Genome mining of labdane-related diterpenoids: Discovery of the two-enzyme pathway leading to (-)-sandaracopimaradiene in the fungus Arthrinium sacchari.

Labdane-related diterpenoids (LRDs) in fungi are a pharmaceutically important, but underexplored family of natural products. In the biosynthesis of fungal LRDs, bifunctional terpene cyclases (TCs) consisting of αßγ domains are generally used to synthesize the polycyclic skeletones of LRDs. Herein, we conducted genome mining of LRDs in our fungal genome database and identified a unique pair of TCs, AsPS and AsCPS, in the fungus Arthrinium sacchari. AsPS consists of catalytically active α and inactive ß domains, whereas AsCPS contains ßγ domains and a truncated α domain. Heterologous expression in Aspergillus oryzae and biochemical characterization of recombinant proteins demonstrated that AsCPS synthesized copalyl diphosphate and that AsPS then converted it to (-)-sandaracopimaradiene. Since AsPS and AsCPS have distinct domain organizations from those of known fungal TCs and are likely generated through fusion or loss of catalytic domains, our findings provide insight into the evolution of TCs in fungi.

Biosynthesis of indole diterpenes: a reconstitution approach in a heterologous host.

Covering: 2013 to 2022In this review, we provide an overview elucidating the biosynthetic pathway and heterologous production of fungal indole diterpenes (IDTs). Based on the studies of six IDT biosynthesis, we extracted nature's strategy: (1) two-stage synthesis for the core scaffold and platform intermediates, and (2) late-stage modifications for installing an additional cyclic system on the indole ring. Herein, we describe reconstitution studies applying this strategy to the synthesis of highly elaborated IDTs. We also discuss its potential for future biosynthetic engineering.

Identification and Functional Characterization of Fungal Chalcone Synthase and Chalcone Isomerase.

By mining fungal genomic information, a noncanonical iterative type I PKS fused with an N-terminal adenylation-thiolation didomain, which catalyzes the formation of naringenin chalcone, was found. Structural prediction and molecular docking analysis indicated that a C-terminal thioesterase domain was involved in the Claisen-type cyclization. An enzyme responsible for formation of (2S)-flavanone in the biosynthesis of fungal flavonoids was also identified. Collectively, these findings demonstrate unprecedented fungal biosynthetic machinery leading to plant-like metabolites.

Structural diversification of fungal natural products by oxidative enzymes.

Ascomycota and basidiomycota fungi are prolific producers of biologically active natural products. Fungal natural products exhibit remarkable structural diversity and complexity, which are generated by the enzymes involved in their biosynthesis. After the formation of core skeletons, oxidative enzymes play a critical role in converting them into mature natural products. Besides simple oxidations, more complex transformations, such as multiple oxidations by single enzymes, oxidative cyclization, and skeletal rearrangement, are often observed. Those oxidative enzymes are of significant interest for the identification of new enzyme chemistry and have the potential to be biocatalysts for the synthesis of complex molecules. This review presents selected examples of unique oxidative transformations that have been found in the biosynthesis of fungal natural products. The development of strategies for refactoring the fungal biosynthetic pathways with an efficient genome-editing method is also introduced.

Total Biosynthesis of Melleolides from Basidiomycota Fungi: Mechanistic Analysis of the Multifunctional GMC Oxidase Mld7.

Mushroom terpenoids are biologically and chemically diverse fungal metabolites. Among them, melleolides are representative sesquiterpenoids with a characteristic protoilludane skeleton. In this study, we applied a recently established hot spot knock-in method to elucidate the biosynthetic pathway leading to 1α-hydroxymelleolide. The biosynthesis of the sesquiterpene core involves the cytochrome P450 catalyzing stepwise hydroxylation of the Δ6 -protoilludene framework and a stereochemical inversion process at the C5 position catalyzed by short-chain dehydrogenase/reductase family proteins. The highlight of the biosynthesis is that the flavoprotein Mld7 catalyzes an oxidation-triggered double-bond shift accompanying dehydration and acyl-group-assisted substitution with two different nucleophiles at the C6 position to afford the Δ7 -protoilludene derivatives, such as melleolide and armillarivin. The complex reaction mechanism was proposed by DFT calculations. Of particular importance is that product distribution is regulated by interaction with the cell membrane.

Elucidation of Late-Stage Biosynthesis of Phomoidride: Proposal of Cyclization Mechanism Affording Characteristic Nine-Membered Ring of Fungal Dimeric Anhydride.

Antihypercholesterolemic agent phomoidride (PMD) B has a highly elaborated bicyclo[4.3.1]deca-1,6-diene core scaffold derived from dimeric anhydride with a nine-membered ring. This report elucidated the late stage transformation from an anhydride monomer to PMD B through the heterologous expression of three enzyme genes, TstC, TstK, and TstE. Additional in vitro studies of TstK and TstE provided evidence on the formation of PMD via dimerization, three-step oxidation, and unusual methylation-triggered bicyclic ketal formation. Elucidation of the function of cyclase TstC prompts us to examine the cyclization mechanism of TstC by using a computational approach. Computational analytical data on PMD and structurally related glaucanic acid indicated that the initial decarboxylation of monomer results in enolate and subsequent double Michael reactions of another monomer, followed by an optional aldol reaction proceeding in an endo-selective manner to give cycloadducts, supporting the fact that the starting orientation of two monomers is directly transferred to the product configurations.

Biosynthetic machineries of anthraquinones and bisanthraquinones in Talaromyces islandicus.

Talaromyces islandicus is a unique fungus that produces more than 20 numbers of anthraquinones (AQs) and their dimeric natural products, bisanthraquinones (BQs). These compounds share a 9,10-anthracenedione core derived from emodin. The biosynthetic pathway of emodin has been firmly established, while that of other AQs and BQs is still unclear. In this study, we identified the biosynthetic gene clusters for chrysophanol and skyrin. The function of key modification enzymes was examined by performing biotransformation experiments and in vitro enzymatic reactions with emodin and its derivatives, allowing us to propose a mechanism for the modification reactions. The present study provides insight into the biosynthesis of AQs and BQs in T. islandicus.

Structure and biosynthesis of the ribosomal lipopeptide antibiotic albopeptins.

Albopeptins produced by Streptomyces albofaciens JC-82-120 were isolated as effective antibiotics for plant pathogenetic disease in 1986. However, their unusual physicochemical properties hampered the determination of their chemical structures. In this report, we describe our efforts to elucidate their structures. Initially, the structure of an unusual C13-fatty acid with an N-hydroxyguanidyl group was determined using degradation and chemical synthesis. After the linear portion of the octapeptide core was constructed based on the 2D-NMR data, the final assembly of the unusual structure, including the sulfoxide bridge, was achieved through the analysis of detailed NMR data. The proposed structure of albopeptin B was supported by MS/MS data, which also enabled us to determine the structure of 5 albopeptin family members. Bioinformatics analysis of the genomic data of the producer strain further led us to propose that their biosynthetic pathway is similar to the ribosomally derived lanthipeptides possessing a long-chain fatty acid.

Heterologous expression of a polyketide synthase ACRTS2 in Aspergillus oryzae produces host-selective ACR toxins: coproduction of minor metabolites.

Previously, we succeeded to produce the core structure of the host-selective ACR toxin (1) on brown leaf spot on rough lemon when the polyketide synthase ACRTS2 gene was heterologously expressed in Aspergillus oryzae (AO). To confirm the production of 1 in AO, the detection limit and suppressing decarboxylation were improved, and these efforts led us to conclude the direct production of 1 instead of its decarboxylation product. During this examination, minor ACR-toxin-related metabolites were found. Their structure determination enabled us to propose a decarboxylation mechanism and a novel branching route forming byproducts from the coupling of the dihydropyrone moiety of 1 with the acetaldehyde and kojic acid abundant in AO. The involvement of putative cyclase ACRTS3 in the chain release of linear polyketide was excluded by the coexpression analysis of ACRTS2 and ACRTS3. Taken together, we concluded that the production of 1 in AO is solely responsible for ACRTS2.

Biosynthetic Studies of Phomopsins Unveil Posttranslational Installation of Dehydroamino Acids by UstYa Family Proteins.

UstYa family proteins (DUF3328) are widely and specifically distributed in fungi. They are known to be involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and nonribosomal peptides, and possibly catalyze various reactions, including oxidative cyclization and chlorination. In this study, we focused on phomopsin A, a fungal RiPP consisting of unique nonproteinogenic amino acids. Gene knockout experiments demonstrated that three UstYa homologues, phomYc, phomYd, and phomYe, are essential for the desaturation of amino acid moieties, showing unprecedented function among UstYa family proteins. Sequence similarity network analysis indicated that their amino acid sequences are highly diverged and that most remain uncharacterized, paving the way for genome mining of fungal metabolites with unique modifications.

Biochemistry-Guided Prediction of the Absolute Configuration of Fungal Reduced Polyketides.

Highly reducing polyketide synthases (HR-PKSs) produce structurally diverse polyketides (PKs). The PK diversity is constructed by a variety of factors, including the ß-keto processing, chain length, methylation pattern, and relative and absolute configurations of the substituents. We examined the stereochemical course of the PK processing for the synthesis of polyhydroxy PKs such as phialotides, phomenoic acid, and ACR-toxin. Heterologous expression of a HR-PKS gene, a trans-acting enoylreductase gene, and a truncated non-ribosomal peptide synthetase gene resulted in the formation of a linear PK with multiple stereogenic centers. The absolute configurations of the stereogenic centers were determined by chemical degradation followed by comparison of the degradation products with synthetic standards. A stereochemical rule was proposed to explain the absolute configurations of other reduced PKs and highlights an error in the absolute configurations of a reported structure. The present work demonstrates that focused functional analysis of functionally related HR-PKSs leads to a better understanding of the stereochemical course.

Biosynthetic Machinery of 6-Hydroxymellein Derivatives Leading to Cyclohelminthols and Palmaenones.

Oxygenated cyclopentene systems are unique structural motifs found in fungal polyketides such as terrein, cyclohelminthols, and palmaenones. Here we report the identification of the biosynthetic gene clusters for cyclohelminthols and palmaenones and the functional characterization of the polyketide synthases and halogenases involved in the construction of 6-hydroxymellein derivatives. Heterologous expression in Aspergillus oryzae demonstrated that 6-hydroxymellein is a common biosynthetic intermediate and that chlorination occurs in the early stages of its products' biosynthesis. This was further confirmed by in vitro enzymatic reactions conducted in the presence of recombinant proteins. Plausible means of biogenesis of fungal polyketides from 6-hydroxymellein derivatives, additionally supported by the reported labeling patterns of terrein and structurally related fungal polyketides, are also discussed. This study sets the stage for elucidation of the biosynthetic machinery of fungal polyketides of this type.

Biosynthesis of Indole Diterpene Lolitrems: Radical-Induced Cyclization of an Epoxyalcohol Affording a Characteristic Lolitremane Skeleton.

Lolitrems are tremorgenic indole diterpenes that exhibit a unique 5/6 bicyclic system of the indole moiety. Although genetic analysis has indicated that the prenyltransferase LtmE and the cytochrome P450 LtmJ are involved in the construction of this unique structure, the detailed mechanism remains to be elucidated. Herein, we report the reconstitution of the biosynthetic pathway for lolitrems employing a recently established genome-editing technique for the expression host Aspergillus oryzae. Heterologous expression and bioconversion of the various intermediates revealed that LtmJ catalyzes multistep oxidation to furnish the lolitrem core. We also isolated the key reaction intermediate with an epoxyalcohol moiety. This observation allowed us to establish the mechanism of radical-induced cyclization, which was firmly supported by density functional theory calculations and a model experiment with a synthetic analogue.

Efficient Reconstitution of Basidiomycota Diterpene Erinacine Gene Cluster in Ascomycota Host Aspergillus oryzae Based on Genomic DNA Sequences.

To develop the versatile methodology for genome mining of mushroom metabolites, we examined the production of bioactive diterpenes erinacines using genomic DNA sequences. In this report, we initially identified high expression loci (hot spots) in Aspergillus oryzae by sequencing the genomic DNAs from highly yielding transformants which were obtained in our previous biosynthetic studies. Genome editing knock-in of all erinacine biosynthetic genes directly to the hot spot showed that A. oryzae correctly spliced more than 90% of the introns in the mushroom genomic DNA gene sequences. Then, we reconstituted the erinacine biosynthetic gene cluster using two rounds of knock-in of the cDNAs and newly developed repeatable genetic engineering by plasmid recycling. At 100% transformation rate, we obtained a transformant that successfully produced erinacine Q and its intermediates. In this study, we elucidated a biosynthetic pathway of erinacines involving functionally unique hydroxylation supported by dehydrogenase EriH and xylose-specific glycosylation by introducing plant genes for supplying UDP-xylose. Our newly developed hot spot knock-in and plasmid recycling allowed us to avoid a time-consuming screening process and to use unlimited introduction of biosynthetic genes due to marker-free genome editing.

Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences.

Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining.IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products.

Identification of the common biosynthetic gene cluster for both antimicrobial streptoaminals and antifungal 5-alkyl-1,2,3,4-tetrahydroquinolines.

5-Alkyl-1,2,3,4-tetrahydroquinolines (5aTHQs) and streptoaminals (STAMs) are natural products isolated from the combined-culture of Streptomyces nigrescens HEK616 and Tsukamurella pulmonis TP-B0596. Despite their unique structures, their biosynthetic pathway has yet to be elucidated. In the present study, we conducted a feeding experiment using 13C-labeled acetates and demonstrated that 5aTHQs are likely synthesized by the action of polyketide synthase (PKS). Based on this observation, we identified the biosynthetic gene cluster for 5aTHQs. Interestingly, the same gene cluster was also responsible for the structurally-distinct STAMs. The gene cluster contains nine genes encoding one acyl carrier protein, two sets of ketosynthases (KSs) and chain length factors (CLFs), one aminotransferase/reductase bifunctional protein, two ketoreductases, and one thioesterase. KSs and CLFs are classified into the phylogenetically distinct clades from those of known type II PKSs. Heterologous expression of the biosynthetic genes and subsequent gene inactivation clearly indicated that all of the nine genes were required for the biosynthesis of both compounds. In the proposed biosynthetic pathway, chain elongation by PKS, reductive cleavage of a thioester bond, and subsequent transamination generate the core skeleton of both compounds. Differences in the oxidation states of the products result in a distinct cyclization mode to yield 5aTHQs and STAMs.

Elucidation of biosynthetic pathway of a plant hormone abscisic acid in phytopathogenic fungi.

Abscisic acid (ABA) is one of the plant hormones that regulates physiological functions in various organisms, including plants, sponges, and humans. The biosynthetic machinery in plants is firmly established, while that in fungi is still unclear. Here, we elucidated the functions of the four biosynthetic genes, bcABA1-bcABA4, found in Botrytis cinerea by performing biotransformation experiments and in vitro enzymatic reactions with putative biosynthetic intermediates. The first-committed step is the cyclization of farnesyl diphosphate to give α-ionylideneethane catalyzed by a novel sesquiterpene synthase, BcABA3, which exhibits low amino acid sequence identities with sesquiterpene synthases. Subsequently, two cytochrome P450s, BcABA1 and BcABA2, mediate oxidative modifications of the cyclized product to afford 1',4'-trans-dihydroxy-α-ionylideneacetic acid, which undergoes alcohol oxidation to furnish ABA. Our results demonstrated that production of ABA does not depend on the nucleotide sequence of bcABA genes. The present study set the stage to investigate the role of ABA in infections.

Biosynthetic study of conidiation-inducing factor conidiogenone: heterologous production and cyclization mechanism of a key bifunctional diterpene synthase.

Conidiogenone, a diterpene with a unique structure, is known to induce the conidiation of Penicillium cyclopium. The biosynthetic pathway of (-)-conidiogenone has been fully elucidated by the heterologous expression of biosynthetic genes in Aspergillus oryzae and by in vitro enzyme assay with 13C-labeled substrates. After construction of deoxyconidiogenol by the action of bifunctional terpene synthase, one cytochrome P450 catalyzes two rounds of oxidation to furnish conidiogenone. Notably, similar biosynthetic genes are conserved among more than 10 Penicillium sp., suggesting that conidiogenone is a common conidiation inducer in this genus. The cyclization mechanism catalyzed by terpene synthase, which involves successive 1,2-alkyl shifts, was fully elucidated using 13C-labeled geranylgeranyl pyrophosphate (GGPP) as substrate. During the structural analysis of deoxyconidiogenol, we observed broadening of some of the 13C signals measured at room temperature, which has not been observed with other structurally related compounds. Careful examination using techniques including 13C NMR studies at -80 °C, conformational analysis and prediction of the 13C chemical shifts using density functional theory gave insights into this intriguing phenomenon.

Chemical Interactions of Cryptic Actinomycete Metabolite 5-Alkyl-1,2,3,4-tetrahydroquinolines through Aggregate Formation.

Organisms often produce secondary metabolites as a mixture of biosynthetically related congeners. However, why are metabolites with minor chemical variations produced simultaneously? 5-Alkyl-1,2,3,4-tetrahydroquinolines (5aTHQs) are small, lipophilic metabolites produced by Streptomyces nigrescens HEK616 when cultured with Tsukamurella pulmonis TP-B0596. A mixture of 5aTHQs forms aggregates that show enhanced membrane affinity and biological activity. The ability to form aggregates and membrane-binding activity is regulated by the length of the alkyl chains. Aggregates with long alkyl chains were too stable to fuse with lipid membranes. However, if inactive 5aTHQ congener was mixed with active congener, the mixture showed increased membrane affinity, enabling cellular entry and biological activity. Therefore, it is shown that sloppiness in a biosynthetic pathway, by which minor structural variations can be produced, is functionally rational, as the metabolites show synergistic action.

Unveiling Biosynthesis of the Phytohormone Abscisic Acid in Fungi: Unprecedented Mechanism of Core Scaffold Formation Catalyzed by an Unusual Sesquiterpene Synthase.

Abscisic acid (ABA) is a well-known phytohormone that regulates abiotic stresses. ABA produced by fungi is also proposed to be a virulence factor of fungal pathogens. Although its biosynthetic pathway in fungi was proposed by a series of feeding experiments, the enzyme catalyzing the reaction from farnesyl diphosphate to α-ionylideneethane remains to be identified. In this work, we identified the novel type of sesquiterpene synthase BcABA3 and its unprecedented three-step reaction mechanism involving two neutral intermediates, ß-farnesene and allofarnesene. Database searches showed that BcABA3 has no homology with typical sesquiterpene synthases and that the homologous enzyme genes are found in more than 100 bacteria, suggesting that these enzymes form a new family of sesquiterpene synthases.