Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on 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 basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most 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 complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining. IMPORTANCE The recent large influx of genome sequences from basidiomycetes, 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 basidiomycete genes. For this purpose, we applied the ascomycete 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 basidiomycete natural products.

UstYa family proteins (DUF3328) are widely and specifically distributed in fungi. They are known to be involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and nonribosomal peptides, and possibly catalyze various reactions, including oxidative cyclization and chlorination. In this study, we focused on phomopsin A, a fungal RiPP consisting of unique nonproteinogenic amino acids. Gene knockout experiments demonstrated that three UstYa homologues, phomYc, phomYd, and phomYe, are essential for the desaturation of amino acid moieties, showing unprecedented function among UstYa family proteins. Sequence similarity network analysis indicated that their amino acid sequences are highly diverged and that most remain uncharacterized, paving the way for genome mining of fungal metabolites with unique modifications.Phomopsin A (1, Figure 1) [1] is a mycotoxin produced by the fungal pathogen Phomopsis leptostromiformis (Diaporthe toxica), which infects lupins and causes lupinosis disease in livestock. [2] It is also a potent antimitotic agent that binds to tubulin with high affinity. [3,4] Regarding its chemical structure and biosynthesis, 1 is a hexapeptide classified as a ribosomally synthesized and posttranslationally modified peptide (RiPP) [5,6] consisting of a 13-membered macrocyclic ether, chlorinated tyrosine (Tyr), and dehydroamino acids. [1] In 2016, Ding et al. reported a biosynthetic gene cluster of 1 and

UstYa family proteins (DUF3328) are widely and specifically distributed in fungi. They are known to be involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and nonribosomal peptides, and possibly catalyze various reactions, including oxidative cyclization and chlorination. In this study, we focused on phomopsin A, a fungal RiPP consisting of unique nonproteinogenic amino acids. Gene knockout experiments demonstrated that three UstYa homologues, phomYc, phomYd, and phomYe, are essential for the desaturation of amino acid moieties, showing unprecedented function among UstYa family proteins. Sequence similarity network analysis indicated that their amino acid sequences are highly diverged and that most remain uncharacterized, paving the way for genome mining of fungal metabolites with unique modifications.Phomopsin A (1, Figure 1) [1] is a mycotoxin produced by the fungal pathogen Phomopsis leptostromiformis (Diaporthe toxica), which infects lupins and causes lupinosis disease in livestock. [2] It is also a potent antimitotic agent that binds to tubulin with high affinity. [3,4] Regarding its chemical structure and biosynthesis, 1 is a hexapeptide classified as a ribosomally synthesized and posttranslationally modified peptide (RiPP) [5,6] consisting of a 13-membered macrocyclic ether, chlorinated tyrosine (Tyr), and dehydroamino acids. [1] In 2016, Ding et al. reported a biosynthetic gene cluster of 1 and