Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity

Abstract

Cytochrome P450 enzymes are renowned for their ability to insert oxygen into an enormous variety of compounds with a high degree of chemo- and regio-selectivity under mild conditions. This property has been exploited in Nature for an enormous variety of physiological functions, and representatives of this ancient enzyme family have been identified in all kingdoms of life. The catalytic versatility of P450s makes them well suited for repurposing for the synthesis of fine chemicals such as drugs. Although these enzymes have not evolved in Nature to perform the reactions required for modern chemical industries, many P450s show relaxed substrate specificity and exhibit some degree of activity towards non-natural substrates of relevance to applications such as drug development. Directed evolution and other protein engineering methods can be used to improve upon this low level of activity and convert these promiscuous generalist enzymes into specialists capable of mediating reactions of interest with exquisite regio- and stereo-selectivity. Although there are some notable successes in exploiting P450s from natural sources in metabolic engineering, and P450s have been proven repeatedly to be excellent material for engineering, there are few examples to date of practical application of engineered P450s. The purpose of the present review is to illustrate the progress that has been made in altering properties of P450s such as substrate range, cofactor preference and stability, and outline some of the remaining challenges that must be overcome for industrial application of these powerful biocatalysts.

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

“…Accordingly, they have garnered increasing attention in recent years as potential biocatalysts for the oxidative tailoring of molecules ranging from simple hydrocarbons to complex natural products. 11–20 In addition, since they operate under mild reaction conditions and employ abundant Fe as a cofactor, P450s have the potential to contribute to the realization of environmentally sustainable approaches toward the production of fine and commodity chemicals. 21 Microbes offer a particularly rich source of novel P450s with potential broad applicability in various biocatalytic processes.…”

“…28–30 Because biosynthetic P450s often catalyze oxidation reactions on complex and densely functionalized substrates, they provide an important and underexplored starting point for the development of new biocatalysts. 20,22 Consequently, much of our recent work has focused on the discovery and biochemical/structural characterization of new P450s involved in the biosynthesis of bacterial natural products. 31–35 As we have demonstrated with PikC, 36–38 these fundamental studies serve as a key starting point to guide future efforts to modulate the substrate scope and selectivity properties of P450 enzymes.…”

Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system

“…To date, a majority of efforts have focused on engineering of P450 monooxygenases, which are believed to be the most versatile biocatalysts in nature (19), using either directed evolution and/or rational design (20)(21)(22). Recently, a new strategy of substrate engineering has been explored (23, 24), wherein anchoring/directing groups are chemically linked to nonsubstrate compounds.…”

“…However, a number of problems are persistently associated with the chemical oxidation of aliphatic C-H bonds in alkylphenols: (i) the requirement of protection/deprotection of the phenolic hydroxyl group under reaction conditions that are distinct from those for oxidation complicates the synthetic process; (ii) it is difficult to delicately control the extent of oxidations (i.e., alcohol vs. aldehyde/ketone vs. carboxylic acid); (iii) the oxidation of an aromatic C-H bond may sometimes occur when using strong oxidants; (iv) the regio-and stereoselective oxidation of an unactivated sp 3 C-H bond (in alkylphenols) remains a central challenge despite recent advances in combined use of specialized directing groups with transition metal catalysts (9-11) and in biomimetic supramolecular assemblies (12-14); and (v) chemical oxidants could be associated with significant environmental concerns (2, 3). Given these issues, oxidative enzymes with inherent catalytic selectivity and reduced environmental impact may be developed into alternative catalysts for selective oxidation of organic compounds including alkylphenols (15-18).To date, a majority of efforts have focused on engineering of P450 monooxygenases, which are believed to be the most versatile biocatalysts in nature (19), using either directed evolution and/or rational design (20)(21)(22). Recently, a new strategy of substrate engineering has been explored (23, 24), wherein anchoring/directing groups are chemically linked to nonsubstrate compounds.…”

Iron management and production of electricity by microorganisms

“…As a functional component of heme groups, it participates in oxygen transport by hemoglobin, drug detoxification by cytochrome P450, or peroxides elimination by cytochrome c peroxidases (Atack and Kelly 2007;Behrendorff et al 2015;Pettigrew et al 2006;Poulos 1995). It also plays an important role as part of iron-sulfur cluster proteins, mediating mitochondrial electron transfer with the production of ATP (Vashchenko and MacGillivray 2013) or in DNA repair systems (Benjdia 2012;Folgosa et al 2015).…”