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1.
Xiangquan Qin Yiping Jiang Jie Chen Fuquan Yao Panxia Zhao Longyi Jin Zhiqi Cong 《International journal of molecular sciences》2022,23(14)
We recently developed an artificial P450–H2O2 system assisted by dual-functional small molecules (DFSMs) to modify the P450BM3 monooxygenase into its peroxygenase mode, which could be widely used for the oxidation of non-native substrates. Aiming to further improve the DFSM-facilitated P450–H2O2 system, a series of novel DFSMs having various unnatural amino acid groups was designed and synthesized, based on the co-crystal structure of P450BM3 and a typical DFSM, N-(ω-imidazolyl)-hexanoyl-L-phenylalanine, in this study. The size and hydrophobicity of the amino acid residue in the DFSM drastically affected the catalytic activity (up to 5-fold), stereoselectivity, and regioselectivity of the epoxidation and hydroxylation reactions. Docking simulations illustrated that the differential catalytic ability among the DFSMs is closely related to the binding affinity and the distance between the catalytic group and heme iron. This study not only enriches the DFSM toolbox to provide more options for utilizing the peroxide-shunt pathway of cytochrome P450BM3, but also sheds light on the great potential of the DFSM-driven P450 peroxygenase system in catalytic applications based on DFSM tunability. 相似文献
2.
Semirational Protein Engineering of CYP153AM.aq.‐CPRBM3 for Efficient Terminal Hydroxylation of Short‐ to Long‐Chain Fatty Acids
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Dr. Sandra Notonier Dr. Łukasz Gricman Prof. Dr. Jürgen Pleiss Prof. Dr. Bernhard Hauer 《Chembiochem : a European journal of chemical biology》2016,17(16):1550-1557
The regioselective terminal hydroxylation of alkanes and fatty acids is of great interest in a variety of industrial applications, such as in cosmetics, in fine chemicals, and in the fragrance industry. The chemically challenging activation and oxidation of non‐activated C?H bonds can be achieved with cytochrome P450 enzymes. CYP153AM.aq.‐CPRBM3 is an artificial fusion construct consisting of the heme domain from Marinobacter aquaeolei and the reductase domain of CYP102A1 from Bacillus megaterium. It has the ability to hydroxylate medium‐ and long‐chain fatty acids selectively at their terminal positions. However, the activity of this interesting P450 construct needs to be improved for applications in industrial processes. For this purpose, the design of mutant libraries including two consecutive steps of mutagenesis is demonstrated. Targeted positions and residues chosen for substitution were based on semi‐rational protein design after creation of a homology model of the heme domain of CYP153AM.aq., sequence alignments, and docking studies. Site‐directed mutagenesis was the preferred method employed to address positions within the binding pocket, whereas diversity was created with the aid of a degenerate codon for amino acids located at the substrate entrance channel. Combining the successful variants led to the identification of a double variant—G307A/S233G—that showed alterations of one position within the binding pocket and one position located in the substrate access channel. This double variant showed twofold increased activity relative to the wild type for the terminal hydroxylation of medium‐chain‐length fatty acids. This variant furthermore showed improved activity towards short‐ and long‐chain fatty acids and enhanced stability in the presence of higher concentrations of fatty acids. 相似文献
3.
Peter Meinhold MatthewW. Peters Adam Hartwick AlishaR. Hernandez FrancesH. Arnold 《Advanced Synthesis \u0026amp; Catalysis》2006,348(6):763-772
Enzymes that catalyze the terminal hydroxylation of alkanes could be used to produce more valuable chemicals from hydrocarbons. Cytochrome P450 BM3 from Bacillus megaterium hydroxylates medium‐chain fatty acids at subterminal positions at high rates. To engineer BM3 for terminal alkane hydroxylation, we performed saturation mutagenesis at selected active‐site residues of a BM3 variant that hydroxylates alkanes. Recombination of beneficial mutations generated a library of BM3 mutants that hydroxylate linear alkanes with a wide range of regioselectivities. Mutant 77‐9H exhibits 52% selectivity for the terminal position of octane. This regioselectivity is octane‐specific and does not transfer to other substrates, including shorter and longer hydrocarbons or fatty acids. These results show that BM3 can be readily molded for regioselective oxidation. 相似文献
4.
Covalent immobilization of cytochrome P450 BM3 (R966D/W1046S) on glutaraldehyde activated SPIONs
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5.
Stabilization of the Reductase Domain in the Catalytically Self‐Sufficient Cytochrome P450BM3 by Consensus‐Guided Mutagenesis
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Prof. Dr. Gloria Saab‐Rincón Hanan Alwaseem Dr. Valeria Guzmán‐Luna Leticia Olvera Prof. Dr. Rudi Fasan 《Chembiochem : a European journal of chemical biology》2018,19(6):622-632
The multidomain, catalytically self‐sufficient cytochrome P450 BM‐3 from Bacillus megaterium (P450BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus‐guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450BM3. Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild‐type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half‐lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD‐binding domain and that the optimal temperature (Topt) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts. 相似文献
6.
There are two types of cytochrome P450 enzymes in nature, namely, the monooxygenases and the peroxygenases. Both enzyme classes participate in substrate biodegradation or biosynthesis reactions in nature, but the P450 monooxygenases use dioxygen, while the peroxygenases take H2O2 in their catalytic cycle instead. By contrast to the P450 monooxygenases, the P450 peroxygenases do not require an external redox partner to deliver electrons during the catalytic cycle, and also no external proton source is needed. Therefore, they are fully self-sufficient, which affords them opportunities in biotechnological applications. One specific P450 peroxygenase, namely, P450 OleTJE, reacts with long-chain linear fatty acids through oxidative decarboxylation to form hydrocarbons and, as such, has been implicated as a suitable source for the biosynthesis of biofuels. Unfortunately, the reactions were shown to produce a considerable amount of side products originating from Cα and Cβ hydroxylation and desaturation. These product distributions were found to be strongly dependent on whether the substrate had substituents on the Cα and/or Cβ atoms. To understand the bifurcation pathways of substrate activation by P450 OleTJE leading to decarboxylation, Cα hydroxylation, Cβ hydroxylation and Cα–Cβ desaturation, we performed a computational study using 3-phenylpropionate and 2-phenylbutyrate as substrates. We set up large cluster models containing the heme, the substrate and the key features of the substrate binding pocket and calculated (using density functional theory) the pathways leading to the four possible products. This work predicts that the two substrates will react with different reaction rates due to accessibility differences of the substrates to the active oxidant, and, as a consequence, these two substrates will also generate different products. This work explains how the substrate binding pocket of P450 OleTJE guides a reaction to a chemoselectivity. 相似文献
7.
P450 BM3‐Catalyzed Regio‐ and Stereoselective Hydroxylation Aiming at the Synthesis of Phthalides and Isocoumarins
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Claudia Holec Ute Hartrampf Dr. Katharina Neufeld Prof. Dr. Jörg Pietruszka 《Chembiochem : a European journal of chemical biology》2017,18(7):676-684
Cytochrome P450 BM3 monooxygenases are able to catalyze the regio‐ and stereoselective oxygenation of a broad range of substrates, with promising potential for synthetic applications. To study the suitability of P450 BM3 variants for stereoselective benzylic hydroxylation of 2‐alkylated benzoic acid esters, the biotransformation of methyl 2‐ethylbenzoate, resulting in both enantiomeric forms of 3‐methylphthalide, was investigated. In the case of methyl 2‐propylbenzoate as a substrate the regioselectivity of the reaction was shifted towards β‐hydroxylation, resulting in the synthesis of enantioenriched R‐ and S‐configured 3‐methylisochroman‐1‐one. The potential of P450 BM3 variants for regio‐ and stereoselective synthesis of phthalides and isocoumarins offers a new route to a class of compounds that are valuable synthons for a variety of natural compounds. 相似文献
8.
Characterisation of CYP102A25 from Bacillus marmarensis and CYP102A26 from Pontibacillus halophilus: P450 Homologues of BM3 with Preference towards Hydroxylation of Medium‐Chain Fatty Acids
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Dr. Joanne L. Porter Jack Manning Selina Sabatini Michele Tavanti Prof. Nicholas J. Turner Prof. Sabine L. Flitsch 《Chembiochem : a European journal of chemical biology》2018,19(5):513-520
Cytochrome P450 monooxygenases are highly desired biocatalysts owing to their ability to catalyse a wide variety of chemically challenging C?H activation reactions. The CYP102A subfamily of enzymes are natural catalytically self‐sufficient proteins consisting of a haem and FMN‐FAD reductase domain fused in a single‐component system. They catalyse the oxygenation of saturated and unsaturated fatty acids to produce primarily ω?1, ω?2 and ω?3 hydroxy acids. These monooxygenases have potential applications in biotechnology; however, their substrate range is still limited and there is a continued need to add diversity to this class of biocatalysts. Herein, we present the characterisation of two new members of this class of enzymes, CYP102A25 (BMar) from Bacillus marmarensis and CYP102A26 (PHal) from Pontibacillus halophilus, both of which express readily in a recombinant bacterial host. BMar exhibits the highest activity toward myristic acid and shows moderate activity towards unsaturated fatty acids. PHal exhibits broader activity towards mid‐chain‐saturated (C14–C18) and unsaturated fatty acids. Furthermore, PHal shows good regioselectivity for the hydroxylation of myristic acid, targeting the ω?2 position for C?H activation. 相似文献
9.
Fatty acid α-hydroxylase from Sphingomonas paucimobilis is an unusual cytochrome P450 enzyme that hydroxylates the α-carbon of fatty acids in the presence of H2O2. Herein, we describe our investigation concerning the utilization of various substrates and the optical configuration of
the α-hydroxyl product using a recombinant form of this enzyme. This enzyme can metabolize saturated fatty acids with carbon
chain lengths of more than 10. The K
m value for pentadecanoic acid (C15) was the smallest among the saturated fatty acids tested (C10–C18) and that for myristic acid (C14) showed similar enzyme kinetics to those seen for C15. As shorter or longer carbon chain lengths were used, K
m values increased. The turnover numbers for fatty acids with carbon chain lengths of more than 11 were of the same order of
magnitude (103 min−1), but the turnover number for undecanoic acid (C11) was less. Dicarboxylic fatty acids and methyl myristate were not metabolized, but monomethyl hexadecanedioate and ω-hydroxypalmitic
acid were metabolized, though with lower turnover values. Arachidonic acid was a good substrate, comparable to C14 or C15. The metabolite of arachidonic acid was only α-hydroxyarachidonic acid. Alkanes, fatty alcohols, and fatty aldehydes were
not utilized as substrates. Analysis of the optical configurations of the α-hydroxylated products demonstrated that the products
were S-enantiomers (more than 98% enantiomerically pure). These results suggested that this P450 enzyme is strictly responsible
for fatty acids and catalyzes highly stereo- and regioselective hydroxylation, where structure of ω-carbon and carboxyl carbon
as well as carbon chain length of fatty acids are important for substrate-enzyme interaction. 相似文献
10.
The hydroxylation activity of the Thr268Ala mutant of P450(BM3) has been shown to occur to varying degrees with small alterations in the structure of a fatty-acid substrate. Ten substrates were investigated, including straight chain, branched chain and cis-cyclopropyl substituted fatty acids with a straight-chain length that varied between 12 and 16 carbon atoms. The efficacy of the hydroxylation activity appeared to be governed by the chain length of the substrate. Substrates possessing 14 to 15 carbons afforded the highest levels of activity, which were comparable with the wild-type enzyme. Outside of this window, straight-chain fatty acids showed reduced activity over the other substrate types. These results provide a cautionary tale concerning the loss of ferryl activity in such cytochrome P450 threonine to alanine mutants, as the nature of the substrate can determine the extent to which hydroxylation chemistry is abolished. 相似文献
11.
Discovery of a Novel Linoleate Dioxygenase of Fusarium oxysporum and Linoleate Diol Synthase of Colletotrichum graminicola
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Fungal pathogens constitute serious threats for many forms of life. The pathogenic fungi Fusarium and Colletotrichum and their formae speciales (f. spp.) infect many types of crops with severe consequences and Fusarium oxysporum can also induce keratitis and allergic conditions in humans. These fungi code for homologues of dioxygenase–cytochrome P450 (DOX–CYP) fusion proteins of the animal heme peroxidase (cyclooxygenase) superfamily. The objective was to characterize the enzymatic activities of the DOX–CYP homologue of Colletotrichum graminicola (EFQ34869) and the DOX homologue of F. oxysporum (EGU79548). The former oxidized oleic and linoleic acids in analogy with 7,8‐linoleate diol synthases (LDSs), but with the additional biosynthesis of 8,11‐dihydroxylinoleic acid. The latter metabolized fatty acids to hydroperoxides with broad substrate specificity. It oxidized 20:4n‐6 and 18:2n‐6 to hydroperoxides with an R configuration at the (n‐10) positions, and other n‐6 fatty acids in the same way. [11S‐2H]18:2n‐6 was oxidized with retention and [11R‐2H]18:2n‐6 with loss of deuterium, suggesting suprafacial hydrogen abstraction and oxygen insertion. Fatty acids of the n‐3 series were oxidized less efficiently and often to hydroperoxides with an R configuration at both (n‐10) and (n‐7) positions. The enzyme spans 1426 amino acids with about 825 residues in the N‐terminal domain with DOX homology and 600 residues at the C‐terminal domain without homology to other enzymes. We conclude that fungal oxylipins can be formed by two novel subfamilies of cyclooxygenase‐related DOX. 相似文献
12.
Yoshiro Miura 《Lipids》1981,16(10):721-725
Frog liver microsomes catalyzed the hydroxylation of 1-dodecanol into the corresponding ω- and (ω-1)-hydroxy derivatives.
The hydroxylation rate for 1-dodecanol was much lower than that for lauric acid. Both NADPH and O2 were required for hydroxylation activity. NADH had no effect on the hydroxylation. The hydroxylating system was inhibited
49% by CO at a CO∶O2 ratio of 4.0. The formation of ω-hydroxydodecanol was more sharply inhibited by CO than was the formation of (ω-1)-hydroxydodecanol,
implying that more than one cytochrome P-450 was involved in the hydroxylation of 1-dodecanol and that CO has a higher affinity
for the P-450 catalyzing the ω-hydroxylation. The formation of laurate during the incubation of 1-dodecanol with frog liver
microsomes suggests that a fatty alcohol oxidation system is also present in the microsomes. NAD+ was the most effective cofactor for the oxidation of 1-dodecanol and NADP+ had a little effect. Pyrazole (an inhibitor of alcohol dehydrogenase) had a slight inhibitory effect on the oxidation and
sodium azide (an inhibitor of catalase) had no effect. 相似文献
13.
The liver microsomes of the Mongolian gerbilMeriones unguiculatus catalyzed the hydroxylation of various saturated fatty acids (C8−C18), alcohols (C12 and C16) and hydrocarbon (C12) to the corresponding ω- and (ω-1)-hydroxy derivatives. Lauric acid was hydroxylated most effectively among saturated fatty
acids and the order of activity as hydroxylation substrates was C12>C14>C13>C16>C10>C18>C8. The specific activity of laurate hydroxylation (5.99 nmol/mg microsomal protein/min) in gerbil liver microsomes was higher
than that observed in other species. 1-Dodecanol was also hydroxylated very effectively (4.58 nmol/mg microsomal protein/min)
by gerbil liver microsomes, but in general the hydroxylation rates for fatty alcohols were much lower than those for the corresponding
acids. It was found from both inhibitor and cofactor studies that the enzyme catalyzing the hydroxylation of fatty acids and
alcohols in the liver microsomes of the Mongolian gerbil was a typical cytochrome P-450-linked monooxygenase, and at least
two different cytochrome P-450 species were involved in the hydroxylation.
Presented in part at the AOCS annual meeting (a joint meeting with the Japan Oil Chemists' Society), Honolulu, Hawaii, May
1986. 相似文献
14.
Ya-Li Dong Gang-Gang Chong Dr. Chun-Xiu Li Prof. Dr. Qi Chen Dr. Jiang Pan Prof. Ai-Tao Li Prof. Jian-He Xu 《Chembiochem : a European journal of chemical biology》2022,23(9):e202200063
The P450-mediated terminal hydroxylation of non-activated C−H bonds is a chemically challenging reaction. CYP153A7 monooxygenase, discovered in Sphingomonas sp. HXN200, belongs to the CYP153A subfamily and shows a pronounced terminal selectivity. Herein, we report the significantly improved terminal hydroxylation activity of CYP153A7 by redesign of the substrate binding pocket based on molecular docking of CYP153A7−C8:0 and sequence alignments. Some of the resultant single mutants were advantageous over the wild-type enzyme with higher reaction rates, achieving a complete conversion of n-octanoic acid (C8:0, 1 mM) in a shorter time period. Especially, a single-mutation variant, D258E, showed 3.8-fold higher catalytic efficiency than the wild type toward the terminal hydroxylation of medium-chain fatty acid C8:0 to the high value-added product 8-hydroxyoctanoic acid. 相似文献
15.
To bring out the potential of wild-type cytochrome P450s, we have developed a series of “decoy molecules” to change their high substrate specificity without any mutagenesis. Decoy molecules are inert dummy substrates with structures that are very similar to those of natural substrates. The decoy molecules force long-alkyl-chain fatty acid hydroxylases (P450BSβ, P450SPα, and P450BM3) to generate the active species and to catalyze oxidation of various substrates other than fatty acids. Interestingly, the catalytic activity was highly dependent on the structure of decoy molecules. Furthermore, the enantioselectivity of reactions catalyzed by P450BSβ and P450SPα was also dependent on the structure of decoy molecules. The decoy molecule system allows us to control reactions catalyzed by wild-type enzymes by designing decoy molecules. 相似文献
16.
Patricia Molina‐Espeja Marina Cañellas Prof. Francisco J. Plou Prof. Martin Hofrichter Dr. Fatima Lucas Prof. Victor Guallar Prof. Miguel Alcalde 《Chembiochem : a European journal of chemical biology》2016,17(4):341-349
There is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to convert naphthalene selectively into 1‐naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene together with quenching of the undesired peroxidative activity on 1‐naphthol (one‐electron oxidation). The resulting double mutant (G241D‐R257K) obtained from this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate's catalytic position. Powered exclusively by catalytic concentrations of H2O2, this soluble and stable biocatalyst has a total turnover number of 50 000, with high regioselectivity (97 %) and reduced peroxidative activity. 相似文献
17.
Nadler A Koch C Brodhun F Wehland JD Tittmann K Feussner I Diederichsen U 《Chembiochem : a European journal of chemical biology》2011,12(5):728-737
PpoA is a bifunctional enzyme that catalyzes the dioxygenation of unsaturated C18 fatty acids. The products of this reaction are termed psi factors and have been shown to play a crucial role in conferring a balance between sexual and asexual spore development as well as production of secondary metabolites in the fungus Aspergillus nidulans. Studies on the reaction mechanism revealed that PpoA uses two different heme domains to catalyze two subsequent reactions. Initially, the fatty acid substrate is dioxygenated at C8, yielding an 8‐hydroperoxy fatty acid at the N‐terminal domain. This reaction is catalyzed by a peroxidase/dioxygenase‐type domain that exhibits many similarities to prostaglandin H2 synthases and involves a stereospecific homolytic hydrogen abstraction from C8 of the substrate. The C terminus harbors a heme thiolate P450 domain in which rearrangement of the 8‐hydroperoxide to the final product, a 5,8‐dihydroxy fatty acid, takes place. To obtain further information about the intrinsic kinetics and reaction mechanism of PpoA, we synthesized C5‐dideutero‐ and C8‐dideutero‐oleic acid by a novel protocol that offers a straightforward synthesis without employing the toxic additive hexamethylphosphoramide (HMPA) during C? C coupling reactions or mercury salts upon thioketal deprotection. These deuterated fatty acids were then employed for kinetic analysis under multiple‐turnover conditions. The results indicate that the hydrogen abstraction at C8 is the rate‐determining step of the overall reaction because we observed a KIE (VH/VD) of ~33 at substrate saturation that suggests extensive nuclear tunneling contributions for hydrogen transfer. Deuteration of the substrate at C5, however, had little effect on VH/VD but resulted in a different product pattern presumably due to an altered lifetime and partitioning of a reaction intermediate. 相似文献
18.
Dr. Li Zong Dr. Yan Zhang Zhengkang Shao Dr. Anita Ljubic Prof. Charlotte Jacobsen Prof. Renjun Gao Dr. Bekir Engin Eser Dr. Yingwu Wang Prof. Zheng Guo 《Chembiochem : a European journal of chemical biology》2023,24(18):e202300368
Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T50 >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs. 相似文献
19.
Fatty acid α-hydroxylase, a cytochrome P450 enzyme, from Sphingomonas paucimobilis, utilizes various straight-chain fatty acids as substrates. We investigated whether a recombinant fatty acid α-hydroxylase
is able to metabolize phytanic acid, a methyl-branched fatty acid. When phytanic acid was incubated with the recombinant enzyme
in the presence of H2O2, a reaction product was detected by gas chromatography, whereas a reaction product was not detected in the absence of H2O2. When a heat-inactivated enzyme was used, a reaction product was not detected with any concentration of H2O2. Analysis of the methylated product by gas chromatography-mass spectrometry revealed a fragmentation pattern of 2-hydroxyphytanic
acid methyl ester. By single-ion monitoring, the mass ion and the characteristic fragmentation ions of 2-hydroxyphytanic acid
methyl ester were detected at the retention time corresponding to the time of the product observed on the gas chromatogram.
The K
m
value for phytanic acid was approximately 50 μM, which was similar to that for myristic acid, although the calculated V
max for phytanic acid was about 15-fold lower than that for myristic acid. These results indicate that a bacterial cytochrome
P450 is able to oxidize phytanic acid to form 2-hydroxyphytanic acid. 相似文献
20.
Dr. Julia Schückel Dr. Elizabeth L. Rylott Dr. Gideon Grogan Prof. Dr. Neil C. Bruce 《Chembiochem : a European journal of chemical biology》2012,13(18):2758-2763
Cytochromes P450 from plants have the potential to be valuable catalysts for industrial hydroxylation reactions, but their application is hindered by poor solubility, the lack of suitable expression systems and the requirement of P450s for auxiliary redox‐transport proteins for the delivery of reducing equivalents from NAD(P)H. In the interests of enabling useful P450 activity from plants, we have developed a suite of vectors for the expression of plant P450s as non‐natural genetic fusions with reductase proteins. First, we have fused the P450 isoflavone synthase (IFS) from Glycine max with the bacterial P450 reductase domain (Rhf‐RED) from Rhodococcus sp., by using our LICRED vector developed previously (F. Sabbadin, R. Hyde, A. Robin, E.‐M. Hilgarth, M. Delenne, S. Flitsch, N. Turner, G. Grogan, N. C. Bruce, ChemBioChem 2010 , 11, 987–994) creating the first active bacterial–plant fusion P450 enzyme. We have then created a complementary vector, ACRyLIC for the fusion of selected plant P450 enzymes to the P450 reductase ATR2 from Arabidopsis thaliana. The applicability of this vector to the creation of active P450 fusion enzymes was demonstrated using both IFS1 and the cinnamate‐4‐hydroxylase (C4H) from A. thaliana. Overall the fusion vector systems will allow the rapid creation of libraries of plant P450s with the aim of identifying enzyme activities with possible applications in industrial biocatalysis. 相似文献