Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.

Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism.

A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD-CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.

Exploring linker's sequence diversity to fuse carotene cyclase and hydroxylase for zeaxanthin biosynthesis.

Fusion of catalytic domains can accelerate cascade reactions by bringing enzymes in close proximity. However, the design of a protein fusion and the choice of a linker are often challenging and lack of guidance. To determine the impact of linker parameters on fusion proteins, a library of linkers featuring various lengths, secondary structures, extensions and hydrophobicities was designed. Linkers were used to fuse the lycopene cyclase (crtY) and ß-carotene hydroxylase (crtZ) from Pantoea ananatis to create fusion proteins to produce zeaxanthin. The fusion efficiency was assessed by comparing the carotenoids content in a carotenoid-producer Escherichia coli strain. It was shown that in addition to the orientation of the enzymes and the size of the linker, the first amino acid of the linker is also a key factor in determining the efficiency of a protein fusion. The wide range of sequence diversity in our linker library enables the fine tuning of protein fusion and this approach can be easily transferred to other enzyme couples.

Inferring assembly-curving trends of bacterial micro-compartment shell hexamers from crystal structure arrangements.

Bacterial microcompartments (BMC) are complex macromolecular assemblies that participate in varied chemical processes in about one fourth of bacterial species. BMC-encapsulated enzymatic activities are segregated from other cell contents by means of semipermeable shells, justifying why BMC are viewed as prototype nano-reactors for biotechnological applications. Herein, we undertook a comparative study of bending propensities of BMC hexamers (BMC-H), the most abundant shell constituents. Published data show that some BMC-H, like ß-carboxysomal CcmK, tend to assemble flat whereas other BMC-H often build curved objects. Inspection of available crystal structures presenting BMC-H in tiled arrangements permitted us to identify two major assembly modes with a striking connection with experimental trends. All-atom molecular dynamics (MD) supported that BMC-H bending is triggered robustly only from the arrangement adopted in crystals by BMC-H that experimentally form curved objects, leading to very similar arrangements to those found in structures of recomposed BMC shells. Simulations on triplets of planar-behaving hexamers, which were previously reconfigured to comply with such organization, confirmed that bending propensity is mostly defined by the precise lateral positioning of hexamers, rather than by BMC-H identity. Finally, an interfacial lysine was pinpointed as the most decisive residue in controlling PduA spontaneous curvature. Globally, results presented herein should contribute to improve our understanding of the variable mechanisms of biogenesis characterized for BMC, and of possible strategies to regulate BMC size and shape.

ß-Cryptoxanthin Production in Escherichia coli by Optimization of the Cytochrome P450 CYP97H1 Activity.

Cytochromes P450, forming a superfamily of monooxygenases containing heme as a cofactor, show great versatility in substrate specificity. Metabolic engineering can take advantage of this feature to unlock novel metabolic pathways. However, the cytochromes P450 often show difficulty being expressed in a heterologous chassis. As a case study in the prokaryotic host Escherichia coli, the heterologous synthesis of ß-cryptoxanthin was addressed. This carotenoid intermediate is difficult to produce, as its synthesis requires a monoterminal hydroxylation of ß-carotene whereas most of the classic carotene hydroxylases are dihydroxylases. This study was focused on the optimization of the in vivo activity of CYP97H1, an original P450 ß-carotene monohydroxylase. Engineering the N-terminal part of CYP97H1, identifying the matching redox partners, defining the optimal cellular background and adjusting the culture and induction conditions improved the production by 400 times compared to that of the initial strain, representing 2.7 mg/L ß-cryptoxanthin and 20% of the total carotenoids produced.

Cytochrome P450 Surface Domains Prevent the ß-Carotene Monohydroxylase CYP97H1 of Euglena gracilis from Acting as a Dihydroxylase.

Molecular biodiversity results from branched metabolic pathways driven by enzymatic regioselectivities. An additional complexity occurs in metabolites with an internal structural symmetry, offering identical extremities to the enzymes. For example, in the terpene family, ß-carotene presents two identical terminal closed-ring structures. Theses cycles can be hydroxylated by cytochrome P450s from the CYP97 family. Two sequential hydroxylations lead first to the formation of monohydroxylated ß-cryptoxanthin and subsequently to that of dihydroxylated zeaxanthin. Among the CYP97 dihydroxylases, CYP97H1 from Euglena gracilis has been described as the only monohydroxylase. This study aims to determine which enzymatic domains are involved in this regioselectivity, conferring unique monohydroxylase activity on a substrate offering two identical sites for hydroxylation. We explored the effect of truncations, substitutions and domain swapping with other CYP97 members and found that CYP97H1 harbours a unique N-terminal globular domain. This CYP97H1 N-terminal domain harbours a hydrophobic patch at the entrance of the substrate channel, which is involved in the monohydroxylase activity of CYP97H1. This domain, at the surface of the enzyme, highlights the role of distal and non-catalytic domains in regulating enzyme specificity.

Optogenetic control of beta-carotene bioproduction in yeast across multiple lab-scales.

Optogenetics arises as a valuable tool to precisely control genetic circuits in microbial cell factories. Light control holds the promise of optimizing bioproduction methods and maximizing yields, but its implementation at different steps of the strain development process and at different culture scales remains challenging. In this study, we aim to control beta-carotene bioproduction using optogenetics in Saccharomyces cerevisiae and investigate how its performance translates across culture scales. We built four lab-scale illumination devices, each handling different culture volumes, and each having specific illumination characteristics and cultivating conditions. We evaluated optogenetic activation and beta-carotene production across devices and optimized them both independently. Then, we combined optogenetic induction and beta-carotene production to make a light-inducible beta-carotene producer strain. This was achieved by placing the transcription of the bifunctional lycopene cyclase/phytoene synthase CrtYB under the control of the pC120 optogenetic promoter regulated by the EL222-VP16 light-activated transcription factor, while other carotenogenic enzymes (CrtI, CrtE, tHMG) were expressed constitutively. We show that illumination, culture volume and shaking impact differently optogenetic activation and beta-carotene production across devices. This enabled us to determine the best culture conditions to maximize light-induced beta-carotene production in each of the devices. Our study exemplifies the stakes of scaling up optogenetics in devices of different lab scales and sheds light on the interplays and potential conflicts between optogenetic control and metabolic pathway efficiency. As a general principle, we propose that it is important to first optimize both components of the system independently, before combining them into optogenetic producing strains to avoid extensive troubleshooting. We anticipate that our results can help designing both strains and devices that could eventually lead to larger scale systems in an effort to bring optogenetics to the industrial scale.

A tripartite carbohydrate-binding module to functionalize cellulose nanocrystals.

The development of protein and microorganism engineering have led to rising expectations of biotechnology in the design of emerging biomaterials, putatively of high interest to reduce our dependence on fossil carbon resources. In this way, cellulose, a renewable carbon based polysaccharide and derived products, displays unique properties used in many industrial applications. Although the functionalization of cellulose is common, it is however limited in terms of number and type of functions. In this work, a Carbohydrate-Binding Module (CBM) was used as a central core to provide a versatile strategy to bring a large diversity of functions to cellulose surfaces. CBM3a from Clostridium thermocellum, which has a high affinity for crystalline cellulose, was flanked through linkers with a streptavidin domain and an azide group introduced through a non-canonical amino acid. Each of these two extra domains was effectively produced and functionalized with a variety of biological and chemical molecules. Structural properties of the resulting tripartite chimeric protein were investigated using molecular modelling approaches, and its potential for the multi-functionalization of cellulose was confirmed experimentally. As a proof of concept, we show that cellulose can be labelled with a fluorescent version of the tripartite protein grafted to magnetic beads and captured using a magnet.

The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments.

Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories.

Multiplicity of carotene patterns derives from competition between phytoene desaturase diversification and biological environments.

Phytoene desaturases catalyse from two to six desaturation reactions on phytoene, generating a large diversity of molecules that can then be cyclised and produce, depending on the organism, many different carotenoids. We constructed a phylogenetic tree of a subset of phytoene desaturases from the CrtI family for which functional data was available. We expressed in a bacterial system eight codon optimized CrtI enzymes from different clades. Analysis of the phytoene desaturation reactions on crude extracts showed that three CrtI enzymes can catalyse up to six desaturations, forming tetradehydrolycopene. Kinetic data generated using a subset of five purified enzymes demonstrate the existence of characteristic patterns of desaturated molecules associated with various CrtI clades. The kinetic data was also analysed using a classical Michaelis-Menten kinetic model, showing that variations in the reaction rates and binding constants could explain the various carotene patterns observed. Competition between lycopene cyclase and the phytoene desaturases modified the distribution between carotene intermediates when expressed in yeast in the context of the full ß-carotene production pathway. Our results demonstrate that the desaturation patterns of carotene molecules in various biological environments cannot be fully inferred from phytoene desaturases classification but is governed both by evolutionary-linked variations in the desaturation rates and competition between desaturation and cyclisation steps.

Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding.

The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is degenerated. Recently, we demonstrated that specific CPR mutations in the FMN-domain (FD) may induce a gain in activity for a specific CYP isoform. In the current report, we confirm the CYP isoform dependence of CPR's degenerated binding by demonstrating that the effect of four of the formerly studied FD mutants are indeed exclusive of a specific CYP isoform, as verified by cytochrome c inhibition studies. Moreover, the nature of CYP's substrate seems to have a modulating role in the CPR:CYP interaction. In silico molecular dynamics simulations of the FD evidence that mutations induces very subtle structural alterations, influencing the characteristics of residues formerly implicated in the CPR:CYP interaction or in positioning of the FMN moiety. CPR seems therefore to be able to form effective interaction complexes with its structural diverse partners via a combination of specific structural features of the FD, which are functional in a CYP isoform dependent manner, and dependent on the substrate bound.

Corrigendum: The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

[This corrects the article DOI: 10.3389/fphar.2020.00299.].

An artificial chromosome ylAC enables efficient assembly of multiple genes in Yarrowia lipolytica for biomanufacturing.

The efficient use of the yeast Yarrowia lipolytica as a cell factory is hampered by the lack of powerful genetic engineering tools dedicated for the assembly of large DNA fragments and the robust expression of multiple genes. Here we describe the design and construction of artificial chromosomes (ylAC) that allow easy and efficient assembly of genes and chromosomal elements. We show that metabolic pathways can be rapidly constructed by various assembly of multiple genes in vivo into a complete, independent and linear supplementary chromosome with a yield over 90%. Additionally, our results reveal that ylAC can be genetically maintained over multiple generations either under selective conditions or, without selective pressure, using an essential gene as the selection marker. Overall, the ylACs reported herein are game-changing technology for Y. lipolytica, opening myriad possibilities, including enzyme screening, genome studies and the use of this yeast as a previous unutilized bio-manufacturing platform.

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b 5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches - formerly indicated to be involved in CPR:CYP interactions - or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

Synthetic Derivatives of (+)-epi-α-Bisabolol Are Formed by Mammalian Cytochromes P450 Expressed in a Yeast Reconstituted Pathway.

Identification of the enzyme(s) involved in complex biosynthetic pathways can be challenging. An alternative approach might be to deliberately diverge from the original natural enzyme source and use promiscuous enzymes from other organisms. In this paper, we have tested the ability of a series of human and animal cytochromes P450 involved in xenobiotic detoxification to generate derivatives of (+)-epi-α-bisabolol and attempt to produce the direct precursor of hernandulcin, a sweetener from Lippia dulcis for which the last enzymatic steps are unknown. Screening steps were implemented in vivo in S. cerevisiae optimized for the biosynthesis of oxidized derivatives of (+)-epi-α-bisabolol by coexpressing two key enzymes: the (+)-epi-α-bisabolol synthase and the NADPH cytochrome P450 reductase. Five out of 25 cytochromes P450 were capable of producing new hydroxylated regioisomers of (+)-epi-α-bisabolol. Of the new oxidized bisabolol products, the structure of one compound, 14-hydroxy-(+)-epi-α-bisabolol, was fully elucidated by NMR while the probable structure of the second product was determined. In parallel, the production of (+)-epi-α-bisabolol derivatives was enhanced through the addition of a supplementary genomic copy of (+)-epi-α-bisabolol synthase that augmented the final titer of hydroxylated product to 64 mg/L. We thus demonstrate that promiscuous drug metabolism cytochromes P450 can be used to produce novel compounds from a terpene scaffold.

Occurrence and stability of hetero-hexamer associations formed by ß-carboxysome CcmK shell components.

The carboxysome is a bacterial micro-compartment (BMC) subtype that encapsulates enzymatic activities necessary for carbon fixation. Carboxysome shells are composed of a relatively complex cocktail of proteins, their precise number and identity being species dependent. Shell components can be classified in two structural families, the most abundant class associating as hexamers (BMC-H) that are supposed to be major players for regulating shell permeability. Up to recently, these proteins were proposed to associate as homo-oligomers. Genomic data, however, demonstrated the existence of paralogs coding for multiple shell subunits. Here, we studied cross-association compatibilities among BMC-H CcmK proteins of Synechocystis sp. PCC6803. Co-expression in Escherichia coli proved a consistent formation of hetero-hexamers combining CcmK1 and CcmK2 or, remarkably, CcmK3 and CcmK4 subunits. Unlike CcmK1/K2 hetero-hexamers, the stoichiometry of incorporation of CcmK3 in associations with CcmK4 was low. Cross-interactions implicating other combinations were weak, highlighting a structural segregation of the two groups that could relate to gene organization. Sequence analysis and structural models permitted the localization of interactions that would favor formation of CcmK3/K4 hetero-hexamers. The crystallization of these CcmK3/K4 associations conducted to the elucidation of a structure corresponding to the CcmK4 homo-hexamer. Yet, subunit exchange could not be demonstrated in vitro. Biophysical measurements showed that hetero-hexamers are thermally less stable than homo-hexamers, and impeded in forming larger assemblies. These novel findings are discussed within the context of reported data to propose a functional scenario in which minor CcmK3/K4 incorporation in shells would introduce sufficient local disorder as to allow shell remodeling necessary to adapt rapidly to environmental changes.

Functional analysis of isoprenoid precursors biosynthesis by quantitative metabolomics and isotopologue profiling.

INTRODUCTION: Isoprenoids are amongst the most abundant and diverse biological molecules and are involved in a broad range of biological functions. Functional understanding of their biosynthesis is thus key in many fundamental and applicative fields, including systems biology, medicine and biotechnology. However, available methods do not yet allow accurate quantification and tracing of stable isotopes incorporation for all the isoprenoids precursors. OBJECTIVES: We developed and validated a complete methodology for quantitative metabolomics and isotopologue profiling of isoprenoid precursors in the yeast Saccharomyces cerevisiae. METHODS: This workflow covers all the experimental and computational steps from sample collection and preparation to data acquisition and processing. It also includes a novel quantification method based on liquid chromatography coupled to high-resolution mass spectrometry. Method validation followed the Metabolomics Standards Initiative guidelines. RESULTS: This workflow ensures accurate absolute quantification (RSD < 20%) of all mevalonate and prenyl pyrophosphates intermediates with a high sensitivity over a large linear range (from 0.1 to 50 pmol). In addition, we demonstrate that this workflow brings crucial information to design more efficient phytoene producers. Results indicate stable turnover rates of prenyl pyrophosphate intermediates in the constructed strains and provide quantitative information on the change of the biosynthetic flux of phytoene precursors. CONCLUSION: This methodology fills one of the last technical gaps for functional studies of isoprenoids biosynthesis and should be applicable to other eukaryotic and prokaryotic (micro)organisms after adaptation of some organism-dependent steps. This methodology also opens the way to 13C-metabolic flux analysis of isoprenoid biosynthesis.

Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae.

Spatial clustering of enzymes has proven an elegant approach to optimize metabolite transfer between enzymes in synthetic metabolic pathways. Among the multiple methods used to promote colocalisation, enzyme fusion is probably the simplest. Inspired by natural systems, we have explored the metabolic consequences of spatial reorganizations of the catalytic domains of Xanthophyllomyces dendrorhous carotenoid enzymes produced in Saccharomyces cerevisiae. Synthetic genes encoding bidomain enzymes composed of CrtI and CrtB domains from the natural CrtYB fusion were connected in the two possible orientations, using natural and synthetic linkers. A tridomain enzyme (CrtB, CrtI, CrtY) harboring the full ß-carotene producing pathway was also constructed. Our results demonstrate that domain order and linker properties considerably impact both the expression and/or stability of the constructed proteins and the functionality of the catalytic domains, all concurring to either diminish or boost specific enzymatic steps of the metabolic pathway. Remarkably, the yield of ß-carotene production doubled with the tridomain fusion while precursor accumulation decreased, leading to an improvement of the pathway efficiency, when compared to the natural system. Our data strengthen the idea that fusion of enzymatic domains is an appropriate technique not only to achieve spatial confinement and enhance the metabolic flux but also to produce molecules not easily attainable with natural enzymatic configurations, even with membrane bound enzymes.

Development of a Biosensor for Detection of Benzoic Acid Derivatives in Saccharomyces cerevisiae.

4-hydroxybenzoic acid (pHBA) is an important industrial precursor of muconic acid and liquid crystal polymers whose production is based on the petrochemical industry. In order to decrease our dependency on fossil fuels and improve sustainability, microbial engineering is a particularly appealing approach for replacing traditional chemical techniques. The optimization of microbial strains, however, is still highly constrained by the screening stage. Biosensors have helped to alleviate this problem by decreasing the screening time as well as enabling higher throughput. In this paper, we constructed a synthetic biosensor, named sBAD, consisting of a fusion of the pHBA-binding domain of HbaR from R. palustris, the LexA DNA binding domain at the N-terminus and the transactivation domain B112 at the C-terminus. The response of sBAD was tested in the presence of different benzoic acid derivatives, with cell fluorescence output measured by flow cytometry. The biosensor was found to be activated by the external addition of pHBA in the culture medium, in addition to other carboxylic acids including p-aminobenzoic acid (pABA), salicylic acid, anthranilic acid, aspirin, and benzoic acid. Furthermore, we were able to show that this biosensor could detect the in vivo production of pHBA in a genetically modified yeast strain. A good linearity was observed between the biosensor fluorescence and pHBA concentration. Thus, this biosensor would be well-suited as a high throughput screening tool to produce, via metabolic engineering, benzoic acid derivatives.

Accurate Determination of Human CPR Conformational Equilibrium by smFRET Using Dual Orthogonal Noncanonical Amino Acid Labeling.

Conjugation of fluorescent dyes to proteins-a prerequisite for the study of conformational dynamics by single-molecule (sm) FRET-can lead to substantial changes in a dye's photophysical properties, ultimately biasing the determination of inter-dye distances. In particular, cyanine dyes and their derivatives, the most commonly used dyes in smFRET experiments, exhibit such behavior. To overcome this, we developed a general strategy to equip proteins site-specifically with FRET pairs through chemoselective reactions with two distinct noncanonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli. Application of this technique to human NADPH-cytochrome P450 reductase (CPR) demonstrated the importance of homogenously labeled samples for accurate determination of FRET efficiencies and unveiled the effect of NADP+ on the ionic-strength-dependent modulation of the conformational equilibrium of CPR. Thanks to its generality and accuracy, the presented methodology establishes a new benchmark for deciphering of complex molecular dynamics in single molecules.