Bioinformatics-Guided Reconstitution of Biosynthetic Machineries of Fungal Eremophilane Sesquiterpenes.

Eremophilanes exhibit diverse biological activities and chemical structures. This study reports the bioinformatics-guided reconstitution of the biosynthetic machinery of fungal eremophilanes, eremofortin C and sporogen-AO1, to elucidate their biosynthetic pathways. Their biosyntheses include P450-catalyzed multistep oxidation and enzyme-catalyzed isomerization by the DUF3237 family protein. Successful characterization of six P450s enabled us to discuss the functions of eremophilane P450s in putative eremophilane biosynthetic gene clusters, providing opportunities to understand the oxidative modification pathways of fungal eremophilanes.

A fungal sesquiterpene biosynthesis gene cluster critical for mutualist-pathogen transition in Colletotrichum tofieldiae.

Plant-associated fungi show diverse lifestyles from pathogenic to mutualistic to the host; however, the principles and mechanisms through which they shift the lifestyles require elucidation. The root fungus Colletotrichum tofieldiae (Ct) promotes Arabidopsis thaliana growth under phosphate limiting conditions. Here we describe a Ct strain, designated Ct3, that severely inhibits plant growth. Ct3 pathogenesis occurs through activation of host abscisic acid pathways via a fungal secondary metabolism gene cluster related to the biosynthesis of sesquiterpene metabolites, including botrydial. Cluster activation during root infection suppresses host nutrient uptake-related genes and changes mineral contents, suggesting a role in manipulating host nutrition state. Conversely, disruption or environmental suppression of the cluster renders Ct3 beneficial for plant growth, in a manner dependent on host phosphate starvation response regulators. Our findings indicate that a fungal metabolism cluster provides a means by which infectious fungi modulate lifestyles along the parasitic-mutualistic continuum in fluctuating environments.

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.

Recent advances in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides of fungal origin.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are growing class of natural products with potent biological activities. Although the core scaffolds of RiPPs are composed of proteinogenic amino acids, remarkable structural diversity is generated through posttranslational modifications (PTMs) of precursor peptides. In addition, ribosomal origin of biosynthetic precursors enables supply of its analogs through genetic approach such as site-directed mutagenesis on corresponding genes. As PTM enzymes often exhibit substrate tolerance, RiPP biosynthetic machineries are considered as efficient tools for generation of unique peptide derivatives. RiPP pathways are distributed among all domains of life and those derived from bacteria and plants have been known for decades. In contrast, fungal RiPPs (F-RiPPs) have fewer examples. Amatoxins and omphalotins are F-RiPPs produced by Basidiomycota fungi. In the biosynthesis of these compounds, macrocyclization by prolyl oligopeptidase homologs and N-methylations of back bone amides have been characterized, respectively. Ustiloxins and related compounds are another group of F-RiPPs with characteristic macrocyclic ethers. UstYa family proteins, which are fungi-specific putative oxidases, have been identified as common proteins involved in PTMs of these compounds. Despite a limited number of characterized examples, recent progress in sequencing of fungal genomes indicated that a number of RiPP pathways are hidden in fungal resources, making F-RiPPs as attractive target for genome mining studies while more detailed understandings of key biosynthetic enzymes are still necessary. This review seeks to describe recent advances on the F-RiPP biosynthesis with slight emphasis on the function of UstYa family proteins.

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.

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.

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.

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.

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.

Genome-Based Discovery of Enantiomeric Pentacyclic Sesterterpenes Catalyzed by Fungal Bifunctional Terpene Synthases.

Genome-based discovery of two previously unreported fungal bifunctional terpene synthases (BFTSs) from phytopathogenic fungi are reported: FoFS catalyzing the formation of fusoxypenes A-C (1-3) and (-)-astellatene (4) and AtAS capable of synthesizing preaspterpenacid I (6). Interestingly, FoFS and AtAS catalyzed the formation of enantiomeric sesterterpenes with a 5-6-7-3-5 ring system. C22-oxidative modification of preaspterpenacid I by AtP450 was characterized as well. Plausible cyclization pathways of the fusoxypenes were illustrated by DFT calculations.

Biosynthesis of Cyclochlorotine: Identification of the Genes Involved in Oxidative Transformations and Intramolecular O,N-Transacylation.

Mycotoxin cyclochlorotine (1) and structurally related astins are cyclic pentapeptides containing unique nonproteinogenic amino acids, such as ß-phenylalanine, l-allo-threonine, and 3,4-dichloroproline. Herein, we report the biosynthetic pathway for 1, which involves intriguing tailoring processes mediated by DUF3328 proteins, including stereo- and regiospecific chlorination and hydroxylation and intramolecular O,N-transacylation. Our findings demonstrate that DUF3328 proteins, which are known to be involved in oxidative cyclization of fungal ribosomal peptides, have much higher functional diversity than previously expected.

Heterologous production of fungal natural products: Reconstitution of biosynthetic gene clusters in model host Aspergillus oryzae.

While exploring phytotoxic metabolites from phytopathogenic fungi in the 1970s, we became interested in biosynthetic enzymes that catalyze Diels-Alder reactions involving biosynthesis of several phytotoxins that we isolated. Target enzymes were successfully characterized, and this triggered the identification of various Diels-Alderases in a recent decade. Through our Diels-Alderase project in 1990s, we recognized a highly efficient expression system of various biosynthetic genes with Aspergillus oryzae as a host. With the development of tools such as genomic data and bioinformatics analysis to identify biosynthetic gene clusters for natural products, we developed a highly reliable methodology such as hot spot knock-in to elucidate the biosynthetic pathways of representative fungal metabolites including phytotoxic substances. This methodology allows total biosynthesis of natural products and genome mining using silent biosynthetic gene clusters to obtain novel bioactive metabolites. Further applications of this technology are discussed.

Fungal-derived brevianamide assembly by a stereoselective semipinacolase.

Fungal bicyclo[2.2.2]diazaoctane indole alkaloids represent an important family of natural products with a wide-spectrum of biological activities. Although biomimetic total syntheses of representative compounds have been reported, the details of their biogenesis, especially the mechanisms for assembly of diastereomerically distinct and enantiomerically antipodal metabolites, have remained largely uncharacterized. Brevianamide A represents a basic form of the sub-family bearing a dioxopiperazine core and a rare 3-spiro-ψ-indoxyl skeleton. Here, we identified the Brevianamide A biosynthetic gene cluster from Penicillium brevicompactum NRRL 864 and elucidated the metabolic pathway. BvnE was revealed to be an essential isomerase/semi-pinacolase that specifies selective production of the natural product. Structural elucidation, molecular modeling, and mutational analysis of BvnE, and quantum chemical calculations provided mechanistic insights into the diastereoselective formation of the 3-spiro-ψ-indoxyl moiety in Brevianamide A. This occurs through a BvnE-controlled semi-pinacol rearrangement and a subsequent spontaneous intramolecular [4+2] hetero-Diels-Alder cycloaddition.

Predicting the chemical space of fungal polyketides by phylogeny-based bioinformatics analysis of polyketide synthase-nonribosomal peptide synthetase and its modification enzymes.

Fungal polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrids are key enzymes for synthesizing structurally diverse hybrid natural products (NPs) with characteristic biological activities. Predicting their chemical space is of particular importance in the field of natural product chemistry. However, the unexplored programming rule of the PKS module has prevented prediction of its chemical structure based on amino acid sequences. Here, we conducted a phylogenetic analysis of 884 PKS-NRPS hybrids and a modification enzyme analysis of the corresponding biosynthetic gene cluster, revealing a hidden relationship between its genealogy and core structures. This unexpected result allowed us to predict 18 biosynthetic gene cluster (BGC) groups producing known carbon skeletons (number of BGCs; 489) and 11 uncharacterized BGC groups (171). The limited number of carbon skeletons suggests that fungi tend to select PK skeletons for survival during their evolution. The possible involvement of a horizontal gene transfer event leading to the diverse distribution of PKS-NRPS genes among fungal species is also proposed. This study provides insight into the chemical space of fungal PKs and the distribution of their biosynthetic gene clusters.

Rapid and Systematic Exploration of Chemical Space Relevant to Artemisinins: Anti-malarial Activities of Skeletally Diversified Tetracyclic Peroxides and 6-Aza-artemisinins.

To achieve both structural changes and rapid synthesis of the tetracyclic scaffold relevant to artemisinins, we explored two kinds of de novo synthetic approaches that generate both skeletally diversified tetracyclic peroxides and 6-aza-artemisinins. The anti-malarial activities of the tetracyclic peroxides with distinct skeletal arrays, however, were moderate and far inferior to artemisinins. Given the privileged scaffold of artemisinins, we next envisioned element implantation at the C6 position with a nitrogen without the trimmings of substituents and functional groups. This molecular design allowed the deep-seated structural modification of the hitherto unexplored cyclohexane moiety (C-ring) while keeping the three-dimensional structure of artemisinins. Notably, this approach induced dramatic changes of retrosynthetic transforms that allow an expeditious catalytic asymmetric synthesis with generation of substitutional variations at three sites (N6, C9, and C3) of the 6-aza-artemisinins. These de novo synthetic approaches led to the lead discovery with substantial intensification of the in vivo activities, which undermine the prevailing notion that the C-ring of artemisinins appears to be merely a structural unit but to be a functional area as the anti-malarial pharmacophore. Furthermore, we unexpectedly found that racemic 6-aza-artemisinin (33) exerted exceedingly potent in vivo efficacies superior to the chiral one and the first-line drug, artesunate.

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.

FAD-dependent enzyme-catalysed intermolecular [4+2] cycloaddition in natural product biosynthesis.

The Diels-Alder reaction is one of the most powerful and widely used methods in synthetic chemistry for the stereospecific construction of carbon-carbon bonds. Despite the importance of Diels-Alder reactions in the biosynthesis of numerous secondary metabolites, no naturally occurring stand-alone Diels-Alderase has been demonstrated to catalyse intermolecular Diels-Alder transformations. Here we report a flavin adenine dinucleotide-dependent enzyme, Morus alba Diels-Alderase (MaDA), from Morus cell cultures, that catalyses an intermolecular [4+2] cycloaddition to produce the natural isoprenylated flavonoid chalcomoracin with a high efficiency and enantioselectivity. Density functional theory calculations and preliminary measurements of the kinetic isotope effects establish a concerted but asynchronous pericyclic pathway. Structure-guided mutagenesis and docking studies demonstrate the interactions of MaDA with the diene and dienophile to catalyse the [4+2] cycloaddition. MaDA exhibits a substrate promiscuity towards both dienes and dienophiles, which enables the expedient syntheses of structurally diverse natural products. We also report a biosynthetic intermediate probe (BIP)-based target identification strategy used to discover MaDA.

Total synthesis of alkaloids using both chemical and biochemical methods.

Covering: 2000 to 2019Rapid access to genomic data has facilitated the identification of the biosynthetic enzyme genes of alkaloid natural products and elucidation of their biosynthetic pathways. Enzymes for the rapid construction of molecular scaffolds and versatile modifications during the late-stage biosynthesis of complex molecular skeletons constitute unique features of biosynthetic machineries. For example, enzymes involved in an alkaloid biosynthesis. In this review, we discuss three types of useful enzymes and enzymatic reactions that have been found in the biosynthetic studies of several alkaloids, and discuss their applications for the total synthesis of natural alkaloids and their derivatives. The selected examples include a single non-ribosomal peptide synthetase SfmC that catalyzes key Pictet-Spengler reactions, which construct a characteristic tetrahydroisoquinoline skeleton in antitumor antibiotics such as saframycin, prenylation-oxidative modification enzymes involved in the biosynthesis of fungal tremorgenic mycotoxins such as penitrem as well as versatile Diels-Alderases recently discovered in the biosynthesis of plant monoterpene indole alkaloids of iboga and aspidosperma type.