Polyketide Synthase-nonribosomal Peptide Synthetase Research Articles

Genome Mining of a Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase Hybrid Megasynthetase Pathway to Synthesize a Phytotoxic N-Acyl Amino Acid.

Guided by the retrobiosynthesis hypothesis, we characterized a fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid megasynthetase pathway to generate 2-trans-4-trans-2-methylsorbyl-d-leucine (1), a polyketide amino acid conjugate that inhibits Arabidopsis root growth. The biosynthesis of 1 includes a PKS-NRPS enzyme to assemble an N-acyl amino alcohol intermediate, which is further oxidized to an N-acyl amino acid (NAAA), demonstrating a new biosynthetic logic for synthesizing NAAAs and expanding the chemical space of products encoded by fungal PKS-NRPS clusters.

Reductive Release from a Hybrid PKS-NRPS during the Biosynthesis of Pyrichalasin H.

Three central steps during the biosynthesis of cytochalasan precursors, including reductive release, Knoevenagel cyclisation and Diels Alder cyclisation are not yet understood at a detailed molecular level. In this work we investigated the reductive release step catalysed by a hybrid polyketide synthase non-ribosomal peptide synthetase (PKS-NRPS) from the pyrichalasin H pathway. Synthetic thiolesters were used as substrate mimics for in vitro studies with the isolated reduction (R) and holo-thiolation (T) domains of the PKS-NRPS hybrid PyiS. These assays demonstrate that the PyiS R-domain mainly catalyses an NADPH-dependent reductive release of an aldehyde intermediate that quickly undergoes spontaneous Knoevenagel cyclisation. The R-domain can only process substrates that are covalently bound to the phosphopantetheine thiol of the upstream T-domain, but it shows little selectivity for the polyketide.

Abi Family Protein, DidK, Is Involved in the Maturation of Anticancer Depsipeptide, Didemnin B.

Didemnin B is a marine-derived depsipeptide with potent antiviral and anticancer activities. A prodrug activation mechanism was previously proposed for the biosynthesis of didemnin B by the nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly line, but the enzyme involved in the maturation process remained unknown. Herein, we demonstrated that DidA, a dimodular NRPS predicted with unrelated activity to didemnin B structure assembly, was indispensable to produce didemnin B, confirming the prodrug mechanism in didemnin B biosynthesis. We further identified an Abi family transmembrane protease, DidK, that functioned as an esterase in the maturation step of didemnin B by in vivo gene knockout and heterologous expression. DidK is structurally distinct from other known hydrolytic enzymes involved in the maturation of bacterial nonribosomal peptides and is the first Abi family protein known to participate in NRPS/PKS-derived natural product production. Further bioinformatic analysis revealed more than 20 DidK homologues encoded in bacterial NRPS/PKS BGCs, suggesting that the involvement of Abi family proteins in natural product biosynthesis might not be rare. These results not only clarify the priming and maturation steps of didemnin B biosynthesis but also expand the function scope of Abi family proteins.

Human-associated bacteria adopt an unusual route for synthesizing 3-acetylated tetramates for environmental adaptation

BackgroundTetramates or tetramic acid-containing compounds (TACs) are a group of bioactive natural products featuring a pyrrolidine-2,4-dione ring acknowledged being closed via Dieckmann cyclization. The cariogenic Streptococcus mutans strains bearing a muc biosynthetic gene cluster (BGC) can synthesize mutanocyclin (MUC), a 3-acetylated TAC that can inhibit both leukocyte chemotaxis and filamentous development in Candida albicans. Some strains can also accumulate reutericyclins (RTCs), the intermediates of MUC biosynthesis with antibacterial activities. However, the formation mechanism of the pyrrolidine-2,4-dione ring of MUC and the distribution of muc-like BGCs along with their ecological functions has not been explored extensively.ResultsWe demonstrated that a key intermediate of MUC biosynthesis, M-307, is installed by a hybrid nonribosomal peptide synthetase-polyketide synthase assembly line and its pyrrolidine-2,4-dione ring is closed via an unprecedented lactam bond formation style. Subsequent C-3 acetylation will convert M-307 to RTCs, which is then hydrolyzed by a deacylase, MucF, to remove the N-1 fatty acyl appendage to generate MUC. Distribution analysis showed that the muc-like BGCs distribute predominantly in human-associated bacteria. Interestingly, most of the muc-like BGCs possessing a mucF gene were isolated from human or livestock directly, indicating their involvement in alleviating the host’s immune attacks by synthesizing MUC; while those BGCs lacking mucF gene distribute mainly in bacteria from fermented products, suggesting that they tend to synthesize RTCs to compete with neighboring bacteria. It is noteworthy that many bacteria in the same habitats (e.g., the oral cavity) lack the muc-like BGC, but possess functional MucF homologues to “detoxify” RTCs to MUC, including several competitive bacteria of S. mutans. We also comparably studied the distribution of TAS1, a fungal enzyme responsible for the production of phytotoxic tenuazonic acids (TeAs), a class of 3-acetylated TACs with similar structure but distinct biosynthetic mechanism to MUC, and found that it mainly exists in plants or crops.ConclusionsThe in vivo and in vitro experiments revealed that the pyrrolidine-2,4-dione ring of MUC is closed via lactam bond formation, which may be adopted by many TACs without 3-acyl decorations. Besides, we found that muc-like BGCs are widespread in human-associated bacteria and their shapes and main products can be influenced by the habitat environment and vice versa. By comparing with TeAs, we provided thought-provoking insights into how ecological and evolutionary forces drive bacteria and fungi to construct a common 3-acetylated pyrrolidine-2,4-dione core through different routes, and how the biosynthetic processes are delicately controlled to generate diverse 3-acetylated TACs for environmental adaptation.2PMNhDpkP3itX1xdtgrn3CVideo

A natural single nucleotide mutation in the small regulatory RNA ArcZ of Dickeya solani switches off the antimicrobial activities against yeast and bacteria.

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.

Somalactams A-D: Anti-inflammatory Macrolide Lactams with Unique Ring Systems from an Arctic Actinomycete Strain.

Four new PKS-NRPS-derived macrolide lactams with three unique ring fusion types were discovered from the Arctic sponge associated actinomycete Streptomyces somaliensis 1107 using a genome mining strategy. Their structures were elucidated by a combination of MS, NMR spectroscopic analysis, and single-crystal X-ray diffraction. Biosynthetically, a novel gene cluster sml consisting of three polyketide synthases and one hybrid polyketide synthase-nonribosomal peptide synthetase together with cytochrome P450s and flavin-containing monooxygenases and oxidoreductases was demonstrated to assemble the unique skeleton. Pharmacological studies revealed that compound 1 displayed a potent anti-inflammatory effect without cytotoxicity. It inhibited IL-6 and TNF-α release in the serum of LPS-stimulated RAW264.7 macrophage cells with IC50 values of 5.76 and 0.18 μM, respectively, and modulated the MAPK pathway. Moreover, compound 1 alleviated LPS-induced systemic inflammation in our transgenic fluorescent zebrafish model.

Discovery of Cyclic Lipopeptides Ralstopeptins A and B from Ralstonia solanacearum Species Complex and Analysis of Biosynthetic Gene Evolution.

Ralstonia solanacearum species complex (RSSC) strains are plant pathogens that produce lipopeptides (ralstonins and ralstoamides) by the polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) enzyme hybrid. Recently, ralstonins were found to be key molecules in the parasitism of RSSC to other hosts, Aspergillus and Fusarium fungi. The PKS-NRPS genes of RSSC strains in the GenBank database suggest the production of additional lipopeptides, although it has not been confirmed to date. Here, we report the genome-driven and mass-spectrometry-guided discovery, isolation, and structural elucidation of ralstopeptins A and B from strain MAFF 211519. Ralstopeptins were found to be cyclic lipopeptides with two amino acid residues less than ralstonins. The partial deletion of the gene encoding PKS-NRPS obliterated the production of ralstopeptins in MAFF 211519. Bioinformatic analyses suggested possible evolutionary events of the biosynthetic genes of RSSC lipopeptides, where intragenomic recombination may have occurred within the PKS-NRPS genes, reducing the gene size. The chlamydospore-inducing activities of ralstopeptins A and B, ralstonins A and B, and ralstoamide A in the fungus Fusarium oxysporum indicated a structural preference for ralstonins. Altogether, we propose a model for the evolutionary processes that contribute to the chemical diversity of RSSC lipopeptides and its relation to the endoparasitism of RSSC in fungi.

Discovery of a Unique Flavonoid Biosynthesis Mechanism in Fungi by Genome Mining

AbstractFlavonoids are important plant natural products with variable structures and bioactivities. All known plant flavonoids are generated under the catalysis of a type III polyketide synthase (PKS) followed by a chalcone isomerase (CHI) and a flavone synthase (FNS). In this study, the biosynthetic gene cluster of chlorflavonin, a fungal flavonoid with acetolactate synthase inhibitory activity, was discovered using a self‐resistance‐gene‐directed strategy. A novel flavonoid biosynthetic pathway in fungi was revealed. A core nonribosomal peptide synthetase‐polyketide synthase (NRPS‐PKS) is responsible for the generation of the key precursor chalcone. Then, a new type of CHI catalyzes the conversion of a chalcone into a flavanone by a histidine‐mediated oxa‐Michael addition mechanism. Finally, the desaturation of flavanone to flavone is catalyzed by a new type of FNS, a flavin mononucleotide (FMN)‐dependent oxidoreductase.

Discovery of a Unique Flavonoid Biosynthesis Mechanism in Fungi by Genome Mining.

Flavonoids are important plant natural products with variable structures and bioactivities. All known plant flavonoids are generated under the catalysis of a type III polyketide synthase (PKS) followed by a chalcone isomerase (CHI) and a flavone synthase (FNS). In this study, the biosynthetic gene cluster of chlorflavonin, a fungal flavonoid with acetolactate synthase inhibitory activity, was discovered using a self-resistance-gene-directed strategy. A novel flavonoid biosynthetic pathway in fungi was revealed. A core nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) is responsible for the generation of the key precursor chalcone. Then, a new type of CHI catalyzes the conversion of a chalcone into a flavanone by a histidine-mediated oxa-Michael addition mechanism. Finally, the desaturation of flavanone to flavone is catalyzed by a new type of FNS, a flavin mononucleotide (FMN)-dependent oxidoreductase.

Genetic responses to adding nitrates to improve hydrophilic yellow pigment in Monascus fermentation.

Nitrates can stimulate the biosynthesis of hydrophilic yellow pigments (HYPs) in Monascus ruber CGMCC 10910. To explore the molecular mechanisms whereby nitrates (NaNO3 and NH4NO3) regulate HYP production, an integrated transcriptomic and proteomic analysis was conducted in this study. Nitrate addition led to an approximately 75% higher HYP production compared with the untreated group, especially compounds Y3 and Y4. Comparative transcriptomic analysis found that mpigsA, H, K, L, and P genes involved in yellow pigment biosynthesis were significantly upregulated. In addition, pigment biosynthesis-related (carbon catabolism, amino acid metabolism, polyketide synthesis, and fatty acid metabolism) genes were upregulated to provide precursors and energy for HYP biosynthesis and cell growth. Secretion-related (cytomembrane ergosterol biosynthetic, and transport) pathways were also noticeably regulated to accelerate transmembrane transport of HYPs. Meanwhile, proteomicanalysis showed that nitrates improved the protein expression of hybrid polyketide synthase-nonribosomal peptide synthetase, oxidoreductase, glucoamylase, endo-1,4-beta-xylanase, O-acetylhomoserine, and isocitrate lyase to enhance HYP production. These findings demonstrated the regulatory mechanism of nitrates for enhancing HYP production in Monascus. KEY POINTS: • Nitrates stimulated the biosynthesis of Monascus hydrophilic yellow pigments (HYPs) • Nitrates affected transcriptional level of pigment biosynthesis- and transport genes • Increased expression of hybrid PKS-NRPS and transporters promoted production of HYPs.

Discovery and biosynthesis of karnamicins as angiotensin converting enzyme inhibitors

Angiotensin-converting enzyme inhibitors are widely used for treatment of hypertension and related diseases. Here, six karnamicins E1-E6 (1–6), which bear fully substituted hydroxypyridine and thiazole moieties are characterized from the rare actinobacterium Lechevalieria rhizosphaerae NEAU-A2. Through a combination of isotopic labeling, genome mining, and enzymatic characterization studies, the programmed assembly of the fully substituted hydroxypyridine moiety in karnamicin is proposed to be due to sequential operation of a hybrid polyketide synthase-nonribosomal peptide synthetase, two regioselective pyridine ring flavoprotein hydroxylases, and a methyltransferase. Based on AlphaFold protein structures predictions, molecular docking, and site-directed mutagenesis, we find that two pyridine hydroxylases deploy active site residues distinct from other flavoprotein monooxygenases to direct the chemo- and regioselective hydroxylation of the pyridine nucleus. Pleasingly, karnamicins show significant angiotensin-converting enzyme inhibitory activity with IC50 values ranging from 0.24 to 5.81 μM, suggesting their potential use for the treatment of hypertension and related diseases.

Discovery and biosynthesis of macrophasetins from the plant pathogen fungus Macrophomina phaseolina.

3-Decalinoyltetramic acids (DTAs) are a class of natural products with chemical diversity and potent bioactivities. In fungal species there is a general biosynthetic route to synthesize this type of compounds, which usually features a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) and a lipocalin-like Diels-Alderase (LLDAse). Using a synthetic biology approach, combining the bioinformatics analysis prediction and heterologous expression, we mined a PKS-NRPS and LLDAse encoding gene cluster from the plant pathogenic fungus Macrophomina phaseolina and characterized the cluster to be responsible for the biosynthesis of novel DTAs, macrophasetins. In addition, we investigated the biosynthesis of these compounds and validated the accuracy of the phylogeny-guided bioinformatics analysis prediction. Our results provided a proof of concept example to this approach, which may facilitate the discovery of novel DTAs from the fungal kingdom.

A fungal NRPS-PKS enzyme catalyses the formation of the flavonoid naringenin

Biosynthesis of the flavonoid naringenin in plants and bacteria is commonly catalysed by a type III polyketide synthase (PKS) using one p-coumaroyl-CoA and three malonyl-CoA molecules as substrates. Here, we report a fungal non-ribosomal peptide synthetase -polyketide synthase (NRPS-PKS) hybrid FnsA for the naringenin formation. Feeding experiments with isotope-labelled precursors demonstrate that FnsA accepts not only p-coumaric acid (p-CA), but also p-hydroxybenzoic acid (p-HBA) as starter units, with three or four malonyl-CoA molecules for elongation, respectively. In vitro assays and MS/MS analysis prove that both p-CA and p-HBA are firstly activated by the adenylation domain of FnsA. Phylogenetic analysis reveals that the PKS portion of FnsA shares high sequence homology with type I PKSs. Refactoring the biosynthetic pathway in yeast with the involvement of fnsA provides an alternative approach for the production of flavonoids such as isorhamnetin and acacetin.

Aspcandine: A Pyrrolobenzazepine Alkaloid Synthesized by a Fungal Nonribosomal Peptide Synthetase-Polyketide Synthase Hybrid.

Characterization of an orphan biosynthetic gene cluster found in the fungus Aspergillus candidus CBS 102.13 resulted in the discovery of a pyrrolobenzazepine alkaloid, aspcandine (1). The unique molecular scaffold of 1 is synthesized by the nonribosomal peptide synthetase-polyketide synthase hybrid AcdB, which unusually incorporates 3-hydroxy-l-kynurenine as a building block. AcdB subsequently performs one round of chain elongation using malonyl-CoA, which is followed by the chain release to furnish the tricyclic system of 1.

Biosynthesis of Odd-Carbon Unsaturated Fatty Dicarboxylic Acids Through Engineering the HSAF Biosynthetic Gene in Lysobacter enzymogenes.

Fatty dicarboxylic acids (FDCA) are useful as starting materials or components for plastics, polyesters, nylons, and fragrances. Most of the commercially available FDCA contain an even number of carbons, and there remain few sustainable methods for production of FDCA with an odd number of carbons (o-FDCA). In this work, we explored a novel biosynthetic route to unsaturated o-FDCA. The approach was based on genetic modifications of hsaf pks-nrps, encoding a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) in Lysobacter enzymogenes, an environmental bacterium emerging as a new biocontrol agent. This single-module PKS-NRPS catalyzes the biosynthesis of lysobacterene A, a polyene-containing precursor of the antifungal natural product Heat-Stable Antifungal Factor (HSAF). We genetically removed the NRPS module from this gene and generated a new strain of L. enzymogenes, in which the PKS module was fused to the thioesterase domain of hsaf pks-nrps. The chimeric gene was verified by DNA sequencing, and its expression in L. enzymogenes was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The total fatty acids were extracted, esterified, and analyzed by GC-MS. The results showed that the engineered strain produced new fatty acids that were absent in the wild type. The main product was identified as hepta-2,4-dienedioic acid, an unsaturated o-FDCA. This work sets the foundation to explore a sustainable and environment-friendly approach toward unsaturated o-FDCA, which could be used as precursors for new compounds that can serve as versatile feedstock for industrial materials.

Xenoacremones D-H, Bioactive Tyrosine-decahydrofluorene Analogues from the Plant-Derived Fungus Xenoacremonium sinensis.

Five novel tyrosine-decahydrofluorene analogues, xenoacremones D–H (1–5), each bearing a fused 6/5/6 tricarbocyclic core and a 13-membered para-cyclophane ring system, were isolated from the endophytic fungus Xenoacremonium sinensis. Compound 1 was a novel polyketide synthase–nonribosomal peptide synthetase (PKS–NRPS) tyrosine-decahydrofluorene hybrid containing a 6/5/6/6/5 ring system. Their structures were elucidated from comprehensive spectroscopic analysis and electronic circular dichroism (ECD) calculations. All compounds were evaluated for their inhibitory activities on LPS-induced NO production in macrophages and their cytotoxicities against the NB4 and U937 cell lines. Compounds 3 and 5 exhibited potent anti-inflammatory activities in vitro. Compounds 1 and 3–5 displayed significant antiproliferative activity against the tumor cell lines (IC50 < 20 µM).

Genome Sequence Resource of Sarocladium terricola TR, an Endophytic Fungus as a Potential Biocontrol Agent Against Meloidogyne incognita.

Genome Sequence Resource of Sarocladium terricola TR, an Endophytic Fungus as a Potential Biocontrol Agent Against Meloidogyne incognita.

Discovery of an Antarctic Ascidian-Associated Uncultivated Verrucomicrobia with Antimelanoma Palmerolide Biosynthetic Potential.

ABSTRACTThe Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially produced hybrid nonribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74-kbp candidate BGC encoding the multimodular enzymatic machinery (hybrid type I-trans-AT polyketide synthase-nonribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide gene targets and a single 16S rRNA gene variant (R = 0.83 to 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a nearly complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as “Candidatus Synoicihabitans palmerolidicus.” The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium.IMPORTANCE Palmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply branching genus, “Candidatus Synoicihabitans palmerolidicus,” in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. Synoicihabitans palmerolidicus 4.29-Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways, a chemotaxis system, flagellar biosynthetic capacity, and other regulatory elements enabling its ascidian-associated lifestyle. The palmerolide producer’s genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants.

Colibrimycins, Novel Halogenated Hybrid Polyketide Synthase-Nonribosomal Peptide Synthetase (PKS-NRPS) Compounds Produced by Streptomyces sp. Strain CS147.

The improvement of genome sequencing techniques has brought to light the biosynthetic potential of actinomycetes due to the large number of gene clusters they present compared to the number of known compounds. Genome mining is a recent strategy in the search for novel bioactive compounds, which involves the analysis of sequenced genomes to identify uncharacterized natural product biosynthetic gene clusters, many of which are cryptic or silent under laboratory conditions, and to develop experimental approaches to identify their products. Owing to the importance of halogenation in terms of structural diversity, bioavailability, and bioactivity, searching for new halogenated bioactive compounds has become an interesting issue in the field of natural product discovery. Following this purpose, a screening for halogenase coding genes was performed on 12 Streptomyces strains isolated from fungus-growing ants of the Attini tribe. Using the bioinformatics tools antiSMASH and BLAST, six halogenase coding genes were identified. Some of these genes were located within biosynthetic gene clusters (BGCs), which were studied by construction of several mutants for the identification of the putative halogenated compounds produced. The comparison of the metabolite production profile of wild-type strains and their corresponding mutants by ultrahigh-performance liquid chromatography-UV and high-performance liquid chromatography-mass spectrometry allowed us the identification of a novel family of halogenated compounds in Streptomyces sp. strain CS147, designated colibrimycins. IMPORTANCE Genome mining has proven its usefulness in the search for novel bioactive compounds produced by microorganisms, and halogenases comprise an interesting starting point. In this work, we have identified a new halogenase coding gene that led to the discovery of novel lipopetide nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS)-derived natural products, the colibrimycins, produced by Streptomyces sp. strain CS147, isolated from the Attini ant niche. Some colibrimycins display an unusual α-ketoamide moiety in the peptide structure. Although its biosynthetic origin remains unknown, its presence might be related to a hypothetical inhibition of virus proteases, and, together with the presence of the halogenase, it represents a feature to be incorporated in the arsenal of structural modifications available for combinatorial biosynthesis.

A Standalone β-Ketoreductase Acts Concomitantly with Biosynthesis of the Antimycin Scaffold.

Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these compounds is well characterized, with the exception of the standalone β-ketoreductase enzyme AntM that is proposed to catalyze the reduction of the C8 carbonyl of the antimycin scaffold. Inactivation of antM and structural characterization suggested that rather than functioning as a post-PKS tailoring enzyme, AntM acts upon the terminal biosynthetic intermediate while it is tethered to the PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 and Phe223) that are proposed to serve as checkpoints controlling substrate access to the AntM active site. Aromatic checkpoint residues are conserved in uncharacterized standalone β-ketoreductases, indicating that they may also act concomitantly with synthesis of the scaffold. These data provide novel mechanistic insights into the functionality of standalone β-ketoreductases and will enable their reprogramming for combinatorial biosynthesis.