substrate COMBINATORIAL OPTIMization strategies to increase Human P450 catalytic Activity in whole cell Biotransformations   Astrid Weninger,1 Anna Alkofer,1 Christian Schmid,1 Monika Pranjic,1 Katharina Ebner,1 Margit Winkler,2 Anton Glieder1,3* 1Institute of Molecular Biotechnology, TU Graz, NAWI Graz, 8010 Graz, Austria 2Austrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14/4, 8010 Graz, Austria 3bisy e.U., Wetzawinkel 20, 8200 Hofstätten/Raab, Austria *Corresponding author: a.glieder@tugraz.at PROTEIN ENGINEERING INTRODUCTION Human cytochrome P450 biotransformations are of great potential for drug metabolite synthesis. However, recombinant production yields and product titers are low, which has been the most limiting factor for their application in large scale processes. In this study, we combined protein engineering with expression-, strain-, and process-optimization to obtain high level human cytochrome P450 producing P. pastoris strains for whole-cell biocatalysis. Error prone PCR (epPCR) random mutagenesis and the multiple site saturation mutagenesis method OmniChange [2] were used to generate P450 variants for improved substrate conversion. We use episomal plasmids, where the library fragment is added to the vector backbone by Gibson assembly. P. pastoris can be directly transformed with a few µl of the reaction mix. STRAIN ENGINEERING For strain engineering purposes we developed a CRISPR/Cas9 system for P. pastoris [3, 4]. The system can be used to delete genes in a less time consuming and labor intensive manner. Cas9 protein gRNA PAM (NGG) target DNA CYP2C9 amino acid residues selected for OmniChange multiple site saturation mutagenesis. Screening of thirty P. pastoris CYP1A2/CPR knockout strains for improved substrate conversion. FINE-TUNING EXPRESSION We developed promoter libraries with different transcription strengths, ratios and regulatory profiles for expression in P. pastoris and E. coli [1]. A set of bidirectional P. pastoris promoters was tested for the expression of human P450s and the associated reductase (CPR). There appeared strong differences depending on the promoter construct used. Equimolar amount epPCR fragment 150 ng vector DNA Assembly for one hour, 50oC strong medium weak constitutive derepressible inducible bidirectional monodirectional Screening landscapes of 21 transformants/construct (not sequenced). P. pastoris parental strain expressing CYP1A2/CPR (31, A344, grey bar). P. pastoris BG11 mutS: negative control. Direct P. pastoris transformation 1.5 – fold Shake flask cultivation of selected transformants. CULTIVATION CONDITIONS In order to increase the production of human P450s we optimized the cultivation media for deep-well plate and shake flask scale. Several parameters were tested including various cultivation temperatures, additives, pH values and types of media as well as the oxygen supply. The activity was increased up to 5-fold at a higher pH value, when rich media was used. CPR CYP2C9 SUMMARY By combining these optimization strategies, the enzyme yields for several human P450s were increased from not-detectible to the highest reported in literature. 2.1 - fold 5.0 - fold REFERENCES [1] T. Vogl et al., “Bidirectional Promoter.,” US 20150011407 A1, 2015. Commercially available from bisy e. U., Austria. [2] A. Dennig et al., “OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons.,” PLoS One, vol. 6, no. 10, pp. e26222, 2011. [3] M. Jinek et al. “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.,” Science, vol. 337, no. 6096, pp. 816–21, 2012. [4] A. Weninger et al., “Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris.,” Journal of Biotechnology, vol. 235, pp. 139-49, 2016. This work received funding from the EU project ROBOX (grant agreement nr. 635734) under EU’s H2020 Programme Research and Innovation actions H2020-LEIT BIO-2014-1. Any statement made herein reflects only the author's views. The European Union is not liable for any use that may be made of the information contained herein.