Effects of Glucose Concentration on Ethanol Fermentation of White-Rot Fungus Phanerochaete sordida YK-624 Under Aerobic Conditions.

White-rot fungi are microorganisms capable of ethanol fermentation; however, the specific conditions activating ethanol fermentation are unclear in contrast to fermentation by yeasts. In this study, we investigated the conditions favoring ethanol fermentation by the white-rot fungus Phanerochaete sordida YK-624, which is able to produce ethanol from woody material. In aerobic stationary cultivation with various concentrations of glucose (0.8-33 g/l), the fungus produced ethanol in media containing an initial glucose concentration of 2.8 g/l or higher. The amount of glucose consumption, mycelial weight, and ethanol production on the second day of culture increased in a concentration-dependent manner at low glucose concentrations; however, these were saturated at high concentrations. Biomass yields (growth/glucose consumption) were decreased until the initial glucose concentration increased to 6.0 g/l, after which the biomass yields showed constant values at higher concentrations (12-33 g/l). On the other hand, ethanol yields increased with decreasing biomass yields. In short shaking cultivation using mycelial suspension, trace amounts of instantaneous aerobic ethanol production were observed with 1.1 and 2.1 g/l glucose, but the relative gene expression levels of key enzymes at the pyruvate branch point showed no significant differences between ethanol production and non-production conditions. From these experimental results, it appears that the white-rot fungus P. sordida YK-624 produces ethanol due to overflow in sugar metabolism under aerobic conditions, although P. sordida YK-624 prioritizes glucose utilization for respiratory growth.

Self-fusion and fusion cell isolation of transformants derived from white rot fungus Phanerochaete sordida YK-624 by simple visual method.

In order to develop a simple method for crossing two transformants, we first attempted to elucidate the fusion type (self-compatibility or -incompatible) of Phanerochaete sordida YK-624. Two transformants expressing green or red fluorescent protein derived from an auxotrophic mutant were constructed. Each recombinant protein fluoresced by expression as a fused protein with glyceraldehyde-3-phosphate dehydrogenase. On co-culture of both transformants, a number of sequential hyphal cells emitting dual fluorescence were formed at the contact areas of both hyphae. Some of the single cells isolated as protoplasts and chlamydospore from the co-cultures also expressed these fluorescent proteins. These results suggest that P. sordida YK-624 possesses a self-compatible fusion system. In addition, transformant strains with different fluorescence derived from this fungus can readily undergo self-fusion and nuclear interchange events by confrontational and mixed cultivation, and we developed a simple method that allows fused cells to be isolated as chlamydospores.

Effect on growth, sugar consumption, and aerobic ethanol fermentation of homologous expression of the sugar transporter gene Pshxt1 in the white rot fungus Phanerochaete sordida YK-624.

Major facilitator superfamily (MFS) transporters are found in all organisms. Although numerous studies have examined the functions of yeast and mold MFS transporters in terms of sugar affinity and metabolic regulation, no functional analyses of MFS sugar transporters in white rot fungi have been reported. This study identified an MFS sugar transporter gene (Pshxt1) of the white rot fungus Phanerochaete sordida YK-624 expressed in liquid culture containing low concentrations of nitrogen source. Homologous expression of Pshxt1 dramatically increased the rates of glucose, fructose, mannose, and xylose consumption. Galactose consumption increased slightly but significantly. These data suggest that Pshxt1 has broad affinity for monosaccharides. In contrast, a transformant homologously expressing Pshxt1 consumed glucose in preference to xylose in wood enzymatic-digestion liquor and liquid culture. Additionally, homologous expression of Pshxt1 improved mycelial growth, aerobic ethanol production, and simultaneous aerobic saccharification and fermentation efficiency, whereas secretion of the ligninolytic enzyme manganese peroxidase was clearly decreased in the presence of glucose by Pshxt1 expression. These results suggest that Pshxt1 is involved in the repression of ligninolytic enzyme activity via carbon catabolite repression at sufficiently high glucose concentrations for activation of primary metabolism.