ИСТИНА

Flavocytochrome P450 BM3 is a fatty acid hydroxylase that is capable of oxidizing inert C-H bonds with high activity [1]. Numerous studies have been conducted to establish mutated variants of P450 BM3 as biocatalysts for the production of various chemicals such as pharmaceutical metabolites or aroma compounds [2-4]. However, the dependence on the nicotinamide cofactor NADPH and low turnover of non-physiological substrates are among the factors that limit broad application of this cytochrome P450. To address these limitations, we created bifunctional enzymatic units via genetic fusion of P450 BM3 with either a formate dehydrogenases or an alcohol dehydrogenase. In the fusions between P450 BM3 and formate dehydrogenase, cofactor regeneration was enabled through the turnover of cheap and readily available formate. Intriguingly, an improved catalytic efficiency was determined for both P450 BM3 and formate dehydrogenase in the fusion constructs and substrate conversions achieved with the fusions were up to 7-fold higher than with the individual enzymes. Fusions of P450 BM3 and an alcohol dehydrogenase were created for the synthesis of the flavour (+)-nootkatone from abundantly available sesquiterpenoid (+)-valencene via two consecutive oxidation steps. The P450-ADH fusions catalyzed the cascade reactions more efficiently with a space-time-yield of up to 21 mg L-1 h-1 leading to 420 mg L-1 (+)-nootkatone compared to 10.2 mg L-1 h-1 resulting in 200 mg L-1 product formed by the separate enzymes. Conclusively, genetic enzyme fusion of P450 BM3 and dehydrogenases is a viable strategy to endow P450 BM3 with efficient cofactor regeneration and to simultaneously enhance the catalytic performance.