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Microbiology Spectrum Jun 2023Eicosapentaenoic acid (EPA) is an omega-3 long-chain polyunsaturated fatty acid (PUFA) essential for human health. is a marine eukaryote that has been widely utilized...
Eicosapentaenoic acid (EPA) is an omega-3 long-chain polyunsaturated fatty acid (PUFA) essential for human health. is a marine eukaryote that has been widely utilized for the synthesis of PUFAs. The current low potency and performance of EPA production by fermentation of spp. limits its prospect in commercial production of EPA. Since the synthesis pathway of EPA in spp. is still unclear, mutagenesis combined with efficient screening methods are still desirable. In this study, a novel screening strategy was developed based on a two-step progressive mutagenesis method based on atmospheric and room temperature plasma (ARTP) and diethyl sulfate (DES) after multiple stresses (sethoxydim, triclosan and 2,2'-bipyridine) compound screening. Finally, the mutant strain DBT-64 with increased lipid (1.57-fold, 31.71 g/L) and EPA (5.64-fold, 1.86 g/L) production was screened from wild-type (W) strains; the docosahexaenoic acid (DHA) content of mutant DBT-64 (M) was 11.41% lower than that of wild-type strains. Comparative transcriptomic analysis showed that the expression of genes related to the polyketide synthase, fatty acid prolongation, and triglyceride synthesis pathways was significantly upregulated in the mutant strain, while the expression of genes involved in the β-oxidation pathway and fatty acid degradation pathway was downregulated in favor of EPA biosynthesis in . This study provides an effective strain improvement method to enhance EPA accumulation in spp. , a marine eukaryotic microorganism, has emerged as a candidate for the commercial production of PUFAs. EPA is an omega-3 PUFA with preventive and therapeutic effects against cardiovascular diseases, schizophrenia, and other disorders. Currently, the low potency and performance of EPA production by spp. limits its commercialization. In this study, we performed two-step progressive mutagenesis based on ARTP and DES and screened multiple stresses (sethoxydim, triclosan, and 2,2'-bipyridine) to obtain the EPA-high-yielding mutant. In addition, high expression of the polyketide synthase pathway, fatty acid elongation pathway, and triglyceride synthesis pathway in the mutants was confirmed by transcriptomic analysis. Therefore, the multistress screening platform established in this study is important for breeding EPA-producing spp. and provides valuable information for regulating the proportion of EPA in microalgal lipids by means of genetic engineering.
Topics: Humans; Eicosapentaenoic Acid; Polyketide Synthases; 2,2'-Dipyridyl; Triclosan; Stramenopiles; Fatty Acids, Unsaturated; Fatty Acids; Gene Expression Profiling; Triglycerides
PubMed: 37093006
DOI: 10.1128/spectrum.00130-23 -
Structure (London, England : 1993) Sep 2022The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KS, that primes polyketide synthesis. Unlike downstream KSs...
The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KS, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups to growing polyketide chains, it performs an extension-decoupled decarboxylation of these groups to generate primer units. When Pik127, a model triketide synthase constructed from modules of the pikromycin synthase, was studied by cryoelectron microscopy (cryo-EM), the dimeric didomain comprised of KS and the neighboring methylmalonyl-selective acyltransferase (AT) dominated the class averages and yielded structures at 2.5- and 2.8-Å resolution, respectively. Comparisons with ketosynthases complexed with their substrates revealed the conformation of the (2S)-methylmalonyl-S-phosphopantetheinyl portion of KS and KS substrates prior to decarboxylation. Point mutants of Pik127 probed the roles of residues in the KS active site, while an AT-swapped version of Pik127 demonstrated that KS can also decarboxylate malonyl groups. Mechanisms for how KS and KS domains catalyze carbon-carbon chemistry are proposed.
Topics: Acyltransferases; Carbon; Cryoelectron Microscopy; Macrolides; Polyketide Synthases
PubMed: 35738283
DOI: 10.1016/j.str.2022.05.021 -
BMC Biology Jun 2022Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and...
BACKGROUND
Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure-function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis.
RESULTS
We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility.
CONCLUSIONS
The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.
Topics: Bacterial Proteins; Mycobacterium tuberculosis; Mycolic Acids; Polyketide Synthases; Polyketides
PubMed: 35729566
DOI: 10.1186/s12915-022-01337-9 -
Environmental Microbiology Jun 2019To date, no demonstration of a direct correlation between the presence of mycoviruses and the quantitative or qualitative modulation of mycotoxins has been shown. In our...
To date, no demonstration of a direct correlation between the presence of mycoviruses and the quantitative or qualitative modulation of mycotoxins has been shown. In our study, we transfected a virus-free ochratoxin A (OTA)-producing isolate of Aspergillus ochraceus with purified mycoviruses from a different A. ochraceus isolate and from Penicillium aurantiogriseum. Among the mycoviruses tested, only Aspergillus ochraceus virus (AoV), a partitivirus widespread in A. ochraceus, caused a specific interaction that led to an overproduction of OTA, which is regulated by the European Commission and is the second most important contaminant of food and feed commodities. Gene expression analysis failed to reveal a specific viral upregulation of the mRNA of genes considered to play a role in the OTA biosynthetic pathway. Furthermore, AoOTApks1, a polyketide synthase gene considered essential for OTA production, is surprisingly absent in the genome of our OTA-producing isolate. The possible biological and evolutionary implications of the mycoviral regulation of mycotoxin production are discussed.
Topics: Aspergillus ochraceus; Biosynthetic Pathways; Fungal Proteins; Fungal Viruses; Ochratoxins; Penicillium; Polyketide Synthases
PubMed: 30289193
DOI: 10.1111/1462-2920.14436 -
Communications Biology Dec 2022Presently, the supply of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) traditionally produced by marine fisheries will be insufficient to meet their market...
Presently, the supply of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) traditionally produced by marine fisheries will be insufficient to meet their market demand in food industry. Thus a sustainable alternative source is urgently required. Schizochytrium sp. is an ideal producer of DHA; however, its ability to co-produce DHA and EPA has not yet been proved. Herein, we first described a cobalamin-independent methionine synthase-like (MetE-like) complex, which contains independent acyltransferase and 3-ketoacyl synthase domains, independent of the traditional polyketide synthase (PKS) system. When the MetE-like complex was activated, the EPA content was increased from 1.26% to 7.63%, which is 6.06-folds higher than that in the inactivated condition. Through lipidomics, we find that EPA is more inclined to be stored as triglyceride. Finally, the EPA production was enhanced from 4.19 to 29.83 (mg/g cell dry weight) using mixed carbon sources, and the final yield reached 2.25 g/L EPA and 9.59 g/L DHA, which means that Schizochytrium sp. has great market potential for co-production of EPA and DHA.
Topics: Eicosapentaenoic Acid; Docosahexaenoic Acids; Polyketide Synthases
PubMed: 36494568
DOI: 10.1038/s42003-022-04334-4 -
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 -
Marine Drugs Jun 2023Marinolides A and B, two new 24- and 26-membered bacterial macrolactones, were isolated from the marine-derived actinobacterium AJS-327 and their stereostructures...
Expanding the Utility of Bioinformatic Data for the Full Stereostructural Assignments of Marinolides A and B, 24- and 26-Membered Macrolactones Produced by a Chemically Exceptional Marine-Derived Bacterium.
Marinolides A and B, two new 24- and 26-membered bacterial macrolactones, were isolated from the marine-derived actinobacterium AJS-327 and their stereostructures initially assigned by bioinformatic data analysis. Macrolactones typically possess complex stereochemistry, the assignments of which have been one of the most difficult undertakings in natural products chemistry, and in most cases, the use of X-ray diffraction methods and total synthesis have been the major methods of assigning their absolute configurations. More recently, however, it has become apparent that the integration of bioinformatic data is growing in utility to assign absolute configurations. Genome mining and bioinformatic analysis identified the 97 kb biosynthetic cluster harboring seven type I polyketide synthases. A detailed bioinformatic investigation of the ketoreductase and enoylreductase domains within the multimodular polyketide synthases, coupled with NMR and X-ray diffraction data, allowed for the absolute configurations of marinolides A and B to be determined. While using bioinformatics to assign the relative and absolute configurations of natural products has high potential, this method must be coupled with full NMR-based analysis to both confirm bioinformatic assignments as well as any additional modifications that occur during biosynthesis.
Topics: Polyketide Synthases; Macrolides; Computational Biology; Bacteria; Biological Products
PubMed: 37367692
DOI: 10.3390/md21060367 -
Microbial Biotechnology Oct 2022Chaetoglobosin A is a complex macrocyclic alkaloid with potent antimycotic, antiparasitic and antitumor properties. However, the low output and high cost of...
Chaetoglobosin A is a complex macrocyclic alkaloid with potent antimycotic, antiparasitic and antitumor properties. However, the low output and high cost of chaetoglobosin A biosynthesis have hampered the application and commercialization of chaetoglobosin A in agriculture and biomedicine. Here, the CgMfs1 gene, which encodes the major facilitator superfamily secondary transporter, was identified based on bioinformatics analysis, and an intensive study of its effects on chaetoglobosin A biosynthesis and secretion was performed using CgMfs1-silencing and CgMfs1-overexpression strategies. Inactivation of CgMfs1 caused a notable decrease in chaetoglobosin A yield from 58.66 mg/L to 19.95 mg/L (MFS1-3) and 17.13 mg/L (MFS1-4). The use of an efficient expression plasmid in Chaetomium globosum W7 to generate the overexpression mutant OEX13 resulted in the highest chaetoglobosin A increase to 298.77 mg/L. Interestingly, the transcription level of the polyketide synthase gene significantly fluctuated with the change in CgMfs1, confirming that the predicted efflux gene CgMfs1 could play a crucial role in chaetoglobosin A transportation. Effective efflux of chaetoglobosin A could possibly alleviate feedback inhibition, resulting in notable increase in the expression of the polyketide synthase gene. Furthermore, we utilized cornstalk as the fermentation substrate to produce chaetoglobosin A, and scanning electron microscopy and Fourier transform-infrared spectroscopy revealed that the strain OEX13 could well degrade cornstalk, presenting significant increases in the chaetoglobosin A yield, when compared with that produced by the wild-type strain (from 40.32 to 191.90 mg/L). Thus, this research provides a novel analogous engineering strategy for the construction of high-yielding strain and offers new insight into large-scale chaetoglobosin A production.
Topics: Alkaloids; Antiparasitic Agents; Chaetomium; Indole Alkaloids; Polyketide Synthases
PubMed: 35930651
DOI: 10.1111/1751-7915.14111 -
Molecules (Basel, Switzerland) Jul 2022Polyesters containing 2,4-dihydroxy-6-(2-hydroxypropyl)benzoate and 3-hydroxybutyrate moieties have been isolated from many fungal species. was previously reported to...
Polyesters containing 2,4-dihydroxy-6-(2-hydroxypropyl)benzoate and 3-hydroxybutyrate moieties have been isolated from many fungal species. was previously reported to produce a similar polyester, talapolyester G. The complete genome sequence and the development of bioinformatics tools have enabled the discovery of the biosynthetic potential of this microorganism. Here, a putative biosynthetic gene cluster (BGC) of the polyesters encoding a highly reducing polyketide synthase (HR-PKS) and nonreducing polyketide synthase (NR-PKS), a cytochrome P450 and a regulator, was identified. Although talapolyester G does not require an oxidative step for its biosynthesis, further investigation into the secondary metabolite production of resulted in isolating two new metabolites called talarodioxadione and talarooxime, in addition to three known compounds, namely 6-hydroxymellein, 15G256α and transtorine that have never been reported from this organism. Interestingly, the biosynthesis of the cyclic polyester 15G256α requires hydroxylation of an inactive methyl group and thus could be a product of the identified gene cluster. The two compounds, talarooxime and transtorine, are probably the catabolic metabolites of tryptophan through the kynurenine pathway. Tryptophan metabolism exists in almost all organisms and has been of interest to many researchers. The biosynthesis of the new oxime is proposed to involve two subsequent N-hydroxylation of 2-aminoacetophenone.
Topics: Multigene Family; Polyesters; Polyketide Synthases; Polyketides; Talaromyces; Tryptophan
PubMed: 35889347
DOI: 10.3390/molecules27144473 -
Proceedings of the National Academy of... Oct 2022Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context...
Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase () inactivation and overproduction strains of the social amoeba . Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.
Topics: Amoeba; Biological Products; Dictyostelium; Polyketide Synthases; Polyketides
PubMed: 36252029
DOI: 10.1073/pnas.2116122119