Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei
Lena Wohlschlager, Florian Csarman, Hucheng Chang, Elisabeth Fitz, Bernhard Seiboth, Roland Ludwig
Abstract
Abstract Background Cellobiose dehydrogenase from Phanerochaete chrysosporium ( Pc CDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus . This study describes the expression of basidiomycetes Pc CDH in T. reesei ( Pc CDH Tr ) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with Pc CDH expressed by P. chrysosporium and P. pastoris ( Pc CDH Pp ). Results Pc CDH Tr was recombinantly produced with a yield of 600 U L −1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium . Pc CDH Tr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for Pc CDH Tr and Pc CDH. The determined FAD cofactor occupancy of 70% for Pc CDH Tr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of Pc CDH. Mass spectrometry showed high mannose-type N -glycans on Pc CDH, but only single N -acetyl- d -glucosamine additions at the six potential N-glycosylation sites of Pc CDH Tr , which indicates the presence of an endo-N-acetyl-β- d -glucosaminidase in the supernatant. Conclusions Heterologous production of Pc CDH Tr is faster and the yield higher than secretion by P. chrysosporium . It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of Pc CDH Tr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of Pc CDH Tr are very similar to those of Pc CDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered Pc CDH for biosensors and biofuel cells.