Cytochrome P450 Monooxygenases in Biotechnology and Synthetic Biology

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Review

Cytochrome P450 Monooxygenases in Biotechnology and Synthetic Biology Vlada B. Urlacher1,2,* and Marco Girhard1,2,* Cytochromes P450 (P450 or CYP) are heme-containing enzymes that catalyze the introduction of one atom of molecular oxygen into nonactivated C–H bonds, often in a regio- and stereoselective manner. This ability, combined with a tremendous number of accepted substrates, makes P450s powerful biocatalysts. Sixty years after their discovery, P450 systems are recognized as essential bio-bricks in synthetic biology approaches to enable production of highvalue complex molecules in recombinant hosts. Recent impressive results in protein engineering led to P450s with tailored properties that are even able to catalyze abiotic reactions. The introduction of P450s in artificial multi-enzymatic cascades reactions and chemo-enzymatic processes offers exciting future perspectives to access novel compounds that cannot be synthesized by nature or by chemical routes.

Highlights Cytochromes P450 are ubiquitous enzymes accepting a tremendous number of substrates and catalyzing a broad range of reactions with potential applications in biotechnology and synthetic biology. P450s were engineered to catalyze abiotic reactions such as carbene or nitrene transfers, opening up completely new perspectives in synthetic chemistry. Lately, P450s have successfully been introduced into artificial multi-enzyme cascades, both in vitro and in vivo, providing alternative routes for retrosynthetic production of high-value oxyfunctionalized compounds.

Cytochrome P450 Monooxygenases: A Short Survey Cytochromes P450 (P450 or CYP) are heme-thiolate proteins (see Glossary) in which the heme prosthetic group is linked to the apoprotein via an axial conserved cysteine. In the presence of molecular oxygen and the reduced cellular cofactors NADH or NADPH, most P450s catalyze monooxygenation reactions. The classical catalytic P450 cycle involves a series of sequential steps, including activation of molecular oxygen, its heterolytic cleavage, and formation of the hydroxylated product (Box 1).

Harnessing the synthetic potential of P450s in chemo-enzymatic processes or as part of reconstituted biosynthetic pathways in microbial hosts provides promising strategies for de novo synthesis of synthons and complex natural products, even though there are still some obstacles to overcome.

Besides hydroxylation of C–H bonds, oxidation reactions catalyzed by P450s include epoxidation of C¼C double bonds, N- and S-oxidations, N-, O-, S-dealkylations, C–C-bond cleavage (Box 1) [1], and some atypical conversions like phenolic coupling and BaeyerVilliger-type oxidations [2]. Recently, CYP102A1 (P450 BM3) from Bacillus megaterium was exploited through enzyme engineering and reaction design to perform abiotic reactions like oxidative deamination of alkyl azides (Figure 1A) [3], olefin cyclopropanation via carbene transfer (Figure 1B) [4], carbene N–H insertion to create C–N bonds (Figure 1C) [5], and intramolecular C–H amination reactions (Figure 1D) [6]. Engineered variants of P450 BM3 with the conserved axial cysteine residue replaced by serine (Figure 1E), designated ‘P411 BM30 [7], were further optimized by protein engineering and successfully exploited to sulfimidation to create new S–N bonds (Figure 1F) [8], aziridination of aryl olefins (Figure 1G) [9], as well as carbene transfer to alkynes (Figure 1H) [10]. Importantly, P411 BM3-based variants were able to catalyze these abiotic reactions in vivo with NADPH as reducing agent when introduced in Escherichia coli [7]. Other reports describe P450-catalyzed abiotic carbon–silicon [11] and carbon–boron bond formation [12]; however, in both cases, the selectivities of the screened P450 variants were lower when compared with engineered variants of cytochrome C from Rhodothermus marinus.

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1

Institute of Biochemistry, HeinrichHeine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany 2 http://www.biochemistry2.hhu.de/

*Correspondence: [email protected] (V.B. Urlacher) and [email protected] (Girhard).

https://doi.org/10.1016/j.tibtech.2019.01.001 © 2019 Elsevier Ltd. All rights reserved.

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Box 1. Cytochromes P450 Catalytic Mechanism and Naturally Catalyzed Reaction Types The classical P450 catalytic cycle starts with substrate binding that induces spin shift, allowing FeIII-to-FeII reduction. The electronic properties of the thiolate-bound heme-Fe enable activation of molecular oxygen, resulting in the formation of a highly reactive heme-FeIV oxo cation radical, so called ‘compound I’, that is responsible for substrate hydroxylation [98]. The two electrons involved in the catalytic cycle are transferred from NAD(P)H to the heme iron via redox partners, such as FAD- or FMN-containing reductases in combination with iron-sulfur cluster-containing ferredoxin or FMN-containing flavodoxins, as well as diflavin cytochrome P450 reductase (CPR). As for every biological system, there are of course exceptions, and various other types of P450 electron transfer chains exist in nature (reviewed in [104]). These include the so-called self-sufficient systems, in which the heme-containing monooxygenase domain is naturally fused to the redox partner domain(s). The best-studied natural self-sufficient CYP102A1 (P450 BM3) from Bacillus megaterium [105] is considered a potent candidate for biotechnological applications due to its high activity, simple handling, and very high evolvability [106]. Alternatively, hydrogen peroxide or organic peroxides can be used as an oxygen source allowing substrate oxidation by bypassing the electron transfer steps, as was described for natural peroxygenases from the CYP152 family [107]. Generally, P450s introduce one oxygen atom into the substrate, whereas the second one is reduced to water: R—H + NAD(P)H + H+ + O2 ! R—OH + NAD(P)+ + H2O After the initial hydroxylation, subsequent reactions can be catalyzed [1]: (i) N-oxidation; (ii) N-dealkylation, O-dealkylation, S-dealkylation; (iii) C-C-bond cleavage. Next to oxidation reactions, CYP55A1 from Fusarium oxysporum (P450nor) was reported to reduce nitric oxide (NO) to nitrous oxide (N2O) using electrons from NADH [108], and CYP1048A1 (TxtE) that uses NO and O2 to catalyze direct nitration of l-tryptophan [109].

Along with a high diversity of catalyzed reactions, P450s act on a broad range of chemically diverse substrates, ranging from rather ‘simple’ molecules such as saturated fatty acids, alkanes, or monoterpenes to ‘complex’ molecules such as eicosanoids, vitamins, steroids, antibiotics, as well as diverse drugs and xenobiotics [13,14]. Similar to the catalyzed reaction types, the repertoire of P450 substrates is steadily extending [15,16].

Current and Emerging Trends Regio-, chemo-, and stereoselective biocatalytic oxyfunctionalizations performed on a broad range of organic molecules are not only indispensable for life on earth but also offer a powerful tool to accomplish chemical transformations that remain a considerable challenge in synthetic chemistry [17]. In our last review on P450s [18], aspects relevant to their synthetic application were summarized with foci on newly identified P450s with novel properties, on the engineering of well-characterized P450s, as well as on aspects of P450 whole-cell biocatalysis. Since then, engineering of P450s for novel properties continued to be a research field of major interest, as verified by the steadily appearing reports on directed evolution and rational design [19–23]. Notably, the focus has now shifted towards improving regio- and stereoselectivity of P450mediated oxidations and their transition into (chemo-)enzymatic multistep cascades for production of valuable compounds. The exponentially growing pool of P450 sequences (350 000 P450 sequences available in databases; systematic nomenclature assigned to >41 000 P450 sequences; status of January 2

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Glossary Abiotic reaction: a reaction that has (so far) not been described as being catalyzed by any known enzyme. Antibiotics: antimicrobial substances that either kill bacteria or inhibit their growth and that are widely used in the treatment and prevention of bacterial infections. Directed evolution: a method of protein engineering that mimics the evolutionary process in nature and combines the generation of large mutant libraries with their rapid and sensitive high-throughput screening. Drugs: chemical substances used to treat diseases or to promote wellbeing. Eicosanoids: signaling molecules made by oxidation of arachidonic acid or other polyunsaturated fatty acids. EMA: European Medicines Agency; an agency of the European Union responsible for the evaluation and supervision of medicines for human and veterinary use. FDA: Food and Drug Administration; an agency of the United States Department of Health and Human Services responsible for protecting and promoting public health through supervision of drugs, vaccines, and biopharmaceuticals. Fingerprint method: a semi-rational method of protein engineering. Based on a mutant library constructed via first-sphere mutagenesis, this method involves mapping of the active site configurations of P450 variants using a set of semisynthetic chromogenic probes, and prioritization of those variants with potential regio- and/or stereoselectivity towards the target substrate. FAD: flavin adenine dinucleotide, a prosthetic group. FMN: flavin mononucleotide, a prosthetic group. Heme: an iron ion coordinated by four nitrogen atoms of porphyrin, a prosthetic group. Heme-thiolate proteins: enzymes in which the heme prosthetic group is linked to the apoprotein via cysteine thiolate axial ligand. Metabolic engineering: the practice of optimizing genetic and regulatory processes within a cell to increase the cells’ production of a substance of interest.

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(A)

P450 BM3

Mutability landscape: a protein engineering technique that allows the discrimination between beneficial and neutral or deleterious mutations. Molecular docking: a computational method to predict the preferred orientation of a substrate in the enzyme active site realized by geometric matching and shape complementarity. Molecular dynamics simulations: a computational method to predict the preferred positioning and state of a substrate in the enzyme active site during catalysis. The method takes into account the conformational space and rigid body transformations (translations and rotations as well as internal changes to the substrate’s structure, for example, torsion angle rotations). NADH: nicotinamide adenine dinucleotide, a cofactor consisting of two nucleotides joined through phosphate groups; one nucleotide contains an adenine base and the other nicotinamide. NADPH: nicotinamide adenine dinucleotide phosphate; differs from NADH in the presence of an additional phosphate group. Prosthetic group: an organic molecule that is tightly, for instance, covalently bound to the enzyme. Rational design: mutagenesis of enzymes based on mechanistic and structural data. Reactive oxygen species: chemically reactive species containing oxygen (e.g., peroxides, superoxide) formed as natural byproducts of the normal metabolism of oxygen. Saturation mutagenesis: a protein engineering technique, in which a DNA codon is randomized to produce all possible amino acids in the protein at that position. Steroids: biologically active organic compounds with a specific core structure composed of 17 carbon atoms bonded in four fused rings. Synthetic biology: an interdisciplinary branch of biology and engineering that combines and applies various disciplines from these domains with the aim of building artificial biological systems for research, engineering, and medical applications. Synthon: a part within a molecule that is related to a possible synthetic

Na2S2O4

anaerobic condions (B)

P450 BM3 Na2S2O4

anaerobic condions (C)

P450 BM3 Na2S2O4 anaerobic condions (D)

P450 BM3 NADPH

(E)

Nave monooxygenaon

P450 BM3

Non-natural cyclopropanaon

Efficient reducon

Poor reducon

NADPH

NADPH

P411 BM3 NADPH

NADPH

Efficient reducon

No reducon

(F)

P411 BM3 NADPH

(G)

P411 BM3 NADPH

(H)

P411 BM3 E. coli

Figure 1. Abiotic Reactions Catalyzed by Engineered P450 BM3 Variants. (A) Oxidative deamination of alkyl (See figure legend on the bottom of the next page.) Trends in Biotechnology, Month Year, Vol. xx, No. yy

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2018; Table 1; [24]) as well as of deciphered biosynthetic pathways involving P450s, together with rapid developments in metabolic engineering and in efficient DNA synthesis play an important role in the emergence of synthetic biology. The trend towards applications of P450s in biotechnology and synthetic biology has further been accelerated by engineering these biocatalysts for abiotic reactions [25], by constructing artificial fusions between P450s and redox partners [26,27], by introducing alternative cofactor regeneration systems [28], and finally by rerouting of natural photosynthetic electron transfer into the biosynthetic production of high-value compounds by P450s [29]. Whereas we will not specifically touch on the methods of P450 engineering and construction of artificial P450-redox partner fusions, which are excellently described in many recent reviews [19–23], we will include a number of examples focusing on synthetic applications of such P450s.

Microbial P450s for Production of Oxyfunctionalized High-Value Compounds Pharmaceutical Agents and Drug Metabolites One of the continuing trends in P450 biotechnology is the synthesis of pharmaceutical agents and their metabolites. This is not surprising when one considers the rising demand for novel medicines or for more effective synthetic routes to existing drugs, and the importance of toxicological evaluation of new drug candidates and their metabolites according to guidelines of the FDA [30] and EMA [31]. In this respect, selective steroid hydroxylations can provide both, metabolites with interesting pharmacological activities and precursors for steroidal drugs. Protein engineering supported by extensive 3D-structure analysis of 15b-steroid hydroxylase CYP106A2 from B. megaterium resulted in 6b- or 11a-hydroxylases with altered stereoselectivity and productivities of up to 5.5 g l1 d1 (Figure 2, Key Figure, panel A) [32]. An iterative saturation mutagenesis based on mutability landscape data and supported by molecular dynamics simulations enabled construction of P450 BM3 variants with high regioselectivity for C16 position of testosterone and four other steroids combined with high diastereoselectivity and activity [33]. Regarding drug metabolite production, in previous years much effort has been put into engineering human P450s for effective expression in recombinant microbial hosts (reviewed in [34]). However, their low efficiency, stability, and restricted scalability might limit the use of recombinant human P450s, particularly if sufficient quantities of minor occurring metabolites need to be isolated. Protein engineering of microbial P450s to mimic human drug metabolism has therefore become the focus of many recent studies. For example, engineered P450 BM3 variants were reported that furnish human metabolites 4ʹ-hydroxydiclofenac, nortriptyline, and norlidocaine [35], or that oxidize omeprazole [36], esomeprazol, lansoprazol (Figure 2A), and rabeprazol [37] in the same manner as human CYP2C19 and CYP3A4. Nonetheless, the direct correlation between metabolite patterns produced by human and engineered microbial P450s still remains a challenge

azides. (B) Olefin cyclopropanation via carbene transfer using dithionite as reducing agent under anaerobic conditions. (C) Generation of C–N bonds via carbene N–H insertion. (D) Cyclization of carbonazidate substrates to yield oxazolidinones via intramolecular C–H amination. (E) Olefin cyclopropanation via carbene transfer by ‘P411 BM30 : P450 BM3 inefficiently catalyzes cyclopropanation using NADPH as a reducing agent because the FeIII-to-FeII reduction potential for the low-spin resting state (E 0 Fe-Cys = 430 mV) is lower than that of NADP+-to-NADPH (E0 NADPH = 320 mV). Mutation of the hemeligating cysteine to serine allows NADPH-driven cyclopropanation while removing native monooxygenation. (F) Sulfimidation generating S–N bonds. (G) Aziridination of aryl olefins. (H) Carbene transfer to alkynes utilizing Escherichia coli whole cell biocatalysts. Abbreviation: Aa, amino acid.

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operation; a retro-synthetic fragmentation structure, often used to mean ‘synthetic building block’. Terpenes: a large and diverse class of secondary metabolites produced mainly by plants. Terpenes consist of different numbers of isoprene units. Terpenes that contain additional functional groups are called terpenoids. Vitamins: organic compounds important to health; an essential nutrient that cannot be produced in the body, but must be procured from food. Xenobiotics: chemical substances found within an organism that are not naturally produced by the organism. Natural compounds can become xenobiotics if they are taken up by another organism.

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Table 1. Cytochrome P450 Diversity in the Tree of Lifea Taxon

P450 sequences with nomenclature assigned

P450 sequences without nomenclature assigned

Totalb

Number of different CYP families

Insects

7426

105

7531

208

Non-insect invertebrates

1925

0

1925

311

Mammals

2419

1334

3753

18

Other vertebrates

1461

83

1544

19

Plants

16 219

168 303

184 522

277

Fungi

7925

77 178

85 103

805

Protozoa

602

0

602

63

Bacteria

2979

59 620

62 599

591

Archaea

64

84

148

14

Viruses

28

0

28

6

Total

41 048

306 707

347 755

2252c

a

Table modified from [24] with permission. P450 sequences available in databases as of January 2018. c Sixty families present in more than one taxon are included only once. b

for protein engineering due to inefficient control of enzyme regioselectivity. Development of new computational tools, their combination, or repurposing for the identification of molecular determinants of P450 regioselectivity could help to solve this problem. Among others, a multistep approach that combines crystallography, molecular docking, molecular dynamics simulation, and binding free-energy calculations was successfully used to evaluate the effect of single mutations in the active site of a P450 BM3 variant capable of producing human metabolites of several nonsteroidal anti-inflammatory drugs (Figure 2A) [38]. Alongside engineering well-characterized P450s, rapid progress in genome sequencing and gene annotation now allows for screening and identification of novel wild type microbial P450s capable of producing human drug metabolites. Recent examples include CYP264A1 from Sorangium cellulosum So ce56, which led to 10-hydroxylated metabolites of the tricyclic antidepressants amitriptyline and imipramine similar to human CYP2D6 [39]. CYP107L from Streptomyces platensis DSM 40041 converted amodiaquine, ritonavir, amitriptyline, and thioridazine to the metabolites produced by human CYPs 3A4, 2C8, 2C19, and 2D6 [40]. These (and many other) examples emphasize the high plasticity of some microbial P450s, making them attractive models for structure–function relationship studies, particularly regarding molecular factors governing enzyme regioselectivity. Late-Stage Oxidation of Complex Natural Products and Their Analogues To date, natural products as well as their derivatives and synthetic analogues remain the main source for new drugs. Natural products with complex molecular architecture often possess multiple chemically equivalent positions, making their regioselective oxidation very difficult. Many P450s, particularly those involved in biosynthesis of secondary metabolites, are highly substrate- and product-specific by nature [41] and have increasingly been exploited for the latestage oxidation of complex natural products [42]. However, their narrow substrate spectrum often limits the application of these P450s in the oxidation of natural product derivatives and synthetic analogues [41,43], whereas oxidations of nonphysiological substrates with engineered P450s often result in undesired mixtures of regio- and stereoisomers.

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Key Figure

Examples for Production of Oxyfunctionalized High-Value Compounds by P450s

(A) (B)

(C)

Figure 2. Regio- and stereoselective oxidations of pharmaceutical agents, drugs, and complex natural products achieved by (A) means of protein engineering (hydroxylation sites are marked with red color and a grey box), and (B) means of substrate engineering (red triangles indicate increasing stereoselectivity). (C) Metabolic (Figure legend continued on the bottom of the next page.)

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Over the past decade, these intrinsic limitations have been approached by both, protein engineering and by well-known but less exploited substrate engineering [44]. For example, oxidation of the sesquiterpenoid cyperenoic acid with anti-angiogenic- effect by a small enriched P450 BM3 library yielded a triple mutant that catalyzed C7-hydroxylation with 94% regioselectivity and a quadruple mutant enabling C9-hydroxylation with 90% regioselectivity and 100% stereoselectivity on a preparative scale (Figure 2A) [45]. In another study, late-stage site-selective hydroxylation of the antimalaria agent artemisinin could be accomplished with P450 BM3 variants constructed using a fingerprint method based on first-sphere mutagenesis [46]. Whereas wild type P450 BM3 has no activity with artemisinin, evolved variants furnished either 7(R)-hydroxy- or 7(S)-hydroxyartemisinin with absolute regio- and enantioselectivity (100% ee), as well as high activity also on a preparative scale (Figure 2A) [47]. The 14-membered macrocycle b-cembrenediol is a noncompetitive inhibitor of nicotinic acetylcholine receptors with potential as a neuroprotective drug. P450 BM3 variants constructed by first-sphere mutagenesis enabled hydroxylation at C9 (100% regioselectivity and 89:11 diastereomeric ratio) and C10 (97% regioselectivity and 74:26 diastereomeric ratio) of b-cembrenediol (Figure 2A) [48]. A substrate engineering approach based on a set of 14membered synthetic cembranoids revealed a strong influence of the polarity and size of the exocyclic substituents at C1-position and the presence of an additional OH-group on the ring on enzyme regioselectivity (Figure 2B) [49]. Following the substrate engineering approach, the regioselectivity of the CYP107L1-catalyzed hydroxylation of the macrolide YC-17 analogues was shifted by introducing N,N-dimethylamino substituents instead of the naturally occurring amino sugar. While CYP107L1-mediated hydroxylation of YC-17 resulted in a 1:1 mixture of C10- and C12-hydroxylated regioisomers, synthetic anchors shifted the regioselectivity of YC-17 analogues’ hydroxylation at C10 and C12 from 20:1 to 1:4 (Figure 2B) [50]. However, the regioselectivity of these hydroxylations remained unpredictable. To achieve tunable site-selective hydroxylation of 11- and 12-membered macrolactones catalyzed by a CYP107L1 variant, the directing dimethylamino groups were temporarily introduced on the substrates via thiazole-containing linkers. Whereas linkers possessing paraor meta-substituted benzene spacers force allylic hydroxylation at a position proximal to the point of anchor connection, shorter linkers enabled hydroxylations at distal positions of the substrate related to the point of anchor connection [51]. This approach, though not leading to targeted regioselectivity, brings us closer to the final goal: to control the regioselectivity of a P450 enzyme. Another elegant method of substrate engineering is based on the use of so-called ‘decoy molecules’ that can change the substrate specificity of P450s without any mutagenesis engineering of Saccharomyces cerevisiae for production of strictosidine; the yeast was optimized for enhanced secondary metabolism towards the precursor GPP by introducing seven additional genes and three gene deletions that enhance secondary metabolism, and it carries 14 plant monoterpene indole alkaloid pathway genes from Catharanthus roseus, including four p450-genes. Low activity of CYP76B6 (marked with a red circle) leading to 8-hydroxygeraniol was recognized as one of the bottlenecks of the recombinant metabolic pathway. Abbreviations: CYP72A1, secologanin synthase; CYP72A224, 7-deoxyloganic acid hydroxylase; CYP76A26, 7deoxyloganetic acid synthase/iridoid oxidase; CYP76B6, geraniol 8-hydroxylase; 7-DLGT, 7-deoxyloganetic acid glucosyl transferase; DMAPP, dimethylallyl pyrophosphate; GES, geranyl synthase; GOR, 8-hydroxygeraniol oxidoreductase; GPP, geranyl pyrophosphate; GPPS, GPP synthase; IPP, isopentenyl pyrophosphate; ISY, iridoid synthase; LAMT, loganic acid-O-methyltransferase; STR, strictosidine synthase.

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(reviewed in [52]). Decoy molecules are inert ‘dummy substrates’ with structures that are very similar to those of the natural substrates of the respective P450. They bind in close proximity to the heme group and force the enzyme to generate the active species and to catalyze oxidation of various substrates other than the native ones. This concept has successfully been employed for medium- and long-alkyl-chain fatty acid hydroxylases, namely the two peroxygenases P450BSb and P450SPa, as well as P450 BM3, forcing them by addition of inert short-alkyl-chain fatty acids (or other decoy molecules) to catalyze oxidation of non-native substrates (e.g., benzene to phenol [53], hydroxylation of gaseous propane and butane [54], or oxidation of various other aromatic and aliphatic hydrocarbons [55]).

P450s in Multistep Cascade Reactions P450s as Components of the Metabolic Engineering Toolbox Recent developments in next-generation sequencing technology, combined with computational methods for the identification of gene clusters, have accelerated the reconstitution of complete plant and microbial biosynthetic pathways or parts thereof in heterologous microorganisms. Stereo- and regiospecific oxyfunctionalization of most natural products, catalyzed by P450s, are pivotal for their biological activities, which makes P450s essential bio-bricks in synthetic biology approaches. Unfortunately, P450s, particularly plant ones, are often the most difficult targets during pathway engineering due to low expression levels, low activity, and low stability [56]. For example, low activity or poor expression of a plant P450 leading to 8-hydroxygeraniol was recognized as one of the bottlenecks to higher production of the central monoterpene indole alkaloid strictosidine in Saccharomyces cerevisiae [57]. This yeast strain was optimized for enhanced secondary metabolism towards the precursor geranyl pyrophosphate and carries 14 plant monoterpene indole alkaloid pathway genes from Catharanthus roseus (Figure 2C) [57]. Often, higher product titers were achieved by linking a plant P450 with its cognate cytochrome P450 reductase (CPR), which ensures their physical proximity, aiming at preventing loss of electrons [58]. Unexpectedly, the CYP725A4-CPR linkage was found to be suboptimal for the production of oxygenated taxanes in E. coli. Moreover, the relatively low expression under the control of a weak promoter was recognized as key for P450 and CPR functionality in this system [59]. Aiming at the synthesis of a mixture of bioactive components of medicinal plant ginseng, three P450s (CYP716A12, CYP716A47, and CYP716A53v2), as well as CPR originating from different plants, were integrated in the yeast genome along with other recombinant genes for the biosynthesis of triterpenoid saponins; this engineered strain produced a mixture of oleanolic acid (21.4 mg l1), protopanaxadiol (17.2 mg l1), and protopanaxatriol (15.9 mg l1) [60]. Replacement of plant P450s by more active and stable microbial P450s with the same substrate/product spectrum as plant P450s or by engineered ones could help to increase productivity of reconstituted plant pathways, as was demonstrated for the reconstituted biosynthesis of the anticancer agent perillyl alcohol in E. coli [61]. While heterologous expression of bacterial P450s in microbial hosts is usually not a problem, sometimes their stereoselectivity does not provide a desired product, as was demonstrated in the recent report on production of the cholesterol-lowering drug pravastatin [62]. The industrial Penicillium chrysogenum DS17690 strain with removed b-lactam biosynthetic genes was used as a chassis. Compactin production was achieved after the introduction of nine heterologous genes and the deletion of an endogenous lipase gene in the P. chrysogenum strain. The final step, 6a-hydroxylation of compactin to pravastatin, was accomplished by an engineered 8

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variant of CYP105AS1 from Amycolatopsis orientalis. CYP105AS1 was fused in frame to the RhF reductase of CYP116B1 and its natural stereoselectivity was inverted by means of directed evolution and rational protein design. A fully fermentative ‘one-pot’ process of recombinant P. chrysogenum provided 6 g l1 pravastatin [62]. Artificial Multi-Enzyme Cascades Besides the reconstitution of naturally occurring biosynthetic pathways, research on completely artificial synthetic cascades is a rapidly developing field that may lead to tailormade molecules with desired properties [63]. While the implementation of P450s in artificial cascades is generally a rather unexplored research area, reports on the combination of P450 with alcohol dehydrogenase (ADH) seem to prevail in the literature [64]. In such two-step cascades, P450s enable the hydroxylation of often chemically inert starting molecules and ADHs oxidize the formed alcohols to the corresponding ketones or aldehydes that can be used as building blocks or as valuable final products. Simultaneously, NAD(P)H consumed by the P450 can be regenerated in the ADH-catalyzed step. This approach has been applied for the production of (+)-nootkatone, a high-value sesquiterpenoid known for its grapefruit smell. A variant of P450 BM3 engineered for high regioselectivity catalyzes the allylic hydroxylation of (+)-valencene to nootkatol, which is subsequently oxidized to (+)-nootkatone by ADH (Figure 3A) [65]. In an in vitro cascade involving a regio- and chemoselective P450 BM3 variant and ADH from Lactobacillus kefiri, inert cyclohexane, cyclooctane, and cyclodecane were converted into corresponding cyclic ketones [66]. This approach was further advanced to furnish all stereoisomers of cyclohexane-1,2-diol [(R,R)-, (S, S)-, and meso-configuration] starting from cyclohexane (Figure 3B); enantiomeric ratios between 96:4 and >99:1, and diastereoselectivities of 80%–93% were achieved [67]. The extension of P450-ADH cascades by introducing an additional enzyme responsible for the production of chiral amines has been exploited by several groups. Recombinant E. coli coexpressing variants of P450cam from Pseudomonas putida fused to the RhF reductase, together with (R)- and (S)-selective ADHs, and (R)-selective v-transaminase from Arthrobacter sp. were used to convert a set of ethylbenzenes to enantiopure (R)-1-phenylethanamines with high enantioselectivity (ee of up to 97.5%) [68]. A similar enzyme combination was successfully applied for the production of v-amino dodecanoic acid, the monomer of Nylon 12 [69]. Instead of v-transaminase, reductive aminase (Figure 3C) [70] or amine dehydrogenase [71] have also been included in three-enzyme one-pot cascades to produce various chiral amines. A P450-ADH cascade complemented with a Bayer-Villiger monooxygenase has been successfully used for transformation of cycloalkanes to their corresponding lactones. Exemplarily, a combination of engineered P450 BM3, ADH, and engineered cyclohexanone monooxygenase, applied in vitro for cycloheptane conversion, resulted in 3 g l1 2-oxocanone (enantholactone) [72]. The use of resting cells of Pseudomonas taiwanensis with an implemented similar cascade for the synthesis of 2.3 g l1 e-caprolactone starting with cyclohexane has also been reported (Figure 3D) [73]. Other types of one-pot cascades including P450s are rather rare but their number is increasing. Among them is a two-step oxidation of fatty acids to a-ketoacids catalyzed by a combination of the peroxygenase CYP152B1 from Clostridium acetobutylicum with two oxidases [74]. Another report describes the synthesis of L-tyrosine derivatives starting with substituted benzenes, pyruvate, and NH3 [75]: firstly, monosubstituted benzenes were regioselectively hydroxylated in the ortho-position by an engineered variant of P450 BM3 to yield the corresponding phenols, Trends in Biotechnology, Month Year, Vol. xx, No. yy

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(A)

(B)

(C)

(D)

Figure 3. Implementation of P450s in Multi-Enzyme Cascade Reactions. (A) P450-ADH cascade for selective oxidation of (+)-valencene to (+)-nootkatone via nootkatol under simultaneous cosubstrate conversion by ADH to regenerate the cofactor NADH. (B) Escherichia coli whole cell biocatalysts for synthesis of (R,R)-5, (S, S)-5, or meso-5 from 1. (C) Multi-enzyme cascade for the amination of cycloalkanes. (D) Pseudomonas putida whole cell biocatalysts for synthesis of lactones from cycloalkanes. Cyclohexane (1); cyclohexanol (2); cyclohexanone (3); 2-hydroxy-cyclohexanone (4); cyclohexane-1,2-diol (5); cyclohexylamine (6); caprolactone (7). Abbreviations: ADH, alcohol dehydrogenase; BVMO, Bayer-Villiger monooxygenase; FDH, formate dehydrogenase; FdR, ferredoxin reductase; Fdx, ferredoxin.

followed by subsequent C–C coupling and simultaneous asymmetric amination using tyrosine phenol lyase. Utilizing this concept L-DOPA surrogates were produced in up to 5.2 g l1 [75]. Chemo-Enzymatic Processes The power of P450-catalyzed oxyfunctionalizations has further been exploited in chemoenzymatic processes. Used together, chemo-catalysts and P450s complement each other ideally, and can be considered a ‘dream team’ for supply of existing and novel oxidized chemicals. Self-sufficient and stable P450 BM3 variants are often used in such approaches. For instance, Ilie and colleagues reported P450 BM3 variants capable of regio- and stereoselective hydroxylation of 6-iodotetralone to (R)-4-hydroxy-6-iodotetralone: a key synthon in subsequent C–C bond-forming transformations leading to various compounds with potential for pharmaceutical applications. With the pure enantiomer in hand, three types of Pd-catalyzed cross-coupling reactions were evaluated as a proof of principle [76]. Following a semisynthetic approach, the highly regioselective P450 BM3-catalyzed hydroxylation of parthenolide at positions C9 or C14 was coupled to chemical modification of the introduced OH-group via, for example, acylation, alkylation, or carbene insertion. A panel of the final new analogues of parthenolide demonstrated improved anticancer properties as compared with the parent 10

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molecule [77–79]. A regioselective variant of P450 BM3 was also successfully used for allylic oxidation of the a,ß-unsaturated ketone function during the total synthesis of the norditerpenoid alkaloid nigelladine A [80]. As mentioned in the previous chapter, P450 from biosynthetic pathways can also be successfully employed in chemo-enzymatic process leading to complex natural products. For example, oxidative P450-mediated diversification of the natural compound tylactone, synthesized via de novo total chemical synthesis, provided access to naturally occurring macrolide antibiotics juvenimicin B1, B3, A3, and A4 [81]. The major hurdle for progress towards more effective hybrid chemo-enzymatic processes is the inherent incompatibility of classical chemo- and biocatalytic reactions. In this regard, a tandem reaction starting with ruthenium-catalyzed alkene metathesis followed by enantioselective epoxidation with engineered P450 BM3 variants to produce aryl epoxides presents one of very few examples for chemo-enzymatic one-pot processes [82]. The incompatibility of both components was overcome by separating them between two phases: the metathesis of alkenes took place in the organic phase and the P450 converted the intermediate into the corresponding epoxide in the aqueous phase. This reaction sequence was used to convert a mixture of stilbene-derived alkenes selectively into a single epoxide [83]. As a conclusion, although only a limited number of chemo-enzymatic cascade reactions with P450s has been reported so far, the summarized examples already demonstrate the manifold scope of P450-catalyzed oxyfunctionalizations when coupled to the toolbox of chemical synthesis.

Alternative Regeneration Systems for P450s Stoichiometric consumption of the costly cofactors NAD(P)H constitutes one of the major challenges for P450 synthetic application. Earlier work on cofactor regeneration predominantly focused on enzyme-mediated and electrochemical approaches. In the past years, harnessing light to drive biocatalysis, and particularly to supply redox equivalents for biocatalytic oxidation reactions, has gained much attention [28]. Transferring light energy to a chemical reaction requires a ‘light harvesting unit’, a so-called photosensitizer, that is either added into the reaction solution, or covalently bound to the P450 [28]. Cadmium sulfide quantum dots [84], eosin Y dye [85], as well as FAD, FMN, riboflavin, or synthetic deazaflavins have been reported as effective photosensitizers [86–89] and supported either P450-catalysis through the peroxide shunt pathway (Figure 4A) or via the reduction of flavin cofactors in redox partners (Figure 4B). In an optimized approach, ruthenium(II)-diimine covalently attached via a cysteine close to the heme group of the P450 BM3 monooxygenase domain promoted direct heme iron reduction without the need for redox partners (Figure 4C). Heme reduction rate was three orders of magnitude faster than that observed with the natural BM3 reductase [90] and supported the P450-catalyzed hydroxylation of lauric acid with a total turnover number >900 and an initial reaction rate of 120 min1 [91]. Natural photosystem I can also be considered as an efficient photosensitizer, enabling electron transfer across the thylakoid membrane to a ferredoxin upon light excitation. Electrons are then transferred to a ferredoxin reductase, which reduces NADP+ to NADPH. Basically, electrons transferred by photosystem I can be redirected to a P450. Indeed, photosystem I can successfully be coupled to P450 catalysis, as was demonstrated in vitro for the membrane-bound CYP79A1 from Sorghum bicolor [92] and the bacterial soluble CYP124 from Trends in Biotechnology, Month Year, Vol. xx, No. yy

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(A)

(C)

DTC

Flavinox 1/2 (DTC)2 Fay acid

EDTA

RuII-complex RuI-complex

Hydroxy fay acid

EDTA+ Flavinred

(B)

Deazaflavinox Fay acid

EDTA

EDTA+

P450 BM3 (holoenzyme) Deazaflavinred

Hydroxy fay acid

Fay acid

P450 BM3 (heme domain)

Hydroxy fay acid

(D) Stroma

Tyrosine

Aldoxime

Thylakoid membrane

Lumen

Figure 4. Light-Harvesting Strategies for Funneling of Electrons to Drive P450 Oxyfunctionalizations. The light-harvesting unit (‘photosensitizer’) is highlighted with red color; ultimate electron suppliers with green color. (A) Formation of reactive oxygen species utilizing naturally occurring P450 peroxygenases (e. g., CYP152 family enzymes) or exploiting the peroxygenase activity of certain P450s. (B) Indirect heme reduction via redox partners (e.g., P450 BM3 holoenzyme). (C) Direct heme reduction with photosensitizer covalently attached to the P450 (e.g., P450 BM3 heme domain). (D) Rerouting of electrons transferred by PS I to P450s (e.g., CYP79A1). CYP fused to Fdx was expressed in the thylakoid membrane of chloroplasts in Nicotiana benthamiana, enabling direct coupling of photosynthetic electron transfer to the heme iron reduction. Broken arrows indicate the physiological pathway, where electrons are transferred via Fdx to FdR to reduce NADP+. Abbreviations: Cytb6f, cytochrome b6f; DTC, diethyldithiocarbamate; EDTA: FdR, ferredoxin reductase; Fdx, ferredoxin; hv, light energy; OEC, oxygen-evolving complex; ox, oxidized; PC, plastocyanin; PQ, plastoquinone pool; PS, photosystem; red, reduced.

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Mycobacterium tuberculosis [93]. Alternatively, CYP79A1 fused to ferredoxin was expressed in the thylakoid membrane of chloroplasts in Nicotiana benthamiana, thus enabling direct coupling of photosynthetic electron transfer to the heme iron reduction (Figure 4D). Importantly, the P450-ferredoxin fusion outperforms nonfused CYP79A1 in vivo [94]. This concept was extended by relocating of CYP79A1, CYP71A1, and a UDP glucosyl transferase from the endoplasmic reticulum of S. bicolor to the chloroplast of transiently transformed tobacco leaves, which were used for biosynthesis of the cyanogenic glucoside dhurrin (D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile) starting with tyrosine [95]. This example demonstrates the potential of plant chloroplasts as a compartment for production of high-value compounds produced via P450 catalysis supported by light-driven electron supply. As a conclusion, novel alternative regeneration systems for P450 oxyfunctionalizations show some potential and have been established as a proof-of-concept, but challenges for broader biotechnological exploitation need to be solved in terms of protein stability, activity, and heme reduction coupling efficiency to rival their monooxygenase counterparts. Among the challenges facing the use of photosystem I in in vivo processes is the competition of recombinant P450s with other proteins reduced by the same ferredoxin, including the endogenous ferredoxin reductase. Moreover, for the reconstitution of complete biosynthetic pathways in chloroplasts, a number of additional genes for precursor production should be included. Generally, this concept could benefit from transferring it to photosynthetic cyanobacteria and its application in whole-cell biocatalysis.

Concluding Remarks and Future Perspectives Sixty years after the discovery of cytochromes P450 in rat liver microsomes by Klingenberg [96], their catalytic mechanism and biophysical properties can now be considered as being well understood [97,98]. The roles of P450s in primary and secondary metabolism of plants and microorganisms, as well as in drug degradation in humans, have been extensively investigated by research groups from academia and industry all over the world.

Outstanding Questions How can the current methods for enzyme characterization be further improved and accelerated in order to close the fast growing gap between the exponential identification of new P450s through rapid genome sequencing and the elucidation of functions and substrate spectra of these new P450s? Which experimental and computational approaches or combinations thereof can enable engineering of P450s with targeted regioselectivity towards a certain site among a multitude of equivalent positions? Is it possible to include the P450-catalyzed abiotic reactions in chemoenzymatic processes or multi-enzymatic cascades in vitro, or to combine them with other enzymes in artificial biosynthetic pathways in microbial recombinant hosts? Can cofactor regeneration by harnessing light be efficiently introduced into recombinant hosts to support the reconstituted natural and artificial biosynthetic pathways?

However, the complicated mode of action of P450s and their generally lower activities in comparison with many other enzyme groups (e.g., lipases or ADH) remained the major factors limiting the implementation of P450s in industrial processes for a long time. Consequently, the roughly 20 years of harnessing P450s in biotechnology has only resulted in a limited number of technical processes, and those are so far dominated by growing cells and native host systems [99,100]. Nevertheless, the understanding of P450s has revolutionized the aspects of drug development where P450s play leading roles in pharmacogenetics, carcinogenesis, and molecular epidemiology, as well as in agriculture where P450s are used in bioremediation, plant breeding, and insect control [101]. In an era when synthetic biology is striving to replace synthetic chemistry for production of highvalue chemicals, P450s have a crucial role. They can catalyze the specific addition of oxygen atoms into complex molecular scaffolds; a challenging task for traditional chemical methods. Design of P450-based bio-bricks and the identification of the bottlenecks that slow down the productivity of reconstituted biosynthetic pathways in recombinant microbes bring us closer to production of high-value compounds with economic viability, like the artemisinin precursors amorphadiene and artemisinic acid [102] and the taxol precursor taxadien-5a-ol [59,103]. De novo biosynthesis of amorphadiene and artemisinic acid by fermentation of engineered S. cerevisiae to produce 40 g l1 and 25 g l1, respectively [102], is not only a successful synthetic biology story, it also demonstrates that methods of synthetic biology combined with process engineering approaches enable the achievement of industrially relevant product concentrations. Trends in Biotechnology, Month Year, Vol. xx, No. yy

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Significant advances in protein engineering have prompted the construction of P450 variants not only with extended substrate scope, or higher activity and stability, but also those catalyzing reactions that do not occur in nature, which opens a completely new perspective for synthetic biology. However, targeted regioselectivity remains an issue and has to be addressed in the near future. Combinations of P450s in multistep cascade reactions with biocatalysts available from an almost exponentially expanding toolbox and/or with chemical steps, offer alternative routes for retro-synthesis of target molecules, as well as exciting future perspectives for access to novel compounds that cannot be synthesized by nature or classical chemical routes. Thus, the concepts provided in this review build a solid foundation for further development of P450s towards applications with efficiencies approaching economic viability, even though not all described systems will be (and should be) implemented on an industrial scale. Moreover, some related questions remain open (see Outstanding Questions). The central question to be answered is whether all the parts of the P450 puzzle can be efficiently combined in one system. Acknowledgements Financial support was kindly provided by the German Federal Ministry of Education and Research (grant number 031B0362A).

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