Opportunities and Challenges

An Overview of P450 Enzymes

Cytochrome P450 enzymes (P450s) containing a heme-iron center, are biocatalysts from all kingdoms, involved in a large variety of reactions. Their potential in catalyzing a broad range of substrates makes perfect candidates for biotechnology applications and the production of high-value compounds.

Biocatalytic reactions performed by P450s have a great interest in the pharmaceutical industry, fine chemicals, cosmetics, and for bioremediation procedures. However, the complex nature of this protein is still a major hurdle in the prospect of using their promising ability for expanding the number of industrial applications. Multiple approaches of protein engineering are currently conducted to improve activity, stability and/or substrate specificity for a given reaction. Furthermore, in combination with the appropriate biocatalyst, a suitable bioengineering process is a key step in the implementation of P450s at the industrial scale. 

Cytochrome P450 monooxygenases (termed CYPs or P450s) are highly promising heme proteins ubiquitously found across all kingdoms, catalyzing a large variety of reactions under mild conditions [1]. Originally discovered in rat liver microsomes, the P450 name is derived from their unusual property to display a maximum peak of absorption at 450 nm of the reduced carbon monoxide gas-bound complex [1,2]. This method also enables the rapid evaluation of protein content [2,3].


P450s have the great ability to catalyze oxido-reduction reactions as e.g hydroxylation, oxidation, sulfoxidation, decarboxylation or dealkylation of a broad range of substrates including alkanes, fatty acids, steroids, terpenes, antibiotics or xenobiotics [4]. A vast majority of P450s are monooxygenases and the transfer of an oxygen atom in a substrate, is the result of the reductive scission of a dioxygen bond and the release of one molecule of water [5]. A catalytic wheel was proposed in early 70’s and described using P450cam, an enzyme isolated from Pseudomonas putida and responsible for the hydroxylation of camphor [6–9]. P450cam was also the first crystal structure solved and was used as reference until the discovery of P450BM3, from Bacillus megaterium, the most catalytically efficient P450 known to date [10–13]. P450BM3 has been extensively studied and engineered to perform a large variety of reactions and still serves as a model for the P450 mechanism [14].

The P450-based reactions occur in the heme domain of CYPs where the substrate binds. However, catalysis also requires the transfer of two electrons from the cofactor nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) (for most of the P450s) usually provided by the reductase domain [5]. Ten different classes of P450 have been described based on the structure of the enzyme and on the redox partners associated e.g. heme domain fused to the reductase domain (P450BM3), membrane bound P450s (mammalian CYPs), or three component soluble system (most of the bacterial P450s) [4,15].

Due to their versatility in catalyzing chemically challenging regio- and stereoselective reactions, P450s are promising candidates for biotechnology use [16]. However their complex nature hampered their potential for establishing cost-competitive industrial processes. The combination as a suitable biocatalyst, host and appropriate process engineering for a given reaction is a crucial study to increase the number of implementation of cytochrome P450 at the industrial scale [17].

We will discuss the common limitations associated to P450- based reactions, at the enzymatic level and during whole-cell biotransformations, the alternatives to address challenges and current industrial biotechnological opportunities and applications of CYPs.

Addressing P450 Challenges 
Substrate specificity, activity, and stability of P450s are key barriers in industrial applications
Protein engineering is the most common approach to expand diversity, tackle low enzyme activity, stability, substrate specificity, and reduce side product formation [18]. Possible strategies for generating improved biocatalysts include gene shuffling, rational protein design or directed evolution [19,20]. 

The increased frequency of available P450 sequences from databases, provides functional and structural information, which in turn greatly facilitates rational protein design strategies [21–24]. As an example, mutant libraries of CYP106A2 isolated from Bacillus megaterium, a difficult protein to crystallize, were based on rational design and docking studies using a homology model and have provided structural and functional information. This led to the generation of improved biocatalysts able to hydroxylate steroids in a more effective manner [25]. Similarly a homology model of the heme domain of CYP153A from Marinobacter aquaeolei (CYP153AM.aq) was created and allowed to facilitate targeted mutagenesis within the binding pocket and at the substrate entrance tunnel. Based on visual assessment, docking studies and high conservation of CYPs sequences alignment analysis, an engineered biocatalyst demonstrated enhanced activity for the terminal hydroxylation of short to long chain fatty acids [26]. The crystal structure of CYP153AM.aq was ultimately solved and revealed a crucial anchoring residue in the active site. Additional mutations demonstrated extended flexibility in the substrate recognition region which enhanced product formation [27].

Directed evolution, generating diversity via random mutagenesis, has the great advantage of identifying improved enzymes in the absence of structural, sequence or functional information [28]. However, the establishment of a high-throughput screening assay or selection method is indispensable to isolate enhanced variants from large mutant libraries [29]. Pioneering work has been establish in Frances Arnold’s lab, including the engineering of P450BM3 for diverse types of reactions and substrates have been accomplished [30,31]. The promiscuity of the heme protein enabled Arnold and coworkers to tailor P450BM3 to catalyze the cyclopropanation reaction [32,33]. 

Besides activity and substrate specificity, P450 instability is a major hurdle for industrial processes. Efforts have been made to design and identify proteins with increased thermostability and high resistance to co-solvents. For instance, P450BM3 was tailored to display high temperature resistance, and stability in the presence of ethanol, acetone, dimethylformamide, dimethyl sulfoxide and acetonitrile, which are commonly utilized to dissolve poorly soluble substrates [34,35]. Due to their complex nature, immobilization of P450s to optimize their stability has proven difficult. Nevertheless, P450BM3 immobilized in a sol-gel matrix displayed increased stability and activity towards three tested substrates including ß-ionone, octane, and naphthalene [36,37]. 

Manipulating redox partners and uncoupling events for optimizing P450s activity
Most P450s receive two electrons essential for the oxygen activation, usually from the electron donor NAD(P)H. Both inefficient electron transfer between the reductase domain and the heme domain and side reactions known as uncoupling events, can result in diminished catalytic rates. The latter encourage formation of reactive oxygen species, leading to heme loss [16]. Protein engineering of P450 has shown to increase coupling efficiency in combination with improved turnover rate but this postulate is not a general case [16]. P450BM3 is a self-sufficient natural protein in which the heme domain is fused to the reductase domain. This results in improved electron transfer rates and high catalytic turnover numbers [38]. Therefore, based on such model, a large number of artificial fusion constructs have been created and the suitable selection of redox protein associated to the heme have generated catalytically enhanced enzymes [39]. Furthermore, the linker region between the heme domain and the reductase domain has been shown to be a decisive element to facilitate electron transfer along with increasing enzyme activity and stability. Generated chimera proteins have resulted in greater activity, simply by modifying the linker with an appropriate amino acid length [40–42]. 

Bioprocess development with P450s
In addition to the challenges identified at the enzyme level, efficient whole-cell P450-based reaction development is a key step for potential industrial applications. For an economically feasible process, various parameters must be carefully considered and evaluated including the choice of the host, of the biocatalyst but also the operating mode (growing cells vs. resting cells) [17]. The potential substrate and/or product inhibition or toxicity to the cells and/or to the enzyme is another factor to examine, as well as substrate transport limitation across the cell membrane and overoxidation and/or degradation of the product [43–46]. 

In the case of a whole-cell biotransformation, the cofactor availability has already been shown as a limiting parameter, resulting in lower P450 activity [45]. Several options can be considered to address this limitation as enzyme engineering. P450cam was tailored into a peroxidase to utilize H2Oin the absence of cofactor for the hydroxylation of naphthalene [47]. The addition of a glucose dehydrogenase for cofactor recycling is a second possibility, which has been demonstrated in Bacillus amyloliquefaciens for the conversion of 1- hexene by P450BM3 [48]. Another strategy involves switching the cofactor from NADPH to the more stable NADH, thereby reducing process costs [49, 50]. If necessary, a larger choice of NAD+-dependent dehydrogenases can also be co-expressed for cofactor regeneration. The chemical synthesis of NADPH or NADH cofactor analogues has also recently demonstrated successful catalysis reactions [51,52]. The utilization of electrochemical way by immobilizing the P450 on electrode surfaces for the electron transfer has also been investigated [53,54]. Another novel approach involved the light-driven catalysis and the creation of an artificial hybrid P450BM3 heme domain and a photosensitizer Ru(II), demonstrating the hydroxylation of dodecanoic acid using light only [55].

Overview of Industrial Applications of P450s
Light of P450s in pharmaceutical industry
P450s have a great interest for the production of drug metabolites, and a vast number of publications demonstrated the successful use of CYPs in the industrial level [16]. As an example, hydrocortisone synthesis is a well-established process via the conversion of the compound Reichstein S steroid by P450 isolated from Curvularia sp., and pravastatin has recently been produced from compactin in a single step fermentation process using an engineered bacterial P450 [56–58]. In addition, CYP1051A from Streptomyces griseolus enables the conversion of vitamin D3 into 1a25-dihydroxyvitamine D3 [59]. Moreover, the company Novartis engineered strains expressing recombinant human P450 for the production of drug metabolites. CYP107 from Saccharopolyspora erythraea and plant P450s are involved in the synthetic steps required for the production of derivatives of erythromycin and Taxol, respectively [60,61]. Production of the anti-cancer drug perilly alcohol from limonene, has been demonstrated in P. putida expressing a P450 from the bacterial CYP153 family [44]. Recently, Sanofi has started producing 25 g L-1 of antimalarial artemisinin from an engineered strain. One biosynthetic route includes the integration of CYP71AV1 isolated from Artemisia annua, executing successive oxidations of the artemisinic acid precursor [62,63].

Deployment of P450s in bioremediation process
P450s can play an essential role in bioremediation processes as environmental contaminants have been shown to be degraded by mammalian and fungal cytochrome P450s. For instance, CYP5145A3 from the white rot fungus Phanerochaete chrysosporium and the rat CYP1A1 showed great activity towards two different polychlorinated dibenzo-p-dioxins [64]. A mammalian cytochrome–expressed in Arabidopsis thaliana, was able to neutralize explosive particulates which remained in soils after a decade of military activity [65]. Furthermore, several P450s were described as able to metabolize polycyclic aromatic hydrocarbons (PAHs) [66]. Besides detoxification of pollutants, engineered plants were also developed to contain mammalian CYPs, conferring a resistance to herbicides [67]. 

Market for renewables
Using the power of cytochrome P450, common dyes including indigo and indirubin were synthesized in cell cultures, which illustrates their potential application within the horticulture industry for the generation of flowers with new colors [68–70].

In the perspective of bioconversion of alkanes for fuels and renewable chemicals, engineered P450BM3 and CYP101 from P. putida have been utilized to produce alcohols from small chain-length alkanes, such as ethane to ethanol or propane to propanol [71–73]. Furthermore, a new type of CYP decarboxylase from the CYP152 family, isolated from Jeotgalicoccus sp., has been shown to generate olefins from fatty acids biosynthesis intermediates [74]. Terminally hydroxylated fatty acids and α, ω-dicarboxylic acids can be used as anticancer agents, but also as precursors for polymers and in the cosmetics area for flavours and fragrances [75–78]. The challenging activation and oxidation of C-H bond can be achieved by the versatile P450 instead of chemically-based processes requiring toxic metals and costly thermodynamic conditions, yielding low regioselectivity [79]. The cultivation of the yeast Candida and Yarrowia sp. on alkanes and fatty acids has demonstrated the production of terminal hydroxy fatty acids and the corresponding dicarboxylic acid due to the expression of CYP52 family [80,81]. The engineered Candida tropicalis was able to produce 174 g L-1of terminal hydroxy fatty acids and 6 g L-1 of diacids from 200 g L-1 methyl tetradecanoate after six days of biotransformation [82]. As alternative to the yeast platform, bacterial CYP153 family was shown to catalyze a large variety of substrates as fatty acids and alkanes but also terpenes and primary alcohols [43,83–87].

Cytochrome P450s are remarkable biocatalysts of great interest for the industry due to their versatility for a wide substrate range and in performing a large number of oxidative reactions (Figure 1). The identification of limitations associated to P450-based catalysis and whole-cell process are the first step towards an industrial application of such powerful proteins. Extensive research and multiple strategies including protein engineering are currently being conducted to circumvent the challenges as low enzyme activity, robustness and/or substrate specificity, required for cost-competitive implementation. With the incessant development of technologies and synthetic biology tools, there is a no doubt that an increased repertoire of industrial applications of P450s will be established in the near future. 

We would like to thank Dr. Lukasz Gricman (Insti- tute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany) for the preparation of 3D structure of Marinobacter aquaeolei CYP153A. 

© 2016 Notonier S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Notonier S, Meyers A, Jayakody LN (2016) An Overview of P450 Enzymes: Opportunities and Challenges in Industrial Applications. Enz Eng 5: 152. doi:10.4172/2329-6674.1000152

Sandra Notonier
Alexander Meyers
Lahiru N Jayakody
National Bioenergy Center
National Renewable Energy Laboratory
15013 Denver (USA)

Corresponding author:
Lahiru N Jayakody
E-mail: Lahiru.Jayakody@nrel.gov

[1] Nelson DR (2013) A world of cytochrome P450s. Philos Trans R Soc B Biol Sci 368: 2012.04.30.
[2] Omura T, Sato R (1964) The carbon monoxide-binding pingement of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239: 2370–2378.
[3] Hannemann F, Bichet A, Ewen KM Bernhardt R (2007) cytochrome P450 systems-biological variations of electron transport chains. Biochim Biophys Acta 1770: 330–344.
[4] Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124: 128–145.
[5] Denisov IG, Makris TM, Sligar SG, Schlichting I (2005) Structure and chemistry of cytochrome P450. Chem Rev 105: 2253–2278.
[6] Gunsalus IC, Pederson TC, Sligar SG (1975) Oxygenase-catalyzed biological hydroxylations. Annu Rev Biochem 44: 377–407.
[7] Guengerich FP (1991) Reactions and significance of cytochrome P-450 enzymes. J Biol Chem 266: 10019–10022.
[8] Katagiri M, Ganguli BN, Gunsalus IC (1968) A soluble cytochrome P-450 functional in methylene hydroxylation. J Biol Chem 243: 3543–3546.
[9] Sono M, Roach MP, Coulter E, Dawson JH (1996) Heme-containing oxygenases. Chem Rev 96: 2841–2888.
[10] Ravichandran KG, Boddupalli SS, Hasermann CA, Peterson JA, Deisenhofer J (1993) Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450’s. Science 261: 731–736.
[11] Narhi L, Fulco A (1986) Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem 261: 7160–7169.
[12] Noble MA, Miles CS, Chapman SK, Lysek DA, MacKay AC, et al. (1999) Roles of key active-site residues in flavocytochrome P450BM3. Biochem J 339: 371–379.
[13] Munro AW, Leys DG, McLean KJ, Marshall KR, Ost TWB et al. (2002) P450BM3: The very model of a modern flavocytochrome. Trends Biochem Sci 27: 250–257.
[14] Wang M, Roberts DL, Paschke R, Shea TM, Masters BS, et al. (1997) Threedimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes. Proc. Natl Acad Sci USA 94: 8411–8416.
[15] Roberts GA, Grogan G, Greter A, Flitsch SL, Turner NJ (2002) Identification of a new class of cytochrome P450 from a Rhodococcus sp. J Bacteriol 184: 3898–3908.
[16] Bernhardt R , Urlacher VB, (2014) Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations, Appl. Microbiol. Biotechnol 98: 6185–203.
[17] Lundemo MT, Woodley JM (2015) Guidelines for development and implementation of biocatalytic P450 processes. Appl Microbiol Biotechnol 99: 2465–2483.
[18] Arnold FH (2015) The nature of chemical innovation: new enzymes by evolution. Q Rev Biophys 48: 404–410.
[19] Rosic NN, Huang W, Johnston WA, DeVoss JJ, Gillam EMJ (2007) Extending the diversity of cytochrome P450 enzymes by DNA family shuffling. Gene 395: 40–48.
[20] Bornscheuer UT, Pohl M (2001). Improved biocatalysts by directed evolution and rational protein design. Curr Opin Chem Biol 5: 137–143.
[21] Gricman L, Vogel C, Pleiss J (2014) Conservation analysis of class-specific positions in cytochrome P450 monooxygenases: Functional and structural relevance. Proteins Struct Funct Bioinforma 82: 491–504.
[22] Fischer M, Knoll M, Sirim D, Wagner F, Funke S, et al. (2007) The cytochrome P450 engineering database: A navigation and prediction tool for the cytochrome P450 protein family. Bioinformatics
[23] 2015–2017. 23. Park J, Lee S, Choi J, Ahn K, Park B, et al. (2008) Fungal cytochrome P450 database. BMC Genomics 9: 402.
[24] Nelson DR (2009) The cytochrome p450 homepage. Hum Genomics 4: 59–65.
[25] Lisurek M, Simgen B, Antes I, Bernhardt R (2008) Theoretical and experimental evaluation of a CYP106A2 low homology model and production of mutants with changed activity and selectivity of hydroxylation. Chembiochem 9: 1439–1449.
[26] Notonier S, Gricman L, Pleiss J, Hauer B (2016) Semi-rational protein engineering of CYP153AM.aq. -CPRBM3 for efficient terminal hydroxylation of short- to long-chain fatty acids. ChemBioChem 16: 1550–1557.
[27] Hoffmann SM, Danesh-azari H, Spandolf C, Weissenborn MJ, Grogan G, et al. (2016) Structure-Guided Redesign of CYP153AM.aq for the Improved Terminal Hydroxylation of Fatty Acids. ChemCatChem 8: 3234–3239.
[28] Dougherty MJ, Arnold FH (2009) Directed evolution: new parts and optimized function. Curr Opin Biotechnol 20: 486–491.
[29] Lutz S (2010) Beyond directed evolution-semi-rational protein engineering and design,” Curr Opin Biotechnol 21: 734–743.
[30] Farinas ET, Schwaneberg U, Glieder A, Arnold FH (2001) Directed evolution of a cytochrome P450 monooxygenase for alkane oxidation. Adv Synth Catal 343: 601–606.
[31] Alcalde M, Farinas ET, Arnold FH (2004) Colorimetric high-throughput assay for alkene epoxidation catalyzed by cytochrome P450 BM-3 variant 139-3. J Biomol Screen 9: 141–146.
[32] Coelho PS, Brustad EM, Kannan A, Arnold FH (2013) Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339: 307–310.
[33] McIntosh JA, Farwell CC, Arnold FH (2014) Expanding P450 catalytic reaction space through evolution and engineering. Curr Opin Chem Biol 19: 126–134.
[34] Salazar O, Cirino PC, Arnold FH (2003) Thermostabilization of a cytochrome P450 peroxygenase. ChemBioChem 4: 891–893.
[35] Wong TS, Arnold FH, Schwaneberg U (2004). Laboratory evolution of cytochrome P450 BM-3 monooxygenase for organic cosolvents. Biotechnol Bioeng 85: 351–358.
[36] Maurer SC, Schulze H, Schmid RD, Urlacher V (2003) Immobilisation of P450BM-3 and an NADP(+) cofactor recycling system: Towards a technical application of heme-containing monooxygenases in fine chemical synthesis. Adv Synth Catal 345: 802–810.
[37] Weber E, Sirim D, Schreiber T, Thomas B, Pleiss J, et al. (2010) Immobilization of P450 BM-3 monooxygenase on mesoporous molecular sieves with different pore diameters. J Mol Catal B Enzym 64: 29–37.
[38] Ortiz de Montellano PR (2005) Cytochrome P450: Structure, mechanism, and biochemistry (3rdedn), New York.
[39] Ortiz de Montellano PR (2015) Cytochrome P450 - Structure, mechanism, and biochemistry (4thedn), New York.
[40] Robinson CR, Sauer RT (1998) Optimizing the stability of single-chain proteins by linker length and composition mutagenesis. Proc Natl Acad Sci USA 95: 5929–5934.
[41] Hoffmann SM, Weissenborn MJ, Gricman L, Notonier S, Pleiss J, et al. (2016) The impact of linker length on P450 fusion constructs: Activity, stability and coupling. ChemCatChem 8: 1591–1597.
[42] Belsare KD, Ruff AJ, Martinez R, Shivange AV, Mundhada H, et al. (2014) P-Link: A method for generating multicomponent cytochrome P450 fusions with variable linker length. Biotechniques 57: 13–20.
[43] Scheps D, Honda Malca S, Richter SM, Marisch K, Nestl BM,et al. (2013). Synthesis of .-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct. Microb. Biotechnol 6: 694–707.
[44] Cornelissen S, Julsing MK, Volmer J, Riechert O, Schmid A, et al. (2013) Whole-cell-based CYP153A6-catalyzed (S)-limonene hydroxylation efficiency depends on host background and profits from monoterpene uptake via AlkL. Biotechnol Bioeng 110: 1282–1292.
[45] Lundemo MT, Notonier S, Striedner G, Hauer B, Woodley JM (2016) Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli. Appl Microbiol Biotechnol 100: 1197–1208.
[46] Kiss FM, Lundemo MT, Zapp J, Woodley JM, Bernhardt R (2015) Process development for the production of 15ß-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst. Microb Cell Fact 14: 28.
[47] Joo H, Lin Z, Arnold FH (1999) Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 399: 670–673.
[48] Siriphongphaew A, Pisnupong P, Wongkongkatep J, Inprakhon P, Vangnai AS, et al. (2012) Development of a whole-cell biocatalyst co-expressing P450 mono-oxygenase and glucose dehydrogenase for synthesis of epoxyhexane. Appl Microbiol Biotechnol 95: 357–367.
[49] Sung C, Jung E, Choi KY, Bae JH, Kim M, et al. (2015) The production of .-hydroxy palmitic acid using fatty acid metabolism and cofactor optimization in Escherichia coli. Appl Microbiol Biotechnol 99: 6667–6676.
[50] Wu JT, Wu LH, Knight JA (1986) Stability of NADPH: effect of various factors on the kinetics of degradation. Clin Chem 32: 314–319.
[51] Ryan JD, Fish RH, Clark DS (2008) Engineering cytochrome P450 enzymes for improved activity towards biomimetic 1,4-NADH cofactors. Chembiochem 9: 2579–2582.
[52] Paul CE, Churakova E, Maurits E, Girhard M, Urlacher VB, et al. (2014) In situ formation of H2O2 for P450 peroxygenases. Bioorg Med Chem 22: 5692–5696.
[53] Fantuzzi A, Meharenna YT, Briscoe PB, Sassone C, Borgia B, et al. (2006) Improving catalytic properties of P450BM3 heme domain electrodes by molecular Lego. Chem Commun (Camb) 12: 1289–1291.
[54] Llaudet EC, Darimont D, Samba R, Matiychyn I, Stelzle M, et al. (2016) Expanding an efficient, electrically driven and CNT-tagged P450 system into the third dimension: a nanowired CNT-containing and enzyme-stabilising 3 D sol-gel electrode. ChemBioChem 17: 1367–1373
[55] Tran NH, Nguyen D, Dwaraknath S, Mahadevan S, Chavez G, et al. (2013) An efficient light-driven P450BM3 biocatalyst. J Am Chem Soc 135: 14484-14487.
[56] Sonomoto K, Hoq MM, Tanaka A, Fukui S (1983) 11beta-hydroxylation of cortexolone (Reichstein compound S) to hydrocortisone by Curvularia lunata entrapped in photo-cross-linked resin gels. Appl Environ Microbiol 45: 436–443.
[57] Fujii T, Fujii Y, Machida K, Ochiai A, Ito M (2009) Efficient biotransformations using Escherichia coli with tolC acrAB mutations expressing cytochrome P450 genes. Biosci Biotechnol Biochem 73: 805–810.
[58] McLean KJ, Hans M, Meijrink B, Van Scheppingen WB, Vollebregt, et al. (2015) Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum. Proc Natl Acad Sci USA 112: 2847–2852.
[59] Sawada N, Sakaki T, Yoneda S, Kusudo T, Shinkyo R, et al. (2004) Conversion of vitamin D3 to 1a,25-dihydroxyvitamin D3 by Streptomyces griseolus cytochrome P450SU-1. Biochem Biophys Res Commun 320: 156–164.
[60] Weber JM, Leung JO, Swanson SJ, Idler KB, McAlpine JB (1991) An erythromycin derivative produced by targeted gene disruption in Saccharopolyspora erythraea. Science 252: 114–117.
[61] Jennewein S, Rithner CD, Williams RM, Croteau RB (2001) Taxol biosynthesis: taxane 13 alpha-hydroxylase is a cytochrome P450-dependent monooxygenase. Proc Natl Acad Sci USA 98: 13595–13600.
[62] Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, et al. (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440: 940–943.
[63] Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, et al. (2013) Highlevel semi-synthetic production of the potent antimalarial artemisinin. Nature 496: 528–532.
[64] Sakaki T, Yamamoto K, Ikushiro S (2013) Possibility of application of cytochrome P450 to bioremediation of dioxins. Biotechnol Appl Biochem 60: 65–70.
[65] Rylott EL, Jackson RG, Edwards J, Womack GL, Seth-Smith HM, et al. (2006) An explosive-degrading cytochrome P450 activity and its targeted application for the phytoremediation of RDX. Nat Biotechnol 24: 216–219.
[66] Shimada T, Fujii-Kuriyama Y (2004) Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1. Cancer Sci 95: 1–6.
[67] Hirose S, Kawahigashi H, Inoue T, Inui H, Ohkawa H, et al. (2005) Enhanced expression of CYP2C9 and tolerance to sulfonylurea herbicides in transgenic rice plants. Plant Biotechnol 22: 89–96.
[68] Gillam EM, Aguinaldo AM, Notley LM, Kim D, Mundkowski RG, et al. (1999) Formation of indigo by recombinant mammalian cytochrome P450 Biochem Biophys Res Commun 265: 469–472.
[69] Warzecha H, Frank A, Peer M, Gillam EM, Guengerich FP, et al. (2007) Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures. Plant Biotechnol J 5: 185–191.
[70] Gillam EM, Guengerich FP (2001) Exploiting the versatility of human cytochrome P450 enzymes: the promise of blue roses from biotechnology. IUBMB Life 52: 271–277.
[71] Xu F, Bell SG, Lednik J, Insley A, Rao Z, et al. (2005) The heme monooxygenase cytochrome P450cam can be engineered to oxidize ethane to ethanol. Angew Chem Int Ed. Engl 44: 4029–4032.
[72] Meinhold P, Peters MW, Chen MMY, Takahashi K, Arnold FH (2005) Direct conversion of ethane to ethanol by engineered cytochrome P450BM3. Chembiochem 6: 1765–1768.
[73] Fasan R, Chen MM, Crook NC, Arnold FH (2007) Engineered alkanehydroxylating cytochrome P450(BM3) exhibiting nativelike catalytic properties. Angew Chem Int Ed Engl 46: 8414–8418.
[74] Rude MA, Baron TS, Brubaker S, Alibhai M, Del Cardayre SB, et al. (2011) Terminal Olefin (1-Alkene) Biosynthesis by a Novel P450 Fatty Acid Decarboxylase from Jeotgalicoccus Species. Appl Environ Microbiol 77: 1718–1727.
[75] Liu C, Liu F, Cai J, Xie W, Long TE, et al. (2011) Polymers from fatty acids: poly(.-hydroxyl tetradecanoic acid) synthesis and physico-mechanical studies. Biomacromolecules 12: 3291–3298.
[76] Huf S, Krügener S, Hirth T, Rupp S, Zibek S (2011) Biotechnological synthesis of long-chain dicarboxylic acids as building blocks for polymers. Eur J Lipid Sci Technol 113: 548–561.
[77] Biermann U, Bornscheuer I, Meier MAR, Metzger JO, Schaefer HJ (2011) Oils and fats as renewable raw materials in chemistry. Angew Chem Int Ed 50: 3854–3871.
[78] Abe A, Sugiyama K (2005) Growth inhibition and apoptosis induction of human melanoma cells by omega hydroxy fatty acids. Anti-Cancer Drug 16: 543–549.
[79] Labinger JA (2004) Selective alkane oxidation: hot and cold approaches to a hot problem. J Mol Catal A Chem 220: 27–35.
[80] Beopoulos A, Verbeke J, Bordes F, Guicherd M, Bressy M, et al. (2014) Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 98: 251–262.
[81] Waché Y, Aguedo M, Nicaud JM, Belin JM (2003) Catabolism of hydroxyacids and biotechnological production of lactones by Yarrowia lipolytica. Appl Microbiol Biotechnol 61: 393–404.
[82] Lu W, Ness JE, Xie W, Zhang X, Minshull J, et al. (2010) Biosynthesis of monomers for plastics from renewable oils. J Am Chem Soc 132: 15451–15455.
[83] Scheps D, Malca SH, Hoffmann H, Nestl BM, Hauer B (2011) Regioselective .-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes from Mycobacterium marinum and Polaromonas sp. strain JS666. Org Biomol Chem 9 : 6727–6733.
[84] Honda Malca S, Scheps D, Kühnel L, Venegas-Venegas E, Seifert A, et al. (2012) Bacterial CYP153A monooxygenases for the synthesis of omegahydroxylated fatty acids. Chem Commun 48: 5115–5117.
[85] Bordeaux M, Galarneau A, Fajula F, Drone J (2011) A regioselective biocatalyst for alkane activation under mild conditions. Angew Chem Int Ed 50: 2075–2079.
[86] Kirtz M, Klebensberger J, Otte KB, Richter SM, Hauer B (2016) Production of .-hydroxy octanoic acid with Escherichia coli. J Biotechnol 230: 30–33.
[87]. Nebel BA, Scheps D, Honda Malca S, Nestl BM, Breuer M, et al. (2014) Biooxidation of n-butane to 1-butanol by engineered P450 monooxygenase under increased pressure. J Biotechnol 191: 86–92.
[88] Notonier S (2015) Development of Highly Efficient CYP153A-Catalysed Terminal Hydroxylation of Fatty Acids. PhD thesis, University of Stuttgart.


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