Total Biosynthesis of Diterpene Aphidicolin, a Specific Inhibitor of DNA Polymerase α: Heterologous Expression of Four Biosynthetic Genes in<i>Aspergillus oryzae</i>

Fujii, Ryuya; Minami, Atsushi; Tsukagoshi, Tae; Sato, Natsuko; Sahara, Takehiko; Ohgiya, Satoru; Gomi, Katsuya; Oikawa, Hideaki

doi:10.1271/bbb.110366

Cited by 81 publications

(66 citation statements)

References 44 publications

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“…Recently, the use of the A. oryzae expression system was further extended to reconstitute the biosynthetic machinery for fungal secondary metabolites. To date, de novo syntheses of tenellin (four genes),12 aphidicolin (four genes),13 paxilline (six genes),14 and aspyridone (three genes)15 in A. oryzae have been reported, as well as the syntheses of the pyripyropene core structure (2+3 genes)16 and andrastin (5+4 genes) 17. The successful demonstration of the total biosynthesis of various types of natural products and stepwise determination of biosynthetic intermediates show the feasibility of using the A. oryzae reconstitution system for the biosynthetic study of fungal metabolites.…”

Section: Methodsmentioning

confidence: 99%

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

et al. 2014

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Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.

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Section: Methodsmentioning

confidence: 99%

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

et al. 2014

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“…172 In addition to the bifunctional diterpene synthase, this cluster encompasses two P450 enzymes that carry out three hydroxylations of the diterpenoid alcohol scaffold, as well as a transporter and the aforementioned GGPP 5 synthase. The two CPS/KS homologs PaDC1 phyllocladen-16 α-ol 82 synthase) and PaDC2 ((+)-CDP synthase) characterized from P. amygdali are each part of separate gene clusters that are involved in the biosynthesis of phyllocladan-11 α,16 α,18-triol 107 .…”

Section: Fungal Diterpenoidsmentioning

confidence: 99%

Traversing the fungal terpenome

2014

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Fungi (Ascomycota and Basidiomycota) are prolific producers of structurally diverse terpenoid compounds. Classes of terpenoids identified in fungi include the sesqui-, di- and triterpenoids. Biosynthetic pathways and enzymes to terpenoids from each of these classes have been described. These typically involve the scaffold generating terpene synthases and cyclases, and scaffold tailoring enzymes such as e.g. cytochrome P450 monoxygenases, NAD(P)+ and flavin dependent oxidoreductases, and various group transferases that generate the final bioactive structures. The biosynthesis of several sesquiterpenoid mycotoxins and bioactive diterpenoids has been well-studied in Ascomycota (e.g. filamentous fungi). Little is known about the terpenoid biosynthetic pathways in Basidiomycota (e.g. mushroom forming fungi), although they produce a huge diversity of terpenoid natural products. Specifically, many trans-humulyl cation derived sesquiterpenoid natural products with potent bioactivities have been isolated. Biosynthetic gene clusters responsible for the production of trans-humulyl cation derived protoilludanes, and other sesquiterpenoids, can be rapidly identified by genome sequencing and bioinformatic methods. Genome mining combined with heterologous biosynthetic pathway refactoring has the potential to facilitate discovery and production of pharmaceutically relevant fungal terpenoids.

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“…Genome walking was used to identify and clone the clustered aphidicolin biosynthetic genes [206] (Fig. 8), and then heterologously express this pathway in A. oryzae [207].…”

Section: Labdane-related Diterpenoids Made By Bifunctional Enzymesmentioning

confidence: 99%

Biosynthesis of Terpenoid Natural Products in Fungi

Schmidt‐Dannert

2014

Advances in Biochemical Engineering/Biotechnology

108

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Tens of thousands of terpenoid natural products have been isolated from plants and microbial sources. Higher fungi (Ascomycota and Basidiomycota) are known to produce an array of well-known terpenoid natural products, including mycotoxins, antibiotics, antitumor compounds, and phytohormones. Except for a few well-studied fungal biosynthetic pathways, the majority of genes and biosynthetic pathways responsible for the biosynthesis of a small number of these secondary metabolites have only been discovered and characterized in the past 5-10 years. This chapter provides a comprehensive overview of the current knowledge on fungal terpenoid biosynthesis from biochemical, genetic, and genomic viewpoints. Enzymes involved in synthesizing, transferring, and cyclizing the prenyl chains that form the hydrocarbon scaffolds of fungal terpenoid natural products are systematically discussed. Genomic information and functional evidence suggest differences between the terpenome of the two major fungal phyla--the Ascomycota and Basidiomycota--which will be illustrated for each group of terpenoid natural products.

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Total Biosynthesis of Diterpene Aphidicolin, a Specific Inhibitor of DNA Polymerase α: Heterologous Expression of Four Biosynthetic Genes inAspergillus oryzae

Cited by 81 publications

References 44 publications

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

Traversing the fungal terpenome

Biosynthesis of Terpenoid Natural Products in Fungi

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