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


RESULTS
First, 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.


CONCLUSIONS
We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.
INRA, UMR1163 BCF, 163 avenue de Luminy, Marseille, France. mathieu.bey@esil.univmed.fr
Mathieu
M
Bey
Jean-Guy
JG
Berrin
Laetitia
L
Poidevin
Jean-Claude
JC
Sigoillot
D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D008031:Lignin / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D055494:Pycnoporus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D005656:Fungal Proteins; D011994:Recombinant Proteins; C036909:lignocellulose; D008031:Lignin; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase