Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences

Abstract

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.

Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production

“…Fungal Biology and Biotechnology *Correspondence: umemura-m@aist.go.jp 1 Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8566, Japan Full list of author information is available at the end of the article Page 2 of 9 Umemura et al Fungal Biol Biotechnol (2020) 7:3 auxotrophic strain (argB − , niaD − , sC − and adeA − ) [12], is used to produce fungal secondary metabolites by simultaneously introducing two to nine genes for biosynthesis of such compounds as pleuromutilin [15], paxilline [16], terretonin [17], helvolic acid [18], menisporopsin A [19] and asperipin-2a [20]. A variety of basidiomycete terpenes have been successfully produced in A. oryzae by heterologous expression of their respective biosynthetic genes using the genome-editing system [21]. Although the number of promoters that can be used for heterologous expression in filamentous fungi is limited in comparison with that in the yeast Saccharomyces cerevisiae, where a well-established set of promoters covers virtually all patterns of expression [22], promoter tools have been developed for filamentous fungi including Trichoderma reesei [23], A. niger [24], Penicillium chrysogenum [25] and Ustilago maydis [26].…”

Recent developments of tools for genome and metabolome studies in basidiomycete fungi and their application to natural product research

“…Among fungi of the phylum Ascomycota, S. cerevisiae has only 1.04 exons per gene (Goffeau et al, 1996), while A. oryzae has 2.98 (Arnaud et al, 2011). Indeed, a recent study led by Oikawa's group showed that A. oryzae is able to remove 87% of the introns from basidiomycete terpene synthase genes (Nagamine et al, 2019). The same group used this host to study the biosynthesis of the diterpene erinacine, where 90% of introns were correctly spliced out by A. oryzae and the remaining introns were removed by PCR prior to achieving correct expression (Liu et al, 2019).…”

“…Besides the engineering of a NP-producing basidiomycete strain, heterologous expression of a candidate NP gene cluster is an established and effective approach to link specialised metabolites to their genetic determinants, discover new bioactive molecules and characterise their biosynthesis (Alberti et al, 2017b). The successful heterologous expression of basidiomycete clusters has been described in several hosts, including S. cerevisiae (Harvey et al, 2018) and, despite the reported incomplete intron-splicing, in A. oryzae (Nagamine et al, 2019). Ascomycete hosts are easier to transform and engineer than basidiomycete fungi, therefore they are the preferred chassis for the study of basidiomycete NP gene clusters.…”

Heterologous production of fungal natural products: Reconstitution of biosynthetic gene clusters in model host <i>Aspergillus oryzae</i>

“…Mushroom-forming basidiomycete fungi are known to be prolific producers of structurally diverse, bioactive NPs. 49 ) However, despite their incredible potential for NP discovery, Basidiomycota fungi are a largely unexplored target for drug discovery compared with Ascomycota fungi. Unfortunately, Basidiomycota have very intron-rich genomes and genes that contain very small and unpredictable exons, and thus currently no reliable tool for automated intron prediction is available.…”

“…In the examination of unpredictable splicing of basidiomycete genes, we used the recently developed technique, a fungal clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system specifically optimized for AO, to ensure the expression of target genes. 49 , 51 ) In addition, the STS genes were introduced (knocked-in) to the hot spot (HS) loci where target genes were integrated in the highly yielding TFs found in our previous studies. Sequence analysis of cDNAs recovered from the TFs revealed that 87% of total introns were correctly spliced, and nonspliced introns were simply skipped with no complicated missplicing.…”