A Screening Method for P450 BM3 Mutant Libraries Using Multiplexed Capillary Electrophoresis for Detection of Enzymatically Converted Compounds.

Capillary electrophoresis (CE) is an analytical method in which charged species are separated by attraction or repulsion performed in submillimeter diameter capillaries or micro- and nanofluidic channels through the application of a high voltage electric field. When capillary electrophoresis is assembled in a multicapillary instrument such as 96-well format (multiplexed), it becomes a powerful high-throughput system with the ability to simultaneously screen several types of samples like genetic mutations, metabolomes, kinase inhibitors, or enzymatic activities to name a few. The usage of a 96-multiplexed capillary electrophoresis system (96-MP-CE) represents a new platform for product-specific high-throughput screening of enzyme mutant libraries from directed evolution campaigns providing a comprehensive view on enzyme activity through the detection of all products formed. We describe the application of 96-MP-CE to screen mutant libraries of P450 BM3. MP-CE was used in directed evolution campaigns toward benzo-1,4-dioxane and α-isophorone.

Engineered P450 BM3 and cpADH5 coupled cascade reaction for ß-oxo fatty acid methyl ester production in whole cells.

Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product.