title: Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris.
abstract: Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L(-1) PDH or 1,330 U L(-1) d(-1) in space-time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown.
authors.affiliation: Department of Food Sciences and Technology, University of Natural Resources and Applied Life Sciences (BOKU), Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Sygmund
authors.forename: Alexander
authors.initials: A
authors.lastname: Gutmann
authors.forename: Iris
authors.initials: I
authors.lastname: Krondorfer
authors.forename: Magdalena
authors.initials: M
authors.lastname: Kujawa
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
authors.forename: Beate
authors.initials: B
authors.lastname: Pscheidt
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.forename: Clemens
authors.initials: C
authors.lastname: Peterbauer
authors.forename: Roman
authors.initials: R
authors.lastname: Kittl
mesh_terms: D000364:Agaricus / Q000201:enzymology* / Q000235:genetics; D050260:Carbohydrate Metabolism; D015870:Gene Expression; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D010088:Oxidoreductases
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