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Synthetic and Systems Biotechnology Sep 2023The bacterial trialkyl-substituted aromatic polyketides are structurally featured with the unusual aromatic core in the middle of polyketide chain such as TM-123 (),...
Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual P450 monooxygenase-mediating aromatization and a putative metallo-beta-lactamase fold hydrolase.
The bacterial trialkyl-substituted aromatic polyketides are structurally featured with the unusual aromatic core in the middle of polyketide chain such as TM-123 (), veramycin A (), NFAT-133 () and benwamycin I (), which were discovered from species and demonstrated with antidiabetic and immunosuppressant activities. Though the biosynthetic pathway of - was reported as a type I polyketide synthase (PKS), the PKS assembly line was interpreted inconsistently, and it remains a mystery how the compound was generated. Herein, the PKS assembly logic of - was revised by site-mutagenetic analysis of the PKS dehydratase domains. Based on gene deletion and complementation, the putative P450 monooxygenase E and metallo-beta-lactamase (MBL) fold hydrolase F were verified as essential genes for the biosynthesis of -. The absence of E led to abolishment of - and accumulation of new products (-). Structural elucidation reveals - as the non-aromatic analogs of , suggesting the NftE-catalyzed aromatic core formation. Deletion of F resulted in disappearance of and with the compounds and unaffected. As a rare MBL-fold hydrolase from type I PKSs, NftF potentially generates the compound through two strategies: catalyze premature chain-offloading as a -acting thioesterase or hydrolyze the lactone-bond of compound as an esterase.
PubMed: 37325182
DOI: 10.1016/j.synbio.2023.05.003 -
ACS Synthetic Biology May 2024spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have...
spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in M1152Δ that could produce aklavinone, 9--aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and M1152Δ expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant hosts.
Topics: Polyketide Synthases; Anthracyclines; Streptomyces coelicolor; Streptomyces; Biosynthetic Pathways; Hydroxylation; Anti-Bacterial Agents
PubMed: 38662967
DOI: 10.1021/acssynbio.4c00043 -
Marine Drugs Sep 2023Coumarins, isocoumarins and their derivatives are polyketides abundant in fungal metabolites. Although they were first discovered over 50 years ago, the biosynthetic...
Coumarins, isocoumarins and their derivatives are polyketides abundant in fungal metabolites. Although they were first discovered over 50 years ago, the biosynthetic process is still not entirely understood. Herein, we report the activation of a silent nonreducing polyketide synthase that encodes a C-methylated isocoumarin, similanpyrone B (), in a marine-derived fungus HDN13-430 by heterologous expression. Feeding studies revealed the host enzymes can change into its hydroxylated derivatives pestapyrone A (). Compounds and showed moderate radical scavenging activities with ED values of 67.4 µM and 104.2 µM. Our discovery fills the gap in the enzymatic elucidation of naturally occurring C-methylated isocoumarin derivatives.
Topics: Isocoumarins; Polyketide Synthases; Coumarins; Hypocreales
PubMed: 37755103
DOI: 10.3390/md21090490 -
BMC Genomics Jun 2024The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer...
BACKGROUND
The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized.
RESULTS
The aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein).
CONCLUSIONS
The results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.
Topics: Hypericum; Multigene Family; Polyketides; Endophytes; Anthraquinones; Fungi; Genome, Fungal; Computer Simulation; Polyketide Synthases; Perylene; Anthracenes; Genomics; Phylogeny
PubMed: 38831295
DOI: 10.1186/s12864-024-10475-z -
Molecular Biology Reports Oct 2023Alkylresorcinols (ARs) are compounds belonging to the class of phenolic lipids. A rich source of ARs are cereal grains such as rye, wheat, triticale or barley. ARs found...
BACKGROUND
Alkylresorcinols (ARs) are compounds belonging to the class of phenolic lipids. A rich source of ARs are cereal grains such as rye, wheat, triticale or barley. ARs found in plants are characterized by a variety of biological properties such as antimicrobial, antifungal and cytotoxic activity. Moreover, they are proven to have a positive influence on human health. Here, we aimed to find and characterize the gene with ARs synthase activity in the species Secale cereale.
METHODS AND RESULTS
Using BAC library screening, two BAC clones containing the gene candidate were isolated and sequenced. Bioinformatic analyses of the resulting contigs were used to examine the structure and other features of the gene, including promoter, intron, 3'UTR and 5'UTR. Mapping using the FISH procedure located the gene on the 4R chromosome. Comparative analysis showed that the gene is highly similar to sequences coding for type III polyketide synthase. The level of gene expression in various parts of the plant was investigated, and the biochemical function of the gene was confirmed by heterologous expression in yeast.
CONCLUSIONS
The conducted analyses contributed to a better understanding of the processes related to ARs synthesis. Although the research concerned the rye model, the knowledge gained may help in understanding the genetic basis of ARs biosynthesis in other species of the Poaceae family as well.
Topics: Humans; Secale; Gene Library; Base Sequence; Introns; Edible Grain
PubMed: 37615923
DOI: 10.1007/s11033-023-08684-y -
Communications Biology May 2024Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the...
Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS's steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.
Topics: Substrate Specificity; Quinolones; Catalytic Domain; Models, Molecular; Polyketide Synthases; Crystallography, X-Ray; Protein Conformation
PubMed: 38745065
DOI: 10.1038/s42003-024-06152-2 -
Nature Communications Nov 2023The surface coatings of cereal plants are dominated by waxy β-diketones crucial for drought resistance and, therefore, grain yield. Here, barley (Hordeum vulgare) wax...
The surface coatings of cereal plants are dominated by waxy β-diketones crucial for drought resistance and, therefore, grain yield. Here, barley (Hordeum vulgare) wax analyses reveal β-diketone and associated 2-alkanol ester profiles suggesting a common C 3-ketoacid precursor. Isotope analysis further shows that the major (C) diketone is synthesized from two plastidial C acyl units. Previous studies identified a gene cluster encoding enzymes responsible for β-diketone formation in barley, but left their biochemical functions unknown. Various assays now characterize one of these enzymes as a thioesterase producing long-chain (mainly C) 3-ketoacids, and another one as a polyketide synthase (PKS) condensing the 3-ketoacids with long-chain (mainly C) acyl-CoAs into β-diketones. The two enzymes are localized to the plastids and Endoplasmic Reticulum (ER), respectively, implying substrate transfer between these two sub-cellular compartments. Overall, our findings define a two-step pathway involving an unprecedented PKS reaction leading directly to the β-diketone products.
Topics: Polyketide Synthases; Hordeum; Ketones
PubMed: 37949901
DOI: 10.1038/s41467-023-42917-9 -
Acta Pharmaceutica Sinica. B Sep 2023Depsides and depsidones have attracted attention for biosynthetic studies due to their broad biological activities and structural diversity. Previous...
Depsides and depsidones have attracted attention for biosynthetic studies due to their broad biological activities and structural diversity. Previous structure‒activity relationships indicated that triple halogenated depsidones display the best anti-pathogenic activity. However, the gene cluster and the tailoring steps responsible for halogenated depsidone nornidulin () remain enigmatic. In this study, we disclosed the complete biosynthetic pathway of the halogenated depsidone through gene disruption, heterologous expression and biochemical experiments. We demonstrated an unusual depside skeleton biosynthesis process mediated by both highly-reducing polyketide synthase and non-reducing polyketide synthase, which is distinct from the common depside skeleton biosynthesis. This skeleton was subsequently modified by two in-cluster enzymes DepG and DepF for the ether bond formation and decarboxylation, respectively. In addition, the decarboxylase DepF exhibited substrate promiscuity for different scaffold substrates. Finally, and interestingly, we discovered a halogenase encoded remotely from the biosynthetic gene cluster, which catalyzes triple-halogenation to produce the active end product nornidulin (). These discoveries provide new insights for further understanding the biosynthesis of depsidones and their derivatives.
PubMed: 37719379
DOI: 10.1016/j.apsb.2023.05.036 -
Frontiers in Plant Science 2023The tropical tree, , is a prominent source of chromone alkaloid rohitukine, which is used in the semi-syntheses of anticancer molecules such as flavopiridol and...
The tropical tree, , is a prominent source of chromone alkaloid rohitukine, which is used in the semi-syntheses of anticancer molecules such as flavopiridol and P-276-00. The biosynthetic pathway of rohitukine or its derivatives is currently unknown in plants. Here, we explored chromone alkaloid biosynthesis in through targeted transcriptome sequencing. Illumina sequencing of leaves and roots of a year-old seedling generated, 42.43 and 38.74 million paired-end short reads, respectively. Quality filtering and assembly of the transcriptome generated 274,970 contigs and 126,788 unigenes with an N50 contig length of 1560 bp. The assembly generated 117,619 translated unigene protein sequences and 51,598 non-redundant sequences. Nearly 80% of these non-redundant sequences were annotated to publicly available protein and nucleotide databases, suggesting the completeness and effectiveness of the transcriptome assembly. Using the assembly, we identified a chalcone synthase (CHS) and three type III polyketide synthases (PKS-III; non-CHS type) that are likely to be involved in the biosynthesis of chromone ring/noreugenin moiety of rohitukine. We also identified key enzymes like lysine decarboxylase in the piperidine pathway that make the piperidine moiety of rohitukine. Besides these, the upstream enzymes in flavonoid biosynthesis like phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H),4-coumarate-CoA ligase (4CL), and chalcone isomerase (CHI) have also been identified. Also, terpene synthases that are likely to be involved in the biosynthesis of various terpenoid scaffolds have been identified. Together, the transcriptome resource forms a basis for further exploration of biosynthetic pathways of these valuable compounds through functional validation of the candidate genes and metabolic engineering in heterologous hosts. Additionally, the transcriptome dataset generated will serve as an important resource for research on functional genomics and enzyme discovery in and comparative analysis with other Meliaceae family members.
PubMed: 37636089
DOI: 10.3389/fpls.2023.1098987 -
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