title: Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris.
abstract: 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.
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18/2 Wien, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Sygmund
authors.forename: Petra
authors.initials: P
authors.lastname: Staudigl
authors.forename: Miriam
authors.initials: M
authors.lastname: Klausberger
authors.forename: Nikos
authors.initials: N
authors.lastname: Pinotsis
authors.forename: Kristina
authors.initials: K
authors.lastname: Djinović-Carugo
authors.forename: Lo
authors.initials: L
authors.lastname: Gorton
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.forename: Roland
authors.initials: R
authors.lastname: Ludwig
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020081:Phyllachorales / Q000737:chemistry / Q000201:enzymology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D042843:Glucose 1-Dehydrogenase