Fig. 2

Effect of CYP79A2 N-terminal modifications on the production of phenylacetaldoxime (Phe-Ox). A N-terminal sequence modifications used to improve function of P450 enzymes include: (1) truncation of the transmembrane domain (ΔTM) or both transmembrane domain with adjacent hydrophilic region (ΔTM+), (2) expression enhancing peptides MALLLAVF (Barnes) or 28 amino acid tag (28aa), (3) transmembrane domain exchange with E. coli native probable protease (SohB), and (4) insertion of leader sequence from outer membrane protein A (OmpA) in front of the full-length protein. B Phe-Ox titres produced from phenylalanine by native and engineered variants of CYP79A2. All strains were grown in 12 biological replicates across two independent experiments. Error bars represent standard deviation from the mean and outliers were removed from the data. Student’s upper-tailed t test denotes a significant increase in Phe-Ox titre compared to native with p value (with Bonferroni adjustment), ** p < 0.01, *** p < 0.001. Exact levels of Phe-Ox, Phe and OD600 are listed in Table S1. C Relative quantification of CYP79A2 levels in strains expressing the native and engineered variants of CYP79A2, showing a representative peptide of CYP79A2 normalized to expression of E. coli isocitrate dehydrogenase. The bars represent the mean of 3–5 biological replicates from one of the experiments in B and error bars represent standard deviation from the mean (Table S2). Additional CYP79A2 peptides can be found in Fig. S1. D Illustration of the CYP79A2-ATR1 operons as cloned into pET52b vector. Full-length cytochrome P450 oxidoreductase (ATR1) was co-expressed to supply electrons for the regeneration of P450 enzymes