The null pigmentation mutant (npgA1) in Aspergillus nidulans results in a phenotype with colorless organs, decreased branching growth, delayed of asexual spore development, and aberrant cell wall structure. The npgA gene was isolated from A. nidulans to investigate these pleiomorphic phenomena of npgA1 mutant. Sequencing analysis of the complementing gene indicated that it contained a 4'-phosphopantetheinyl transferase (PPTase) superfamily domain. Enzymatic assay of the PPTase, encoded by the npgA gene, was implemented in vivo and in vitro. Loss-of-function of LYS5, which encoded a PPTase in Saccharomyces cerevisiae, was functionally complemented by NpgA, and Escherichia coli-derived NpgA revealed phosphopantetheinylation activity with the elaboration of 3'5'-ADP. Deletion of the npgA gene caused perfectly a lethal phenotype and the absence of asexual/sexual sporulation and secondary metabolites such as pigments in A. nidulans. However, a cross feeding effect with A. nidulans wild type allowed recovery from deletion defects, and phased-culture filtrate from the wild type were used to verify that the npgA gene was essential for formation of metabolites needed for development as well as growth. In addition, forced expression of npgA promoted the formation of conidia and cleistothecia as well as growth. These results indicate that the npgA gene is involved in the phosphopantetheinylation required for primary biological processes such as growth, asexual/sexual development, and the synthesis of secondary metabolites in A. nidulans.

A double-stranded RNA (dsRNA) mycovirus was detected in malformed fruiting bodies of Pleurotus ostreatus strain ASI2792, one of bottle cultivated commercial strains of the edible oyster mushroom. The partial RNA-dependent RNA polymerase (RdRp) gene of the P. ostreatus ASI2792 mycovirus (PoV-ASI2792) was cloned, and a cDNA sequences alignment revealed that the sequence was identical to the RdRp gene of a known PoSV found in the P. ostreatus strain. To investigate the symptoms of PoV-ASI2792 infection by comparing the isogenic virus-free P. ostreatus strains with a virus-infected strain, isogenic virus-cured P. ostreatus strains were obtained by the mycelial fragmentation method for virus curing. The absence of virus was verified with gel electrophoresis after dsRNA-specific virus purification and Northern blot analysis using a partial RdRp cDNA of PoV-ASI2792. The growth rate and mycelial dry weight of virus-infected P. ostreatus strain with PoV-ASI2792 mycovirus were compared to those of three virus-free isogenic strains on 10 different media. The virus-cured strains showed distinctly higher mycelial growth rates and dry weights on all kinds of experimental culture media, with at least a 2.2-fold higher mycelial growth rate on mushroom complete media (MCM) and Hamada media, and a 2.7-fold higher mycelial dry weight on MCM and yeastmalt-glucose agar media than those of the virus-infected strain. These results suggest that the infection of PoV mycovirus has a deleterious effect on the vegetative growth of P. ostreatus.

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Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular βglucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiosefermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucosefermenting S. cerevisiae with extracellular β-glucosidase. These results suggest that the cellobiosefermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.

The development of a yeast strain capable of fermenting mixed sugars efficiently is crucial for producing biofuels and value-added materials from cellulosic biomass. Previously, a mutant Pichia stipitis YN14 strain capable of co-fermenting xylose and cellobiose was developed through evolutionary engineering of the wild-type P. stipitis CBS6054 strain, which was incapable of cofermenting xylose and cellobiose. In this study, through genomic and transcriptomic analyses, we sought to investigate the reasons for the improved sugar metabolic performance of the mutant YN14 strain in comparison with the parental CBS6054 strain. Unfortunately, comparative wholegenome sequencing (WGS) showed no mutation in any of the genes involved in the cellobiose metabolism between the two strains. However, comparative RNA sequencing (RNA-seq) revealed that the YN14 strain had 101.2 times and 5.9 times higher expression levels of HXT2.3 and BGL2 genes involved in cellobiose metabolism, and 6.9 times and 75.9 times lower expression levels of COX17 and SOD2.2 genes involved in respiration, respectively, compared with the CBS6054 strain. This may explain how the YN14 strain enhanced cellobiose metabolic performance and shifted the direction of cellobiose metabolic flux from respiration to fermentation in the presence of cellobiose compared with the CBS6054 strain.