Homologous expression of recombinant cellobiose dehydrogenase in Phanerochaete chrysosporium.
Cellobiose dehydrogenase (CDH) is a novel extracellular hemoflavoenzyme from Phanerochaete chrysosporium and is produced only in cultures supplemented with cellulose. In this report, CDH from P. chrysosporium has been homologously expressed in cultures supplemented with glucose as the sole carbon source when no endogenous CDH is expressed. This was achieved by placing the cdh-1 gene under the control of the D-glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter (1.1 kb) fused upstream of the ATG start codon of cdh-1. The gpd promoter-chd-1 construct was inserted into the multiple cloning site of the expression vector pOGI18, which contained the Schizophyllum commune ade5 as a selectable marker. The P. chrysosporium ade1 auxotrophic strain OGC107-1 was transformed with the pAGC1 construct, and the prototrophic transformants were assayed for CDH activity. Approximately 50% of the Ade(+) transformants exhibited CDH activity in the extracellular medium of stationary cultures. At least one of the transformants produced high levels (500-600 U/liter) of recombinant CDH (rCDH). Purification by ammonium sulfate precipitation, Sephacryl S-200 chromatography, and FPLC using a Mono-Q 5/5 column yielded homogeneous rCDH. Physical, spectral, and kinetic characteristics of purified homologously expressed rCDH were similar to those of wild-type CDH. This expression system will enable site-directed mutagenesis studies to be carried out on CDH.
Li
B
B
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 N.W. Walker Road, Beaverton, Oregon 97006-8921, USA.
Rotsaert
F A
FA
Gold
M H
MH
Renganathan
V
V
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002475:Cellobiose / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D005822:Genetic Vectors; D020075:Phanerochaete / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014170:Transformation, Genetic
D003574:Cytochrome c Group; D011994:Recombinant Proteins; D002475:Cellobiose; D015086:2,6-Dichloroindophenol; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase