Abstract
BackgroundThe ability of fungal cellobiose dehydrogenase (CDH) to generate H2O2in-situ is highly interesting for biotechnological applications like cotton bleaching, laundry detergents or antimicrobial functionalization of medical devices. CDH’s ability to directly use polysaccharide derived mono- and oligosaccharides as substrates is a considerable advantage compared to other oxidases such as glucose oxidase which are limited to monosaccharides. However CDH’s low activity with oxygen as electron acceptor hampers its industrial use for H2O2 production. A CDH variant with increased oxygen reactivity is therefore of high importance for biotechnological application. Uniform expression levels and an easy to use screening assay is a necessity to facilitate screening for CDH variants with increased oxygen turnover.ResultsA uniform production and secretion of active Myriococcum thermophilum CDH was obtained by using Saccharomyces cerevisiae as expression host. It was found that the native secretory leader sequence of the cdh gene gives a 3 times higher expression than the prepro leader of the yeast α-mating factor. The homogeneity of the expression in 96-well deep-well plates was good (variation coefficient <15%). A high-throughput screening assay was developed to explore saturation mutagenesis libraries of cdh for improved H2O2 production. A 4.5-fold increase for variant N700S over the parent enzyme was found. For production, N700S was expressed in P. pastoris and purified to homogeneity. Characterization revealed that not only the kcat for oxygen turnover was increased in N700S (4.5-fold), but also substrate turnover. A 3-fold increase of the kcat for cellobiose with alternative electron acceptors indicates that mutation N700S influences the oxidative- and reductive FAD half-reaction.ConclusionsSite-directed mutagenesis and directed evolution of CDH is simplified by the use of S. cerevisiae instead of the high-yield-host P. pastoris due to easier handling and higher transformation efficiencies with autonomous plasmids. Twelve clones which exhibited an increased H2O2 production in the subsequent screening were all found to carry the same amino acid exchange in the cdh gene (N700S). The sensitive location of the five targeted amino acid positions in the active site of CDH explains the high rate of variants with decreased or entirely abolished activity. The discovery of only one beneficial exchange indicates that a dehydrogenase’s oxygen turnover is a complex phenomenon and the increase therefore not an easy target for protein engineering.
Highlights
The ability of fungal cellobiose dehydrogenase (CDH) to generate H2O2 in-situ is highly interesting for biotechnological applications like cotton bleaching, laundry detergents or antimicrobial functionalization of medical devices
Expression of M. thermophilum CDH in S. cerevisiae CDH is a secretory glycosylated fungal protein, and expression has so far only been successful in eukaryotic expression systems
The successful expression of CDH under the control of the GAL1 promoter in S. cerevisiae offers a possibility to produce and screen for genetically engineered CDH variants and maybe other mutated fungal oxidoreductases, which are needed for various biotechnological applications
Summary
The ability of fungal cellobiose dehydrogenase (CDH) to generate H2O2 in-situ is highly interesting for biotechnological applications like cotton bleaching, laundry detergents or antimicrobial functionalization of medical devices. A CDH variant with increased oxygen reactivity is of high importance for biotechnological application. The widespread appearance implies an important function of CDH in the process of wood degradation [2,3,4,5]. It is a monomeric protein consisting of two domains [6,7], which oxidizes several carbohydrates at the flavodehydrogenase domain carrying an FAD cofactor. Re-oxidation of the FAD can be performed by either two-electron acceptors (quinones, 2,6-dichloroindophenol, phenoxazine- and phenothiazine dyes) or by one-electron acceptors (polysaccharide monooxygenases, cytochrome c, ferricyanide and ferrocenium) or very slowly by oxygen [3,4,9]
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