Enhanced secretion of an Agrocybe aegerita peroxygenase variant in K. phaffii using the native signal peptide.

Unspecific Peroxygenases (UPOs) are enzymes with significant potential as catalysts in synthetic chemistry as they catalyse selective oxygenation reactions on organic substrates at the expense of only hydrogen peroxide as the external oxidant. The demand for UPOs has stimulated considerable research into efficient heterologous expression systems for their production, which have included optimisation of the signal peptide (SP) included upstream of the UPO gene. In this study we report a comparison of the production of the prototypical and widely-used UPO variant from the fungus Agrocybe aegerita (rAaeUPO-PaDa-I-H) using both its native SP and variant SPs evolved for improved heterologous expression in yeast hosts. The results show that improvements in protein production identified using variant SPs in S. cerevisiae are not necessarily extended to the yeast Komagataella phaffii. Indeed in K. phaffii we observed a five to sevenfold improvement in the activity of crude secretates produced using the native fungal SP of AaeUPO, compared with SPs that were evolved for improved expression and screened in S. cerevisiae These findings suggest that superior yields of this widely-used UPO can be obtained from scaled production in K. phaffii by using the native SP from the source fungus.