Mechanism Behind the Programmed Biosynthesis of Heterotrimeric Fungal Depside Thielavin A.

Thielavin A (1) is a fungal depside composed of one 3-methylorsellinic acid and two 3,5-dimethylorsellinic acid units. It displays diverse biological activities. However, the mechanism underlying the assembly of the heterotrimeric structure of 1 remains to be clarified. In this study, we identified the polyketide synthase (PKS) involved in the biosynthesis of 1. This PKS, designated as ThiA, possesses an unusual domain organization with the C-methyltransferase (MT) domain situated at the C-terminus following the thioesterase (TE) domain. Our findings indicated that the TE domain is solely responsible for two rounds of ester bond formation, along with subsequent chain hydrolysis. We identified a plausible mechanism for TE-catalyzed reactions and obtained insights into how a single PKS can selectively yield a specific heterotrimeric product. In particular, the tandem acyl carrier protein domains of ThiA are critical for programmed methylation by the MT domain. Overall, this study highlighted the occurrence of highly optimized domain-domain communication within ThiA for the selective synthesis of 1, which can advance our understanding of the programming rules of fungal PKSs.

Production of non-natural 5-methylorsellinate-derived meroterpenoids in Aspergillus oryzae.

Fungal meroterpenoids are diverse structurally intriguing molecules with various biological properties. One large group within this compound class is derived from the aromatic precursor 3,5-dimethylorsellinic acid (DMOA). In this study, we constructed engineered metabolic pathways in the fungus Aspergillus oryzae to expand the molecular diversity of meroterpenoids. We employed the 5-methylorsellinic acid (5-MOA) synthase FncE and three additional biosynthetic enzymes for the formation of (6R,10'R)-epoxyfarnesyl-5-MOA methyl ester, which served as a non-native substrate for four terpene cyclases from DMOA-derived meroterpenoid pathways. As a result, we successfully generated six unnatural 5-MOA-derived meroterpenoid species, demonstrating the effectiveness of our approach in the generation of structural analogues of meroterpenoids.

Ketoreductase Domain-Catalyzed Polyketide Chain Release in Fungal Alkyl Salicylaldehyde Biosynthesis.

Alkyl salicylaldehyde derivatives are polyketide natural products, which are widely distributed in fungi and exhibit great structural diversity. Their biosynthetic mechanisms have recently been intensively studied; however, how the polyketide synthases (PKSs) involved in the fungal alkyl salicylaldehyde biosyntheses release their products remained elusive. In this study, we discovered an orphan biosynthetic gene cluster of salicylaldehyde derivatives in the fungus Stachybotrys sp. g12. Intriguingly, the highly reducing PKS StrA, encoded by the gene cluster, performs a reductive polyketide chain release, although it lacks a C-terminal reductase domain, which is typically required for such a reductive release. Our study revealed that the chain release is achieved by the ketoreductase (KR) domain of StrA, which also conducts cannonical ß-keto reductions during polyketide chain elongation. Furthermore, we found that the cupin domain-containing protein StrC plays a critical role in the aromatization reaction. Collectively, we have provided an unprecedented example of a KR domain-catalyzed polyketide chain release and a clearer image of how the salicylaldehyde scaffold is generated in fungi.

Biosynthetic Characterization, Heterologous Production, and Genomics-Guided Discovery of GABA-Containing Fungal Heptapeptides.

The biosynthetic gene cluster of γ-aminobutyric acid (GABA)-containing fungal cyclic heptapeptides unguisins A (1) and B (2) was identified in the fungus Aspergillus violaceofuscus CBS 115571. In vitro enzymatic reactions and gene deletion experiments revealed that the unguisin pathway involves the alanine racemase UngC to provide d-alanine, which is then accepted by the first adenylation domain of the nonribosomal peptide synthetase (NRPS) UngA. Intriguingly, the hydrolase UngD was found to transform unguisins into previously undescribed linear peptides. Subsequently, heterologous production of these peptides in Aspergillus oryzae was achieved, in which we established a methodology to readily introduce a large NRPS gene into the fungal host. Finally, genome mining revealed new unguisin congeners, each containing a (2R,3R)-ß-methylphenylalanine residue.

Dissection of the Catalytic Mechanisms of Transmembrane Terpene Cyclases Involved in Fungal Meroterpenoid Biosynthesis.

Pyr4-family terpene cyclases are noncanonical transmembrane terpene cyclases involved in the biosynthesis of microbial meroterpenoids and catalyze diverse cyclization reactions. Despite the ubiquity of Pyr4-family terpene cyclases in microorganisms, their three-dimensional structures have never been experimentally determined. Herein, we focused on AdrI, the Pyr4-family enzyme for the andrastin A pathway, and its homologues, and performed a series of mutational experiments using their AlphaFold2-generated structures. Intriguingly, we found that AdrI and InsA7, which both accept the same substrate, use different amino acid residues for the initiation of the cyclization cascade. Furthermore, we obtained several AdrI variants with altered product selectivity, one of which dominantly yielded a new meroterpenoid species. Collectively, our study provides important insights into the catalytic functions of Pyr4-family terpene cyclases and will facilitate the engineering of these enzymes.

Depside Bond Formation by the Starter-Unit Acyltransferase Domain of a Fungal Polyketide Synthase.

Depsides are polyphenolic molecules comprising two or more phenolic acid derivatives linked by an ester bond, which is called a depside bond in these molecules. Despite more than a century of intensive research on depsides, the biosynthetic mechanism of depside bond formation remains unclear. In this study, we discovered a polyketide synthase, DrcA, from the fungus Aspergillus duricaulis CBS 481.65 and found that DrcA synthesizes CJ-20,557 (1), a heterodimeric depside composed of 3-methylorsellinic acid and 3,5-dimethylorsellinic acid. Moreover, we determined that depside bond formation is catalyzed by the starter-unit acyltransferase (SAT) domain of DrcA. Remarkably, this is a previously undescribed form of SAT domain chemistry. Further investigation revealed that 1 is transformed into duricamidepside (2), a depside-amino acid conjugate, by the single-module nonribosomal peptide synthetase DrcB.

Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum.

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

Biosynthetic Elucidation and Structural Revision of Brevione†E: Characterization of the Key Dioxygenase for Pathway Branching from Setosusin Biosynthesis.

Brevione E (1) is a fungal hexacyclic meroditerpenoid with unique oxepane and cycloheptenone moieties. In this study, we identified the biosynthetic gene cluster of 1 and elucidated its biosynthetic pathway via heterologous expression of the biosynthetic genes and in vitro enzymatic reactions. Surprisingly, reexamination of the structure of 1 revealed a substituted tetrahydrofuran ring instead of the previously proposed oxepane system. Moreover, we determined that cycloheptenone synthesis involves skeletal rearrangement catalyzed by the α-ketoglutarate-dependent dioxygenase BrvJ. BrvJ is highly homologous to SetK, which engages in the biosynthesis of another fungal metabolite, setosusin, and accepts the same substrate as BrvJ but performs only simple hydroxylation. Finally, we identified the key amino acid residues critical for product selectivity of BrvJ and SetK, providing insight into how the biosynthesis pathways of 1 and setosusin diverge and how fungi diversify natural products.

Molecular and Computational Bases for Spirofuranone Formation in Setosusin Biosynthesis.

The 3(2H)-furanone unit is observed in many biologically active natural products, as represented by the antifungal medication griseofulvin. Setosusin (1) is a fungal meroditerpenoid featuring a unique spiro-fused 3(2H)-furanone moiety; however, the biosynthetic basis for spirofuranone formation has not been investigated since its isolation. Therefore, in this study we identified the biosynthetic gene cluster of 1 in the fungus Aspergillus duricaulis CBS 481.65 and elucidated its biosynthetic pathway by heterologous reconstitution of related enzyme activities in Aspergillus oryzae. To understand the reaction mechanism to afford spirofuranone, we subsequently performed a series of in vivo and in vitro isotope-incorporation experiments and theoretical calculations. The results indicated that SetF, the cytochrome P450 enzyme that is critical for spirofuranone synthesis, not only performs the epoxidation of the polyketide portion of the substrate but also facilitates the protonation-initiated structural rearrangement to yield 1. Finally, a mutagenesis experiment using SetF identified Lys303 as one of the potential catalytic residues that are important for spirofuranone synthesis.

Heterologous Biosynthesis of Tetrahydroxanthone Dimers: Determination of Key Factors for Selective or Divergent Synthesis.

Tetrahydroxanthone dimers are fungal products, among which secalonic acid D (1) is one of the most studied compounds because of its potent biological activity. Because the biosynthetic gene cluster of 1 has been previously identified, we sought to heterologously produce 1 in Aspergillus oryzae by expressing the relevant biosynthetic genes. However, our initial attempt of the total biosynthesis of 1 failed; instead, it produced four isomers of 1 due to the activity of an endogenous enzyme of A. oryzae. Subsequent overexpression of the Baeyer-Villiger monooxygenase, AacuH, which competes with the endogenous enzyme, altered the product profile and successfully generated 1. Characterization of the key biosynthetic enzymes revealed the surprising substrate promiscuity of the dimerizing enzyme, AacuE, and indicated that efficient synthesis of 1 requires highly selective preparation of the tetrahydroxanthone monomer, which is apparently controlled by AacuH. This study facilitates engineered biosynthesis of tetrahydroxanthone dimers both in a selective and divergent manner.

Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species.

The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus, and A. steynii) have been whole-genome PacBio sequenced to provide genetic references in three Aspergillus sections. A. taichungensis and A. candidus also were sequenced for SM elucidation. Thirteen Aspergillus genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, A. novofumigatus was compared with the pathogenic species A. fumigatus This suggests that A. novofumigatus can produce most of the same allergens, virulence, and pathogenicity factors as A. fumigatus, suggesting that A. novofumigatus could be as pathogenic as A. fumigatus Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in A. ochraceoroseus, A. campestris, and A. steynii, respectively, and novofumigatonin, ent-cycloechinulin, and epi-aszonalenins in A. novofumigatus Our study delivers six fungal genomes, showing the large diversity found in the Aspergillus genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of A. novofumigatus pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.

One Polyketide Synthase, Two Distinct Products: Trans-Acting Enzyme-Controlled Product Divergence in Calbistrin Biosynthesis.

Calbistrins are fungal polyketides consisting of the characteristic decalin and polyene moieties. Although the biosynthetic gene cluster of calbistrin A was recently identified, the pathway of calbistrin A biosynthesis has largely remained uninvestigated. Herein, we investigated the mechanism by which the backbone structures of calbistrins are formed, by heterologous and in vitro reconstitution of the biosynthesis and a structural biological study. Intriguingly, our analyses revealed that the decalin and polyene portions of calbistrins are synthesized by the single polyketide synthase (PKS) CalA, with the aid of the trans-acting enoylreductase CalK and the trans-acting C-methyltransferase CalH, respectively. We also determined that the esterification of the two polyketide parts is catalyzed by the acyltransferase CalD. Our study has uncovered a novel dual-functional PKS and thus broadened our understanding of how fungi synthesize diverse polyketide natural products.

Structural Basis for Isomerization Reactions in Fungal Tetrahydroxanthone Biosynthesis and Diversification.

The novel isomerase NsrQ, from Aspergillus novofumigatus, is a key enzyme in the biosynthesis of fungal tetrahydroxanthones and is responsible for dearomatizing cyclization to provide a tetrahydroxanthone scaffold. NsrQ catalyzes a two-step isomerization reaction, involving the isomerization of allylic alcohol and subsequent inversion of configuration at the methyl group. We report on the biochemical and structural characterizations of NsrQ, and its homologue Dcr3, from Diaporthe longicolla. The crystal structures of NsrQ and Dcr3 revealed their similar overall structures, with a cone-shaped α+ß barrel fold, to those of the nuclear transport factor 2-like superfamily enzymes. Furthermore, the structures of Dcr3 and NsrQ variants complexed with substrate analogues and the site-directed mutagenesis studies identified the catalytic residues and the important hydrophobic residues in shaping the active site pocket for substrate binding. These enzymes thus utilize Glu and His residues as acid-base catalysts. Based on these observations, we proposed a detailed reaction mechanism for NsrQ-catalyzed isomerization reactions.

Molecular Basis for the Biosynthesis of an Unusual Chain-Fused Polyketide, Gregatin A.

Gregatin A (1) is a fungal polyketide featuring an alkylated furanone core, but the biosynthetic mechanism to furnish the intriguing molecular skeleton has yet to be elucidated. Herein, we have identified the biosynthetic gene cluster of gregatin A (1) in Penicillium sp. sh18 and investigated the mechanism that produces the intriguing structure of 1 by in vivo and in vitro reconstitution of its biosynthesis. Our study established the biosynthetic route leading to 1 and illuminated that 1 is generated by the fusion of two different polyketide chains, which are, amazingly, synthesized by a single polyketide synthase GrgA with the aid of a trans-acting enoylreductase GrgB. Chain fusion, as well as chain hydrolysis, is catalyzed by an α/ß hydrolase, GrgF, hybridizing the C11 and C4 carbon chains by Claisen condensation. Finally, structural analysis and mutational experiments using GrgF provided insight into how the enzyme facilitates the unusual chain-fusing reaction. In unraveling a new biosynthetic strategy involving a bifunctional PKS and a polyketide fusing enzyme, our study expands our knowledge concerning fungal polyketide biosynthesis.

BODI-Pt, a Green-Light-Activatable and Carboplatin-Based Platinum(IV) Anticancer Prodrug with Enhanced Activation and Cytotoxicity.

Platinum drugs are widely used in clinics to treat various types of cancer. However, a number of severe side effects induced by the nonspecific binding of platinum drugs to normal tissues limit their clinical use. The conversion of platinum(II) drugs into more inert platinum(IV) derivatives is a promising strategy to solve this problem. Some platinum(IV) prodrugs, such as carboplatin-based tetracarboxylatoplatinum(IV) prodrugs, are not easily reduced to active platinum(II) species, leading to low cytotoxicity in vitro. In this study, we report the design and synthesis of a carboplatin-based platinum(IV) prodrug functionalized with a boron dipyrromethene (bodipy) ligand at the axial position, and the ligand acts as a photoabsorber to photoactivate the platinum(IV) prodrug. This compound, designated as BODI-Pt, is highly stable in the dark but quickly activated under irradiation to release carboplatin and the axial ligands. A cytotoxic study reveals that BODI-Pt is effective under irradiation, with cytotoxicity 11 times higher than that in the dark and 39 times higher than that of carboplatin in MCF-7 cells. Moreover, BODI-Pt has been proven to kill cancer cells by binding to the genomic DNA, arresting the cell cycle at the G2/M phase, inducing oncosis, and generating ROS upon irradiation. In summary, we report a green-light-activatable and carboplatin-based Pt(IV) prodrug with improved cytotoxicity against cancer cells, and our strategy can be used as a promising way to effectively activate carboplatin-based platinum(IV) prodrugs.

Molecular basis for the unusual ring reconstruction in fungal meroterpenoid biogenesis.

Trt14 from Aspergillus terreus is involved in unusual skeletal reconstruction during the biosynthesis of the fungal meroterpenoid terretonin. Detailed in vitro characterization revealed that this novel multifunctional enzyme catalyzes not only the D-ring expansion via intramolecular methoxy rearrangement, but also the hydrolysis of the expanded D-ring. The X-ray crystal structures of Trt14, in complex with substrate or product, and two Trt14 homologs, AusH and PrhC from Aspergillus nidulans and Penicillium brasilianum, respectively, indicated similar overall structures to those of the NTF2-like superfamily of enzymes, despite lacking sequence and functional similarities. Moreover, we gained structural insight into the mechanism of the Trt14-catalyzed ring reconstruction from the in-crystal enzyme reaction and site-directed mutagenesis to show that this reaction involves sequential ester bond cleavage and formation. Structural comparison of Trt14 and its homologs suggests that the enzymes in this new superfamily employ similar acid-base chemistry to diversify the molecular architecture of fungal meroterpenoids.

Structural and Computational Bases for Dramatic Skeletal Rearrangement in Anditomin Biosynthesis.

AndA, an Fe(II)/α-ketoglutarate (αKG)-dependent enzyme, is the key enzyme that constructs the unique and congested bridged-ring system of anditomin (1), by catalyzing consecutive dehydrogenation and isomerization reactions. Although we previously characterized AndA to some extent, the means by which the enzyme facilitates this drastic structural reconstruction have remained elusive. In this study, we have solved three X-ray crystal structures of AndA, in its apo form and in the complexes with Fe(II), αKG, and two substrates. The crystal structures and mutational experiments identified several key amino acid residues important for the catalysis and provided insight into how AndA controls the reaction. Furthermore, computational calculations validated the proposed reaction mechanism for the bridged-ring formation and also revealed the requirement of a series of conformational changes during the transformation.

Unique chemistry of non-heme iron enzymes in fungal biosynthetic pathways.

Covering: up to 2018 Non-heme iron enzymes are a versatile family of oxygenases that catalyze remarkable types of chemistry. This review highlights the intriguing chemistry of non-heme iron enzymes, especially those utilizing α-ketoglutarate (α-KG) as a co-substrate, in fungal secondary metabolism and aims to summarize how nature diversifies and complexifies natural products.

Discovery of Key Dioxygenases that Diverged the Paraherquonin and Acetoxydehydroaustin Pathways in Penicillium brasilianum.

Paraherquonin (1), a fungal meroterpenoid produced by Penicillium brasilianum NBRC 6234, possesses a unique, highly congested hexacyclic molecular architecture. Here we identified the biosynthetic gene cluster of 1 (the prh cluster) and elucidated the pathway up to berkeleydione (2), which serves as the key intermediate for the biosynthesis of 1 as well as many other meroterpenoids. Interestingly, the nonheme iron and α-ketoglutarate-dependent dioxygenase PrhA constructs the cycloheptadiene moiety to afford 2 from preaustinoid A1 (6), probably via the homoallyl-homoallyl radical rearrangement. Additionally, another fungal strain, P. brasilianum MG11, which produces acetoxydehydroaustin instead of 1, was found to have a gene cluster nearly identical to the prh cluster. The dioxygenase encoded by the cluster shares 92% sequence identity with PrhA, and also accepts 6 but produces preaustinoid A3 (17) with a spiro-lactone system, generating a diverging point for the two different meroterpenoid pathways in the same species.

Genome-Based Discovery of an Unprecedented Cyclization Mode in Fungal Sesterterpenoid Biosynthesis.

Sesterterpenoids are a group of terpenoid natural products that are primarily biosynthesized via cyclization of the C25 linear substrate geranylfarnesyl pyrophosphate (GFPP). Although the long carbon chain of GFPP in theory allows for many different cyclization patterns, sesterterpenoids are relatively rare species among terpenoids, suggesting that many intriguing sesterterpenoid scaffolds have been overlooked. Meanwhile, the recent identification of the first sesterterpene synthase has allowed the discovery of new sesterterpenoids by the genome mining approach. In this study, we characterized the unusual fungal sesterterpene synthase EvQS and successfully obtained the sesterterpene quiannulatene (1) with a novel and unique highly congested carbon skeleton, which is further oxidized to quiannulatic acid (2) by the cytochrome P450 Qnn-P450. A mechanistic study of its cyclization from GFPP indicated that the biosynthesis employs an unprecedented cyclization mode, which involves three rounds of hydride shifts and two successive C-C bond migrations to construct the 5-6-5-5-5 fused ring system of 1.