Improving the expression of UPOs using signal peptide domain engineering

Unspecific peroxygenases (UPOs) are enzymes that have great potential for industrial biocatalysis as they are able to perform highly efficient selective oxidations while only needing hydrogen peroxide as the co-substrate. Recombinant expression of UPOs however, has proved to be challenging, with only modest expression levels reported for secreted expression from yeasts such as S. cerevisiae and K. phaffii.
In this study we addressed the challenge behind the production of UPOs by developing a universal protocol for the directed evolution of the signal peptide domain. The platform was developed by using truncated versions of AaeUPO fused to Gaussia luciferase as a screening reporter and expressed under the AaeUPO variant PaDa-I (m55) and native (n55) signal peptides. The signal peptide was mutagenized by epPCR, and the platform was validated when a promising variant (1C1) was found to have enhanced the secretion of AaeUPO in S. cerevisiae. The 1C1 signal peptide proved to outperform secretion of the model recombinant proteins fused to Gaussia luciferase (m55 and n55) and when it was transferred to whole protein production, it enabled enhanced secretion of the whole AaeUPO protein in S. cerevisiae. These findings helped validate the engineered platform and proved that mutagenesis of the signal peptide and screening of mutant libraries with Gaussia luciferase can be a promising method for the identification of favourable variants for enhanced secretion of eukaryotic targets in S. cerevisiae.
The system was then transferred to K. phaffii for enhanced secretion through fermentation, revealing that what constitutes an optimal signal peptide for S. cerevisiae, was not ideal in K. phaffii. These findings highlighted the importance of achieving a harmonious balance between the signal peptide, the target enzyme and the host organism to obtain functional secretion.