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Shiitake (Lentinula edodes) contains various beneficial compounds and possesses several notable properties. However, there are few reports on its molecular breeding due to delay in development of its gene-modifying technology. Therefore, here, strain UV30 (pyrG –) was bred from the UV-irradiated protoplasts of strain M2. Strain UV30 was uracil-auxotrophic, and the phenylalanine residue in the active centre of orotidine-5-phosphate decarboxylase encoded by pyrG in the strain was substituted with a serine residue. Next, a recycling marker consisting of the upstream sequence of ku80 , a repeat sequence (a portion of the downstream sequence of ku80), pyrG , and the downstream sequence of ku80 was introduced into the strain UV30. Consequently, the prototrophic strain ckp2-1, in which ku80 was replaced with the recycling marker, was obtained. After cultivation in complete medium, mycelia from the edges of ckp2-1 colonies were inoculated into a complete medium containing 5-Fluoroorotic acid (5-FOA). A 5-FOA-resistant strain KaM2, in which pyrG sequence was spliced from the recycling marker sequence via homologous recombination, was obtained. In this study, we developed the first marker recycling system for multigene targeting in L. edodes. Moreover, the resulting ∆ ku80 strain may serve as a non-homologous end-joining deficient strain for further genetic manipulations. [ABSTRACT FROM AUTHOR]

RNA-binding protein Nrd1 plays a role in RNA polymerase II transcription termination. In this study, we showed that the orthologous NrdA is important in global mRNA expression and secondary metabolism in Aspergillus species. We constructed an nrdA conditional expression strain using the Tet-On system in Aspergillus luchuenesis mut. kawachii . Downregulation of nrdA caused a severe growth defect, indicating that NrdA is essential for the proliferation of A. kawachii . Parallel RNA-sequencing and RNA immunoprecipitation-sequencing analysis identified potential NrdA-interacting transcripts, corresponding to 32% of the predicted protein-coding genes of A. kawachii . Subsequent gene ontology analysis suggested that overexpression of NrdA affects the production of secondary metabolites. To clarify this, we constructed Aspergillus nidulans , Aspergillus fumigatus , and Aspergillus oryzae strains overexpressing NrdA in the early developmental stage. Overexpression of NrdA reduced the production of sterigmatocystin and penicillin in A. nidulans , as well as that of helvolic acid and pyripyropene A in A. fumigatus . Moreover, it increased the production of kojic acid and reduced the production of penicillin in A. oryzae . These effects were accompanied by almost consistent changes in the mRNA levels of relevant genes. Collectively, these results suggest that NrdA is the essential RNA-binding protein, which plays a significant role in global gene expression and secondary metabolism in Aspergillus species.IMPORTANCENrd1, a component of the Nrd1-Nab3-Sen1 complex, is an essential RNA-binding protein involved in transcriptional termination in yeast. However, its role in filamentous fungi has not been studied. In this study, we characterized an orthologous NrdA in the Aspergillus species, identified potential NrdA-interacting mRNA, and investigated the effect of overexpression of NrdA on mRNA expression in Aspergillus luchuensis mut. kawachii . The results indicated that NrdA controls global gene expression involved in versatile metabolic pathways, including the secondary metabolic process, at least in the early developmental stage. We demonstrated that NrdA overexpression significantly affected the production of secondary metabolites in Aspergillus nidulans , Aspergillus oryzae , and Aspergillus fumigatus . Our findings are of importance to the fungal research community because the secondary metabolism is an industrially and clinically important aspect for the Aspergillus species.