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Background  FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete  Glomerella cingulata  is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by  Glomerella  ( anamorph Colletotrichum) spp .    Results  Heterologous expression of active  G. cingulata  GDH has been achieved in both  Escherichia coli  and  Pichia pastoris , however, the expressed volumetric activity was about 4800-fold higher in  P. pastoris . Expression in  E. coli  resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a  P. pastoris  transformant resulted in an expression of 48,000 U L -1  of GDH activity (57 mg L -1 ). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in  P. pastoris  and the wild-type enzyme from its natural fungal source.    Conclusions  The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in  P. pastoris  facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.

Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris