Upregulation of MAP kinase HOG1 gene of white-rot fungus Phlebia sp. MG-60 inhibits the ethanol fermentation and mycelial growth.

Wood biomass conversion for fossil resource replacement could result in the sustainable production of chemicals, although lignin represents an obstacle to efficient polysaccharide use. White-rot fungus Phlebia sp. MG-60 reportedly selectively and aerobically degrades lignin in hardwood, then it begins cellulose saccharification from the delignified wood to produce ethanol. Environmental conditions might change white-rot fungi-driven biomass conversion. However, how the environmental response sensor affects ethanol fermentation in white-rot fungi remains elusive. In this study, we focused on MGHOG1, the yeast Hog1 homolog in Phlebia sp. MG-60, a presumably important player in osmoresponse. We generated MGHOG1 overexpressing (OE) transformants in Phlebia sp. MG-60, exhibiting slower mycelial growth compared with the wild-type under salinity stress. MGHOG1 overexpressing liquid cultures displayed suppressed mycelial growth and ethanol fermentation. Therefore, MGHOG1 potentially influences ethanol fermentation and mycelial growth in Phlebia sp. MG-60. This study provides novel insights into the regulation of white-rot fungi-mediated biomass conversion.

A finding of potential coexisting bacteria and characterization of the bacterial communities in the fruiting body of Sarcodon aspratus.

Sarcodon aspratus (Berk.) S. Ito is a Japanese local dish with unique aroma and is effective against allergic diseases. However, its cultivation was still difficult. Recently, coexisting bacteria were regarded as an important factor for mycelium growth and fruiting body formation. Therefore, we performed 16S rRNA amplicon sequencing in the fruiting body of S. aspratus and its adhered soil to understand the bacterial communities in the fruiting body of S. aspratus. The fruiting body group showed lower alpha diversities and a significant difference in the structure of bacterial communities compared to the soil group. In addition, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium had the highest relative abundance in the fruiting body group, and it was also a potential coexisting bacterium in the fruiting body of S. aspratus by linear discriminant analysis effect size (LEfSe) analysis. This highest relative abundance phenomenon in Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium clade was also found in the fruiting body of Cantharellus cibarius. These findings suggested that Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium plays a key role in the bacterial communities in the fruiting body of S. aspratus. Bacteria in the fruit bodies of S. aspratus and C. cibarius probably present a similar coexistence model.

Selective Homologous Expression of Recombinant Manganese Peroxidase Isozyme of Salt-Tolerant White-Rot Fungus Phlebia sp. MG-60, and Its Salt-Tolerance and Thermostability.

Phlebia sp. MG-60 is the salt-tolerant, white-rot fungus which was isolated from a mangrove forest. This fungus expresses three kinds of manganese peroxidase (MGMnP) isozymes, MGMnP1, MGMnP2 and MGMnP3 in low nitrogen medium (LNM) or LNM containing NaCl. To date, there have been no reports on the biochemical salt-tolerance of these MnP isozymes due to the difficulty of purification. In present study, we established forced expression transformants of these three types of MnP isozymes. In addition, the fact that this fungus hardly produces native MnP in a high-nitrogen medium (HNM) was used to perform isozyme-selective expression and simple purification in HNM. The resulting MGMnPs showed high tolerance for NaCl compared with the MnP of Phanerochaete chrysosporium. It was worth noting that high concentration of NaCl (over 200 mM to 1200 mM) can enhance the activity of MGMnP1. Additionally, MGMnP1 showed relatively high thermo tolerance compared with other isozymes. MGMnPs may have evolved to adapt to chloride-rich environments, mangrove forest.

Down-regulation of pyruvate decarboxylase gene of white-rot fungus Phlebia sp. MG-60 modify the metabolism of sugars and productivity of extracellular peroxidase activity.

Ethanologenic white-rot fungus Phlebia sp. MG-60-P2 produces ethanol directly from several lignocelluloses. Efficient gene silencing methods are needed for metabolic engineering of this fungus for biorefinery use. In this study, we evaluated the effectiveness of RNAi-mediated silencing of the pyruvate decarboxylase gene of Phlebia sp. MG-60-P2 (MGpdc1). The RNAi lines generated showed a variety of suppression levels of ethanol production and MGpdc1 expression, and two selected strains led to different metabolic fluxes, resulting in rapid accumulation of xylitol from xylose. Knockdown lines KD2 and KD10 showed different strength of silencing. The moderate-inhibition line (KD10) showed faster xylitol accumulation from xylose than the severe-inhibition line (KD2). KD2, KD10 and knockout line KO77 showed higher extracellular peroxidase activity than the wild-type. Gene silencing using RNAi for MGpdc1 in the ethanologenic white-rot fungus Phlebia sp. MG-60-P2 is an effective first step in metabolic engineering to produce other chemicals besides ethanol. This high efficiency of transformation and silencing effect will make it possible to cotransform with multiple expression vectors which enhance the minor metabolic pathway or introduce exogenous metabolic reaction in Phlebia sp. MG-60-P2.