Bi-directional promoter systems facilitate expression of unspecific peroxygenase in K. phaffii (known as P. pastoris) BSYBG11

Abstract

The majority of recombinant proteins is produced using monodirectional promoter systems (MDPs). MDPs are well-established, available for a variety of inducers, characterized for tunability but, unfortunately, lack in flexibility and versatility. Bidirectional promoter systems (BDPs) are able to boost recombinant protein production via multi-gene expression. Thereby, the implementation of BDPs would allow co-expression of chaperones increasing the amount of correctly folded protein. In this study, we compared MDPs and BDPs, trying to express an unspecific peroxygenase in the yeast Komagataella phaffii BSYBG11. To assist the folding of our target enzyme, we introduced the protein disulfide isomerase in the BDP construct. Both genes were under control of a derepressed promotor, with additional promotor boost for the Heme containing protein. We characterized BDPs and MDPs in fed-batch cultivations under (i) derepressed feeding and (ii) methanol-glycerol mixed feed induction. Results show that higher enzyme productivity can be achieved (i) when applying BDPs instead of MDPs and (ii) that derepressed feeding outperforms methanol addition for the expression of unspecific peroxygenase. We present that BDPs are a promising solution for recombinant enzyme expression.