초록
<P><B>Abstract</B><P>Background<P>Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest <I>Komagataella phaffi</I> to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (<I>Ano</I>UPO), we aimed to apply a bi-directionalized expression strategy with <I>Komagataella phaffii</I>.</P></P><P>Results<P>In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in <I>K. phaffi</I>. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system.</P></P><P>Conclusions<P>In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in <I>K. phaffii</I>, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.</P></P></P>