Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes

Abstract

BackgroundCellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass.ResultsFirst, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i) the production of a large amount of gluconic acid, (ii) increased hemicellulose degradation, and (iii) increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH) expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM). Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome.ConclusionsWe demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

“…This might be due to glycosylation of the linker regions between the catalytic and the CBM1 modules that contain multiple glycosylation sites. In parallel, CDH was produced in a bioreactor as described by Bey et al (28).…”

“…The activity of CDH was determined by monitoring the reduction of 0.2 mM 2,6-dichlorophenol indophenol (DCPIP) in 100 mM sodium acetate buffer (pH 4.8) containing 10 mM cellobiose, as described by Bey et al (28).…”

“…The heme is involved in intramolecular electron transfer from FAD to the heme and from the heme to another electron acceptor, such as Fe 3ϩ (24,25). It is now established that CDHs are secreted by fungi under cellulolytic conditions and are involved in cellulose/lignin degradation (26)(27)(28)(29)(30).…”

“…For this purpose, we cloned and heterologously expressed two family GH61 enzymes from the coprophilic ascomycete Podospora anserina. The comparative action of the two GH61 enzymes was evaluated through the identification of oxidized products generated in combination with the CDH of Pycnoporus cinnabarinus that we have recently characterized (28). ; the pH was adjusted to 7 with KH 2 PO 4 ).…”

Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

“…CDHs are secreted by a variety of fungi, including white-rot, brown-rot, and soft-rot fungi, as well as molds (1), upon growth on cellulose and plant biomass (3,4). The first CDHs, initially named cellobiose oxidase, were discovered as enzymes able to catalyze the cellobiose-dependent reduction of quinones (5,6).…”