Abstract
Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C-H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.
Highlights
Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism
We focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals
The results showed that the pair Fdx4/FdR1 functioned as the preferred redox partner system for this bacterial P450 enzyme in vitro [72]
Summary
Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. A P450 catalytic system includes four components: the substrate, a P450 enzyme for substrate binding and oxidative catalysis, the redox partner(s) that functions as an electron transfer shuttle, and the cofactor (NAD(P)H), which provides the reducing equivalents. P450 protein engineering has been playing a vital role in developing biocatalysts for industrial applications, as exemplified by the heterologous production of artemisinic acid (Fig. 3, compound 10), an important synthetic precursor for the potent antimalarial drug artemisinin [49]. By applying different synthetic biology strategies, including the introduction of the cognate reductase CPR1 of CYP71AV1 and a cytochrome b5 protein (CYB5, an electron transfer component for CYP71AV1 from A. annua), the titer of artemisinic acid was dramatically improved to 25 g/liter on an industrial scale, which successfully reduced the price and provided a stable artemisinin supply for the market [28]. The oxidation of sesquiterpene (ϩ)-valencene to high value-added flavor (ϩ)-nootkatone with P450 enzymes was first accomplished in the Wong group by rationally designed mutants of P450BM3 and P450cam [50] (Fig. 3, compound 11)
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