Two Cytochrome P450 Enzymes Form the Tricyclic Nested Skeleton of Meroterpenoids by Sequential Oxidative Reactions.

Meroterpenoid clavilactones feature a unique benzo-fused ten-membered carbocyclic ring unit with an α,ß-epoxy-γ-lactone moiety, forming an intriguing 10/5/3 tricyclic nested skeleton. These compounds are good inhibitors of the tyrosine kinase, attracting a lot of chemical synthesis studies. However, the natural enzymes involved in the formation of the 10/5/3 tricyclic nested skeleton remain unexplored. Here, we identified a gene cluster responsible for the biosynthesis of clavilactone A in the basidiomycetous fungus Clitocybe clavipes. We showed that a key cytochrome P450 monooxygenase ClaR catalyzes the diradical coupling reaction between the intramolecular hydroquinone and allyl moieties to form the benzo-fused ten-membered carbocyclic ring unit, followed by the P450 ClaT that exquisitely and stereoselectively assembles the α,ß-epoxy-γ-lactone moiety in clavilactone biosynthesis. ClaR unprecedentedly acts as a macrocyclase to catalyze the oxidative cyclization of the isopentenyl to the nonterpenoid moieties to form the benzo-fused macrocycle, and a multifunctional P450 ClaT catalyzes a ten-electron oxidation to accomplish the biosynthesis of the 10/5/3 tricyclic nested skeleton in clavilactones. Our findings establish the foundation for the efficient production of clavilactones using synthetic biology approaches and provide the mechanistic insights into the macrocycle formation in the biosynthesis of fungal meroterpenoids.

An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid.

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.

Overproduction of medicinal ergot alkaloids based on a fungal platform.

Privileged ergot alkaloids (EAs) produced by the fungal genus Claviceps are used to treat a wide range of diseases. However, their use and research have been hampered by the challenging genetic engineering of Claviceps. Here we systematically refactored and rationally engineered the EA biosynthetic pathway in heterologous host Aspergillus nidulans by using a Fungal-Yeast-Shuttle-Vector protocol. The obtained strains allowed the production of diverse EAs and related intermediates, including prechanoclavine (PCC, 333.8 mg/L), chanoclavine (CC, 241.0 mg/L), agroclavine (AC, 78.7 mg/L), and festuclavine (FC, 99.2 mg/L), etc. This fungal platform also enabled the access to the methyl-oxidized EAs (MOEAs), including elymoclavine (EC), lysergic acid (LA), dihydroelysergol (DHLG), and dihydrolysergic acid (DHLA), by overexpressing a P450 enzyme CloA. Furthermore, by optimizing the P450 electron transfer (ET) pathway and using multi-copy of cloA, the titers of EC and DHLG have been improved by 17.3- and 9.4-fold, respectively. Beyond our demonstration of A. nidulans as a robust platform for EA overproduction, our study offers a proof of concept for engineering the eukaryotic P450s-contained biosynthetic pathways in a filamentous fungal host.

Beyond the cyclopropyl ring formation: fungal Aj_EasH catalyzes asymmetric hydroxylation of ergot alkaloids.

Ergot alkaloids (EAs) are among the most important bioactive natural products. FeII/α-ketoglutarate-dependent dioxygenase Aj_EasH from Aspergillus japonicus is responsible for the formation of the cyclopropyl ring of the ergot alkaloid (EA) cycloclavine (4). Herein we reconstituted the biosynthesis of 4 in vitro from prechanoclavine (1) for the first time. Additionally, an unexpected activity of asymmetric hydroxylation at the C-4 position of EA compound festuclavine (5) for Aj_EasH was revealed. Furthermore, Aj_EasH also catalyzes the hydroxylation of two more EAs 9,10-dihydrolysergol (6) and elymoclavine (7). Thus, our results proved that Aj_EasH is a promiscuous and bimodal dioxygenase that catalyzes both the formation of cyclopropyl ring in 4 and the asymmetric hydroxylation of EAs. Molecular docking (MD) revealed the substrate-binding mode as well as the catalytic mechanism of asymmetric hydroxylation, suggesting more EAs could potentially be recognized and hydroxylated by Aj_EasH. Overall, the newly discovered activity empowered Aj_EasH with great potential for producing more diverse and bioactive EA derivatives. KEY POINTS: • Aj_EasH was revealed to be a promiscuous and bimodal FeII/α-ketoglutarate-dependent dioxygenase. • Aj_EasH converted festuclavine, 9,10-dihydrolysergol, and elymoclavine to their hydroxylated derivatives. • The catalytic mechanism of Aj_EasH for hydroxylation was analyzed by molecular docking.

Genome mining and biosynthesis of a polyketide from a biofertilizer fungus that can facilitate reductive iron assimilation in plant.

Fungi have the potential to produce a large repertoire of bioactive molecules, many of which can affect the growth and development of plants. Genomic survey of sequenced biofertilizer fungi showed many secondary metabolite gene clusters are anchored by iterative polyketide synthases (IPKSs), which are multidomain enzymes noted for generating diverse small molecules. Focusing on the biofertilizer Trichoderma harzianum t-22, we identified and characterized a cryptic IPKS-containing cluster that synthesizes tricholignan A, a redox-active ortho-hydroquinone. Tricholignan A is shown to reduce Fe(III) and may play a role in promoting plant growth under iron-deficient conditions. The construction of tricholignan by a pair of collaborating IPKSs was investigated using heterologous reconstitution and biochemical studies. A regioselective methylation step is shown to be a key step in formation of the ortho-hydroquinone. The responsible methyltransferase (MT) is fused with an N-terminal pseudo-acyl carrier protein (ψACP), in which the apo state of the ACP is essential for methylation of the growing polyketide chain. The ψACP is proposed to bind to the IPKS and enable the trans MT to access the growing polyketide. Our studies show that a genome-driven approach to discovering bioactive natural products from biofertilizer fungi can lead to unique compounds and biosynthetic knowledge.

Tuning an Imine Reductase for the Asymmetric Synthesis of Azacycloalkylamines by Concise Structure-Guided Engineering.

Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm , and a significant increase in the e.e. value from 78 % (R) to >99 % (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.

De novo biosynthesis and gram-level production of m-cresol in Aspergillus nidulans.

The industrially important meta-cresol (m-cresol, 3-methylphenol) is mainly produced from fossil resources by chemical methods. The microbial production of m-cresol was rarely investigated. Herein, we constructed a platform for the overproduction of m-cresol in a modified fungus Aspergillus nidulans FGSC no. A1145∆ST∆EM, which gave a gram-level titer using starch as carbon resource. For the biosynthesis of m-cresol, the 6-methyl salicylic acid synthase (MSAS)-encoding gene patK and 6-methyl salicylic acid decarboxylase-encoding gene patG from A. clavatus were co-expressed in the host A. nidulans. Multiple strategies, including promotor engineering, gene multiplication, and fed-batch fermentation, were applied to raise the production of m-cresol, which resulted in the titers of 1.29 g/L in shaking flasks and 2.03 g/L in fed-batch culture. The chassis cell A. nidulans A1145∆ST∆EM was proved to possess better tolerance to m-cresol than yeast, as it could grow in the liquid medium containing up to 2.5 g/L of m-cresol. These results showed that A. nidulans has great potential to be further engineered for industrial production of m-cresol.Key points• m-Cresol was de novo biosynthesized by a fungal chassis cell Aspergillus nidulans.• Promoter engineering and gene multiplication implemented the fine-tuned genes expression.• The titer of m-cresol reached 2.03 g/L via fed-batch culture.

Catalase Involved in Oxidative Cyclization of the Tetracyclic Ergoline of Fungal Ergot Alkaloids.

A dedicated enzyme for the formation of the central C ring in the tetracyclic ergoline of clinically important ergot alkaloids has never been found. Herein, we report a dual role catalase (EasC), unexpectedly using O2 as the oxidant, that catalyzes the oxidative cyclization of the central C ring from a 1,3-diene intermediate. Our study showcases how nature evolves the common catalase for enantioselective C-C bond construction of complex polycyclic scaffolds.

Biosynthesis of Heptacyclic Duclauxins Requires Extensive Redox Modifications of the Phenalenone Aromatic Polyketide.

Duclauxins are dimeric and heptacyclic fungal polyketides with notable bioactivities. We characterized the cascade of redox transformations in the biosynthetic pathway of duclauxin from Talaromyces stipitatus. The redox reaction sequence is initiated by a cupin family dioxygenase DuxM that performs an oxidative cleavage of the peri-fused tricyclic phenalenone and affords a transient hemiketal-oxaphenalenone intermediate. Additional redox enzymes then morph the oxaphenoalenone into either an anhydride or a dihydrocoumarin-containing monomeric building block that is found in dimeric duxlauxins. Oxidative coupling between the monomers to form the initial C-C bond was shown to be catalyzed by a P450 monooxygenase, although the enzyme responsible for the second C-C bond formation was not found in the pathway. Collectively, the number and variety of redox enzymes used in the duclauxin pathway showcase Nature's strategy to generate structural complexity during natural product biosynthesis.

Recent examples of α-ketoglutarate-dependent mononuclear non-haem iron enzymes in natural product biosyntheses.

Covering: up to 2018 α-Ketoglutarate (αKG, also known as 2-oxoglutarate)-dependent mononuclear non-haem iron (αKG-NHFe) enzymes catalyze a wide range of biochemical reactions, including hydroxylation, ring fragmentation, C-C bond cleavage, epimerization, desaturation, endoperoxidation and heterocycle formation. These enzymes utilize iron(ii) as the metallo-cofactor and αKG as the co-substrate. Herein, we summarize several novel αKG-NHFe enzymes involved in natural product biosyntheses discovered in recent years, including halogenation reactions, amino acid modifications and tailoring reactions in the biosynthesis of terpenes, lipids, fatty acids and phosphonates. We also conducted a survey of the currently available structures of αKG-NHFe enzymes, in which αKG binds to the metallo-centre bidentately through either a proximal- or distal-type binding mode. Future structure-function and structure-reactivity relationship investigations will provide crucial information regarding how activities in this large class of enzymes have been fine-tuned in nature.

Enzyme-Catalyzed Intramolecular Enantioselective Hydroalkoxylation.

Hydroalkoxylation is a powerful and efficient method of forming C-O bonds and cyclic ethers in synthetic chemistry. In studying the biosynthesis of the fungal natural product herqueinone, we identified an enzyme that can perform an intramolecular enantioselective hydroalkoxylation reaction. PhnH catalyzes the addition of a phenol to the terminal olefin of a reverse prenyl group to give a dihydrobenzofuran product. The enzyme accelerates the reaction by 3 × 105-fold compared to the uncatalyzed reaction. PhnH belongs to a superfamily of proteins with a domain of unknown function (DUF3237), of which no member has a previously verified function. The discovery of PhnH demonstrates that enzymes can be used to promote the enantioselective hydroalkoxylation reaction and form cyclic ethers.

Collaborative Biosynthesis of Maleimide- and Succinimide-Containing Natural Products by Fungal Polyketide Megasynthases.

Fungal polyketide synthases (PKSs) can function collaboratively to synthesize natural products of significant structural diversity. Here we present a new mode of collaboration between a highly reducing PKS (HRPKS) and a PKS-nonribosomal peptide synthetase (PKS-NRPS) in the synthesis of oxaleimides from the Penicillium species. The HRPKS is recruited in the synthesis of an olefin-containing free amino acid, which is activated and incorporated by the adenylation domain of the PKS-NRPS. The precisely positioned olefin from the unnatural amino acid is proposed to facilitate a scaffold rearrangement of the PKS-NRPS product to forge the maleimide and succinimide cores of oxaleimides.

Selected Mutations Reveal New Intermediates in the Biosynthesis of Mupirocin and the Thiomarinol Antibiotics.

Thiomarinol and mupirocin are assembled on similar polyketide/fatty acid backbones and exhibit potent antibiotic activity against methicillin-resistant Staphylococcus aureus (MRSA). They both contain a tetrasubstituted tetrahydropyran (THP) ring that is essential for biological activity. Mupirocin is a mixture of pseudomonic acids (PAs). Isolation of the novel compound mupirocin P, which contains a 7-hydroxy-6-keto-substituted THP, from a ΔmupP strain and chemical complementation experiments confirm that the first step in the conversion of PA-B into the major product PA-A is oxidation at the C6 position. In addition, nine novel thiomarinol (TM) derivatives with different oxidation patterns decorating the central THP core were isolated after gene deletion (tmlF). These metabolites are in accord with the THP ring formation and elaboration in thiomarinol following a similar order to that found in mupirocin biosynthesis, despite the lack of some of the equivalent genes. Novel mupirocin-thiomarinol hybrids were also synthesized by mutasynthesis.

Phenalenone Polyketide Cyclization Catalyzed by Fungal Polyketide Synthase and Flavin-Dependent Monooxygenase.

Phenalenones are polyketide natural products that display diverse structures and biological activities. The core of phenalenones is a peri-fused tricyclic ring system cyclized from a linear polyketide precursor via an unresolved mechanism. Toward understanding the unusual cyclization steps, the phn biosynthetic gene cluster responsible for herqueinone biosynthesis was identified from the genome of Penicillium herquei. A nonreducing polyketide synthase (NR-PKS) PhnA was shown to synthesize the heptaketide backbone and cyclize it into the angular, hemiketal-containing naphtho-γ-pyrone prephenalenone. The product template (PT) domain of PhnA catalyzes only the C4-C9 aldol condensation, which is unprecedented among known PT domains. The transformation of prephenalenone to phenalenone requires an FAD-dependent monooxygenase (FMO) PhnB, which catalyzes the C2 aromatic hydroxylation of prephenalenone and ring opening of the γ-pyrone ring simultaneously. Density functional theory calculations provide insights into why the hydroxylated intermediate undergoes an aldol-like phenoxide-ketone cyclization to yield the phenalenone core. This study therefore unveiled new routes and biocatalysts for polyketide cyclization.

Biochemical Characterization of a Eukaryotic Decalin-Forming Diels-Alderase.

The trans-decalin structure formed by intramolecular Diels-Alder cycloaddition is widely present among bioactive natural products isolated from fungi. We elucidated the concise three-enzyme biosynthetic pathway of the cytotoxic myceliothermophin and biochemically characterized the Diels-Alderase that catalyzes the formation of trans-decalin from an acyclic substrate. Computational studies of the reaction mechanism rationalize both the substrate and stereoselectivity of the enzyme.

Rhizovarins A-F, Indole-Diterpenes from the Mangrove-Derived Endophytic Fungus Mucor irregularis QEN-189.

Genome mining of the fungus Mucor irregularis (formerly known as Rhizomucor variabilis) revealed the presence of various gene clusters for secondary metabolite biosynthesis, including several terpene-based clusters. Investigation into the chemical diversity of M. irregularis QEN-189, an endophytic fungus isolated from the fresh inner tissue of the marine mangrove plant Rhizophora stylosa, resulted in the discovery of 20 structurally diverse indole-diterpenes including six new compounds, namely, rhizovarins A-F (1-6). Among them, compounds 1-3 represent the most complex members of the reported indole-diterpenes. The presence of an unusual acetal linked to a hemiketal (1) or a ketal (2 and 3) in an unprecedented 4,6,6,8,5,6,6,6,6-fused indole-diterpene ring system makes them chemically unique. Their structures and absolute configurations were elucidated by spectroscopic analysis, modified Mosher's method, and chemical calculations. Each of the isolated compounds was evaluated for antitumor activity against HL-60 and A-549 cell lines.

Biosynthesis of mupirocin by Pseudomonas fluorescens NCIMB 10586 involves parallel pathways.

Mupirocin, a clinically important antibiotic produced via a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens, consists of a mixture of mainly pseudomonic acids A, B, and C. Detailed metabolic profiling of mutant strains produced by systematic inactivation of PKS and tailoring genes, along with re-feeding of isolated metabolites to mutant stains, has allowed the isolation of a large number of novel metabolites, identification of the 10,11-epoxidase, and full characterization of the mupirocin biosynthetic pathway, which proceeds via major (10,11-epoxide) and minor (10,11-alkene) parallel pathways.

Cytotoxic anthranilic acid derivatives from deep sea sediment-derived fungus Penicillium paneum SD-44.

Five new anthranilic acid derivatives, penipacids A-E (1-5), together with one known analogue (6), which was previously synthesized, were characterized from the ethyl acetate extract of the marine sediment-derived fungus Penicillium paneum SD-44. Their structures were elucidated mainly by extensive NMR spectroscopic and mass spectrometric analysis. The cytotoxicity and antimicrobial activity of the isolated compounds were evaluated. Compounds 1, and 5 exhibited inhibitory activity against human colon cancer RKO cell line, while compound 6 displayed cytotoxic activity against Hela cell line.

Structure-guided semi-rational design of an imine reductase for enantio-complementary synthesis of pyrrolidinamine.

In this study, engineered imine reductases (IREDs) of IRED M5, originally from Actinoalloteichus hymeniacidonis, were obtained through structure-guided semi-rational design. By focusing on mutagenesis of the residues that directly interact with the ketone donor moiety, we identified two residues W234 and F260, playing essential roles in enhancing and reversing the stereoselectivity, respectively. Moreover, two completely enantio-complementary variants S241L/F260N (R-selectivity up to 99%) and I149D/W234I (S-selectivity up to 99%) were achieved. Both variants showed excellent stereoselectivity toward the tested substrates, offering valuable biocatalysts for synthesizing pyrrolidinamines. Its application was demonstrated in a short synthesis of the key intermediates of potential drug molecules leniolisib and JAK1 inhibitor 4, from cheap and commercially available pro-chiral N-Boc-piperidone 1 (2 and 3 steps, respectively).