The cytochrome P450 monooxygenase (P450) enzyme superfamily contains over 300,000 P450 sequences spanning across all kingdoms of life [1]. P450s are responsible for oxyfunctionalization of substrates as carbon sources; for clearance of xenobiotic or endobiotic chemicals; and for introducing structural complexity into endogenous substrates in complex chemical communication pathways. They are especially prominent in chemical warfare between organisms, where they both produce secondary metabolites to counter other organisms (e.g., in plants and microorganisms) and defend against xenobiotic compounds (e.g., in herbivores feeding on plants) [[2], [3], [4]]. P450s are classified based on amino acid sequence identity and denoted by a prefix, CYP, for cytochrome P450, then a number, letter and second number defining in order the family, subfamily and individual form under consideration.

Due to their vast substrate range and the ability to perform diverse chemistry, P450s are some of the most attractive natural biocatalysts for industrial applications [5,6]. They are best-known for performing oxyfunctionalization of sp3 bonds in organic molecules in a regio- and stereospecific manner [7], a reaction which has been of interest for over 50 years [8] yet remains challenging for organic synthesis [[9], [10], [11]]. However, there are several limitations to the practical application of P450s. Aside from slow kinetics on non-natural substrates, most P450-mediated reactions are NADPH-dependent, and co-expression of redox partners is often required to use this essential cofactor [[12], [13], [14]]. This is particularly true of membrane-bound class II systems that use the diflavin reductase, cytochrome P450 reductase (CPR), as redox partner. Several strategies have been pursued to circumvent the need to constantly supply NADPH, such as by using NADPH-regenerating systems based on glucose, formate, or alcohol dehydrogenases [[15], [16], [17], [18], [19]]. However, this does not eliminate the cofactor dependency and a heterologous redox partner is still required, which adds to the metabolic burden on the host cells when P450s are expressed in bacteria and yeast. Peroxides and other oxygen surrogates e.g., tert-butyl hydroperoxide, cumene hydroperoxide, bis(acetoxy)iodobenzene, and H2O2 have also been employed to circumvent NADPH dependence, taking advantage of the peroxide shunt pathway [20]. However, oxygen surrogates such as peroxides damage the enzyme leading to instability and reducing total turnovers (TTN) [21]. Moreover, only a handful of native and engineered P450s have been investigated and shown to be supported effectively by oxygen surrogates to date [[21], [22], [23], [24]]. Thus, a cost effective and efficient support system for practical application of P450s is still needed and the characteristics of each P450 need to be taken into consideration to determine the most suitable approach.