Self-sufficient Cytochrome P450 Research Articles

Overexpression of chaperones GroEL/ES from Escherichia coli enhances indigo biotransformation production of cytochrome P450 BM3 mutant.

The self-sufficient cytochrome P450 BM3 mutant (A74G/F87V/D168H/L188Q) can serve as a biocatalyst for whole-cell catalysis process of indigo. Nevertheless, the bioconversion yield of indigo is generally low under normal cultivation conditions (37°C, 250rpm). In this study, a recombinant E. coli BL21(DE3) strain was constructed to co-express the P450 BM3 mutant gene and GroEL/ES genes to investigate whether GroEL/ES can promote the indigo bioconversion yield in E. coli. The results revealed that the GroEL/ES system could significantly increase the indigo bioconversion yield, and the indigo bioconversion yield of the strain co-expressing P450 BM3 mutant and GroEL/ES was about 21-fold that of the strain only expressing the P450 BM3 mutant. In addition, the P450 BM3 enzyme content and in vitro indigo bioconversion yield were determined to explore the underlying mechanism for the improvement of indigo bioconversion yield. The results revealed that GroEL/ES did not increase indigo bioconversion yield by increasing the content of P450 BM3 enzyme and its enzymatic transformation efficiency. Moreover, GroEL/ES could improve the intracellular nicotinamide adenine dinucleotide phosphate (NADPH)/NADP+ ratio. Given that NADPH is an important coenzyme in the catalytic process of indigo, the underlying mechanism for the improvement of indigo bioconversion yield is probably related to an increase in the intracellular NADPH/NADP+ ratio.

Biocatalytic Synthesis of Chiral Benzylic Alcohols via Enantioselective Hydroxylation by a Self-Sufficient Cytochrome P450 from Deinococcus gobiensis

AbstractEnzymatic asymmetric benzylic hydroxylation represents a green synthesis of valuable chiral benzylic alcohols. The stereoselective C–H direct hydroxylation profile possessed by cytochrome P450 monooxygenases (P450s) makes it more attractive. Here, we successfully expressed a self-sufficient cytochrome P450 monooxygenase from Deinococcus gobiensis in Escherichia coli BL21(DE3) host. The recombinant E. coli (P450DG) strain performed well in terms of functionality and has stereoselective benzylic hydroxylation ability for propylbenzene substrates. Asymmetric benzylic hydroxylation of various aromatic compounds was further investigated using the recombinant E. coli (P450DG) strain based on the optimal conditions, producing the corresponding enantioenriched pharmaceutically relevant benzylic alcohols in moderate yields with good to excellent enantioselectivity.

A P450 Harboring Manganese Protoporphyrin IX Generates a Manganese Analogue of Compound I by Activating Dioxygen

The self-sufficient cytochrome P450 102A1 (P450BM3), known for its highly efficient activation of molecular oxygen, was reconstituted with manganese protoporphyrin IX. This artificial enzyme (Mn-BM3) was found to catalyze the monooxygenation of various substrates by activating molecular oxygen. Investigation using mechanistic probes (i.e., radical clock substrates) revealed that hydroxylation by Mn-BM3 proceeds via a radical intermediate of the substrate, supporting the involvement of an “H atom abstraction/•OH recombination” mechanism, which is the commonly accepted mechanism for hydroxylation by P450s. A DFT study also indicated that the activation barrier of Mn-porphine in the rebound step is higher than that of Fe-porphine, consistent with the experimental results obtained in the radical clock experiment, wherein Mn-BM3 presents a slower rebound rate than that of heme-bound BM3.

Hydrophobic Outer Membrane Pores Boost Testosterone Hydroxylation by Cytochrome P450 BM3 Containing Cells

The implementation of biocatalytic steroid hydroxylation processes at an industrial scale still suffers from low conversion rates. In this study, we selected variants of the self-sufficient cytochrome P450 monooxygenase BM3 from Bacillus megaterium (BM3) for the hydroxylation of testosterone either at the 2β- or 15β-position. Recombinant Escherichia coli cells were used as biocatalysts to provide a protective environment for recombinant enzymes and to ensure continuous cofactor recycling via glucose catabolism. However, only low initial whole-cell testosterone conversion rates were observed for resting cells. Results obtained with different biocatalyst formats (permeabilized cells, cell-free extracts, whole cells) indicated a limitation in substrate uptake, most likely due to the hydrophilic character of the outer membrane of E. coli. Thus, we co-expressed nine genes encoding hydrophobic outer membrane proteins potentially facilitating steroid uptake. Indeed, the application of four candidates led to increased initial testosterone hydroxylation rates. Respective whole-cell biocatalysts even exceeded activities obtained with permeabilized cells or cell-free extracts. The highest activity of 34 U gCDW−1 was obtained for a strain containing the hydrophobic outer membrane protein AlkL from Pseudomonas putida GPo1 and the BM3 variant KSA14m. Overall, we show that the straightforward application of hydrophobic outer membrane pores can boost whole-cell steroid conversion rates and thus be game-changing with regard to industrial steroid production efficiency.

EPR characterization of the heme domain of a self-sufficient cytochrome P450 (CYP116B5)

CYP116B5 is a self-sufficient cytochrome P450 (CYP450) with interesting catalytic properties for synthetic purposes. When isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Here, by means of continuous wave and advanced EPR techniques, the coordination environment of iron in the isolated CYP116B5 heme domain (CYP116b5hd) is characterized. The ligand-free protein shows the characteristic EPR spectrum of a low-spin (S = 1/2) FeIII-heme with [gz = 2.440 ± 0.005, gy = 2.25 ± 0.01, gx = 1.92 ± 0.01]. These g-values reflect an electronic ground state very similar to classical P450 monooxygenases rather than P450 peroxygenases. Binding of imidazole results in g-values very close to the ones reported for CYP152 peroxygenases. The detection of hyperfine interactions through HYperfine Sub-level CORrElation (HYSCORE) Spectroscopy experiments, shows that this is due to a nitrogen-mediated axial coordination. This work adds a piece of experimental evidence to the research, aimed at elucidating the features that distinguish the classical P450 enzymes from peroxygenases. It shows that the electronic environment of heme iron of CYP116B5 in the resting state is similar to the classical P450 monooxygenases. Therefore, it is not the critical factor that confers to CYP116B5hd its peroxygenase-like activity, suggesting a crucial role of the protein matrix.

Identification and characterization of a novel class of self-sufficient cytochrome P450 hydroxylase involved in cyclohexanecarboxylate degradation in Paraburkholderia terrae strain KU-64.

Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylate-degrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD(P)H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain (in that order from the N terminus). Reverse transcription-polymerase chain reaction results indicated that the P450CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450CHC largely preferred NADH (Km=0.011 m m) over NADPH (Km=0.21 m m). The Kd, Km, and kcat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s-1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.

Identification of a self-sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2-hydroxyphenylacetic acid catabolism, via homogentisate pathway.

SummaryThe self‐sufficient cytochrome P450 RhF and its homologues belonging to the CYP116B subfamily have attracted considerable attention due to the potential for biotechnological applications based in their ability to catalyse an array of challenging oxidative reactions without requiring additional protein partners. In this work, we showed for the first time that a CYP116B self‐sufficient cytochrome P450 encoded by the ohpA gene harboured by Cupriavidus pinatubonensis JMP134, a β‐proteobacterium model for biodegradative pathways, catalyses the conversion of 2‐hydroxyphenylacetic acid (2‐HPA) into homogentisate. Mutational analysis and HPLC metabolite detection in strain JMP134 showed that 2‐HPA is degraded through the well‐known homogentisate pathway requiring a 2‐HPA 5‐hydroxylase activity provided by OhpA, which was additionally supported by heterologous expression and enzyme assays. The ohpA gene belongs to an operon including also ohpT, coding for a substrate‐binding subunit of a putative transporter, whose expression is driven by an inducible promoter responsive to 2‐HPA in presence of a predicted OhpR transcriptional regulator. OhpA homologues can be found in several genera belonging to Actinobacteria and α‐, β‐ and γ‐proteobacteria lineages indicating a widespread distribution of 2‐HPA catabolism via homogentisate route. These results provide first time evidence for the natural function of members of the CYP116B self‐sufficient oxygenases and represent a significant input to support novel kinetic and structural studies to develop cytochrome P450‐based biocatalytic processes.

Optimization and Engineering of a Self-Sufficient CYP102 Enzyme from Bacillus amyloliquefaciens towards Synthesis of In-Chain Hydroxy Fatty Acids

Cytochrome P450 (CYP) mediated enzymatic hydroxylation of fatty acids present a green alternative to chemical synthesis of hydroxy fatty acids (HFAs), which are high-value oleochemicals with various uses in materials industry and medical field. Although many CYPs require the presence of additional reductase proteins for catalytic activity, self-sufficient CYPs have their reductase partner naturally fused into their catalytic domain, leading to a greatly simplified biotransformation process. A recently discovered self-sufficient CYP, BAMF2522 from Bacillus amyloliquefaciens DSM 7, exhibits novel regioselectivity by hydroxylating in-chain positions of palmitic acid generating ω-1 to ω-7 HFAs, a rare regiodiversity profile among CYPs. Besides, F89I mutant of BAMF2522 expanded hydroxylation up to ω-9 position of palmitic acid. Here, we further characterize this enzyme by determining optimum temperature and pH as well as thermal stability. Moreover, using extensive site-directed and site-saturation mutagenesis, we obtained BAMF2522 variants that demonstrate greatly increased regioselectivity for in-chain positions (ω-4 to ω-9) of various medium to long chain fatty acids. Remarkably, when a six-residue mutant was reacted with palmitic acid, 84% of total product content was the sum of ω-7, ω-8 and ω-9 HFA products, the highest in-chain selectivity observed to date with a self-sufficient CYP. In short, our study demonstrates the potential of a recently identified CYP and its mutants for green and sustainable production of a variety of in-chain hydroxy enriched HFAs.

Front Cover: Advanced Understanding of the Electron Transfer Pathway of Cytochrome P450s (ChemBioChem 8/2021)

The landmark building of our school is the place where we started to solve the first full-length crystal structure of a self-sufficient cytochrome P450 (CYP116B46). The building has three main parts that are in an alignment similar to that of the three-domain CYP116B46. Cytochrome P450s are powerful biocatalysts with potential in many biotechnological applications. The majority of P450s demand redox equivalents that travel from pyridine nucleotides through the electron-transfer path to the catalytic center, like the lightning descending. In our review, we summarize the current understanding of the P450 electron-transfer route, with particular focus on the recently reported structures of two types of self-sufficient P450s. This information should provide important insights into the P450 mechanism studies and engineering. More information can be found in the review by X. Yu, R.-T. Guo, et al.

Expression and characterization of a novel cytochrome P450 enzyme from Variovorax paradoxus S110

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.

Structural insight into the electron transfer pathway of a self-sufficient P450 monooxygenase

Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.

CYP505E3: A Novel Self-Sufficient ω-7 In-Chain Hydroxylase.

The self-sufficient cytochrome P450 monooxygenase CYP505E3 from Aspergillus terreus catalyzes the regioselective in-chain hydroxylation of alkanes, fatty alcohols, and fatty acids at the ω-7 position. It is the first reported P450 to give regioselective in-chain ω-7 hydroxylation of C10-C16 n-alkanes, thereby enabling the one step biocatalytic synthesis of rare alcohols such as 5-dodecanol and 7-tetradecanol. It shows more than 70 % regioselectivity for the eighth carbon from one methyl terminus, and displays remarkably high activity towards decane (TTN≈8000) and dodecane (TTN≈2000). CYP505E3 can be used to synthesize the high-value flavour compound δ-dodecalactone via two routes: 1) conversion of dodecanoic acid into 5-hydroxydodecanoic acid (24 % regioselectivity), which at low pH lactonises to δ-dodecalactone, and 2) conversion of 1-dodecanol into 1,5-dodecanediol (55 % regioselectivity), which can be converted into δ-dodecalactone by horse liver alcohol dehydrogenase.

Characterization of a Self-Sufficient Cytochrome P450 Monooxygenase from Deinococcus apachensis for Enantioselective Benzylic Hydroxylation.

A self-sufficient cytochrome P450 monooxygenase from Deinococcus apachensis (P450DA) was identified and successfully overexpressed in Escherichia coli BL21(DE3). P450DA would be a member of the CYP102D subfamily and assigned as CYP102D2 according to the phylogenetic tree and sequence alignment. Purification and characterization of the recombinant P450DA indicated both NADH and NADPH could be used by P450DA as a reducing cofactor. The recombinant E. coli (P450DA) strain was functionally active, showing excellent enantioselectivity for benzylic hydroxylation of methyl 2-phenylacetate. Further substrate scope studies revealed that P450DA is able to catalyze benzylic hydroxylation of a variety of compounds, affording the corresponding chiral benzylic alcohols in 86-99 % ee and 130-1020 total turnover numbers.

Influence of inter-domain dynamics and surrounding environment flexibility on the direct electrochemistry and electrocatalysis of self-sufficient cytochrome P450 3A4-BMR chimeras

The linker region of multi-domain enzymes has a very important role for the interconnection of different enzyme modules and for the efficiency of catalytic activity. This is particularly evident for artificial chimeric systems. We characterised an artificial self-sufficient enzyme developed by genetic fusion of the catalytic domain of cytochrome P450 3A4 and reductase domain of Bacillus megaterium BM3 (BMR).Here we report the direct electrochemistry of 3A4-BMR chimeras immobilised on glassy carbon electrodes and we investigated the effect of inter-domain loop length and immobilising environment flexibility on both redox properties and electrocatalysis. We observe that redox potential can be modulated by the linker length and the immobilising layer flexibility. In addition, enzyme inter-domain dynamics and environment flexibility also modulate 3A4-BMR turnover efficiency on electrode system. Vmax values are increased up to about 100% in the presence of testosterone and up to about 50% in presence of tamoxifen by decreasing immobilising film rigidity. The effect on 3A4-BMR Vmax values is dependent on inter-domain loop length with 3A4-5GLY-BMR chimera being the more affected. The underlying reason for these observations is the potential motion of the FMN domain that is the key to shuttle electrons from FAD to haem.

Stabilization of the Reductase Domain in the Catalytically Self-Sufficient Cytochrome P450BM3 by Consensus-Guided Mutagenesis.

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.

In vitro characterization of CYP102G4 from Streptomyces cattleya: A self-sufficient P450 naturally producing indigo

Self-sufficient CYP102As possess outstanding hydroxylating activity to fatty acids such as myristic acid. Other CYP102 subfamily members share substrate specificity of CYP102As, but, occasionally, unusual characteristics of its own subfamily have been found. In this study, only one self-sufficient cytochrome P450 from Streptomyces cattleya was renamed from CYP102A_scat to CYP102G4, purified and characterized. UV–Vis spectrometry pattern, FAD/FMN analysis, and protein sequence comparison among CYP102s have shown that CYP102 from Streptomyces cattleya belongs to CYP102G subfamily. It showed hydroxylation activity toward fatty acids generating ω-1, ω-2, and ω-3-hydroxyfatty acids, which is similar to the general substrate specificity of CYP102 family. Unexpectedly, however, expression of CYP102G4 showed indigo production in LB medium batch flask culture, and high catalytic activity (kcat/Km) for indole was measured as 6.14±0.10min−1mM−1. Besides indole, CYP102G4 was able to hydroxylate aromatic compounds such as flavone, benzophenone, and chloroindoles. Homology model has shown such ability to accept aromatic compounds is due to its bigger active site cavity. Unlike other CYP102s, CYP102G4 did not have biased cofactor dependency, which was possibly determined by difference in NAD(P)H binding residues (Ala984, Val990, and Tyr1064) compared to CYP102A1 (Arg966, Lys972 and Trp1046). Overall, a self-sufficient CYP within CYP102G subfamily was characterized using purified enzymes, which appears to possess unique properties such as an only prokaryotic CYP naturally producing indigo.

Partial fusion of a cytochrome P450 system by carboxy-terminal attachment of putidaredoxin reductase to P450cam (CYP101A1).

Cytochrome P450 (CYP) enzymes catalyze the insertion of oxygen into carbon-hydrogen bonds and have great potential for enzymatic synthesis. Application development of class I CYPs is hampered by their dependence on two redox partners (a ferredoxin and ferredoxin reductase), slowing catalysis compared to self-sufficient CYPs such as CYP102A1 (P450BM3). Previous attempts to address this have fused all three components in several permutations and geometries, with much reduced activity compared to the native system. We report here the new approach of fusing putidaredoxin reductase (PdR) to the carboxy-terminus of CYP101A1 (P450cam) via a linker peptide and reconstituting camphor hydroxylase activity with free putidaredoxin (Pdx). Initial purification of a P450cam-PdR fusion yielded 2.0% heme incorporation. Co-expression of E. coli ferrochelatase, lengthening the linker from 5 to 20 residues, and altering culture conditions for enzyme production furnished 85% heme content. Fusion co-expression with Pdx gave a functional system with comparable in vivo camphor oxidation activity as the native system. In vitro, the fused system's steady state NADH oxidation rate was two-fold faster than that of the native system. In contrast to the native system, NADH oxidation rates for the fusion enzyme showed non-hyperbolic dependence on Pdx concentration, suggesting a role for the PdR domain; these data were consistent with a kinetic model based on two-site binding of Pdx by P450cam-PdR and inactive dimer formation of the fusion. P450cam-PdR is the first example of a class I P450 fusion that exhibits significantly more favorable behavior than that of the native system.

CYP116B5: a new class VII catalytically self-sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes.

A gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N- and C-terminal domains suggests an evolutionary model involving a plasmid-mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer-Villiger monooxygenase, allowed the recombinant bacteria to grow on long- and medium-chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium-chain alkanes overlapping AlkB and in the first step of sub-terminal oxidation of long-chain alkanes. It was also demonstrated that CYP116B5 is a self-sufficient cytochrome P450 consisting of a heme domain (aa 1-392) involved in the oxidation step of n-alkanes degradation, and its reductase domain (aa 444-758) comprising the NADPH-, FMN- and [2Fe2S]-binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.

A broad-range yeast expression system revealsArxula adeninivoransexpressing a fungal self-sufficient cytochrome P450 monooxygenase as an excellent whole-cell biocatalyst

The feasibility of using a single vector to clone a cytochrome P450 monooxygenase (P450) in different yeasts and then compare whole-cell hydroxylase activity was investigated. A broad-range yeast expression vector using the ylTEFp to drive expression of the cloned gene and the scTEFp to drive the hygromycin resistance marker gene was used to clone the genes encoding two self-sufficient P450s, CYP102A1 and CYP505A1. Both genes were cloned into Saccharomyces cerevisiae, Kluyveromyces marxianus, Yarrowia lipolytica (two strains) and Arxula adeninivorans. 4-Hexylbenzoic acid (HBA), which is subterminally hydroxylated by both CYP102A1 and CYP505A1, was used to compare whole-cell hydroxylase activity of transformants. Kluyveromyces marxianus and A.adeninivorans exhibited activity with both CYP102A1 and CYP505A1, while S.cerevisiae only displayed CYP102A1 activity and Y.lipolytica only CYP505A1 activity. The highest CYP102A1 activity (0.8mM HBA converted in 24h) was observed with concentrated resting-cell suspensions of S.cerevisiae. The CYP505A1 activity observed with growing cultures of A.adeninivorans was however at least 12 times higher than the CYP102A1 activity of S.cerevisiae with up to 2mM HBA converted within 6h. The use of K.marxianus and A.adeninivorans for P450 expression has not previously been reported.

Phosphite‐driven Self‐sufficient Cytochrome P450

AbstractCytochrome P450s (P450s) are attractive for biotechnology applications because P450s can catalyze regio‐ and stereo‐selective oxidations of non‐activated hydrocarbons. Many bacterial P450s require electron transfer from ferredoxins, which are in turn reduced by specific ferredoxin reductases and NAD(P)H. Stoichiometric consumption of NADH, an expensive reagent, is a major challenge for practical applications. A phosphite dehydrogenase (PTDH) that catalyzes the oxidation of phosphite to phosphate using NAD+, is an attractive choice of enzyme for NADH regeneration. In this study, we incorporated PTDH into an artificial heterotrimeric complex of Pseudomonas putida P450, putidaredoxin and putidaredoxin reductase, and the resulting complex almost completely hydroxylated 1 mm d‐camphor using 10 mM phosphite in the presence of 20 μM NAD+. This phosphite‐driven self‐sufficient P450 complex could provide a useful prototype for industry‐scale oxidizing biocatalysts.