Takayoshi Awakawa

Pentaketide Resorcylic Acid Synthesis by Type III Polyketide Synthase from Neurospora crassa

Type III polyketide synthases (PKSs) are responsible for aromatic polyketide synthesis in plants and bacteria. Genome analysis of filamentous fungi has predicted the presence of fungal type III PKSs, although none have thus far been functionally characterized. In the genome of Neurospora crassa, a single open reading frame, NCU04801.1, annotated as a type III PKS was found. In this report, we demonstrate that NCU04801.1 is a novel type III PKS catalyzing the synthesis of pentaketide alkylresorcylic acids. NCU04801.1, hence named 2′-oxoalkylresorcylic acid synthase (ORAS), preferred stearoyl-CoA as a starter substrate and condensed four molecules of malonyl-CoA to give a pentaketide intermediate. For ORAS to yield pentaketide alkylresorcylic acids, aldol condensation and aromatization of the intermediate, which is still attached to the enzyme, are presumably followed by hydrolysis for release of the product as a resorcylic acid. ORAS is the first type III PKS that synthesizes pentaketide resorcylic acids.

Physically Discrete β-Lactamase-Type Thioesterase Catalyzes Product Release in Atrochrysone Synthesis by Iterative Type I Polyketide Synthase

ATEG_08451 in Aspergillus terreus, here named atrochrysone carboxylic acid synthase (ACAS), is a nonreducing, iterative type I polyketide synthase that contains no thioesterase domain. In vitro, reactions of ACAS with malonyl-CoA yielded a polyketide intermediate, probably attached to its acyl carrier protein (ACP). The addition of ATEG_08450, here named atrochrysone carboxyl ACP thioesterase (ACTE), to the reaction resulted in the release of products derived from atrochrysone carboxylic acid, such as atrochrysone and endocrocin. ACTE, belonging to the beta-lactamase superfamily, thus appears to be a novel type of thioesterase responsible for product release in polyketide biosynthesis. These findings show that ACAS synthesizes the scaffold of atrochrysone carboxylic acid from malonyl-CoA, and that ACTE hydrolyzes the thioester bond between the ACP of ACAS and the intermediate to release atrochrysone carboxylic acid as the reaction product.

Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C22–C26 fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C22–C26 fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.

Complete biosynthetic pathway of anditomin: nature's sophisticated synthetic route to a complex fungal meroterpenoid.

Anditomin and its precursors, andilesins, are fungal meroterpenoids isolated from Aspergillus variecolor and have unique, highly oxygenated chemical structures with a complex bridged-ring system. Previous isotope-feeding studies revealed their origins as 3,5-dimethylorsellinic acid and farnesyl pyrophosphate and suggested the possible involvement of a Diels-Alder reaction to afford the congested bicyclo[2.2.2]octane core structure of andilesins. Here we report the first identification of the biosynthetic gene cluster of anditomin and the determination of the complete biosynthetic pathway by characterizing the functions of 12 dedicated enzymes. The anditomin pathway actually does not employ a Diels-Alder reaction, but involves the nonheme iron-dependent dioxygenase AndA to synthesize the bridged-ring by an unprecedented skeletal reconstruction. Another dioxygenase, AndF, is also responsible for the structural complexification, generating the end product anditomin by an oxidative rearrangement.

Multiplexing of Combinatorial Chemistry in Antimycin Biosynthesis: Expansion of Molecular Diversity and Utility

Diversity-oriented biosynthesis of a library of antimycin-like compounds (380 altogether) was accomplished by using multiplex combinatorial biosynthesis. The core strategy depends on the use of combinatorial chemistry at different biosynthetic stages. This approach is applicable for the diversification of polyketides, nonribosomal peptides, and the hybrids that share a similar biosynthetic logic.

Uncovering the unusual D-ring construction in terretonin biosynthesis by collaboration of a multifunctional cytochrome P450 and a unique isomerase.

Terretonin (1) is a fungal meroterpenoid isolated from Aspergillus terreus, and possesses a highly oxygenated and unique tetracyclic structure. Although the biosynthetic gene cluster for 1 has been identified and the biosynthesis has recently been studied by heterologous reconstitution and targeted-gene deletion experiments, the last few steps of the terretonin pathway after terrenoid (6) have yet to be elucidated. Notably, the mechanism for the D-ring expansion to afford the terretonin scaffold has been a long-standing mystery to solve. Here we report the characterization of three enzymes that convert 6 into 1, as well as the complete biosynthetic pathway of 1. In the proposed terretonin pathway, the cytochrome P450 Trt6 catalyzes three successive oxidations to transform 6 into an unstable intermediate, which then undergoes the D-ring expansion and unusual rearrangement of the methoxy group to afford the core skeleton of 1. This unprecedented rearrangement is catalyzed by a novel isomerase Trt14. Finally, the nonheme iron-dependent dioxygenase Trt7 accomplishes the last two oxidation reactions steps to complete the biosynthesis.

Terretonin Biosynthesis Requires Methylation as Essential Step for Cyclization

The fungal meroterpenoids have tremendous structural diversity. This group includes several medicinally important compounds, such as the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, pyripyropene A, the selective acetylcholinesterase inhibitor, arisugacin A, and the protein farnesyltransferase inhibitor, andrastin A (Scheme 1). Many of these compounds have polyketide and terpenoid origins. The diversities of these two groups contribute to the structural diversity of fungal meroterpenoids. Understanding the biosynthetic routes of meroterpenoids is important for utilizing the pathways for combinatorial biosynthesis. We have previously reported the functions of the enzymes catalyzing pyripyropene A biosynthesis, consisting of CoA-ligase, polyketide synthase (PKS), prenyltransferase (PT), flavin-dependent monooxygenase (FMO) and terpene cyclase (CYC). In the biosynthesis of pyripyropene A, the terpenoid moiety is cyclized by Pyr4, a novel transmembrane protein with very low similarity to the known terpene cyclases. The cyclization reaction in fungal meroterpenoid biosynthesis is one of the key steps that generate the structural diversity of this class of compounds. As exemplified by terretonin, austinol, andrastin A and anditomin, some compounds are derived from the same polyketide core and differently cyclized terpenoid moieties. These compounds are all derived from 3,5-dimethylorsellinic acid (DMOA, 1, Scheme 2), but they have various cyclic terpenoid moieties. These differences are due to the presence of CYCs with diverse cyclization activities. Terretonin is a toxic compound isolated from Aspergillus terreus. 8] The intriguing structure of terretonin has tempted many researchers to study its biosynthetic route. A previous isotope-feeding experiment revealed that terretonin is derived from 1 and farnesyl diphosphate (FPP). Recently, we identified the biosynthetic gene cluster of terretonin (8) in the A. terreus NIH2624 genome and revealed, using a fungal heterologous expression system, that the PKS (trt4), PT (trt2) and FMO (trt8) genes, which are homologous to the pyripyropene A biosynthetic genes, are responsible for the production of epoxyfarnesyl-DMOA (3), a precursor of terretonin (Scheme 2). However, the CYC gene responsible for the cyclization of the farnesyl moiety of 3 remained unknown. Therefore, we set out to clarify the cyclization step of terretonin biosynthesis. Since there are many DMOA-derived meroterpenoids, significant knowledge about a number of compounds should be obtained by such a study. We first focused on trt1, which is homologous to the pyr4 gene and encoded upstream of trt2, as the CYC gene responsible for the cyclization of 3. To characterize the function of Trt1, we expressed trt1 along with trt4, trt2 and trt8 in the heterologous fungal host A. oryzae NSAR1, a quadruple auxotrophic mutant strain (niaD , sC , DargB, adeA ). By using this host and four expression vectors—pTAex3 harboring a argB marker, pPTRI harboring a ptrA marker, pUSA harboring a sC marker and pAdeA harboring a adeA marker —the five genes were coexpressed under the control of amyB promoter. The transformant was cultured in Czapek–Dox (CD) medium, supplemented with starch to induce expression. After three days, the culture supernatant and the mycelial extract were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). 1 and dihydroxyfarnesyl-DMOA (4), a hydrolyzed compound derived from 3, were observed, but in contrast to our expectaScheme 1. Representative fungal meroterpenoids.

Biosynthetic Pathway for High Structural Diversity of a Common Dilactone Core in Antimycin Production

We herein report comparative analysis of two versions of the biosynthetic gene clusters of antimycins, a natural product family possessing up to 44 distinct entities. The biosynthetic pathway of antimycins is amenable to the high structural variation of the substrates, supported by successes in heterologous expression of the ant cluster and in fluorine incorporation. The latter facilitated the investigation of the structure-activity relationship into the usually invariable 3-formamidosalicylic acid moiety of the molecules.

A methyltransferase initiates terpene cyclization in teleocidin B biosynthesis.

Teleocidin B is an indole terpenoid isolated from Streptomyces. Due to its unique chemical structure and ability to activate protein kinase C, it has attracted interest in the areas of organic chemistry and cell biology. Here, we report the identification of genes encoding enzymes for teleocidin B biosynthesis, including nonribosomal peptide synthetase (tleA), P-450 monooxygenase (tleB), prenyltransferase (tleC), and methyltransferase (tleD). The tleD gene, which is located outside of the tleABC cluster on the chromosome, was identified by transcriptional analysis and heterologous expression. Remarkably, TleD not only installs a methyl group on the geranyl moiety of the precursor but also facilitates the nucleophilic attack from the electron-rich indole to the resultant cation, to form the indole-fused six-membered ring. This is the first demonstration of a cation, generated from methylation, triggering successive terpenoid ring closure.

Pyranonigrin E: a PKS-NRPS hybrid metabolite from Aspergillus niger identified by genome mining.

Induced production of PKS-NRPS metabolites: the genome mining approach is useful for obtaining new compounds. We activated a dormant PKS-NRPS gene cluster in Aspergillus niger ATCC 1015 by expressing its dedicated transcriptional activator (PynR). As a result, the transformant expressing pynR produced a new pyranonigrin compound that we have named pyranonigirin E.