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Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

  • Zhong Li
    Footnotes
    Affiliations
    Shandong Provincial Key Laboratory of Synthetic Biology and CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China

    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, China

    University of Chinese Academy of Sciences, Beijing 100049, China
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  • Yuanyuan Jiang
    Footnotes
    Affiliations
    Shandong Provincial Key Laboratory of Synthetic Biology and CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China

    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, China

    University of Chinese Academy of Sciences, Beijing 100049, China
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  • F. Peter Guengerich
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Li Ma
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, China
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  • Shengying Li
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, China

    Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237 Shandong, China
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  • Wei Zhang
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, China

    Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237 Shandong, China
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  • Author Footnotes
    1 Both authors contributed equally to this work.
Open AccessPublished:December 06, 2019DOI:https://doi.org/10.1016/S0021-9258(17)49939-X
      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.

      Introduction

      Cytochrome P450 enzymes (P450s)
      The abbreviations used are: P450 or CYP
      cytochrome P450
      FdR
      ferredoxin reductase
      Fdx
      ferredoxin
      Adx
      adrenodoxin
      AdR
      adrenodoxin reductase
      CPR
      cytochrome P450 reductase
      Cpd 0
      I, and II, compound 0, I, and II
      epPCR
      error-prone polymerase chain reaction
      1α,25(OH)2D3
      1α,25-dihydroxyvitamin D3
      PDB
      Protein Data Bank
      SRS
      substrate recognition site.
      are a superfamily of heme-thiolate–containing proteins named for the characteristic state of the reduced, carbon monoxide (CO)-bound complex displaying a maximum UV-visible absorption band at 450 nm, due to the heme iron group being linked to the apoprotein via an axial conserved cysteine (
      • Guengerich F.P.
      Mechanisms of cytochrome P450-catalyzed oxidations.
      ,
      • Meunier B.
      • de Visser S.P.
      • Shaik S.
      Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes.
      ).
      Since the first discovery of P450 as a pigment (the P denoting “pigment”) in rat liver microsomes in 1958 (
      • Klingenberg M.
      Pigments of rat liver microsomes.
      ), more than 370,000 P450 sequences have been released (UniProt), which are found in human, animals, plants, microbes, and even viruses, demonstrating their incredible and significant diversity in nature (
      • Nelson D.R.
      Cytochrome P450 diversity in the tree of life.
      ). P450s play important roles in biosynthetic pathways for natural products, degradation of xenobiotics, biosynthesis of steroid hormones, and drug metabolism (
      • Zhang X.
      • Li S.
      Expansion of chemical space for natural products by uncommon P450 reactions.
      ,
      • Guengerich F.P.
      Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity.
      ). P450s are considered to be the most versatile biocatalysts in nature (
      • Coon M.J.
      Cytochrome P450: nature's most versatile biological catalyst.
      ) and are involved in more than 20 different types of chemical oxidation reactions, including hydroxylation, epoxidation, decarboxylation, N- and O-dealkylation, nitration, and C–C bond coupling or cleavage, to name a few (
      • Zhang X.
      • Li S.
      Expansion of chemical space for natural products by uncommon P450 reactions.
      ,
      • Guengerich F.P.
      • Munro A.W.
      Unusual cytochrome P450 enzymes and reactions.
      ) (plus some reductions). Furthermore, the substrate diversity of P450s covers almost all classes of organic structures found in nature (e.g. terpenoids, polyketides, fatty acids, alkaloids, and polypeptides) (
      • Zhang X.
      • Li S.
      Expansion of chemical space for natural products by uncommon P450 reactions.
      ,
      • Podust L.M.
      • Sherman D.H.
      Diversity of P450 enzymes in the biosynthesis of natural products.
      ,
      • Rudolf J.D.
      • Chang C.Y.
      • Ma M.
      • Shen B.
      Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function.
      ). The ubiquitous distribution and the multiplicity of reactions and substrates demonstrate the plasticity of P450 enzyme systems, providing a limitless space for mining, engineering, and designing P450 systems for practical catalysis.
      Among diverse functionalities, the most important is that P450s are capable of catalyzing the regio- and stereoselective oxidation of inert C–H bonds in complex molecular scaffolds under mild conditions, making them superior to many chemical catalysts and of great interest for pharmaceutical, chemical, and biotechnological applications. However, the narrow substrate scope of some P450s, low catalytic efficiency, low stability, dependence on redox partners, high cost of cofactors, and electron uncoupling have limited the industrial applications of P450s (
      • Sakaki T.
      Practical application of cytochrome P450.
      ,
      • Bernhardt R.
      • Urlacher V.B.
      Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations.
      ). More recently, innovative P450 systems have been developed to fuel industrial projects with the use of a number of new engineering strategies (e.g. interactions of essential elements, including P450 itself, redox partner, substrate, and cofactor). These include the powerful directed evolution approach pioneered by the Nobel Laureate Frances H. Arnold, used to build unnatural but more robust P450 systems (
      • Arnold F.H.
      Design by directed evolution.
      ).
      Several excellent reviews have covered the diversity, functions, novel chemistry, and applications of P450s (
      • Zhang X.
      • Li S.
      Expansion of chemical space for natural products by uncommon P450 reactions.
      ,
      • Rudolf J.D.
      • Chang C.Y.
      • Ma M.
      • Shen B.
      Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function.
      ,
      • Urlacher V.B.
      • Girhard M.
      Cytochrome P450 monooxygenases in biotechnology and synthetic biology.
      ,
      • Xu L.H.
      • Du Y.L.
      Rational and semi-rational engineering of cytochrome P450s for biotechnological applications.
      ,
      • Wei Y.
      • Ang E.L.
      • Zhao H.
      Recent developments in the application of P450 based biocatalysts.
      ,
      • Schmitz L.M.
      • Rosenthal K.
      • Lütz S.
      Recent advances in heme biocatalysis engineering.
      ). For more insight into intriguing P450-related mechanisms and to deeply understand the strategies related to the practical application of P450 catalysis, we will focus on recent advances in P450 protein engineering, particularly engineering strategies for optimization of the interaction between P450s and redox partners. We will also consider substrate engineering, cofactor (NAD(P)H) regeneration, and several atypical strategies for engineering the electron transport system. Finally, a brief summary of P450-related metabolic engineering will be provided.

      P450 catalytic system

      In general, 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.
      Most P450s share a common sophisticated catalytic cycle (Fig. 1) (
      • Meunier B.
      • de Visser S.P.
      • Shaik S.
      Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes.
      ,
      • Zhang X.
      • Li S.
      Expansion of chemical space for natural products by uncommon P450 reactions.
      ,
      • Jiang Y.
      • Li S.
      Catalytic function and application of cytochrome P450 enzymes in biosynthesis and organic synthesis.
      ), using the typical hydroxylation reaction as a paradigm, as shown in Fig. 1. The ferric resting state (generally) of a P450 (A) first accepts a substrate (RH), which displaces an active-site water molecule but does not bond directly to the iron. The ferric iron (FeIII) of the high-spin, substrate-bound complex (B) is then reduced to ferrous iron (FeII) (C) by one electron, transferred via a redox partner. Next, binding of dioxygen to FeII results in the [FeII O2] complex (D). The complex D is reduced by the second electron to form complex E, which uses a proton from solvent to generate a ferric hydroperoxo species [FeIII–OOH] (F), referred as to Compound 0 (Cpd 0). The O–O bond of Cpd 0 is cleaved upon the addition of the second proton and releases a molecule of water to generate the high-valent porphyrin π radical cation tetravalent iron [FeIV=O] (i.e. Compound I (Cpd I; G)). This highly reactive complex abstracts a hydrogen atom from the substrate, leading to the formation of the ferryl-hydroxo compound II (Cpd II; H). Subsequently, the hydroxylated product (R-OH) is formed by the reaction of the substrate radical with the hydroxyl group of Cpd II and released from the active site of complex I. Finally, a molecule of water returns to coordinate with FeIII, restoring the resting state A. The same catalytic cycle is initiated repeatedly as substrate molecules bind to the heme-centered active site of P450.
      Figure thumbnail gr1
      Figure 1The catalytic cycle of P450s (dashed arrows indicate the peroxide shunt pathway and P450 uncoupling).
      It is worth noting that some P450s are capable of directly utilizing H2O2 as the sole electron and proton donor to form Cpd 0 and do catalysis via the so-called peroxide shunt pathway (Fig. 1, dashed arrows). However, this shunt pathway is greatly limited by the low efficiency and the low H2O2 tolerance of most P450s, except P450 peroxygenases (e.g. CYP152 subfamily) (
      • Matthews S.
      • Belcher J.D.
      • Tee K.L.
      • Girvan H.M.
      • McLean K.J.
      • Rigby S.E.
      • Levy C.W.
      • Leys D.
      • Parker D.A.
      • Blankley R.T.
      • Munro A.W.
      Catalytic determinants of alkene production by the cytochrome P450 peroxygenase OleTJE.
      ). The well-studied and established catalytic cycle provides a theoretical basis and roadmap to understand and manipulate this P450 peroxygenase subfamily by protein and substrate engineering.
      Maintenance of the P450 catalytic cycle relies on continuous electron transport to the heme-iron by redox partners, which are complicated electron-transfer systems. Based on the types of redox partners and the P450-redox partner interaction relationships, P450 systems can be divided into five main classes (
      • Rudolf J.D.
      • Chang C.Y.
      • Ma M.
      • Shen B.
      Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function.
      ,
      • Sakaki T.
      Practical application of cytochrome P450.
      ,
      • Xu L.H.
      • Du Y.L.
      Rational and semi-rational engineering of cytochrome P450s for biotechnological applications.
      ) (Fig. 2). The Class I P450 system present in most bacterial and mitochondrial P450s has a two-component redox partner system, comprised of an FAD-containing ferredoxin reductase (FdR) and a small iron-sulfur-containing ferredoxin (Fdx) (
      • Sevrioukova I.F.
      • Poulos T.L.
      Structural biology of redox partner interactions in P450cam monooxygenase: a fresh look at an old system.
      ,
      • Kido T.
      • Kimura T.
      The formation of binary and ternary complexes of cytochrome P-450scc with adrenodoxin and adrenodoxin reductase·adrenodoxin complex: the implication in ACTH function.
      ). The Class II P450 system employed by eukaryotic organisms has a single-component redox partner, which is a membrane-bound protein containing both an FAD and an FMN domain, termed cytochrome P450 reductase (CPR). Class III P450 systems have a eukaryotic-like CPR naturally fused to the C terminus of the P450 domain through a flexible linker, represented by Bacillus megaterium P450BM3 (CYP102A1) (
      • Whitehouse C.J.C.
      • Bell S.G.
      • Wong L.-L.
      P450BM3(CYP102A1): connecting the dots.
      ). Class IV P450 systems are exemplified by P450 RhF from Rhodococcus sp. NCIMB 9784, whose FMN/Fe2S2-containing reductase domain forms a natural fusion with the P450 domain (
      • Roberts G.A.
      • Celik A.
      • Hunter D.J.
      • Ost T.W.
      • White J.H.
      • Chapman S.K.
      • Turner N.J.
      • Flitsch S.L.
      A self-sufficient cytochrome P450 with a primary structural organization that includes a flavin domain and a [2Fe-2S] redox center.
      ). Interestingly, a few P450s can directly interact with their electron donors and are independent of additional redox partner proteins to accomplish the catalytic reactions; these Class V P450s include P450Nor (
      • Daiber A.
      • Shoun H.
      • Ullrich V.
      Nitric oxide reductase (P450nor) from Fusarium oxysporum.
      ) and P450 TxA (
      • Hsu P.-Y.
      • Tsai A.-L.
      • Kulmacz R.J.
      • Wang L.-H.
      Expression, purification, and spectroscopic characterization of human thromboxane synthase.
      ). Class III–V P450s are independent of redox partner proteins and are often called self-sufficient P450s. Notably, these single-component P450 systems provide very desirable scaffolds for engineering P450 systems, due to their self-sufficiency and hence the significantly increased electron transport efficiency. It is worth noting that other classification systems also exist: Munro et al. (
      • Munro A.W.
      • Girvan H.M.
      • McLean K.J.
      Cytochrome P450-redox partner fusion enzymes.
      ) have categorized five other novel P450-fused redox partner systems in addition to the classical Class I and Class II types, and Bernhardt et al. (
      • Hannemann F.
      • Bichet A.
      • Ewen K.M.
      • Bernhardt R.
      Cytochrome P450 systems—biological variations of electron transport chains.
      ) classified 10 types for P450s based on the topology of protein components involved in the electron transfer chains of P450 enzymes.
      Figure thumbnail gr2
      Figure 2Classification of the P450 systems based on redox partner proteins.

      Successful applications of P450 catalytic systems

      The incomparable diversity of P450s regarding substrates and reaction types provides nearly limitless application potential for production of chemicals and pharmaceuticals (
      • Paddon C.J.
      • Westfall P.J.
      • Pitera D.J.
      • Benjamin K.
      • Fisher K.
      • McPhee D.
      • Leavell M.D.
      • Tai A.
      • Main A.
      • Eng D.
      • Polichuk D.R.
      • Teoh K.H.
      • Reed D.W.
      • Treynor T.
      • Lenihan J.
      • et al.
      High-level semi-synthetic production of the potent antimalarial artemisinin.
      ,
      • McLean K.J.
      • Hans M.
      • Meijrink B.
      • van Scheppingen W.B.
      • Vollebregt A.
      • Tee K.L.
      • van der Laan J.M.
      • Leys D.
      • Munro A.W.
      • van den Berg M.A.
      Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.
      ), biosensor-based analysis (
      • Bistolas N.
      • Wollenberger U.
      • Jung C.
      • Scheller F.W.
      Cytochrome P450 biosensors—a review.
      ), chemoenzymatic synthesis (
      • Lowell A.N.
      • DeMars 2nd, M.D.
      • Slocum S.T.
      • Yu F.
      • Anand K.
      • Chemler J.A.
      • Korakavi N.
      • Priessnitz J.K.
      • Park S.R.
      • Koch A.A.
      • Schultz P.J.
      • Sherman D.H.
      Chemoenzymatic total synthesis and structural diversification of tylactone-based macrolide antibiotics through late-stage polyketide assembly, tailoring, and C–H functionalization.
      ), and pollutant biodegradation (
      • Du L.
      • Dong S.
      • Zhang X.
      • Jiang C.
      • Chen J.
      • Yao L.
      • Wang X.
      • Wan X.
      • Liu X.
      • Wang X.
      • Huang S.
      • Cui Q.
      • Feng Y.
      • Liu S.J.
      • Li S.
      Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system.
      ). For instance, the Saccharopolyspora erythraea EryF and EryK P450s are involved in the production of the antibacterial agent erythromycin (Fig. 3, compound 1) (
      • Minas W.
      • Brünker P.
      • Kallio P.T.
      • Bailey J.E.
      Improved erythromycin production in a genetically engineered industrial strain of Saccharopolyspora erythraea.
      ); Streptomyces fradiae TylI and TylHI P450s are involved in the biosynthesis of the antimycoplasma drug tylosin (Fig. 3, compound 2) (
      • Bate N.
      • Cundliffe E.
      The mycinose-biosynthetic genes of Streptomyces fradiae, producer of tylosin.
      ); and Aspergillus terreus LovA is responsible for biosynthesizing monacolin J acid (Fig. 3, compound 3), the precursor of a series of cholesterol-lowering statin drugs (
      • Barriuso J.
      • Nguyen D.T.
      • Li J.W.
      • Roberts J.N.
      • MacNevin G.
      • Chaytor J.L.
      • Marcus S.L.
      • Vederas J.C.
      • Ro D.K.
      Double oxidation of the cyclic nonaketide dihydromonacolin L to monacolin J by a single cytochrome P450 monooxygenase, LovA.
      ,
      • Huang X.
      • Tang S.
      • Zheng L.
      • Teng Y.
      • Yang Y.
      • Zhu J.
      • Lu X.
      Construction of an efficient and robust Aspergillus terreus cell factory for monacolin J production.
      ). The production of high value-added chemical intermediates from phenolic environmental pollutants has been achieved with P450 biodegradation systems (
      • Du L.
      • Dong S.
      • Zhang X.
      • Jiang C.
      • Chen J.
      • Yao L.
      • Wang X.
      • Wan X.
      • Liu X.
      • Wang X.
      • Huang S.
      • Cui Q.
      • Feng Y.
      • Liu S.J.
      • Li S.
      Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system.
      ,
      • Sulistyaningdyah W.T.
      • Ogawa J.
      • Li Q.S.
      • Maeda C.
      • Yano Y.
      • Schmid R.D.
      • Shimizu S.
      Hydroxylation activity of P450 BM-3 mutant F87V towards aromatic compounds and its application to the synthesis of hydroquinone derivatives from phenolic compounds.
      ,
      • Du L.
      • Ma L.
      • Qi F.
      • Zheng X.
      • Jiang C.
      • Li A.
      • Wan X.
      • Liu S.J.
      • Li S.
      Characterization of a unique pathway for 4-cresol catabolism initiated by phosphorylation in Corynebacterium glutamicum.
      ), and soluble P450s have been used in bacterial cell libraries to mimic human P450 drug metabolic profiles (
      • Otey C.R.
      • Bandara G.
      • Lalonde J.
      • Takahashi K.
      • Arnold F.H.
      Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450.
      ,
      • Parikh A.
      • Gillam E.M.
      • Guengerich F.P.
      Drug metabolism by Escherichia coli expressing human cytochromes P450.
      ).
      Figure thumbnail gr3
      Figure 3Structures involved in practical catalysis of diverse P450 systems. Red-colored groups are introduced by P450s.
      Genome mining and high-throughput screening of P450s have proven to be effective and successful strategies for seeking suitable and robust biocatalysts in industry. P450sca-2 (CYP105A3), screened from Streptomyces carbophilus, is able to catalyze the 6β-hydroxylation of compactin produced by Penicillium citrinum, generating the cholesterol-lowering drug pravastatin (Fig. 3, compound 4) (
      • Hosobuchi M.
      • Kurosawa K.
      • Yoshikawa H.
      Application of computer to monitoring and control of fermentation process: microbial conversion of ML-236B Na to pravastatin.
      ), considered to be one of the most successful instances of practical P450 catalysis in industry (
      • Sakaki T.
      Practical application of cytochrome P450.
      ,
      • Yasuda K.
      • Sugimoto H.
      • Hayashi K.
      • Takita T.
      • Yasukawa K.
      • Ohta M.
      • Kamakura M.
      • Ikushiro S.
      • Shiro Y.
      • Sakaki T.
      Protein engineering of CYP105s for their industrial uses.
      ). The bioconversion of 11-deoxycortisol into hydrocortisol by the P450lun-containing fungus Curvularia lunata has been launched by Bayer on an industrial scale (
      • Sakaki T.
      Practical application of cytochrome P450.
      ,
      • Suzuki K.
      • Sanga K.
      • Chikaoka Y.
      • Itagaki E.
      Purification and properties of cytochrome P-450 (P-450lun) catalyzing steroid 11β-hydroxylation in Curvularia lunata.
      ) (Table 1 and Fig. 3, compound 5). The industrially relevant P450 VD25 (CYP105A2) from Amycolata autotrophica (later renamed as Pseudonocardia autotrophica) is capable of transforming vitamin D3 into its most bioactive form, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) (
      • Kawauchi H.
      • Sasaki J.
      • Adachi T.
      • Hanada K.
      • Beppu T.
      • Horinouchi S.
      Cloning and nucleotide sequence of a bacterial cytochrome P-450VD25 gene encoding vitamin D-3 25-hydroxylase.
      ) (Table 1 and Fig. 3, compound 6). The P450 hydroxylase CYP-sb21 from the rare actinomycetes Sebekia benihana and CYP-pa1 from P. autotrophica are candidate biocatalysts for site-selective hydroxylation of the immunosuppressive drug cyclosporin A to two hair-stimulating agents with significantly decreased immunosuppressant activity, γ-hydroxy-N-methyl-l-Leu4-cyclosporin A and γ-hydroxy-N-methyl-l-Leu9-cyclosporin A, respectively (
      • Ma L.
      • Du L.
      • Chen H.
      • Sun Y.
      • Huang S.
      • Zheng X.
      • Kim E.S.
      • Li S.
      Reconstitution of the in vitro activity of the cyclosporine-specific P450 hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system.
      ,
      • Ban J.G.
      • Woo M.W.
      • Lee B.R.
      • Lee M.J.
      • Choi S.S.
      • Kim E.S.
      A novel regiospecific cyclosporin hydroxylase gene revealed through the genome mining of Pseudonocardia autotrophica.
      ,
      • Sun Y.
      • Ma L.
      • Han D.
      • Du L.
      • Qi F.
      • Zhang W.
      • Sun J.
      • Huang S.
      • Kim E.S.
      • Li S.
      In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins.
      ,
      • Lee M.-J.
      • Kim H.-B.
      • Yoon Y.J.
      • Han K.
      • Kim E.-S.
      Identification of a cyclosporine-specific P450 hydroxylase gene through targeted cytochrome P450 complement (CYPome) disruption in Sebekia benihana.
      ) (Table 1 and Fig. 3, compounds 7–9).
      Table 1Selected P450s involved in production of pharmaceuticals and chemical intermediates
      P450PDB codeOriginWT/mutantFunctionSubstrateReference
      EryF (CYP107A1)1OXASaccharopolyspora erythraeaWT6-Hydroxylation6-Deoxyerythronolide B
      • Minas W.
      • Brünker P.
      • Kallio P.T.
      • Bailey J.E.
      Improved erythromycin production in a genetically engineered industrial strain of Saccharopolyspora erythraea.
      EryK (CYP113A1)2JJNS. erythraeaWT12-HydroxylationErythromycin D
      • Minas W.
      • Brünker P.
      • Kallio P.T.
      • Bailey J.E.
      Improved erythromycin production in a genetically engineered industrial strain of Saccharopolyspora erythraea.
      TylIStreptomyces fradiaeWTC20 hydroxylation/dehydrogenation5-Mycaminosyl tylacone
      • Bate N.
      • Cundliffe E.
      The mycinose-biosynthetic genes of Streptomyces fradiae, producer of tylosin.
      TylHI6B11S. fradiaeWTC23 Hydroxylation23-Deoxy-5-omycaminosyl-tylonolide
      • Bate N.
      • Cundliffe E.
      The mycinose-biosynthetic genes of Streptomyces fradiae, producer of tylosin.
      LovAAspergillus terreusWT4a/5-Dehydrogenation, 8-hydroxylationDihydromonacolin L
      • Barriuso J.
      • Nguyen D.T.
      • Li J.W.
      • Roberts J.N.
      • MacNevin G.
      • Chaytor J.L.
      • Marcus S.L.
      • Vederas J.C.
      • Ro D.K.
      Double oxidation of the cyclic nonaketide dihydromonacolin L to monacolin J by a single cytochrome P450 monooxygenase, LovA.
      CYP71AV1Artemisia annuaWT12-CarboxylationAmorphadiene
      • Ro D.K.
      • Paradise E.M.
      • Ouellet M.
      • Fisher K.J.
      • Newman K.L.
      • Ndungu J.M.
      • Ho K.A.
      • Eachus R.A.
      • Ham T.S.
      • Kirby J.
      • Chang M.C.
      • Withers S.T.
      • Shiba Y.
      • Sarpong R.
      • Keasling J.D.
      Production of the antimalarial drug precursor artemisinic acid in engineered yeast.
      P450sca-2 (CYP105A3)Streptomyces carbophilusWT6β-HydroxylationCompactin
      • Hosobuchi M.
      • Kurosawa K.
      • Yoshikawa H.
      Application of computer to monitoring and control of fermentation process: microbial conversion of ML-236B Na to pravastatin.
      Semi-rational designR8–5C/T85F/ T119S/V194N/N363Y6β-HydroxylationCompactin
      • Ba L.
      • Li P.
      • Zhang H.
      • Duan Y.
      • Lin Z.
      Semi-rational engineering of cytochrome P450sca-2 in a hybrid system for enhanced catalytic activity: insights into the important role of electron transfer.
      P450lunCurvularia lunataWT11-Hydroxylation11-Deoxycortisol
      • Sakaki T.
      Practical application of cytochrome P450.
      ,
      • Suzuki K.
      • Sanga K.
      • Chikaoka Y.
      • Itagaki E.
      Purification and properties of cytochrome P-450 (P-450lun) catalyzing steroid 11β-hydroxylation in Curvularia lunata.
      P450VD25 (CYP105A2)Pseudonocardia autotrophicaWT25-HydroxylationVitamin D3
      • Kawauchi H.
      • Sasaki J.
      • Adachi T.
      • Hanada K.
      • Beppu T.
      • Horinouchi S.
      Cloning and nucleotide sequence of a bacterial cytochrome P-450VD25 gene encoding vitamin D-3 25-hydroxylase.
      CYP-sb21 (CYP107Z14)Sebekia benihanaWTHydroxylation at the 4th N-methyl leucineCyclosporin A
      • Ma L.
      • Du L.
      • Chen H.
      • Sun Y.
      • Huang S.
      • Zheng X.
      • Kim E.S.
      • Li S.
      Reconstitution of the in vitro activity of the cyclosporine-specific P450 hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system.
      CYP-pa1P. autotrophicaWTHydroxylation at the 9th N-methyl leucineCyclosporin A
      • Sun Y.
      • Ma L.
      • Han D.
      • Du L.
      • Qi F.
      • Zhang W.
      • Sun J.
      • Huang S.
      • Kim E.S.
      • Li S.
      In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins.
      P450BM3 (CYP102A1)1JPZBacillus megateriumWTHydroxylationFatty acids
      • Whitehouse C.J.C.
      • Bell S.G.
      • Wong L.-L.
      P450BM3(CYP102A1): connecting the dots.
      2X7YSite-directed mutagenesisF87A2β-/15β-Hydroxylation (1:1)Testosterone
      • Kille S.
      • Zilly F.E.
      • Acevedo J.P.
      • Reetz M.T.
      Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution.
      Directed evolutionR47Y/T49F/V78L/A82M/F87A15β-Hydroxylation (96%)Testosterone
      • Kille S.
      • Zilly F.E.
      • Acevedo J.P.
      • Reetz M.T.
      Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution.
      Directed evolutionA330W/F87A2β-Hydroxylation (97%)Testosterone
      • Kille S.
      • Zilly F.E.
      • Acevedo J.P.
      • Reetz M.T.
      Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution.
      Heme domain of P450BM3Directed evolution9C1 A74V4′-Hydroxypropranolol and 5′-hydroxypropranololPropranolol
      • Otey C.R.
      • Bandara G.
      • Lalonde J.
      • Takahashi K.
      • Arnold F.H.
      Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450.
      P450 Vdh (CYP107BR1)3A4GP. autotrophicaWT1α-/25-HydroxylationVitamin D3
      • Fujii Y.
      • Kabumoto H.
      • Nishimura K.
      • Fujii T.
      • Yanai S.
      • Takeda K.
      • Tamura N.
      • Arisawa A.
      • Tamura T.
      Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica.
      Directed evolutionT70R/V156L/E216M/E384R1α-/25-HydroxylationVitamin D3
      • Yasutake Y.
      • Fujii Y.
      • Nishioka T.
      • Cheon W.-K.
      • Arisawa A.
      • Tamura T.
      Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase.
      CYP105A12ZBX

      2ZBZ
      Streptomyces griseolusWT1α-/25-HydroxylationVitamin D3
      • Hayashi K.
      • Sugimoto H.
      • Shinkyo R.
      • Yamada M.
      • Ikeda S.
      • Ikushiro S.
      • Kamakura M.
      • Shiro Y.
      • Sakaki T.
      Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1.
      Rational designR73A/R84A1α-/25-HydroxylationVitamin D3
      • Hayashi K.
      • Yasuda K.
      • Sugimoto H.
      • Ikushiro S.
      • Kamakura M.
      • Kittaka A.
      • Horst R.L.
      • Chen T.C.
      • Ohta M.
      • Shiro Y.
      • Sakaki T.
      Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1: enzymes and catalysis.
      CYP105AS14OQRAmycolatopsis orientalisWT6-epi-Hydroxylation (97%)Compactin
      • McLean K.J.
      • Hans M.
      • Meijrink B.
      • van Scheppingen W.B.
      • Vollebregt A.
      • Tee K.L.
      • van der Laan J.M.
      • Leys D.
      • Munro A.W.
      • van den Berg M.A.
      Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.
      P450PravaDirected evolutionI95T/Q127R/A180V/L263I/A265N6β-Hydroxylation (100%)Compactin
      • McLean K.J.
      • Hans M.
      • Meijrink B.
      • van Scheppingen W.B.
      • Vollebregt A.
      • Tee K.L.
      • van der Laan J.M.
      • Leys D.
      • Munro A.W.
      • van den Berg M.A.
      Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.
      PikC2C6H

      2C7X
      Streptomyces venezuelae ATCC 15439C10/C12 Hydroxylation, C12/C14 hydroxylationYC-17/Narbomycin
      • Sherman D.H.
      • Li S.
      • Yermalitskaya L.V.
      • Kim Y.
      • Smith J.A.
      • Waterman M.R.
      • Podust L.M.
      The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae.
      Rational designD50NC10/C12 Hydroxylation, C12/C14 hydroxylationYC-17/Narbomycin
      • Sherman D.H.
      • Li S.
      • Yermalitskaya L.V.
      • Kim Y.
      • Smith J.A.
      • Waterman M.R.
      • Podust L.M.
      The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae.
      2C6H

      2C7X
      Redox parent engineeringWTC10/C12 Hydroxylation, C12/C14 hydroxylationYC-17/Narbomycin
      • Li S.
      • Podust L.M.
      • Sherman D.H.
      Engineering and analysis of a self-sufficient biosynthetic cytochrome P450 PikC fused to the RhFRED reductase domain.
      Substrate engineeringD50NRegio-selective hydroxylationUnnatural substrates
      • Li S.
      • Chaulagain M.R.
      • Knauff A.R.
      • Podust L.M.
      • Montgomery J.
      • Sherman D.H.
      Selective oxidation of carbolide C–H bonds by an engineered macrolide P450 mono-oxygenase.
      MycG5UHUMicromonospora griseorubidaWTC12/C13 Epoxidation, C14 hydroxylationMycinamicin IV
      • Anzai Y.
      • Li S.
      • Chaulagain M.R.
      • Kinoshita K.
      • Kato F.
      • Montgomery J.
      • Sherman D.H.
      Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics.
      Redox parent engineeringN-DemethylationMycinamicin IV
      • Zhang W.
      • Liu Y.
      • Yan J.
      • Cao S.
      • Bai F.
      • Yang Y.
      • Huang S.
      • Yao L.
      • Anzai Y.
      • Kato F.
      • Podust L.M.
      • Sherman D.H.
      • Li S.
      New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.
      CYP725A4Taxus cuspidataWT5α-HydroxylationTaxadiene
      • Biggs B.W.
      • Lim C.G.
      • Sagliani K.
      • Shankar S.
      • Stephanopoulos G.
      • De Mey M.
      • Ajikumar P.K.
      Overcoming heterologous protein interdependency to optimize P450-mediated taxol precursor synthesis in Escherichia coli.
      Redox parent engineeringN-terminal hydrophilic modifications5α-HydroxylationTaxadiene
      • Biggs B.W.
      • Lim C.G.
      • Sagliani K.
      • Shankar S.
      • Stephanopoulos G.
      • De Mey M.
      • Ajikumar P.K.
      Overcoming heterologous protein interdependency to optimize P450-mediated taxol precursor synthesis in Escherichia coli.
      CYP2C91OG2HumanSelf-sufficientN-DemethylationErythromycin
      • Dodhia V.R.
      • Fantuzzi A.
      • Gilardi G.
      Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular lego.
      CY2C19HumanSelf-sufficient4-HydroxylationDiclofenac
      • Dodhia V.R.
      • Fantuzzi A.
      • Gilardi G.
      Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular lego.
      CYP3A46DA2HumanSelf-sufficient5-HydroxylationOmeprazole
      • Dodhia V.R.
      • Fantuzzi A.
      • Gilardi G.
      Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular lego.
      1,2-Dehydroreticuline synthase (CYP82Y2-like)Papaver bracteatumPbDRS-DRRDehydrogenation(S)-Reticuline
      • Galanie S.
      • Thodey K.
      • Trenchard I.J.
      • Filsinger Interrante M.
      • Smolke C.D.
      Complete biosynthesis of opioids in yeast.
      SalSynPapaver somniferumyPbSalSyn92–504C–C Coupling(R)-Reticuline
      • Galanie S.
      • Thodey K.
      • Trenchard I.J.
      • Filsinger Interrante M.
      • Smolke C.D.
      Complete biosynthesis of opioids in yeast.
      CYP76AH1Salvia miltiorrhizaWTHydroxylation and dehydrogenationMiltiradiene
      • Guo J.
      • Zhou Y.J.
      • Hillwig M.L.
      • Shen Y.
      • Yang L.
      • Wang Y.
      • Zhang X.
      • Liu W.
      • Peters R.J.
      • Chen X.
      • Zhao Z.K.
      • Huang L.
      CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.
      P450 protopanaxadiol synthasePanax ginsengWTHydroxylationDammarenediol-II
      • Guo J.
      • Zhou Y.J.
      • Hillwig M.L.
      • Shen Y.
      • Yang L.
      • Wang Y.
      • Zhang X.
      • Liu W.
      • Peters R.J.
      • Chen X.
      • Zhao Z.K.
      • Huang L.
      CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.
      CYP11A13N9YHumanWTC–C CleavageEgrosta-5-eneol/ergosta-5,22-dieneol
      • Szczebara F.M.
      • Chandelier C.
      • Villeret C.
      • Masurel A.
      • Bourot S.
      • Duport C.
      • Blanchard S.
      • Groisillier A.
      • Testet E.
      • Costaglioli P.
      • Cauet G.
      • Degryse E.
      • Balbuena D.
      • Winter J.
      • Achstetter T.
      • et al.
      Total biosynthesis of hydrocortisone from a simple carbon source in yeast.
      CYP17A13RUKHumanWT17α-HydroxylationProgesterone
      • Szczebara F.M.
      • Chandelier C.
      • Villeret C.
      • Masurel A.
      • Bourot S.
      • Duport C.
      • Blanchard S.
      • Groisillier A.
      • Testet E.
      • Costaglioli P.
      • Cauet G.
      • Degryse E.
      • Balbuena D.
      • Winter J.
      • Achstetter T.
      • et al.
      Total biosynthesis of hydrocortisone from a simple carbon source in yeast.
      CYP21A1HumanWT21-Hydroxylation17-Hydroxyprogesterone
      • Szczebara F.M.
      • Chandelier C.
      • Villeret C.
      • Masurel A.
      • Bourot S.
      • Duport C.
      • Blanchard S.
      • Groisillier A.
      • Testet E.
      • Costaglioli P.
      • Cauet G.
      • Degryse E.
      • Balbuena D.
      • Winter J.
      • Achstetter T.
      • et al.
      Total biosynthesis of hydrocortisone from a simple carbon source in yeast.
      CYP11B1HumanWT11β-Hydroxylation11-Deoxycortisol
      • Szczebara F.M.
      • Chandelier C.
      • Villeret C.
      • Masurel A.
      • Bourot S.
      • Duport C.
      • Blanchard S.
      • Groisillier A.
      • Testet E.
      • Costaglioli P.
      • Cauet G.
      • Degryse E.
      • Balbuena D.
      • Winter J.
      • Achstetter T.
      • et al.
      Total biosynthesis of hydrocortisone from a simple carbon source in yeast.
      P450 box AStreptomyces sp. TM-7WT6β-HydroxylationCompactin
      • Fujii T.
      • Fujii Y.
      • Machida K.
      • Ochiai A.
      • Ito M.
      Efficient biotransformations using Escherichia coli with tolC acrAB mutations expressing cytochrome P450 genes.
      CYP106A24YT3B. megaterium ATCC 13368WT15β-HydroxylationProgesterone, testosterone
      • Janocha S.
      • Carius Y.
      • Hutter M.
      • Lancaster C.R.
      • Bernhardt R.
      Crystal structure of CYP106A2 in substrate-free and substrate-bound form.
      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 (
      • Ro D.K.
      • Paradise E.M.
      • Ouellet M.
      • Fisher K.J.
      • Newman K.L.
      • Ndungu J.M.
      • Ho K.A.
      • Eachus R.A.
      • Ham T.S.
      • Kirby J.
      • Chang M.C.
      • Withers S.T.
      • Shiba Y.
      • Sarpong R.
      • Keasling J.D.
      Production of the antimalarial drug precursor artemisinic acid in engineered yeast.
      ). Traditional production of the anti-malarial drug artemisinin from the Chinese medicinal plant Artemisia annua L. is low-yield, unsustainable, and too expensive for millions of individuals suffering from malaria. A recombinant Saccharomyces cerevisiae strain with a heavily engineered mevalonate pathway, an amorphadiene synthase, and a key CYP71AV1 (from A. annua) produced 100 mg of artemisinic acid per liter (
      • Ro D.K.
      • Paradise E.M.
      • Ouellet M.
      • Fisher K.J.
      • Newman K.L.
      • Ndungu J.M.
      • Ho K.A.
      • Eachus R.A.
      • Ham T.S.
      • Kirby J.
      • Chang M.C.
      • Withers S.T.
      • Shiba Y.
      • Sarpong R.
      • Keasling J.D.
      Production of the antimalarial drug precursor artemisinic acid in engineered yeast.
      ) (Table 1 and Fig. 3, compound 10). 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 (
      • Paddon C.J.
      • Westfall P.J.
      • Pitera D.J.
      • Benjamin K.
      • Fisher K.
      • McPhee D.
      • Leavell M.D.
      • Tai A.
      • Main A.
      • Eng D.
      • Polichuk D.R.
      • Teoh K.H.
      • Reed D.W.
      • Treynor T.
      • Lenihan J.
      • et al.
      High-level semi-synthetic production of the potent antimalarial artemisinin.
      ).
      The P450 systems have also been engineered for the fragrance production. For instance, the oxidation of sesquiterpene (+)-valencene to high value-added flavor (+)-nootkatone with P450 enzymes was first accomplished in the Wong group by rationally designed mutants of P450BM3 and P450cam (
      • Sowden R.J.
      • Yasmin S.
      • Rees N.H.
      • Bell S.G.
      • Wong L.-L.
      Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3.
      ) (Fig. 3, compound 11).

      Strategies and progress of engineering P450 enzyme systems

      Although P450s have demonstrated amazing catalytic diversities and great prospects for application, the aforementioned limitations in industrial applications of P450s are also significant. To overcome these limitations, versatile engineering strategies have been proposed and developed to satisfy different application requirements, including protein engineering of P450s and redox partners, substrate engineering, cofactor regeneration, and P450-related metabolic engineering (Fig. 4).
      Figure thumbnail gr4
      Figure 4Engineering strategies for P450 systems discussed in the current review. DHase and OXase, dehydrogenase and oxidase, respectively.

      Protein engineering

      Protein engineering involves modification of the residues based on the folding principles and molecular structure of proteins, with the goal of obtaining the desired mutated proteins with enhanced properties to compensate for the poor stability, low selectivity, slow catalytic rates, and limited application space of the native proteins (
      • Woodley J.M.
      Protein engineering of enzymes for process applications.
      ). Directed evolution and rational and semi-rational design are routinely used methods in P450 engineering and play very important roles in the development of pharmaceutical catalysts (
      • Xu L.H.
      • Du Y.L.
      Rational and semi-rational engineering of cytochrome P450s for biotechnological applications.
      ,
      • Turner N.J.
      Directed evolution drives the next generation of biocatalysts.
      ).

      Directed evolution

      Directed evolution has been widely applied to engineer P450s, the structures of which are often unknown, for desired properties under artificial selective pressure, including random mutagenesis and screening (
      • Arnold F.H.
      Design by directed evolution.
      ). To obtain the desired mutant proteins, a protein library with a large number of mutants covering sufficient molecular diversity is usually generated by error-prone PCR (epPCR), combinatorial saturation mutagenesis, or DNA-shuffling methods (
      • Turner N.J.
      Directed evolution drives the next generation of biocatalysts.
      ). In addition, directed evolution is also an effective tool for understanding relationships between key amino acid residues surrounding catalytic pockets and catalytic abilities toward different unnatural substrates for well-characterized P450s (e.g. P450BM3 and P450cam) (
      • Poulos T.L.
      • Finzel B.C.
      • Howard A.J.
      High-resolution crystal structure of cytochrome P450cam.
      ).
      Reetz and co-workers (
      • Kille S.
      • Zilly F.E.
      • Acevedo J.P.
      • Reetz M.T.
      Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution.
      ) found that the simple P450BM3 mutant F87A, with reduced steric hindrance for substrate, was able to catalyze the nonselective 2β- and 15β-hydroxylation of testosterone to generate a 1:1 mixture of products. To alter the regioselectivity, iterative saturation mutagenesis of 20 selected residues lining the substrate-binding pocket was done, leading to two effective mutants (A330W/F87A and R47Y/T49F/V78L/A82M/F87A) that achieved specific regio-selective production of the 15β (96%) or 2β products (97%), respectively (
      • Kille S.
      • Zilly F.E.
      • Acevedo J.P.
      • Reetz M.T.
      Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution.
      ) (Table 1 and Fig. 3, compounds 12–14). P450 Vdh (CYP107BR1, Protein Data Bank (PDB) entry 3A4G) from P. autotrophica was also reported to produce 1α,25(OH)2D3 from vitamin D3 (
      • Fujii Y.
      • Kabumoto H.
      • Nishimura K.
      • Fujii T.
      • Yanai S.
      • Takeda K.
      • Tamura N.
      • Arisawa A.
      • Tamura T.
      Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica.
      ) (Table 1 and Fig. 3, compound 6). A Vdh-K1 mutant (T70R/V156L/E216M/E384R) was generated with 6-fold higher specific activity than WT P450 through high-throughput screening of a site-saturated mutagenesis library (
      • Yasutake Y.
      • Fujii Y.
      • Nishioka T.
      • Cheon W.-K.
      • Arisawa A.
      • Tamura T.
      Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase.
      ) (Table 1 and Fig. 3, compound 6).
      Directed evolution of P450s has also been applied in the generation of drug metabolites as an effective strategy for further pharmaceutical studies. Here, the strategy is to use the bacterial P450s to generate larger amounts of drug metabolites to facilitate structural analysis of the small quantities of drug metabolites, which is a regulatory requirement for further drug development. Arnold and associates (
      • Otey C.R.
      • Bandara G.
      • Lalonde J.
      • Takahashi K.
      • Arnold F.H.
      Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450.
      ) constructed a mutant library of P450BM3 using epPCR and combinatorial saturation mutagenesis of seven active-site residues surrounding the heme domain. The mutants selectively oxidized the antiarrhythmic drug propranolol to its active human metabolites, including 4′-hydroxypropranolol and 5′-hydroxypropranolol, via the “H2O2” shunt pathway (
      • Otey C.R.
      • Bandara G.
      • Lalonde J.
      • Takahashi K.
      • Arnold F.H.
      Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450.
      ) (Table 1 and Fig. 3, compounds 15–17). Subsequently, a small panel of P450BM3 variants was further subjected to site-directed mutagenesis of the active-site residues, leading to a set of metabolites of the antihypertensive drug verapamil and the antiallergic astemizole that are the same as those metabolized by mammalian P450s (
      • Sawayama A.M.
      • Chen M.M.
      • Kulanthaivel P.
      • Kuo M.S.
      • Hemmerle H.
      • Arnold F.H.
      A panel of cytochrome P450 BM3 variants to produce drug metabolites and diversify lead compounds.
      ).
      The Guengerich laboratory screened a series of CYP1A2 mutants generated by random mutagenesis at six substrate recognition sites (SRSs), and the obtained variants had 2–4-fold increases in kcat/Km (specificity constant) toward the analgesic and antipyretic drug phenacetin compared with the parent enzyme (
      • Parikh A.
      • Josephy P.D.
      • Guengerich F.P.
      Selection and characterization of human cytochrome P450 1A2 mutants with altered catalytic properties.
      ). Similarly, human CYP2A6 mutants were screened based on the production of indole oxidation products, which could find application in production of dyestuffs or as protein kinase inhibitors (
      • Nakamura K.
      • Martin M.V.
      • Guengerich F.P.
      Random mutagenesis of human cytochrome P450 2A6 and screening with indole oxidation products.
      ).
      The protein stability of P450s, another important factor for practical applications to enhance the total turnover numbers, can also be improved via directed evolution, as exemplified by solvent tolerance optimization of the P450BM3 variant F87A/T235A/R471A/E494K/S1024E, which was obtained from libraries constructed by saturation mutagenesis and random mutagenesis (
      • Wong T.S.
      • Arnold F.H.
      • Schwaneberg U.
      Laboratory evolution of cytochrome P450 BM-3 monooxygenase for organic cosolvents.
      ) (the substrates are generally hydrophobic and dissolved in organic solvents). The conversion of p-nitrophenoxydodecanoic acid to p-nitrophenol was enhanced 5.5-fold in the presence of 25% (v/v) DMSO and 10-fold in 2% THF (v/v) compared with the parental P450BM3 F87A mutant (
      • Wong T.S.
      • Arnold F.H.
      • Schwaneberg U.
      Laboratory evolution of cytochrome P450 BM-3 monooxygenase for organic cosolvents.
      ).

      Rational and semi-rational design

      Major disadvantages of directed evolution include the dependence on a high-throughput screening method, which is not always available, the requirement of automated instruments, and high cost. Rational or semi-rational design, based on the well-characterized protein tertiary structure and the mechanistic understanding of structure-activity relationships, is regarded as an effective alternate strategy. Generally (but not always), “hot spot” residues for rational and semi-rational design are usually located within the SRS, the substrate access channel, and the P450-redox partner interaction sites (
      • Xu L.H.
      • Du Y.L.
      Rational and semi-rational engineering of cytochrome P450s for biotechnological applications.
      ,
      • Urlacher V.B.
      • Girhard M.
      Cytochrome P450 monooxygenases: an update on perspectives for synthetic application.
      ).
      CYP105AS1 (from Amycolatopsis orientalis) catalyzes the conversion of compactin to the cholesterol-lowering drug pravastatin plus its ineffective epimer 6-epi-pravastatin, in a ratio of 3:97. Based on the crystal structure of CYP105AS1 (PDB entry 4OQR), a single round of epPCR mutagenesis of selected residues led to a mutant with a ratio of pravastatin/6-epi-pravastatin of 48:52. A further two rounds of site-saturated mutagenesis produced a mutant P450Prava (I95T/Q127R/A180V/L263I/A265N), in which the stereoselectivity was completely inverted into a P450 pravastatin synthase with a 21-fold lower Km value for compactin (
      • McLean K.J.
      • Hans M.
      • Meijrink B.
      • van Scheppingen W.B.
      • Vollebregt A.
      • Tee K.L.
      • van der Laan J.M.
      • Leys D.
      • Munro A.W.
      • van den Berg M.A.
      Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.
      ) (Table 1).
      CYP105A1 (S. griseolus) hydroxylates vitamin D3 to form 1α,25(OH)2 vitamin D3. Site-directed mutagenesis of CYP105A1 based on its three-dimensional structure (PDB entries 2ZBX and 2ZBZ) was performed. Three arginine residues (Arg-73, Arg-84, and Arg-193) located along the substrate access channel of CYP105A1 were mutated to nonpolar alanines on the basis of their important roles in substrate binding and catalysis, delineated from a crystal structure of CYP105A1 with its enzymatic product 1α,25-OH vitamin D3 (
      • Sugimoto H.
      • Shinkyo R.
      • Hayashi K.
      • Yoneda S.
      • Yamada M.
      • Kamakura M.
      • Ikushiro S.-I.
      • Shiro Y.
      • Sakaki T.
      Crystal structure of CYP105A1 (P450SU-1) in complex with 1α,25-dihydroxyvitamin D3.
      ). As hypothesized, the double mutant R73A/R84A exhibited ∼400- and 100-fold increased activity for 25-hydroxylation and 1α-hydroxylation of vitamin D3 compared with the WT enzyme (
      • Hayashi K.
      • Sugimoto H.
      • Shinkyo R.
      • Yamada M.
      • Ikeda S.
      • Ikushiro S.
      • Kamakura M.
      • Shiro Y.
      • Sakaki T.
      Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1.
      ,
      • Hayashi K.
      • Yasuda K.
      • Sugimoto H.
      • Ikushiro S.
      • Kamakura M.
      • Kittaka A.
      • Horst R.L.
      • Chen T.C.
      • Ohta M.
      • Shiro Y.
      • Sakaki T.
      Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1: enzymes and catalysis.
      ) (Table 1).
      The co-crystal structures of multifunctional P450 PikC (PDB entries 2C6H and 2C7X) bound to its native substrates (narbomycin and YC-17) suggested that Asp-50, Glu-85, and Glu-94 (located in the catalytic pocket) might be critical for substrate binding and catalytic activity (Table 1 and Fig. 3, compounds 18–23). Accordingly, a series of mutants was constructed, and PikC D50N displayed significantly higher hydroxylation activities toward both narbomycin and YC-17 than did the WT enzyme (
      • Sherman D.H.
      • Li S.
      • Yermalitskaya L.V.
      • Kim Y.
      • Smith J.A.
      • Waterman M.R.
      • Podust L.M.
      The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae.
      ) (Table 1 and Fig. 3, compounds 1823).
      The availability of a tertiary protein structure is considered to be a major limitation in rational and semi-rational design, in that experimental structural information is not often available for the >370,000 P450 sequences. Homology modeling has often been used to bridge the “structure knowledge gap,” based on the general observation that proteins with homologous sequences share similar structures (
      • Muhammed M.T.
      • Aki-Yalcin E.
      Homology modeling in drug discovery: overview, current applications, and future perspectives.
      ,
      • Schwede T.
      Protein modeling: what happened to the “protein structure gap”?.
      ). A well-known example of P450 semi-rational design for industrial application is the development of a highly efficient mutant of P450sca-2 involved in the production of pravastatin (
      • Ito S.
      • Matsuoka T.
      • Watanabe I.
      • Kagasaki T.
      • Serizawa N.
      • Hata T.
      Crystallization and preliminary X-ray diffraction analysis of cytochrome P450sca-2 from Streptomyces carbophilus involved in production of pravastatin sodium, a tissue-selective inhibitor of HMG-CoA reductase.
      ). Based on homology modeling analysis of an active mutant (R8-5C) of P450sca-2 generated from directed evolution, five sites located in the substrate binding pocket (Arg-77/Thr-85), the substrate access channel (Val-194), and the redox partner interface (Asn-363/Thr-119) were selected for systematic site-directed saturation mutagenesis and three rounds of mutagenesis (
      • Ba L.
      • Li P.
      • Zhang H.
      • Duan Y.
      • Lin Z.
      Semi-rational engineering of cytochrome P450sca-2 in a hybrid system for enhanced catalytic activity: insights into the important role of electron transfer.
      ). As a result, a more active mutant (R8-5C/T85F/T119S/V194N/N363Y) was obtained, with 7-fold higher whole-cell biotransformation activity and a 10-fold higher kcat value than that of R8-5C (
      • Ba L.
      • Li P.
      • Zhang H.
      • Duan Y.
      • Lin Z.
      Semi-rational engineering of cytochrome P450sca-2 in a hybrid system for enhanced catalytic activity: insights into the important role of electron transfer.
      ) (Table 1). Based on the X-ray crystal structure of the high-activity progesterone hydroxylase rabbit CYP2C5 (PDB entry 1DT6), the low progesterone hydroxylase activity of CYP2B1 was re-engineered by changing active-site residues in the three-dimensional structural model of CYP2B1 to the corresponding residues of CYP2C5 (
      • Williams P.A.
      • Cosme J.
      • Sridhar V.
      • Johnson E.F.
      • McRee D.E.
      Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity.
      ,
      • Kumar S.
      • Scott E.E.
      • Liu H.
      • Halpert J.R.
      A rational approach to re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5.
      ). Finally, a CYP2B1 mutant (I114A/F206V/F297G/V363L/V130I/S294D/I477F) exhibited a 3-fold higher kcat value than that of CYP2C5 for progesterone 21-hydroxylation, with 80% regioselectivity (
      • Kumar S.
      • Scott E.E.
      • Liu H.
      • Halpert J.R.
      A rational approach to re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5.
      ).

      Redox partner engineering

      Most P450s require redox partner proteins to sequentially transfer two electrons from NAD(P)H to the heme-iron reactive center to activate O2 for substrate oxygenation (
      • Guengerich F.P.
      Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity.
      ), which can often be the rate-limiting step of the P450 catalytic cycle (Fig. 1). However, the reconstitution of a P450 catalytic system in vitro or in a recombinant host is often hampered by the lack of information about its cognate redox partners or inaccessibility of optimal surrogate redox partners. Therefore, protein-protein interactions between P450 and surrogate redox partners have been optimized to enhance the electron transfer efficiency of P450 systems, which we will term “redox partner engineering.”
      A comprehensive screening of redox partners to identify the best electron transport pathway for supporting the CYP105D5 activity was done in the Guengerich laboratory (
      • Chun Y.J.
      • Shimada T.
      • Sanchez-Ponce R.
      • Martin M.V.
      • Lei L.
      • Zhao B.
      • Kelly S.L.
      • Waterman M.R.
      • Lamb D.C.
      • Guengerich F.P.
      Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2).
      ). Briefly, all four FdRs and six Fdxs encoded by the genome of Streptomyces coelicolor A3(2) were heterogeneously expressed and purified. A total of 24 native redox partner combinations were assembled and screened with a specific S. coelicolor P450, CYP105D5, which had been shown to hydroxylate free fatty acids (
      • Chun Y.J.
      • Shimada T.
      • Sanchez-Ponce R.
      • Martin M.V.
      • Lei L.
      • Zhao B.
      • Kelly S.L.
      • Waterman M.R.
      • Lamb D.C.
      • Guengerich F.P.
      Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2).
      ). The results showed that the pair Fdx4/FdR1 functioned as the preferred redox partner system for this bacterial P450 enzyme in vitro (
      • Chun Y.J.
      • Shimada T.
      • Sanchez-Ponce R.
      • Martin M.V.
      • Lei L.
      • Zhao B.
      • Kelly S.L.
      • Waterman M.R.
      • Lamb D.C.
      • Guengerich F.P.
      Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2).
      ).
      Adrenodoxin and adrenodoxin reductase (Adx/AdR) were characterized as optimal redox partners in supporting the in vitro hydroxylation of lauric acid by CYP109D5 from Sorangium cellulosum So ce56, the catalytic efficiency of which was 3–4-fold higher than that of CYP109D5 supported by endogenous redox partners (Fdx2/FdR_B and Fdx8/FdR_B) (
      • Khatri Y.
      • Hannemann F.
      • Ewen K.M.
      • Pistorius D.
      • Perlova O.
      • Kagawa N.
      • Brachmann A.O.
      • Müller R.
      • Bernhardt R.
      The CYPome of Sorangium cellulosum So ce56 and identification of CYP109D1 as a new fatty acid hydroxylase.
      ). Interestingly, the combination of Fdx8/FdR_B was reported to be a much better pair of redox partners of P450 EpoK in the bioconversion of epothilone D to epothilone B compared with the spinach Fdx/FdR redox pair (
      • Kern F.
      • Dier T.K.
      • Khatri Y.
      • Ewen K.M.
      • Jacquot J.P.
      • Volmer D.A.
      • Bernhardt R.
      Highly efficient CYP167A1 (EpoK) dependent epothilone B formation and production of 7-ketone epothilone D as a new epothilone derivative.
      ) (Table 1 and Fig. 3, compound 24). Thus, a certain P450 enzyme may have a differentially preferred combination of Fdx and FdR among multiple combinations, although alternative redox partners could be functionally complementary (
      • McLean K.J.
      • Luciakova D.
      • Belcher J.
      • Tee K.L.
      • Munro A.W.
      Biological diversity of cytochrome P450 redox partner systems.
      ). It is also worth noting that surrogate redox partners may be superior to the cognate ones; thus, it can be helpful to apply a redox partner interchange approach to determine optimal electron transfer pathways, particularly in bacterial systems, to fully exploit P450 applications.
      To determine whether there are any principles for guiding the screening of optimal redox partners for a given Class I bacterial P450, Zhang et al. (
      • Zhang W.
      • Du L.
      • Li F.
      • Zhang X.
      • Qu Z.
      • Han L.
      • Li Z.
      • Sun J.
      • Qi F.
      • Yao Q.
      • Sun Y.
      • Geng C.
      • Li S.
      Mechanistic insights into interactions between bacterial class I P450 enzymes and redox partners.
      ) constituted a reaction matrix network based on 16 Fdxs, eight FdRs, and six P450s toward seven substrates. By analyzing the reactivity profiles of 896 reactions, plastidic-type FdR and Fe2S2 Fdx were found to be the favored types of redox partners by Class I P450 systems. Based on the empirically derived rules, the optimal cognate Fdx of PikC from Streptomyces venezuelae ATCC 15439 was predicted and confirmed in vitro to be SveFdx1948 (
      • Zhang W.
      • Du L.
      • Li F.
      • Zhang X.
      • Qu Z.
      • Han L.
      • Li Z.
      • Sun J.
      • Qi F.
      • Yao Q.
      • Sun Y.
      • Geng C.
      • Li S.
      Mechanistic insights into interactions between bacterial class I P450 enzymes and redox partners.
      ). This work has provided information about the P450-preferred redox partners, and we envision that the findings will benefit future practical applications of P450 enzymes.
      Notably, the protein pair SelFdx1499 (Fe2S2)/SelFdR0978 (plastidic-type FdR) from the cyanobacterial strain Synechococcus elongatus PCC 7942 has been shown to be an optimal combination for supporting in vitro reactions of prokaryotic P450s, including MycG, PikC, P450sca-2 and others (
      • Zhang W.
      • Du L.
      • Li F.
      • Zhang X.
      • Qu Z.
      • Han L.
      • Li Z.
      • Sun J.
      • Qi F.
      • Yao Q.
      • Sun Y.
      • Geng C.
      • Li S.
      Mechanistic insights into interactions between bacterial class I P450 enzymes and redox partners.
      ). Besides the above-mentioned P450 reaction matrix network, the protein pair SelFdx1499/SelFdR0978 has also been shown to be optimal for the site-selective hydroxylation of CsA by CYP-sb21 and CYP-pa1 (
      • Ma L.
      • Du L.
      • Chen H.
      • Sun Y.
      • Huang S.
      • Zheng X.
      • Kim E.S.
      • Li S.
      Reconstitution of the in vitro activity of the cyclosporine-specific P450 hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system.
      ,
      • Sun Y.
      • Ma L.
      • Han D.
      • Du L.
      • Qi F.
      • Zhang W.
      • Sun J.
      • Huang S.
      • Kim E.S.
      • Li S.
      In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins.
      ), the uncommon ester-to-ether transformation catalyzed by Rif16 in rifamycin biosynthesis (
      • Qi F.
      • Lei C.
      • Li F.
      • Zhang X.
      • Wang J.
      • Zhang W.
      • Fan Z.
      • Li W.
      • Tang G.L.
      • Xiao Y.
      • Zhao G.
      • Li S.
      Deciphering the late steps of rifamycin biosynthesis.
      ), the tandem ether installation and hydroxylation by AmbV involved in neoabyssomicin/abyssomicin biosynthesis (
      • Li Q.
      • Ding W.
      • Yao Z.
      • Tu J.
      • Wang L.
      • Huang H.
      • Li S.
      • Ju J.
      AbmV catalyzes tandem ether installation and hydroxylation during neoabyssomicin/abyssomicin biosynthesis.
      ), and the biosynthesis of phenylserine (β-OH-Phe) unit in atratumycin by Atr27 (
      • Sun C.
      • Yang Z.
      • Zhang C.
      • Liu Z.
      • He J.
      • Liu Q.
      • Zhang T.
      • Ju J.
      • Ma J.
      Genome mining of Streptomyces atratus SCSIO ZH16: discovery of atratumycin and identification of its biosynthetic gene cluster.
      ).
      In addition to the screening and prediction of optimal redox partners, optimization of interaction modes between P450s and redox partners through redox partner engineering provides another effective strategy for P450 activity improvement. The residues located at the P450-Fdx (Fdx directly interacts with P450) interaction interface play important roles in affecting the catalytic activity of a P450. Screening of Adx derivatives modified at N-terminal or C-terminal polypeptide sequences led to the finding that Adx(4–108) truncated at N-terminal amino acids 1–3 and C-terminal amino acids 109–128 supported the 11β-hydroxylation of 11-deoxycortisol to cortisol by CYP11B1 with a higher electron transfer rate, and the specificity constant (kcat/Km) was increased 21-fold relative to that of WT Adx (
      • Uhlmann H.
      • Kraft R.
      • Bernhardt R.
      C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P450.
      ,
      • Müller A.
      • Müller J.J.
      • Muller Y.A.
      • Uhlmann H.
      • Bernhardt R.
      • Heinemann U.
      New aspects of electron transfer revealed by the crystal structure of a truncated bovine adrenodoxin, Adx(4–108).
      ,
      • Ewen K.M.
      • Kleser M.
      • Bernhardt R.
      Adrenodoxin: the archetype of vertebrate-type [2Fe-2S] cluster ferredoxins.
      ). The availability of co-crystal structures of P450s and their redox partners will facilitate engineering of the P450/Fdx interface, as exemplified by the artificial fusion CYP11A1-Adx (
      • Strushkevich N.
      • MacKenzie F.
      • Cherkesova T.
      • Grabovec I.
      • Usanov S.
      • Park H.-W.
      Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system.
      ) and the cross-linked CYP101A1-Pdx complex (
      • Tripathi S.
      • Li H.
      • Poulos T.L.
      Structural basis for effector control and redox partner recognition in cytochrome P450.
      ). Based on the interaction analysis, the amino acids of ferredoxin PuxB interacting with P450 were swapped (site-directed mutagenesis) to mimic the biogenic ferredoxin Pux of CYP199A2 from Rhodopseudomonas palustris CGA009. A PuxB variant with seven mutations was generated, and the rate of demethylation of 4-methoxybenzoic acid by CYP199A2 was increased 12-fold compared with WT PuxB (
      • Bell S.G.
      • McMillan J.H.
      • Yorke J.A.
      • Kavanagh E.
      • Johnson E.O.
      • Wong L.L.
      Tailoring an alien ferredoxin to support native-like P450 monooxygenase activity.
      ). However, the semi-rational engineering approach remains challenging due to the lack of comprehensive understanding of the dynamic mechanisms for protein-protein recognition and intermolecular electron transfer. Thus, further work is needed for understanding P450-Fdx complex structures at the molecular level to address this challenge.
      Inspired by the paradigm of self-sufficient P450 enzymes (Class III and IV) that contain both P450 and redox partner domains in one polypeptide chain, the construction of “unnatural” self-sufficient enzymes by making variant versions of P450-redox partner fusion proteins has been pursued (
      • Sadeghi S.J.
      • Gilardi G.
      Chimeric P450 enzymes: activity of artificial redox fusions driven by different reductases for biotechnological applications.
      ). A self-sufficient PikC-RhFRED fusion was generated, and its catalytic activity toward YC-17 was increased ∼4-fold compared with that of a three-component system (PikC + spinach Fdx/FdR) in vitro, likely due to enhanced intramolecular electron transfer efficiency compared with the intermolecular reaction (
      • Li S.
      • Podust L.M.
      • Sherman D.H.
      Engineering and analysis of a self-sufficient biosynthetic cytochrome P450 PikC fused to the RhFRED reductase domain.
      ). Another striking example is the construction of self-sufficient P450Prava-RhFRED. The introduction of P450Prava-RhFRED into the compactin-producing P. chrysogenum delivered more than 6 g of pravastatin per liter in a one-step fermentation (
      • McLean K.J.
      • Hans M.
      • Meijrink B.
      • van Scheppingen W.B.
      • Vollebregt A.
      • Tee K.L.
      • van der Laan J.M.
      • Leys D.
      • Munro A.W.
      • van den Berg M.A.
      Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.
      ). High-throughput generation of self-sufficient P450 libraries by fusing P450 heme domains to RhFRED via a ligation-independent cloning vector, “LICRED,” was developed (
      • Sabbadin F.
      • Hyde R.
      • Robin A.
      • Hilgarth E.M.
      • Delenne M.
      • Flitsch S.
      • Turner N.
      • Grogan G.
      • Bruce N.C.
      LICRED: a versatile drop-in vector for rapid generation of redox-self-sufficient cytochrome P450s.
      ). Self-sufficient mammalian P450-reductase fusion enzymes have been prepared, mimicking the precedent of the efficient P450BM3, including CYP2C9, CY2C19, and CYP3A4 for drug metabolism studies (
      • Dodhia V.R.
      • Fantuzzi A.
      • Gilardi G.
      Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular lego.
      ). However, establishing the optimal design and length of the linker has not been trivial (
      • Robin A.
      • Roberts G.A.
      • Kisch J.
      • Sabbadin F.
      • Grogan G.
      • Bruce N.
      • Turner N.J.
      • Flitsch S.L.
      Engineering and improvement of the efficiency of a chimeric [P450cam-RhFRed reductase domain] enzyme.
      ). Among seven fused P450cam-RhFRED (L1–L7) enzymes with varying linker regions, L4 was the most optimal, with 100% conversion of 3 mm (+)-camphor under the conditions tested (
      • Robin A.
      • Roberts G.A.
      • Kisch J.
      • Sabbadin F.
      • Grogan G.
      • Bruce N.
      • Turner N.J.
      • Flitsch S.L.
      Engineering and improvement of the efficiency of a chimeric [P450cam-RhFRed reductase domain] enzyme.
      ).
      Other chimeras have been made with diverse P450s from mammals, plants, and bacteria, including P450cam (
      • Robin A.
      • Köhler V.
      • Jones A.
      • Ali A.
      • Kelly P.P.
      • O'Reilly E.
      • Turner N.J.
      • Flitsch S.L.
      Chimeric self-sufficient P450cam-RhFRed biocatalysts with broad substrate scope.
      ), P450 TxtE (
      • Zuo R.
      • Zhang Y.
      • Huguet-Tapia J.C.
      • Mehta M.
      • Dedic E.
      • Bruner S.D.
      • Loria R.
      • Ding Y.
      An artificial self-sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio-selectivity.
      ), CYP257A1 (
      • Kulig J.K.
      • Spandolf C.
      • Hyde R.
      • Ruzzini A.C.
      • Eltis L.D.
      • Grönberg G.
      • Hayes M.A.
      • Grogan G.
      A P450 fusion library of heme domains from Rhodococcus jostii RHA1 and its evaluation for the biotransformation of drug molecules.
      ), OleTJE (
      • Liu Y.
      • Wang C.
      • Yan J.
      • Zhang W.
      • Guan W.
      • Lu X.
      • Li S.
      Hydrogen peroxide-independent production of α-alkenes by OleTJE P450 fatty acid decarboxylase.
      ,
      • Lu C.
      • Shen F.
      • Wang S.
      • Wang Y.
      • Liu J.
      • Bai W.-J.
      • Wang X.
      An engineered self-sufficient biocatalyst enables scalable production of linear α-olefins from carboxylic acids.
      ), P450 isoflavone synthase (
      • Schückel J.
      • Rylott E.L.
      • Grogan G.
      • Bruce N.C.
      A gene-fusion approach to enabling plant cytochromes P450 for biocatalysis.
      ), and CYP2E1 (
      • Fairhead M.
      • Giannini S.
      • Gillam E.M.
      • Gilardi G.
      Functional characterisation of an engineered multidomain human P450 2E1 by molecular lego.
      ). In principle these fusion proteins can improve catalytic activity, coupling efficiency, and other electron transfer properties by simplifying the overall P450 redox system and process suitability (
      • McLean K.J.
      • Luciakova D.
      • Belcher J.
      • Tee K.L.
      • Munro A.W.
      Biological diversity of cytochrome P450 redox partner systems.
      ,
      • Ciaramella A.
      • Minerdi D.
      • Gilardi G.
      Catalytically self-sufficient cytochromes P450 for green production of fine chemicals.
      ). More challenging are engineering and expression of eukaryotic P450s in prokaryotic systems. First, compartmentalization is one consideration for Class II P450 systems, in that interaction between P450 and CPR typically occurs in the endoplasmic reticulum. Second, the molar ratio of P450 and its redox partner in a chimeric system is fixed at 1:1, instead of 15:1 with membrane-bounded P450s and CPR in the liver (
      • Shephard E.A.
      • Phillips I.R.
      • Bayney R.M.
      • Pike S.F.
      • Rabin B.R.
      Quantification of NADPH cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique.
      ). The construction of a chimeric protein will hamper the flexibility of modulating P450/CPR ratios. These shortcomings were circumvented during the heterologous production of oxygenated taxanes with engineered Taxus cuspidata P450 CYP725A4 and its native CPR in Escherichia coli. By optimizing the relative expression level of the CPR, physically unlinked to CYP725A4, the optimal ratio of P450 to CPR was shown to be ∼12 (
      • Biggs B.W.
      • Lim C.G.
      • Sagliani K.
      • Shankar S.
      • Stephanopoulos G.
      • De Mey M.
      • Ajikumar P.K.
      Overcoming heterologous protein interdependency to optimize P450-mediated taxol precursor synthesis in Escherichia coli.
      ) (Table 1 and Fig. 3, compound 25). This information may be useful in further studies on the efficient redox partner engineering system of eukaryotic P450s in E. coli in vivo.
      A change of redox partners may not only influence catalytic efficiency and product distribution (
      • Bernhardt R.
      • Urlacher V.B.
      Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations.
      ) but also affect the type and selectivity of a P450 reaction (
      • Zhang W.
      • Liu Y.
      • Yan J.
      • Cao S.
      • Bai F.
      • Yang Y.
      • Huang S.
      • Yao L.
      • Anzai Y.
      • Kato F.
      • Podust L.M.
      • Sherman D.H.
      • Li S.
      New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.
      ). For example, the multifunctional P450 MycG interacted with a free form of the reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx, supporting unnatural reactions leading to the production of seven novel demethylated mycinamicin products (in addition to the physiological hydroxylation/epoxidation reactions), which were not observed with either the chimeric fusion MycG-RhFRED or the spinach Fdx/FdR- supported reaction (
      • Zhang W.
      • Liu Y.
      • Yan J.
      • Cao S.
      • Bai F.
      • Yang Y.
      • Huang S.
      • Yao L.
      • Anzai Y.
      • Kato F.
      • Podust L.M.
      • Sherman D.H.
      • Li S.
      New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.
      ,
      • Anzai Y.
      • Li S.
      • Chaulagain M.R.
      • Kinoshita K.
      • Kato F.
      • Montgomery J.
      • Sherman D.H.
      Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics.
      ) (Table 1 and Fig. 3, compounds 26 and 27). Of particular importance, these findings highlight the potential role of redox partners in modulating the function of P450 enzymes and also suggest that P450 enzymes could be made even more versatile through interaction with a variety of redox partners to gain alternative functionalities.

      Substrate engineering

      Limited substrate scope is a general problem with biocatalysts. The eukaryotic Class II P450s, with high substrate promiscuity, are generally not particularly suitable for synthetic and biotechnological applications due to their membrane-bound nature. To expand the substrate repertoire of prokaryotic soluble P450s, the strategy of “substrate engineering” has been practiced more often in recent years (
      • Urlacher V.B.
      • Girhard M.
      Cytochrome P450 monooxygenases in biotechnology and synthetic biology.
      ,
      • Xu J.
      • Wang C.
      • Cong Z.
      Strategies for substrate-regulated P450 catalysis: from substrate engineering to co-catalysis.
      ,
      • Shoji O.
      • Watanabe Y.
      Bringing out the potential of wild-type cytochrome P450s using decoy molecules: oxygenation of nonnative substrates by bacterial cytochrome P450s.
      ).
      Typical substrate engineering is aimed toward modification of a nonnative substrate by covalently linking an anchoring/directing group to enable the productive binding of the engineered substrate. Some pioneering work on P450 substrate engineering involved PikC, based on extensive structural studies (
      • Sherman D.H.
      • Li S.
      • Yermalitskaya L.V.
      • Kim Y.
      • Smith J.A.
      • Waterman M.R.
      • Podust L.M.
      The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae.
      ). The hydrogen bond network and strong ionic interactions between the desosamine moiety (a common 2-deoxy sugar in the two native PikC substrates YC-17 and narbomycin) and several residues in the P450 BC loop and FG helices were identified as key determinants in substrate recognition (
      • Uhlmann H.
      • Kraft R.
      • Bernhardt R.
      C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P450.
      ). Thus, a series of substrates was chemically engineered to contain the desosamine anchoring group, and selective C–H bond hydroxylation of a series of unnatural carbolide substrates was achieved and mechanistically interpreted (
      • Li S.
      • Chaulagain M.R.
      • Knauff A.R.
      • Podust L.M.
      • Montgomery J.
      • Sherman D.H.
      Selective oxidation of carbolide C–H bonds by an engineered macrolide P450 mono-oxygenase.
      ). The regio-selectivity of PikC hydroxylation was further probed by testing the chemically modified YC-17 analogs with varied synthetic anchoring groups. As a result, the regioselectivity of PikC could be changed significantly (
      • Negretti S.
      • Narayan A.R.
      • Chiou K.C.
      • Kells P.M.
      • Stachowski J.L.
      • Hansen D.A.
      • Podust L.M.
      • Montgomery J.
      • Sherman D.H.
      Directing group-controlled regioselectivity in an enzymatic C–H bond oxygenation.
      ). Furthermore, PikCD50NRhFRED (a superior self-sufficient PikC mutant) was utilized to catalyze oxidation of nonactivated methylene C–H bonds of small nonnative substrates with further simplified synthetic anchors containing a dimethyl amino group (e.g. menthol and several bicyclic and bridged bicyclic compounds) (
      • Narayan A.R.
      • Jiménez-Osés G.
      • Liu P.
      • Negretti S.
      • Zhao W.
      • Gilbert M.M.
      • Ramabhadran R.O.
      • Yang Y.F.
      • Furan L.R.
      • Li Z.
      • Podust L.M.
      • Montgomery J.
      • Houk K.N.
      • Sherman D.H.
      Enzymatic hydroxylation of an unactivated methylene C–H bond guided by molecular dynamics simulations.
      ). A substrate engineering approach was also successfully applied to the major drug-metabolizing human P450 CYP3A4 toward theobromine analogues (
      • Polic V.
      • Cheong K.J.
      • Hammerer F.
      • Auclair K.
      Regioselective epoxidations by cytochrome P450 3A4 using a theobromine chemical auxiliary to predictably produce N-protected β- or γ-amino epoxides.
      ), CYP2E1 toward nicotinate esters (
      • Ménard A.
      • Fabra C.
      • Huang Y.
      • Auclair K.
      Type II ligands as chemical auxiliaries to favor enzymatic transformations by P450 2E1.
      ), and P450BM3 on mono- and polysaccharides, with predictable control of the regio- and stereoselectivity (
      • Lewis J.C.
      • Bastian S.
      • Bennett C.S.
      • Fu Y.
      • Mitsuda Y.
      • Chen M.M.
      • Greenberg W.A.
      • Wong C.H.
      • Arnold F.H.
      Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450BM3 demethylases.
      ).
      Recently, based on the understanding of the structural basis for substrate recognition in 4-cresol biodegradation by Corynebacterium glutamicum P450 CreJ, the biocatalytically installed phosphate group (attached by a ATP-dependent two-subunit phosphatase CreHI) was harnessed as an anchoring/directing group to deliver a group of p- and m-alkyphenols into the active site of P450 CreJ, achieving the highly challenging selective oxidation of the aliphatic C–H bonds of the tested alkylphenols in a controlled manner (
      • Du L.
      • Dong S.
      • Zhang X.
      • Jiang C.
      • Chen J.
      • Yao L.
      • Wang X.
      • Wan X.
      • Liu X.
      • Wang X.
      • Huang S.
      • Cui Q.
      • Feng Y.
      • Liu S.J.
      • Li S.
      Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system.
      ). This biosynthetic approach, without any chemical modification steps, may find useful applications in the pharmaceutical, biomanufacturing, and environmental remediation industries.
      Distinct from typical substrate engineering using chemically or biologically modified substrates, Watanabe and his associates have systematically developed an atypical substrate engineering strategy, “decoy” substrate engineering, in which an inactive “dummy” substrate (decoy molecule) is used to trigger the P450-catalyzed reaction on the real nonnative substrate (
      • Xu J.
      • Wang C.
      • Cong Z.
      Strategies for substrate-regulated P450 catalysis: from substrate engineering to co-catalysis.
      ,
      • Shoji O.
      • Watanabe Y.
      Bringing out the potential of wild-type cytochrome P450s using decoy molecules: oxygenation of nonnative substrates by bacterial cytochrome P450s.
      ,
      • Shoji O.
      • Watanabe Y.
      Monooxygenation of nonnative substrates catalyzed by bacterial cytochrome P450s facilitated by decoy molecules.
      ). Notably, there is no covalent linkage between the decoy and real substrates. A decoy molecule has a similar chemical structure to native substrate, so that it can be recognized and accommodated by the P450 enzyme, and its binding can reshape the substrate-binding pocket for the binding of a nonnative substrate, which can then be oxidized more efficiently. The first generation of decoy substrates for P450BSβ (CYP152A1) were short-chain fatty acids (
      • Lee D.-S.
      • Yamada A.
      • Sugimoto H.
      • Matsunaga I.
      • Ogura H.
      • Ichihara K.
      • Adachi S.
      • Park S.-Y.
      • Shiro Y.
      Substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome P450 from Bacillus subtilis crystallographic, spectroscopic, and mutational studies.
      ), followed by different types of perfluorinated fatty acids bearing shorter alkyl chains (
      • Kawakami N.
      • Shoji O.
      • Watanabe Y.
      Use of perfluorocarboxylic acids to trick cytochrome P450BM3 into initiating the hydroxylation of gaseous alkanes.
      ), N-perfluoroacyl amino acids (
      • Cong Z.
      • Shoji O.
      • Kasai C.
      • Kawakami N.
      • Sugimoto H.
      • Shiro Y.
      • Watanabe Y.
      Activation of wild-type cytochrome P450BM3 by the next generation of decoy molecules: enhanced hydroxylation of gaseous alkanes and crystallographic evidence.
      ), and nonfluorinated N-acyl amino acids (
      • Shoji O.
      • Yanagisawa S.
      • Stanfield J.K.
      • Suzuki K.
      • Cong Z.
      • Sugimoto H.
      • Shiro Y.
      • Watanabe Y.
      Direct hydroxylation of benzene to phenol by cytochrome P450BM3 triggered by amino acid derivatives.
      ) for P450BM3. Four generations of decoy molecules have been developed, not only for expanding the substrate capabilities of P450s but also for exploring the stereoselectivity and enantioselectivity toward various substrates (e.g. styrene and ethylbenzene), leading to diverse chemical scaffolds that can be applied in the pharmaceutical industry (
      • Shoji O.
      • Fujishiro T.
      • Nakajima H.
      • Kim M.
      • Nagano S.
      • Shiro Y.
      • Watanabe Y.
      Hydrogen peroxide dependent monooxygenations by tricking the substrate recognition of cytochrome P450BSβ.
      ).
      Recently, a class of dual-functional small molecules containing an anchoring group for binding to the P450 and a basic group for H2O2 activation was elegantly designed and successfully transformed the P450BM3 monooxygenase into a peroxygenase. N-(ω-Imidazolyl)-hexanoyl-l-phenylalanine (Im-C6-Phe) was the optimal co-catalyst supporting the P450BM3-H2O2 system (
      • Ma N.
      • Chen Z.
      • Chen J.
      • Chen J.
      • Wang C.
      • Zhou H.
      • Yao L.
      • Shoji O.
      • Watanabe Y.
      • Cong Z.
      Dual-functional small molecules for generating an efficient cytochrome P450BM3 peroxygenase.
      ). The rate of epoxidation of (R)-(+)-styrene and the enantiomeric specificity (ee value) of the product were dramatically increased (to ee 91%) by this innovative substrate engineering approach. This engineered peroxide-driven P450BM3 system was further utilized to hydroxylate small alkanes with the assistance of Im-C6-Phe (
      • Chen J.
      • Kong F.
      • Ma N.
      • Zhao P.
      • Liu C.
      • Wang X.
      • Cong Z.
      Peroxide-driven hydroxylation of small alkanes catalyzed by an artificial P450BM3 peroxygenase system.
      ) (Fig. 3, compound 28).
      As an alternative strategy to protein engineering, the observed exquisite specificity and selectivity introduced by substrate engineering of P450 enzymes has highlighted the profound influence of the substrate-anchoring groups on the functional plasticity of P450s (
      • Xu J.
      • Wang C.
      • Cong Z.
      Strategies for substrate-regulated P450 catalysis: from substrate engineering to co-catalysis.
      ). Thus, this strategy has the potential to improve the synthetic utility of P450s. For example, it could be used for building a library of chemical structures that bear hydroxyl groups at various positions as functional group handles for further synthetic transformations (e.g. attachment of sugars).

      Electron source engineering

      Almost all natural P450s are cofactor-dependent enzymes, which are often expensive and must be recycled or circumvented from a process engineering perspective. To resolve this problem, several methods have been established on a laboratory scale over several decades, including cofactor regeneration systems, peroxide replacement, electrochemical approaches, and light-activated systems (
      • Urlacher V.B.
      • Girhard M.
      Cytochrome P450 monooxygenases in biotechnology and synthetic biology.
      ,
      • Urlacher V.B.
      • Lutz-Wahl S.
      • Schmid R.D.
      Microbial P450 enzymes in biotechnology.
      ,
      • Shumyantseva V.V.
      • Bulko T.V.
      • Archakov A.I.
      Electrochemical reduction of cytochrome P450 as an approach to the construction of biosensors and bioreactors.
      ).

      Cofactor engineering

      NAD(P)H regeneration is a popular method in cell-free biocatalysis and biotransformation, in which constant supply of reducing equivalents is achieved by introducing a second reaction system to reduce NAD(P)+. Many cost-effective approaches have been widely developed in industry not only for P450s but also for many NAD(P)H-dependent oxidoreductases, including glucose dehydrogenase/glucose (
      • Lu Y.
      • Mei L.
      Co-expression of P450 BM3 and glucose dehydrogenase by recombinant Escherichia coli and its application in an NADPH-dependent indigo production system.
      ), glucose-6-phosphate dehydrogenase/glucose 6-phosphate (
      • Ding Y.
      • Seufert W.H.
      • Beck Z.Q.
      • Sherman D.H.
      Analysis of the cryptophycin P450 epoxidase reveals substrate tolerance and cooperativity.
      ), isocitrate dehydrogenase/isocitrate (
      • Meinhold P.
      • Peters M.W.
      • Hartwick A.
      • Hernandez A.R.
      • Arnold F.H.
      Engineering cytochrome P450 BM3 for terminal alkane hydroxylation.
      ), formate dehydrogenase/formate (
      • Taylor M.
      • Lamb D.C.
      • Cannell R.J.
      • Dawson M.J.
      • Kelly S.L.
      Cofactor recycling with immobilized heterologous cytochrome P450 105D1 (CYP105D1).
      ), ethanol dehydrogenase/ethanol (
      • Sandberg M.
      • Yasar Ü.
      • Strömberg P.
      • Höög J.O.
      • Eliasson E.
      Oxidation of celecoxib by polymorphic cytochrome P450 2C9 and alcohol dehydrogenase.
      ), and engineered phosphite dehydrogenase/phosphite (
      • Johannes T.W.
      • Woodyer R.D.
      • Zhao H.
      Efficient regeneration of NADPH using an engineered phosphite dehydrogenase.
      ).
      The “peroxide shunt pathway” (Fig. 1) has also been successfully engineered through directed evolution because it could be industrially relevant in making P450s use the cheaper peroxides (e.g. H2O2) rather than NAD(P)H as the electron donor (
      • Du L.
      • Ma L.
      • Qi F.
      • Zheng X.
      • Jiang C.
      • Li A.
      • Wan X.
      • Liu S.J.
      • Li S.
      Characterization of a unique pathway for 4-cresol catabolism initiated by phosphorylation in Corynebacterium glutamicum.
      ). For instance, an efficient H2O2 regeneration system was recently applied to the catalytic reaction of a P450 peroxygenase by coupling with an oxidase, as demonstrated by an enzyme cascade comprised of the P450 peroxygenase P450CLA or P450Spα and the enantioselective α-hydroxyacid oxidase (S)-α-HAO from Aerococcus viridans or d-lactate oxidase GO-LOX from Gluconobacter oxydans. This enzyme cascade efficiently converted fatty acids of various chain length (C6:0 to C10:0) into the chemical intermediate α-ketoacids in the presence of an internal H2O2-recycling system (
      • Gandomkar S.
      • Dennig A.
      • Dordic A.
      • Hammerer L.
      • Pickl M.
      • Haas T.
      • Hall M.
      • Faber K.
      Biocatalytic oxidative cascade for the conversion of fatty acids into α-ketoacids via internal H2O2 recycling.
      ). Moreover, a novel P450 monooxygenase-peroxygenase cascade consisting of P450BM3 and OleTJE was recently developed for asymmetric catalysis in the conversion of 3-phenylpropionic acid to (R)-phenyl glycol without an external supply of H2O2 (
      • Yu D.
      • Wang J.B.
      • Reetz M.T.
      Exploiting designed oxidase-peroxygenase mutual benefit system for asymmetric cascade reactions.
      ).

      Electrochemical approaches

      Electrochemical reductions have been used to circumvent the requirement for redox partners in shuttling electrons from NADPH to P450, with the electrode being the source of reducing equivalents. Progress with electrode-adsorbed/immobilization of P450 enzymes on various electrodes has been accomplished by engineering of both electrodes and enzymes, including layer-by-layer films with polyions (
      • Krishnan S.
      • Abeykoon A.
      • Schenkman J.B.
      • Rusling J.F.
      Control of electrochemical and ferryloxy formation kinetics of cyt P450s in polyion films by heme iron spin state and secondary structure.
      ,
      • Krishnan S.
      • Schenkman J.B.
      • Rusling J.F.
      Bioelectronic delivery of electrons to cytochrome P450 enzymes.
      ), a cobalt(III) sepulchrate (Zn/CoIIIsep) mediator (
      • Belsare K.D.
      • Horn T.
      • Ruff A.J.
      • Martinez R.
      • Magnusson A.
      • Holtmann D.
      • Schrader J.
      • Schwaneberg U.
      Directed evolution of P450cin for mediated electron transfer.
      ,
      • Tosstorff A.
      • Dennig A.
      • Ruff A.J.
      • Schwaneberg U.
      • Sieber V.
      • Mangold K.-M.
      • Schrader J.
      • Holtmann D.
      Mediated electron transfer with monooxygenases—insight in interactions between reduced mediators and the co-substrate oxygen.
      ), covalent immobilization to a gold (Au) self-assembled monolayer (
      • Mak L.H.
      • Sadeghi S.J.
      • Fantuzzi A.
      • Gilardi G.
      Control of human cytochrome P450 2E1 electrocatalytic response as a result of unique orientation on gold electrodes.
      ), and nanomaterial-modified electrodes (
      • Lu J.
      • Zhang Y.
      • Li H.
      • Yu J.
      • Liu S.
      Electrochemically driven drug metabolism via a CYP1A2-UGT1A10 bienzyme confined in a graphene nano-cage.
      ,
      • Lu J.
      • Cui D.
      • Li H.
      • Zhang Y.
      • Liu S.
      Cytochrome P450 bienzymes assembled on Au/chitosan/reduced graphene oxide nanosheets for electrochemically-driven drug cascade metabolism.
      ). Due to the limitations of applying purified soluble P450s on various electrodes, protein film electrochemistry has been considered in electrocatalytic studies. Some of the studies include engineered membrane-bound human P450s with the reductase protein CPR added to a modified gold (Au) electrode (
      • Mie Y.
      • Suzuki M.
      • Komatsu Y.
      Electrochemically driven drug metabolism by membranes containing human cytochrome P450.
      ,
      • Nerimetla R.
      • Walgama C.
      • Singh V.
      • Hartson S.D.
      • Krishnan S.
      Mechanistic insights into voltage-driven biocatalysis of a cytochrome P450 bactosomal film on a self-assembled monolayer.
      ), (membrane-bound) liver microsomes with rat and human P450s immobilized on carbon electrodes and carbon nanostructures (
      • Walgama C.
      • Nerimetla R.
      • Materer N.F.
      • Schildkraut D.
      • Elman J.F.
      • Krishnan S.
      A simple construction of electrochemical liver microsomal bioreactor for rapid drug metabolism and inhibition assays.
      ,
      • Wasalathanthri D.P.
      • Malla S.
      • Faria R.C.
      • Rusling J.F.
      Electrochemical activation of the natural catalytic cycle of cytochrome P450s in human liver microsomes.
      ,
      • Nerimetla R.
      • Krishnan S.
      Electrocatalysis by subcellular liver fractions bound to carbon nanostructures for stereoselective green drug metabolite synthesis.
      ), and purified P450s assembled with membrane-bound CPR on pyrolytic graphite electrodes (
      • Krishnan S.
      • Wasalathanthri D.
      • Zhao L.
      • Schenkman J.B.
      • Rusling J.F.
      Efficient bioelectronic actuation of the natural catalytic pathway of human metabolic cytochrome P450s.
      ).

      Light-activated systems

      Systems have been developed by utilizing energy from light to drive the P450 catalytic cycle. Three main pathways have been designed based on the catalytic nature of P450 enzymes. The first takes advantage of the peroxide shunt pathway, with controlled generation of the reactive oxygen species in situ, mainly limited to the CYP152 P450 family with peroxygenase activity (e.g. P450BSβ, CYPC1a, and OleTJE) (
      • Girhard M.
      • Kunigk E.
      • Tihovsky S.
      • Shumyantseva V.V.
      • Urlacher V.B.
      Light-driven biocatalysis with cytochrome P450 peroxygenases.
      ,
      • Zachos I.
      • Gaβmeyer S.K.
      • Bauer D.
      • Sieber V.
      • Hollmann F.
      • Kourist R.
      Photobiocatalytic decarboxylation for olefin synthesis.
      ). The second approach mimics the native electron transfer pathway by employment of redox partners to transfer electrons from a photosensitizer instead of cofactor, exemplified by a deazaflavin-dependent photoregeneration system (
      • Zilly F.E.
      • Taglieber A.
      • Schulz F.
      • Hollmann F.
      • Reetz M.T.
      Deazaflavins as mediators in light-driven cytochrome P450 catalyzed hydroxylations.
      ) and photosystem I with ferredoxin as an electron mediator (
      • Wlodarczyk A.
      • Gnanasekaran T.
      • Nielsen A.Z.
      • Zulu N.N.
      • Mellor S.B.
      • Luckner M.
      • Thøfner J.F.B.
      • Olsen C.E.
      • Mottawie M.S.
      • Burow M.
      • Pribil M.
      • Feussner I.
      • Møller B.L.
      • Jensen P.E.
      Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803.
      ,
      • Nielsen A.Z.
      • Ziersen B.
      • Jensen K.
      • Lassen L.M.
      • Olsen C.E.
      • Møller B.L.
      • Jensen P.E.
      Redirecting photosynthetic reducing power toward bioactive natural product synthesis.
      ,
      • Mellor S.B.
      • Nielsen A.Z.
      • Burow M.
      • Motawia M.S.
      • Jakubauskas D.
      • Møller B.L.
      • Jensen P.E.
      Fusion of ferredoxin and cytochrome P450 enables direct light-driven biosynthesis.
      ,
      • Jensen K.
      • Jensen P.E.
      • Møller B.L.
      Light-driven cytochrome P450 hydroxylations.
      ). The third simply involves direct shuttling of electrons to the heme active site and circumvention of redox partners by the employment of a fluorescent dye, eosin Y (
      • Di Nardo G.
      • Gilardi G.
      Optimization of the bacterial cytochrome P450 BM3 system for the production of human drug metabolites.
      ,
      • Park J.H.
      • Lee S.H.
      • Cha G.S.
      • Choi D.S.
      • Nam D.H.
      • Lee J.H.
      • Lee J.K.
      • Yun C.H.
      • Jeong K.J.
      • Park C.B.
      Cofactor-free light-driven whole-cell cytochrome P450 catalysis.
      ), and covalently attached Ru(II)-diimine complexes (
      • Lam Q.
      • Kato M.
      • Cheruzel L.
      Ru(II)-diimine functionalized metalloproteins: from electron transfer studies to light-driven biocatalysis.
      ,
      • Tran N.H.
      • Huynh N.
      • Chavez G.
      • Nguyen A.
      • Dwaraknath S.
      • Nguyen T.A.
      • Nguyen M.
      • Cheruzel L.
      A series of hybrid P450 BM3 enzymes with different catalytic activity in the light-initiated hydroxylation of lauric acid.
      ). However, there are still many challenges for photobased strategies in practical P450 catalysis, including the low efficiency of light conversion, weak coupling efficiency, low protein stability and activity, and technical difficulties.
      Readers are referred to details in the individual research and reviewed publications regarding enzymatic regeneration systems (
      • Liu W.
      • Wang P.
      Cofactor regeneration for sustainable enzymatic biosynthesis.
      ), reactive oxygen species (
      • Shoji O.
      • Watanabe Y.
      Peroxygenase reactions catalyzed by cytochromes P450.
      ), electrochemical reduction (
      • Sadeghi S.J.
      • Fantuzzi A.
      • Gilardi G.
      Breakthrough in P450 bioelectrochemistry and future perspectives.
      ), and light-activated approaches (
      • Shalan H.
      • Kato M.
      • Cheruzel L.
      Keeping the spotlight on cytochrome P450.
      ,
      • Mellor S.B.
      • Vavitsas K.
      • Nielsen A.Z.
      • Jensen P.E.
      Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins.
      ). It is fairly important to find alternative economical electron sources for development of sustainable P450 catalytic systems to reduce the production costs. Except for the NAD(P)H- and H2O2-regenerating systems that have found practical applications in industry, other strategies are still in the developmental phase due to several critical problems (e.g. low coupling efficiency and redox potential management). Advances in biotechnology, discovery, and design of novel catalytic units and more interdisciplinary approaches may help overcome these challenges.

      P450-related metabolic engineering

      Rapid development of synthetic biology has led to more and more P450-related metabolic engineering work that has integrated protein, substrate, and cofactor engineering of P450 systems. These efforts have enabled cost-effective bioproduction of many commercial compounds as “natural” products for variant purposes.
      As some of the most important enzymes in natural product biosynthesis, numerous natural and engineered P450s have already been included in the metabolic engineering toolbox. For example, the aforementioned CYP71AV1 has been used for bioproduction of the antimalarial drug precursor artemisinic acid (
      • Ro D.K.
      • Paradise E.M.
      • Ouellet M.
      • Fisher K.J.
      • Newman K.L.
      • Ndungu J.M.
      • Ho K.A.
      • Eachus R.A.
      • Ham T.S.
      • Kirby J.
      • Chang M.C.
      • Withers S.T.
      • Shiba Y.
      • Sarpong R.
      • Keasling J.D.
      Production of the antimalarial drug precursor artemisinic acid in engineered yeast.
      ), and CYP75 enzymes have been used for the hydroxylation on the B-ring of anthocyanidins to produce commercial blue roses and carnations (
      • Tanaka Y.
      • Brugliera F.
      Flower colour and cytochromes P450.
      ). However, in both cases P450-related metabolic engineering has not been a straightforward process. In addition to a robust P450 with the desired activity, other requirements include high-level heterologous expression, optimization of metabolic fluxes, choice of a suitable heterologous host, and the deletion or silencing of competing pathways.
      P450 SalSyn from Papaver somniferum is a key element in the complete biosynthesis of two opioid drugs in S. cerevisiae. Expression of an engineered P450 SalSyn with increased activity in generating the pro-morphinan scaffold (salutaridine) (Table 1 and Fig. 3, compound 29), together with co-expression of 19 of 21 heterologous enzymes and two native enzymes and deletion of one native yeast gene, resulted in the microbial production of thebaine/hydrocodone (
      • Galanie S.
      • Thodey K.
      • Trenchard I.J.
      • Filsinger Interrante M.
      • Smolke C.D.
      Complete biosynthesis of opioids in yeast.
      ). The critical bioconversion of (S)-reticuline to (R)-reticuline was mediated by a CYP82Y2-like P450 (1,2-dehydroreticuline synthase) from Papaver bracteatum fused with 1,2-dehydroreticuline reductase, which was achieved by complementary approaches including gene mining, protein mutagenesis, codon optimization, and heterologous expression in yeast (
      • Galanie S.
      • Thodey K.
      • Trenchard I.J.
      • Filsinger Interrante M.
      • Smolke C.D.
      Complete biosynthesis of opioids in yeast.
      ) (Table 1 and Fig. 3, compound 30).
      Szczebara et al. (
      • Szczebara F.M.
      • Chandelier C.
      • Villeret C.
      • Masurel A.
      • Bourot S.
      • Duport C.
      • Blanchard S.
      • Groisillier A.
      • Testet E.
      • Costaglioli P.
      • Cauet G.
      • Degryse E.
      • Balbuena D.
      • Winter J.
      • Achstetter T.
      • et al.
      Total biosynthesis of hydrocortisone from a simple carbon source in yeast.
      ) fully designed a de novo biosynthetic pathway involving 13 engineered enzymes in recombinant S. cerevisiae strains, in which the total biosynthesis of hydrocortisol and several steroids was achieved. First, recombinant S. cerevisiae was engineered to overproduce egrosta-5-eneol and ergosta-5,22-dieneol, which was further converted into pregnenolone by CYP11A1 (Table 1, Fig. 3, compound 31). Finally, the oxidation steps that are sequentially catalyzed by 3β-hydroxysteroid dehydrogenase/isomerase, CYP17A1, CYP21A1, and CYP11B1 were reconstituted, giving rise to the production of progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, and the final product hydrocortisol (
      • Szczebara F.M.
      • Chandelier C.
      • Villeret C.
      • Masurel A.
      • Bourot S.
      • Duport C.
      • Blanchard S.
      • Groisillier A.
      • Testet E.
      • Costaglioli P.
      • Cauet G.
      • Degryse E.
      • Balbuena D.
      • Winter J.
      • Achstetter T.
      • et al.
      Total biosynthesis of hydrocortisone from a simple carbon source in yeast.
      ) (Table 1, compounds 3234).
      Optimization of redox partners in vivo is also important in P450-related metabolic engineering. Huang and co-workers (
      • Guo J.
      • Zhou Y.J.
      • Hillwig M.L.
      • Shen Y.
      • Yang L.
      • Wang Y.
      • Zhang X.
      • Liu W.
      • Peters R.J.
      • Chen X.
      • Zhao Z.K.
      • Huang L.
      CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.
      ) reconstituted the catalytic activity of CYP76AH1 in the bioconversion of miltiradiene to ferruginol, a key bioactive component of the Chinese medicinal plant Salvia miltiorrhiza, in a miltiradiene-overproducing yeast strain. The production of 10.5 mg of ferruginol per liter was enabled with a surrogate redox partner protein, smCPR1, from Salvia miltiorrhiza Bungefor (Table 1 and Fig. 3, compound 35). Zhao et al. (
      • Zhao F.
      • Bai P.
      • Liu T.
      • Li D.
      • Zhang X.
      • Lu W.
      • Yuan Y.
      Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae.
      ) designed an artificial biosynthetic pathway of protopanaxadiol (Table 1 and Fig. 3, compound 36), the precursor of bioactive ginsenosides of Panax ginseng, in an engineered S. cerevisiae strain. The self-sufficient P450 protopanaxadiol synthase was constructed by fusing it with an AtCPR from Arabidopsis thaliana, which resulted in a 71% increase in protopanaxadiol production (>1400 mg/liter) compared with co-expression of the two stand-alone components, protopanaxadiol synthase and AtCPR (
      • Zhao F.
      • Bai P.
      • Liu T.
      • Li D.
      • Zhang X.
      • Lu W.
      • Yuan Y.
      Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae.
      ) (Table 1 and Fig. 3, compound 36).
      Distinct from the de novo biosynthesis of high value-added compounds in recombinant cells from sugar sources, engineering a P450 system into a robust whole-cell biocatalyst is also a useful strategy. For instance, when P450 boxA from Streptomyces sp. TM-7 was introduced into an efflux pump inactivation mutant of E. coli, the production of 1.7 g of pravastatin per liter (from compactin) was achieved, which was 7-fold higher than that using WT E. coli (
      • Fujii T.
      • Fujii Y.
      • Machida K.
      • Ochiai A.
      • Ito M.
      Efficient biotransformations using Escherichia coli with tolC acrAB mutations expressing cytochrome P450 genes.
      ) (Table 1). When this system was expressed in the pravastatin-tolerant actinomycetes strain P. autotrophica, accumulation of pravastatin reached a level of 14 g/liter, 8-fold higher than in its E. coli counterpart and 3-fold higher than in the original Streptomyces sp. TM-7 (
      • Fujii Y.
      • Norihisa K.
      • Fujii T.
      • Aritoku Y.
      • Kagawa Y.
      • Sallam K.I.
      • Johdo O.
      • Arisawa A.
      • Tamura T.
      Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor.
      ). These results indicate the importance of a suitable heterologous host for construction of robust whole-cell biocatalysts.
      Cofactor regeneration and cofactor-free P450 systems have also found applications in whole-cell biocatalysts. Watanabe and associates developed E. coli as a whole-cell biocatalyst vehicle to mediate the hydroxylation of benzene into phenol by WT P450BM3 in the presence of decoy molecules (
      • Karasawa M.
      • Stanfield J.K.
      • Yanagisawa S.
      • Shoji O.
      • Watanabe Y.
      Whole-cell biotransformation of benzene to phenol catalysed by intracellular cytochrome P450BM3 activated by external additives.
      ). A novel whole-cell P450 photobiocatalysis system driven by the electrons from eosin Y instead of redox partners and cofactors was used for the bioconversion of pharmaceuticals with engineered bacterial P450s and human P450s (
      • Park J.H.
      • Lee S.H.
      • Cha G.S.
      • Choi D.S.
      • Nam D.H.
      • Lee J.H.
      • Lee J.K.
      • Yun C.H.
      • Jeong K.J.
      • Park C.B.
      Cofactor-free light-driven whole-cell cytochrome P450 catalysis.
      ). Different cofactor regeneration systems were also applied in many cases of whole-cell biotransformation, such as CYPsb-21 (
      • Ma L.
      • Du L.
      • Chen H.
      • Sun Y.
      • Huang S.
      • Zheng X.
      • Kim E.S.
      • Li S.
      Reconstitution of the in vitro activity of the cyclosporine-specific P450 hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system.
      ), P450 SMO from Rhodococcus sp. (
      • Zhang J.D.
      • Li A.T.
      • Yu H.L.
      • Imanaka T.
      • Xu J.H.
      Synthesis of optically pure S-sulfoxide by Escherichia coli transformant cells coexpressing the P450 monooxygenase and glucose dehydrogenase genes.
      ), and CYP106A2 (PDB entry 4YT3) from B. megaterium ATCC 13368 (
      • Janocha S.
      • Carius Y.
      • Hutter M.
      • Lancaster C.R.
      • Bernhardt R.
      Crystal structure of CYP106A2 in substrate-free and substrate-bound form.
      ) (Table 1).

      Conclusions and future prospects

      Compared with some robust and widely applied commercial enzymes (e.g. hydrolases and ligases), P450 biocatalysts are still very limited by practical disadvantages, including low activity, poor stability, narrow substrate scope, and cofactor and redox partner dependence for most P450s. However, the irresistible regio- and stereoselectivity inherent in P450s continues to attract extensive efforts to deliver more P450 systems for industrial applications in production of pharmaceuticals, fine chemicals, flavors, and fragrances.
      Exciting new biotechnology approaches have contributed to breakthroughs in P450 system engineering for practical catalysis in the past decade (
      • Urlacher V.B.
      • Girhard M.
      Cytochrome P450 monooxygenases in biotechnology and synthetic biology.
      ,
      • Wei Y.
      • Ang E.L.
      • Zhao H.
      Recent developments in the application of P450 based biocatalysts.
      ,
      • Yasuda K.
      • Sugimoto H.
      • Hayashi K.
      • Takita T.
      • Yasukawa K.
      • Ohta M.
      • Kamakura M.
      • Ikushiro S.
      • Shiro Y.
      • Sakaki T.
      Protein engineering of CYP105s for their industrial uses.
      ,
      • Xu J.
      • Wang C.
      • Cong Z.
      Strategies for substrate-regulated P450 catalysis: from substrate engineering to co-catalysis.
      ). The multiple engineering strategies mentioned in this review have significantly improved the substrate scope, stability (
      • Wong T.S.
      • Arnold F.H.
      • Schwaneberg U.
      Laboratory evolution of cytochrome P450 BM-3 monooxygenase for organic cosolvents.
      ), catalytic efficiency (
      • McLean K.J.
      • Hans M.
      • Meijrink B.
      • van Scheppingen W.B.
      • Vollebregt A.
      • Tee K.L.
      • van der Laan J.M.
      • Leys D.
      • Munro A.W.
      • van den Berg M.A.
      Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.
      ), and reaction specificity (
      • Kille S.
      • Zilly F.E.
      • Acevedo J.P.
      • Reetz M.T.
      Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution.
      ) of P450s (Fig. 4). Moreover, P450-related metabolic engineering has opened a door for industrial application of the low-stability P450 systems (
      • Urlacher V.B.
      • Girhard M.
      Cytochrome P450 monooxygenases in biotechnology and synthetic biology.
      ,
      • Urlacher V.B.
      • Girhard M.
      Cytochrome P450 monooxygenases: an update on perspectives for synthetic application.
      ). A very recent development is the application of mammalian P450s selected by mining sequences of their relatives and prediction of primordial precursors, which unexpectedly has yielded more thermostable catalysts. These have broad specificity and can be used to generate useful products at much higher temperatures, increasing their efficiency (
      • Gumulya Y.
      • Baek J.-M.
      • Wun S.-J.
      • Thomson R.E.S.
      • Harris K.L.
      • Hunter D.J.B.
      • Behrendorff J.B.Y.H.
      • Kulig J.
      • Zheng S.
      • Wu X.
      • Wu B.
      • Stok J.E.
      • De Voss J.J.
      • Schenk G.
      • Jurva U.
      • et al.
      Engineering highly functional thermostable proteins using ancestral sequence reconstruction.
      ). Lately, the integration of P450 catalytic systems into multienzyme cascades has been shown to be useful (
      • Yu D.
      • Wang J.B.
      • Reetz M.T.
      Exploiting designed oxidase-peroxygenase mutual benefit system for asymmetric cascade reactions.
      ).
      Versatile P450s are vital elements in the enzyme toolbox gifted from nature, and they will become much more powerful in the era of synthetic biology. In the future, we envision that functional mining of new P450s, construction of systematic libraries of P450s and redox partners, design of new electron-sourcing systems, the development of stable and highly efficient redox partner–independent P450 systems, and perhaps even the de novo design of P450s on demand will be the frontiers of P450 system engineering. Close collaboration between biologists, chemists, physicists, engineers, computer scientists, and mathematicians will be needed for engineering future new-concept P450 systems, which can create new exciting opportunities in practical catalysis for this most versatile superfamily of enzymes.

      Acknowledgments

      We thank Dr. Stella A. Child for comments on the manuscript.

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