Transcriptional Response of the Cellobiose Dehydrogenase Gene to Cello- and Xylooligosaccharides in the Basidiomycete Phanerochaete chrysosporium

Abstract

Cellobiose dehydrogenase (CDH) gene transcripts were quantified by reverse transcription-PCR (RT-PCR) in cultures ofPhanerochaete chrysosporium supplemented with various cello-and xylooligosaccharides in order to elucidate the mechanism of enhanced CDH production in xylan/cellulose culture. Cellotriose and cellotetraose induced cdh expression, while xylobiose and xylotriose induced expression of cellobiohydrolase genes, especially cel7C. The basidiomycete Phanerochaete chrysosporium produces various extracellular carbohydrate-degrading enzymes (14,24) and oxidative enzymes (13) that degrade wood, which contains cellulose, hemicellulose, and lignin as major components. Cellulose is a ␤-1,4-linked homopolymer of glucose, and the cellulolytic system of this fungus involves multiple endoglucanases (EGs) and cellobiohydrolases (CBHs) (20, 21), which hydrolyze cellulose into cellobiose and cellooligosaccharides. In addition to hydrolytic enzymes, redox enzymes such as cellobiose dehydrogenase (CDH) are involved in cellulose degradation (1,3,4,23). CDH oxidizes cellobiose and cellooligosaccharides into the corresponding 1,5-␦-lactones (5, 15). The role of CDH in cellulose degradation has been widely discussed (7,8,10,25), and its relationship with polysaccharides monooxygenase was recently pointed out (12,16,22). A study by Dumonceaux and coworkers on a CDHdeficient mutant of the wood-rotting basidiomycete Trametes versicolor indicated that CDH is also involved in degradation of other plant cell wall components (2). We have recently reported that arabinoxylan, the major hemicellulose of monocots, enhances fungal growth and CDH production when it is supplemented into cellulose-degrading cultures, although it could not serve as a carbon source for P. chrysosporium by itself (6). In the present study, a similar response of the fungus was observed when glucuronoxylan (from beech wood) was added to cellulolytic culture, suggesting that the main-chain structure of xylan affects fungal growth, extracellular protein production and CDH activity. To elucidate the mechanism of these effects, we used reverse transcription-PCR (RT-PCR) to quantify CDH gene transcripts in cultures supplemented with xylose or xylooligosaccharides as well as glucose or cellooligosaccharides.Phanerochaete chrysosporium strain K-3 (9) was cultivated in Kremer and Wood medium (11) for 4 days with cellulose (CF11; Whatman, Fairfield, NJ), with or without xylan (from oat spelt, Serva Electrophoresis, Heidelberg, Germany; from beech, SigmaAldrich, St. Louis, MO). Briefly, xylan from beech wood has side chains of 4-O-methyl-␤-D-glucuronic acid whereas that from oat spelt has additional side chains of ␣-L-arabinose. The packed volume of fungal mycelium and protein concentration of the culture filtrate were estimated as described previously (6). Cellobiose dehydrogenase (CDH) activity of the culture filtrates was assayed using cellobiose and cytochrome c as described previously (17). For transcriptional analysis in cultures with purified xylooligosac-

Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence‐assisted carbohydrate electrophoresis

“…Taking into account the order of different endoglucanases secreted by microbes growing on various carbon resources , it could be concluded that the oligosaccharide profiles under different growth conditions should be dynamic, and some enzyme components, such as An EglA, could be hydrolyzed to produce a series of oligosaccharides that would induce additional components of GHs . Because water‐insoluble substrates cannot be transported into the cell, cellulolytic microbes require a fine‐response mechanism to regulate the secretion of additional cellulases .…”

Oxidoreductases and Reactive Oxygen Species in Conversion of Lignocellulosic Biomass

“…When the white-rot P. chrysosporium is grown on spruce wood, expression of genes encoding ligninolytic enzymes (MOX and GLOX) is upregulated, whereas cellulase genes are expressed in a more constitutive manner (209). Earlier reports have shown that CDH expression is also induced early, particularly by products of cellulase action (280). In a recent comprehensive study, Miyauchi et al used an integrative omics approach to show that the saprophytic white-rot fungus Pycnoporus coccineus simultaneously secretes GHs, esterases, and oxidoreductases when grown on various lignocellulosic substrates (148).…”

Cellobiose dehydrogenase: An essential enzyme for lignocellulose degradation in nature – A review / Cellobiosedehydrogenase: Ein essentielles Enzym für den Lignozelluloseabbau in der Natur – Eine Übersicht

“…To date, the literature reports the characterization of about 30 CDHs from their native fungal producers. The formation of CDH in vitro requires the presence of insoluble cellulose or soluble cello-oligosaccharides, which act as an 148 Daniel Kracher, Roland Ludwi g inducer (Hori et al, 2012). CDH is secreted into the extracellular space and typically represents about 0.5-2.5% of the total secreted protein on a mass basis (Zámocký et al, 2006).…”