Promising optimization of bacterial cytochrome P450BM3 enzyme production by engineered Escherichia coli BL21

Abstract P450BM3 monooxygenase enzymes are used as biocatalysts in biotechnology and engineering with an array of applications including drug and food ingredients synthesis. However, enzyme production efficiency is still not enough for catalysis reactions in large scale applications. Hence, it is important to optimize the culture conditions to increase maximum titers. The vast characterization of P450BM3 monooxygenases in the literature shows the great interest in their industrial applications. This work aims to provide a practical approach for improvement of biotechnological processes and accurate model parameters based on the optimum conditions. This study depicts the optimization of recombinant P450BM3 enzyme in engineered Escherichia coli BL21 in batch flasks and bioreactor, in which agitation, aeration and nutrient conditions were controlled. P450BM3 production in bioreactor was 41%-fold higher than in batch flasks. Our findings showed that P450BM3 production has been the highest ever reported for this enzyme. P450BM3 activity was improved by batch fermentations using glycerol 0.8%, FeCl3 0.12 g/L and 4 mL trace elements. The kinetic bioreactor parameters for enzyme production, KLa, R2 and PO2 were 0.025s−1, 0.99 and 21.17 mM O2/g, respectively. Therefore, this bioreactor cultivation allows providing promising experimental conditions for P450BM3 production in engineered E. coli, enabling this enzyme to be more applicable in many fields.