: Botrytis; Secondary Metabolism; Peptide Synthases; Polyketide Synthases; Fungal Proteins; Computational Biology; Multigene Family; Genes, Fungal
PubMed: 38892087
DOI: 10.3390/ijms25115900 -
Chembiochem : a European Journal of... Jun 2024Hitachimycin is a bicyclic macrolactam antibiotic with (S)-β-phenylalanine (β-Phe) at the starter position of the polyketide skeleton. While the enzymes that recognize...
Hitachimycin is a bicyclic macrolactam antibiotic with (S)-β-phenylalanine (β-Phe) at the starter position of the polyketide skeleton. While the enzymes that recognize β-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.
PubMed: 38849317
DOI: 10.1002/cbic.202400405 -
Metabolic Engineering Communications Jun 2024Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial...
Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast , by fusing carbon-source responsive elements of the native promoter to the strong or native promoter cores. Two hybrids, P and P , were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of P increased TAL more than 50% relative to the native promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the 6-methylsalicylic acid synthase (6-MSAS) under the control of P resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used P to express the IaaM and IaaH proteins and the sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for , allowing greater control over heterologous protein expression and improved production of toxic metabolites.
PubMed: 38845682
DOI: 10.1016/j.mec.2024.e00238 -
Scientific Reports Jun 2024Some 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 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.
Topics: Cell-Free System; Promoter Regions, Genetic; Streptomyces griseus; Escherichia coli; Multigene Family; Bacterial Proteins; Polyketide Synthases; Codon; Acyltransferases
PubMed: 38839808
DOI: 10.1038/s41598-024-61376-w