Ascomycota Aspergillus oryzae is an efficient expression host for production of Basidiomycota terpenes using genomic DNA sequences.

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Ascomycota Aspergillus oryzae is an efficient expression host for production of Basidiomycota terpenes using genomic DNA sequences.

Appl Environ Microbiol. 2019 May 17;:

Authors: Nagamine S, Liu C, Nishishita J, Kozaki T, Sogahata K, Sato Y, Minami A, Ozaki T, Schmidt-Dannert C, Maruyama JI, Oikawa H

Basidiomycota fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects of mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for Basidiomycota fungi. To develop a versatile host for Basidiomycota genes, we examined gene expression using genomic DNA sequences in the robust Ascomycota host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from Basidiomycota Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most of introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complimentary use of AO-expression and automated prediction will be a powerful tool for genome mining.ImportanceThe recent large influx of genome sequences from Basidiomycota, which are prolific producers of bioactive natural products may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of Basidiomycota genes. For this purpose, we applied the Ascomycota Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron-prediction, this Aspergillus oryzae system represents a practical method for the production of Basidiomycota natural products.

PMID: 31101615 [PubMed – as supplied by publisher]

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