Reconstitution of Polyketide-Derived Meroterpenoid Biosynthetic Pathway in Aspergillus oryzae

Awakawa, Takayoshi; Abe, Ikuro

doi:10.3390/jof7060486

Cited by 18 publications

(13 citation statements)

References 41 publications

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“…To identify the biosynthetic reactions in the early stage biosynthesis, different gene combinations were heterologously expressed in A. oryzae NSAR1. − First, tlxH was expressed, and the transformant produced (3 R )-6-hydroxymellein ( 8 ) (Table S12 and Figures B, S1A, S23, and S24). Coexpression of tlxH and tlxE led to the production of verruculide C ( 9 ) (Table S13 and Figures B, S1A, S25, and S26).…”

Section: Resultsmentioning

confidence: 99%

Heterodimeric Non-heme Iron Enzymes in Fungal Meroterpenoid Biosynthesis

Awakawa

Mori

et al. 2021

J. Am. Chem. Soc.

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hydroxymellein and 4,5-seco-drimane substructures, isolated from the marine fungus Talaromyces purpureogenus. We have identified the biosynthetic gene cluster tlxA-J by heterologous expression in Aspergillus, in vitro enzyme assays, and CRISPR-Cas9-based gene inactivation. Remarkably, the heterodimer of non-heme iron (NHI) enzymes, TlxJ-TlxI, catalyzes three steps of oxidation including a key reaction, hydroxylation at C-5 and C-9 of 12, the intermediate with 3-ketohydroxydrimane scaffold, to facilitate a retro-aldol reaction, leading to the construction of the 4,5secodrimane skeleton and characteristic ketal scaffold of 1−6. The products of TlxJ-TlxI, 1 and 4, were further hydroxylated at C-4′β by another NHI heterodimer, TlxA-TlxC, and acetylated by TlxB to yield the final products, 3 and 6. The X-ray structural analysis coupled with site-directed mutagenesis provided insights into the heterodimer TlxJ-TlxI formation and its catalysis. This is the first report to show that two NHI proteins form a heterodimer for catalysis and utilizes a novel methodology to create functional oxygenase structures in secondary metabolite biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Heterodimeric Non-heme Iron Enzymes in Fungal Meroterpenoid Biosynthesis

Awakawa

Mori

et al. 2021

J. Am. Chem. Soc.

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“…The planar structure of 4′ was identified as 10′,11′-dihydroxyfarnesyl-DHMP by MS and NMR analyses (electronic supplementary material, figure S5 and table S4). Compound 4′ was likely to be generated through the hydrolysis of the epoxide group of 4 in A. oryzae , as often seen in other meroterpenoid biosynthetic studies [5]. These data assigned the role of AstF as an FMO, while the role of AstL remained unclear because no methylated product was detected.…”

Section: Resultsmentioning

confidence: 62%

“…Compound 6 was identified as a known compound, austalide V, by MS/NMR spectral analyses and a comparison of its optical rotation value with published data (figure 1 a ; electronic supplementary material, table S6) [25]. The keto group at C-3 in 6 indicates that AstJ is responsible for the oxidation of OH-3, as expected from the annotation of this enzyme as a short-chain reductase/dehydrogenase (electronic supplementary material, figure S3) [5]. The NMR data of 5 (electronic supplementary material, table S5) were almost identical to those of a previously known compound 17 S -dihydroaustalide K ( 5′ ) (electronic supplementary material, table S8) [26], except that the data of 5 lacked a signal corresponding to the methyl group at OMe-6′.…”

Section: Resultsmentioning

confidence: 86%

Orthoester formation in fungal meroterpenoid austalide F biosynthesis

Awakawa

Liu

Bai

et al. 2023

Phil. Trans. R. Soc. B

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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 Aspergillus oryzae and in vitro 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’.

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“…NSAR1 is a quadruple auxotrophic mutant that allows the transformation of at least four expression vectors. Furthermore, the development of multiple expression cassettes in a single vector enabled the expression of multiple genes of interest in one transformation using only one selectable marker. , Reconstitution of meroterpenoid biosynthesis pathways in A. oryzae has recently been reviewed …”

Section: Expression Of Heterologous Genes In Aspergillusmentioning

confidence: 99%

Total Heterologous Biosynthesis of Fungal Natural Products in Aspergillus nidulans

2022

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Fungal natural products comprise a wide range of bioactive compounds including important drugs and agrochemicals. Intriguingly, bioinformatic analyses of fungal genomes have revealed that fungi have the potential to produce significantly more natural products than what have been discovered so far. It has thus become widely accepted that most biosynthesis pathways of fungal natural products are silent or expressed at very low levels under laboratory cultivation conditions. To tap into this vast chemical reservoir, the reconstitution of entire biosynthetic pathways in genetically tractable fungal hosts (total heterologous biosynthesis) has become increasingly employed in recent years. This review summarizes total heterologous biosynthesis of fungal natural products accomplished before 2020 using Aspergillus nidulans as heterologous hosts. We review here Aspergillus transformation, A. nidulans hosts, shuttle vectors for episomal expression, and chromosomal integration expression. These tools, collectively, not only facilitate the discovery of cryptic natural products but can also be used to generate high-yield strains with clean metabolite backgrounds. In comparison with total synthesis, total heterologous biosynthesis offers a simplified strategy to construct complex molecules and holds potential for commercial application.

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Reconstitution of Polyketide-Derived Meroterpenoid Biosynthetic Pathway in Aspergillus oryzae

Cited by 18 publications

References 41 publications

Heterodimeric Non-heme Iron Enzymes in Fungal Meroterpenoid Biosynthesis

Heterodimeric Non-heme Iron Enzymes in Fungal Meroterpenoid Biosynthesis

Orthoester formation in fungal meroterpenoid austalide F biosynthesis

Total Heterologous Biosynthesis of Fungal Natural Products in Aspergillus nidulans

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