Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System.

Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.