Andrastins (andrastin A-D), produced by several Penicillium species, exhibit inhibitory activity against ras farnesyltransferase, suggesting that these compounds could be promising leads for ...antitumor agents. Although the genome sequence of Penicillium chrysogenum, an andrastin-producing species, is available, the genetic and molecular bases for the biosynthesis of andrastins have not been elucidated. Here we report the identification and characterization of the gene cluster for andrastin biosynthesis. We reconstituted the biosynthetic pathway in Aspergillus oryzae, a fungal expression host, by the co-expression of five genes, including that of a terpene cyclase, and of four genes encoding the tailoring enzymes, required for the generation of andrastins. Remarkably, we successfully obtained andrastin A, the most complex andrastin molecule, as the metabolite of nine gene products, thus confirming the potential of the fungal expression system to synthesize useful natural products.
Display omitted
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
The Fe- and 2-oxoglutarate-dependent oxygenase SptF is a promising powerful biocatalys with unusual catalytic versatility and promiscuity. The site-specific random substitution of N150, I63, and N65, ...which are involved in substrate interactions, generated three compounds that were not produced by the SptF wild type. The substrate binding mode was dramatically altered by the introduction of only one or two substitutions. These results provide insights into the engineering of Fe- and 2-oxoglutarate-dependent oxygenases for chemoenzymatic syntheses of bioactive compounds.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Endoperoxide natural products are widely distributed in nature and exhibit various biological activities. Due to their chemical features, endoperoxide and endoperoxide-derived secondary metabolites ...have attracted keen attention in the field of natural products and organic synthesis. In this review, we summarize the structural analyses, mechanistic investigations, and proposed reaction mechanisms of endoperoxide-forming oxygenases, including cyclooxygenase, fumitremorgin B endoperoxidase (FtmOx1), and the asnovolin A endoperoxygenase NvfI.
Structurally diverse fungal meroterpenoids are promising drug seed compounds. To obtain unnatural, novel meroterpene scaffolds, we tested combinatorial biosynthesis by co-expressing functionally ...distinct terpene cyclase (TPC) genes, pyr4, ascF, andB, or cdmG, with the biosynthetic genes for the production of a TPC substrate, (10′R)-epoxyfarnesyl-dimethylorsellinic acid-3,5-methyl ester, in Aspergillus oryzae NSAR1 as a heterologous host. As a result, all of the tested TPCs afforded the same two novel mono-cyclization products. This study provides important information on the substrate scope of the TPCs, and will contribute to the production of unnatural, novel molecules for future drug discovery.
Fungal meroterpenoids are important bioactive natural products. Their biosynthetic machineries are highly diverse, and reconstitutions lead to the production of unnatural meroterpenoids. In this ...study, heterologous gene expression in
and
assays elucidated the biosynthetic pathway of the orthoester-containing fungal meroterpenoid austalide F. Remarkably, the α-ketoglutarate-dependent oxygenase AstB produces the hemiacetal intermediate, and the methyltransferase AstL transfers a methyl group on it to construct the orthoester functionality. This study presents the extraordinary orthoester biosynthetic machinery and provides valuable insights into the creation of unnatural novel bioactive meroterpenoids through engineered biosynthesis. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.
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.
Calbistrin biosynthesis involves an unusual dual‐functional polyketide synthase CalA, which synthesizes both of the two structurally distinct moieties of calbistrins. The product divergence is intriguingly controlled by two trans‐acting enzymes, a trans‐acting enoylreductase CalK and a trans‐acting C‐methyltransferase CalH, to yield the decalin and polyene portions, respectively.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Sulfonamides and sulfamates are a group of organosulfur compounds that contain the signature sulfamoyl structural motif. These compounds were initially only known as synthetic antibacterial drugs but ...were later also discovered as natural products. Eight highly potent examples have been isolated from actinomycetes to date, illustrating the large biosynthetic repertoire of this bacterial genus. For the biosynthesis of these compounds, several distinct and unique biosynthetic machineries have been discovered, capable to generate the unique S-N bond. For the creation of novel, second generation natural products by biosynthetic engineering efforts, a detailed understanding of the underlying enzyme machinery toward potent structural motifs is crucial. In this review, we aim to summarize the current state of knowledge on sulfonamide and sulfamate biosynthesis. A detailed discussion for the secondary sulfamate ascamycin, the tertiary sulfonamide sulfadixiamycin A, and the secondary sulfonamide SB-203208 is provided and their bioactivities and mode of actions are discussed.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We describe the photoinduced reductive debromination of phenacyl bromides using pyridoxal 5′-phosphate (PLP). The reaction requires irradiation with cyan or blue light in an anaerobic atmosphere. ...Mechanistic analysis reveals the formation of the phenacyl radical as an intermediate in the reaction, implying a single electron transfer to phenacyl bromides from a PLP-derived species resulting from excitation by illumination.
Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, ...astellifadiene (1), having a 6‐8‐6‐5‐fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope‐incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation‐initiated second cyclization sequence, 1,2‐alkyl migration, and 1,5‐hydride shift to generate the novel scaffold of 1.
A unique tetracyclic fungal sesterterpene: Genome mining and heterologous expression of a cryptic fungal terpene synthase provided astellifadiene (1) with an unprecedented 6‐8‐6‐5‐fused ring system. The structure of 1 was unambiguously determined by the crystalline sponge method coupled with NMR analyses. Further, the biosynthesis of 1 was proposed on the basis of the isotope‐incorporation experiments performed both in vivo and in vitro.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
PDF
