Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10 mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E. coli and investigated and compared both strategies, (a) the production of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the production of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titre of 100 mg L−1 cultivation broth, and were able to produce 48 mg active HRP per litre cultivation broth in the periplasm. In terms of biochemical properties, soluble HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and production. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amounts, which are then refolded.

Lytic polysaccharide monooxygenases (LPMOs) have changed our understanding of lignocellulosic degradation dramatically over the last years. These metalloproteins catalyze oxidative cleavage of recalcitrant polysaccharides and can act on the C1 and/or C4 position of glycosidic bonds. Structural data have led to several hypotheses, but we are still a long way from reaching complete understanding of the factors that determine their divergent regioselectivity. Site-directed mutagenesis enables the investigation of structure-function relationship in enzymes and will be of major importance in unraveling this intriguing matter. In this context, it is crucial to have an enzyme assay or screening approach with a direct correlation with the desired functionality. LPMOs render this search extra challenging due to their insoluble substrates, complex pattern of reaction products and lack of synthetic standards of most oxidized products. Here, we describe a regioselectivity indicator diagram based on the time-course of only 2 HPAEC-PAD signals. The diagram was successfully used to confirm the hypothesis that aromatic surface residues influence the C1/C4 oxidation ratio in Hypocrea jecorina LPMO9A. Consequently, the diagram should become a valuable tool in the search towards better understanding and engineering of regioselectivity in LPMOs.

Auto-inducible promoter systems have been reported to increase soluble product formation in the periplasm of E. coli compared to inducer-dependent systems. In this study, we investigated the phosphate (PO4)-sensitive phoA expression system (pAT) for the production of a recombinant model antigen-binding fragment (Fab) in the periplasm of E. coli in detail. We explored the impact of non-limiting and limiting PO4 conditions on strain physiology as well as Fab productivity. We compared different methods for extracellular PO4 detection, identifying automated colorimetric measurement to be most suitable for at-line PO4 monitoring. We showed that PO4 limitation boosts phoA-based gene expression, however, the product was already formed at non-limiting PO4 conditions, indicating leaky expression. Furthermore, cultivation under PO4 limitation caused physiological changes ultimately resulting in a metabolic breakdown at PO4 starvation. Finally, we give recommendations for process optimization with the phoA expression system. In summary, our study provides very detailed information on the E. coli phoA expression system, thus extending the existing knowledge of this system, and underlines its high potential for the successful production of periplasmic products in E. coli.

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