Comparison of the metabolite profiles in P. pastoris clones expressing the wild type form (PP WT) and the mutant forms (PP W129A, PP W129L, PP W129F, and PP L324G) of CYP5136A3.

aFor each metabolite, the observed HPLC count value was converted into metabolite concentration using the standard curve generated with the known quantity of the metabolite (X-axis) and its HPLC count value (Y-axis). The proportion of each metabolite (%) was calculated from the total amount of metabolites (observed for each oxidation reaction) which was considered as 100%. Values represent means ± standard deviations (in parenthesis) based on three biological replicates. Asterisks indicate values that are statistical significant (P≤0.05) compared to PP WT. Formation of 3-OH Phe is a random event [50], [52] and hence it is not an indication of regio-selectivity by the mutants. All clones (PP WT, PP W129A, PP W129F and PP L324G) showed comparable product recovery (89–92% product recovery) for phenanthrene. For pyrene, HPLC absorbance values were used to quantify the amounts of the oxidation products in PP W129A, PP W129L, and PP W324G compared to the PP WT sample and the product recoveries are shown in the table. Due to low levels of pyrene oxidation by PP W129F, the metabolite level was below detection limit. Abbreviations: 3-OH Phe, 3-phenanthrol; 4-OH Phe, 4-phenanthrol; 9-OH Phe, 9-phenanthrol; 2-OH Pyr, 2-hydroxypyrene; 1-OH Pyr, 1-hydroxypyrene.bThe ratio between 3-phenanthrol and 4-phenanthrol was calculated on a scale of 100. Due to low levels of pyrene oxidation by PP W129F, the metabolite level was below detection limit. The mechanism of oxidation of phenanthrene followed the NIH shift mechanism as all the mutants showed higher levels of 4-phenanthrol compared to 3-phenanthrol. See text for details.cIn light of the commercial non-availability of an authentic standard for 2-pyreneol, we used the molecular mass and the HPLC elution profile criteria to tentatively identify the second peak as 2-pyrenol [15]; both profiles matched with the CYP101 mutants' pyrene oxidation profile [72], where 1-pyrenol had longer retention time as compared to 2-pyrenol.