Abstract
The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450−1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.
Highlights
Monooxygenases are highly valuable enzymes from a synthetic perspective since they perform complex and specific oxidation reactions at ambient temperature using molecular oxygen
After establishment of conditions for screening with a multiplexed capillary electrophoresis (MP-capillary electrophoresis (CE)) platform, two site-saturation mutagenesis (SSM) libraries of P450 BM3 were screened towards increased activity for the oxidation of alpha-isophorone to 4-hydroxy-isophorone (Fig. 1)
P450 catalysis applying P450 BM3 wild type (WT) is the successful production of a vitamin E synthesis intermediate, 4-hydroxy-isophorone, through an enzymatic para-hydroxylation of alpha-isophorone[41]
Summary
Monooxygenases are highly valuable enzymes from a synthetic perspective since they perform complex and specific oxidation reactions at ambient temperature using molecular oxygen. P450 monooxygenases convert a wide range of substrates like fatty acids, terpenes, steroids, prostaglandins, polyaromatic and heteroaromatic compounds, as well as a vast number of drugs, organic solvents, antibiotics, pesticides, carcinogens and toxins Their high chemoselectivity makes them great candidates for biotechnological applications in synthesis. In case of P450s and usage of non-natural substrates, a main limitation of the NADPH screening systems is that cofactor usage is only partially linked to the target product formation. Multichannel systems were employed to perform high throughput screening of genetic mutations[37] and clinical samples, in metabolomic and metabonomic studies[38], of compound libraries to find potential and selective kinase inhibitors[39] and of enzymatic activity under different reaction conditions[40]. A comparison of the MP-CE to the NADPH screening systems was performed to benchmark the MP-CE system
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