Accélération de l'exploration de l'espace chimique du cytochrome P450 BM3 par des méthodes de criblage à haut débit et bio-informatiques

Organic chemistry plays a major role in the pharmaceutical, cosmetic, food and household product industries. Industrial organic synthesis has constantly evolved to be more efficient and less cost-effective. However, those improvements were often made regardless of their impact on the environment, spawning the creation of the discipline of Green Chemistry. One among many strategies to make chemistry more innocuous to the environment is to apply biocatalysis: the use of enzymes for their ‘green’ properties and catalytic efficacy. Cytochrome P450 oxidases are well known for their substrate promiscuity in detoxification of xenobiotics and endogenous molecules in the body. This superfamily of enzymes catalyzes a diversity of oxidation reactions in a single step, including hydroxylations, epoxidations, deaminations, dehalogenations and cyclopropanations. Cytochrome P450 BM3 (P450 BM3) from Bacillus megaterium is one of the most efficient of its family, justifying the growing interest for its industrial applications. This thesis considers the necessity to improve assays in order to increase the throughput of enzyme library screening and to improve the ratio of active variants. Indigo synthesis has been proposed to be an efficient predictor of promiscuity towards novel substrates of P450 BM3. We explore the mutational space of P450 BM3 for indigo synthesis and compare screening methods. We made variant libraries using site-directed saturation mutagenesis to obtain a uniform distribution of mutations at each position and reduce cost and time. We optimized a method to quantify P450 BM3 variants and two different screening methods. We compared a direct colorimetric assay using indigo absorbance and an indirect assay using NADPH fluorescence coupled with mass spectrometry (LC-MS, GC-MS and LC prep). This second approach circumvents the need to develop an assay dependent of the substrate. To increase screening throughput, we optimized methods in a 96-well plate format and use an automated pipetting station. We confirm the discovery of new indigo-producing variants. We further demonstrate that indigo can serve as a strong predictor of raspberry ketone production, a high value industrial compound.  The correlation of the activity of P450 BM3 with indigo and raspberry ketone justifies the exploration of the fitness landscape P450 BM3. Understanding this correlation would accelerate engineering of P450 BM3 for reaction with novel substrates. We attempt to explain the relationship between the structure, dynamism and activity through deep-mutational scanning of P450 BM3. Conventional screening methods are not sufficient to produce large datasets (Big Data); we apply next-generation sequencing (NGS) to identify >104 variants simultaneously and to link the genotype (DNA) and the phenotype (indigo production).  Analysis of NGS for enzyme engineering required the development of statistical and bioinformatics tools such as modeling, molecular dynamics and time-structure independent component analysis (tICA). Those methods allowed us to observe conformational perturbations that were shared between the active variants represented as a shift of equilibrium between two major conformations.  This shift may explain the propensity of diverse P450 BM3 variants to synthesize indigo. This master’s thesis successfully demonstrates the accelerated discovery of engineered variants of P450 BM3 with activity for industrially relevant reactions. The methodologies we have developed contribute to knowledge on enzyme engineering and have the potential to be applied more broadly in other enzyme systems.

Keyphrases: cytochrome P450 BM3, indigo, Automation, Biocatalysis, High-throughput screening, Modeling, Molecular dynamics, Mutagenesis, Next-generation sequencing, Oxidation, Protein engineering, Raspberry ketone, Statistical analysis, Time-structure independent component analysis