Homologous expression of recombinant cellobiose dehydrogenase in Phanerochaete chrysosporium

Cellobiose dehydrogenase (CDH) is a novel extracellular hemoflavoenzyme fro m Phanerochaete chrysosporium and is produced only in cultures supplemented with cellulose. In this report, CDH from P. chrysosporium has been homolog ously expressed in cultures supplemented with glucose as the sole carbon so urce when no endogenous CDH is expressed. This was achieved by placing the cdh-l gene under the control of the D-glyceraldehyde-3-phosphate dehydrogen ase (gpd) promoter (1.1 kb) fused upstream of the ATG start codon of cdh-l. The gpd promoter chd-l construct was inserted into the multiple cloning si te of the expression vector pOGI18, which contained the Schizophyllum commu ne ade5 as a selectable marker. The P, chrysosporium ade1 auxotrophic strai n OGC107-1 was transformed with the pAGC1 construct, and the prototrophic t ransformants were assayed for CDH activity. Approximately 50% of the Ade(+) transformants exhibited CDH activity in the extracellular medium of statio nary cultures. At least one of the transformants produced high levels (500- 600 U/liter) of recombinant CDH (rCDH). Purification by ammonium sulfate pr ecipitation, Sephacryl S-200 chromatography, and FPLC using a Mono-Q 5/5 co lumn yielded homogeneous rCDH. Physical, spectral, and kinetic characterist ics of purified homologously expressed rCDH were similar to those of wild-t ype CDH. This expression system will enable site-directed mutagenesis studi es to be carried out on CDH. (C) 2000 Academic Press.