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Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. Results We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. Conclusion The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative depolymerization of industrially relevant crystalline polysaccharides, such as cellulose, in a reaction that depends on an electron donor and O2 or H2O2. While it is well known that LPMOs can utilize a wide variety of electron donors, the variation in reported efficiencies of various LPMO-reductant combinations remains largely unexplained. Results In this study, we describe a novel two-domain cellulose-active family AA10 LPMO from a marine actinomycete, which we have used to look more closely at the effects of the reductant and copper ions on the LPMO reaction. Our results show that ascorbate-driven LPMO reactions are extremely sensitive to very low amounts (micromolar concentrations) of free copper because reduction of free Cu(II) ions by ascorbic acid leads to formation of H2O2, which speeds up the LPMO reaction. In contrast, the use of gallic acid yields steady reactions that are almost insensitive to the presence of free copper ions. Various experiments, including dose–response studies with the enzyme, showed that under typically used reaction conditions, the rate of the reaction is limited by LPMO-independent formation of H2O2 resulting from oxidation of the reductant. Conclusion The strong impact of low amounts of free copper on LPMO reactions with ascorbic acid and O2, i.e. the most commonly used conditions when assessing LPMO activity, likely explains reported variations in LPMO rates. The observed differences between ascorbic acid and gallic acid show a way of making LPMO reactions less copper-dependent and illustrate that reductant effects on LPMO action need to be interpreted with great caution. In clean reactions, with minimized generation of H2O2, the (O2-driven) LPMO reaction is exceedingly slow, compared to the much faster peroxygenase reaction that occurs when adding H2O2.