Cytochrome P450 BM3 Mutants Research Articles

Bacterial Acyl Homoserine Lactones Triggered Non‐Native Substrate Hydroxylation Catalyzed by Directed‐Evolution‐Derived Cytochrome P450BM3 Mutants

AbstractWe report the directed evolution of cytochrome P450BM3 to efficiently utilize the bacterial quorum sensing signalling molecule N‐decanoyl homoserine lactone (C10‐HSL) as an effective decoy molecule. This represents the first important step in our endeavor to develop of a self‐sufficient decoy‐molecule system in whole‐cells that only necessitates the addition of culture medium and substrate to realize the hydroxylation of non‐native substrates. Following five rounds of directed evolution, mutant P450BM3, in the presence of C10‐HSL, catalyzed the hydroxylation of benzene at a rate of 475 min−1, the highest turnover rate recorded for any P450 enzyme, and achieving a 46% yield in a whole‐cell reaction system. High‐resolution X‐ray crystal structure analysis of a series of mutants narrates the directed evolution process, revealing how C10‐HSL is fixed in the binding pocket to permit binding of non‐native substrates. Finally, introduction of the C10‐HSL synthase gene ExpI into Escherichia coli, enabled the in situ production of C10‐HSL, realizing, for the first time, the hydroxylation of non‐native substrates without the need for the laborious synthesis and addition of decoy molecules.

Regio- and stereo-selective 1β-hydroxylation of lithocholic acid by cytochrome P450 BM3 mutants.

Regio- and stereo-selective hydroxylation of bile acids is a valuable reaction but often lacks suitable catalysts. In the research, semi-rational design in protein engineering techniques had been applied on cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, and a mutation library had been set up for the 1β-hydroxylation of lithocholic acid (LCA) to produce 1β-OH-LCA. After four rounds of mutagenesis, a key residue at W72 was identified to regulate the regio- and stereo-selectivity at C1 of LCA. A quadruple variant (G87A/W72T/A74L/L181M) was identified to reach 99.4% selectivity of 1β-hydroxylation and substrate conversion of 68.1% resulting in a 21.5-fold higher level of 1β-OH-LCA production than the template LG-23. Molecular docking indicated that introducing hydrogen bonds at W72 was responsible for enhancing selectivity and catalytic activity, which gave some insights into the structure-based understanding of Csp3 -H activation by the developed P450 BM3 mutants.

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.

A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

AbstractAssaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high‐throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high‐throughput mass‐spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click‐assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.

A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts.

Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high-throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high-throughput mass-spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click-assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.

Structural analysis of Cytochrome P450 BM3 mutant M11 in complex with dithiothreitol.

The bacterial Cytochrome P450 (CYP) BM3 (CYP102A1) is one of the most active CYP isoforms. BM3 mutants can serve as a model for human drug-metabolizing CYPs and/or as biocatalyst for selective formation of drug metabolites. Hence, molecular and computational biologists have in the last two decades shown strong interest in the discovery and design of novel BM3 variants with optimized activity and selectivity for substrate conversion. This led e.g. to the discovery of mutant M11 that is able to metabolize a variety of drugs and drug-like compounds with relatively high activity. In order to further improve our understanding of CYP binding and reactions, we performed a co-crystallization study of mutant M11 and report here the three-dimensional structure M11 in complex with dithiothreitol (DTT) at a resolution of 2.16 Å. The structure shows that DTT can coordinate to the Fe atom in the heme group. UV/Vis spectroscopy and molecular dynamics simulation studies underline this finding and as first structure of the CYP BM3 mutant M11 in complex with a ligand, it offers a basis for structure-based design of novel mutants.

A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants

The 5′-hydroxymethyl metabolite of the penicillin based antibiotic flucloxacillin (FLX) is considered to be involved in bile duct damage occurring in a small number of patients. Because 5′-hydroxymethyl FLX is difficult to obtain by organic synthesis, biosynthesis using highly active and regioselective biocatalysts would be an alternative approach. By screening an in-house library of Cytochrome P450 (CYP) BM3 mutants, mutant M11 L437E was identified as a regioselective enzyme with relatively high activity in production of 5′-hydroxymethyl FLX as was confirmed by mass spectrometry and NMR. In contrast, incubation of M11 L437E and other mutants with oxacillin (OX, which differs from FLX by a lack of aromatic halogens) resulted in formation of two metabolites. In addition to 5′-hydroxymethyl OX we identified a product resulting from aromatic hydroxylation. In silico studies of both FLX and OX with three CYP BM3 mutants revealed substrate binding poses allowing for 5′-methyl hydroxylation, as well as binding poses with the aromatic moiety in the vicinity of the heme iron for which the corresponding product of aromatic hydroxylation was not observed for FLX. Supported by the (differences in) experimentally determined ratios of product formation for OX hydroxylation by M11 and its L437A variant and M11 L437E, Molecular Dynamics simulations suggest that the preference of mutant M11 L437E to bind FLX in its catalytically active pose over the other binding orientation contributes to its biocatalytic activity, highlighting the benefit of studying effects of active-site mutations on possible alternative enzyme-substrate binding poses in protein engineering.

P450-Catalyzed Regio- and Stereoselective Oxidative Hydroxylation of 6-Iodotetralone: Preparative-Scale Synthesis of a Key Intermediate for Pd-Catalyzed Transformations.

Chiral alcohols are important building blocks for the production of pharmaceuticals, catalytic access being possible by the use of enzymes, transition metal catalysts, or organocatalysts. Herein we report the use of cytochrome P450-BM3 mutants for the oxidative hydroxylation of 6-iodotetralone regio- and enantioselectively at C4 with formation of the ( R)-alcohol. This CH activation is not possible using modern synthetic catalysts. The synthetic utility of this valuable synthon was explored in palladium-catalyzed coupling reactions that occur in the absence of undesired racemization.

Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3

The search of new substrates with pharmaceutical and industrial potential for biocatalysts including cytochrome P450 enzymes is always challenging. Cytochrome P450 BM3 mutant 139-3, a versatile biocatalyst, exhibited hydroxylation activities towards fatty acids and alkanes. However, there were limited reports about its hydroxylation activity towards steroids. Herein, an Escherichia coli–based whole-cell extract containing the recombinant 139-3 protein was used as the biocatalyst to screen 13 steroids. Results revealed that 139-3 was able to specifically hydroxylate androstenedione (1) at 1α-position, generating a hydroxylated steroid 1α-OH-androstenedione (1a). To investigate whether C-1α hydroxylation catalyzed by BM3 mutant 139-3 could be industrially used, an optimization of catalyzing conditions was performed. Accordingly, the BM3 mutant 139-3 enzyme was observed to display maximum activity at 37°C, under pH 7.0 for 4h, with 37% transformation rate. Moreover, four 139-3 variants were generated by random mutagenesis with the aim of improving its activity and expanding substrate scope. Surprisingly, these mutants, sharing a common mutated site R379S, lost their activities towards androstenedione (1). These data clearly indicated that arginine residue located at site 379 played key role in the hydroxylation activities of 139-3. Overall, these new findings broadened the substrate scope of 139-3 enzyme, thereby expanding its potential applications as a biocatalyst on steroids hydroxylation in pharmaceutical industry.

Hydroxylation of anilides by engineered cytochrome P450BM3.

Biocatalytic direct monohydroxylation of anilides has been achieved on preparative scale using mutant cytochrome P450BM3 enzymes. Representative mono- and disubstituted N-trifluoromethanesulfonyl anilides are shown to be converted in most cases to the corresponding 4-hydroxy derivatives, with substituent hydroxylation also occurring in two cases. By mutation variation, it is possible to achieve selective hydroxylation of either ring- or side-chain sites.

Insights into regioselective metabolism of mefenamic acid by cytochrome P450 BM3 mutants through crystallography, docking, molecular dynamics, and free energy calculations.

Cytochrome P450 BM3 (CYP102A1) mutant M11 is able to metabolize a wide range of drugs and drug-like compounds. Among these, M11 was recently found to be able to catalyze formation of human metabolites of mefenamic acid and other nonsteroidal anti-inflammatory drugs (NSAIDs). Interestingly, single active-site mutations such as V87I were reported to invert regioselectivity in NSAID hydroxylation. In this work, we combine crystallography and molecular simulation to study the effect of single mutations on binding and regioselective metabolism of mefenamic acid by M11 mutants. The heme domain of the protein mutant M11 was expressed, purified, and crystallized, and its X-ray structure was used as template for modeling. A multistep approach was used that combines molecular docking, molecular dynamics (MD) simulation, and binding free-energy calculations to address protein flexibility. In this way, preferred binding modes that are consistent with oxidation at the experimentally observed sites of metabolism (SOMs) were identified. Whereas docking could not be used to retrospectively predict experimental trends in regioselectivity, we were able to rank binding modes in line with the preferred SOMs of mefenamic acid by M11 and its mutants by including protein flexibility and dynamics in free-energy computation. In addition, we could obtain structural insights into the change in regioselectivity of mefenamic acid hydroxylation due to single active-site mutations. Our findings confirm that use of MD and binding free-energy calculation is useful for studying biocatalysis in those cases in which enzyme binding is a critical event in determining the selective metabolism of a substrate.

Application of a cocktail approach to screen cytochrome P450 BM3 libraries for metabolic activity and diversity.

In the present study, the validity of using a cocktail screening method in combination with a chemometrical data mining approach to evaluate metabolic activity and diversity of drug-metabolizing bacterial Cytochrome P450 (CYP) BM3 mutants was investigated. In addition, the concept of utilizing an in-house-developed library of CYP BM3 mutants as a unique biocatalytic synthetic tool to support medicinal chemistry was evaluated. Metabolic efficiency of the mutant library towards a selection of CYP model substrates, being amitriptyline (AMI), buspirone (BUS), coumarine (COU), dextromethorphan (DEX), diclofenac (DIC) and norethisterone (NET), was investigated. First, metabolic activity of a selection of CYP BM3 mutants was screened against AMI and BUS. Subsequently, for a single CYP BM3 mutant, the effect of co-administration of multiple drugs on the metabolic activity and diversity towards AMI and BUS was investigated. Finally, a cocktail of AMI, BUS, COU, DEX, DIC and NET was screened against the whole in-house CYP BM3 library. Different validated quantitative and qualitative (U)HPLC-MS/MS-based analytical methods were applied to screen for substrate depletion and targeted product formation, followed by a more in-depth screen for metabolic diversity. A chemometrical approach was used to mine all data to search for unique metabolic properties of the mutants and allow classification of the mutants. The latter would open the possibility of obtaining a more in-depth mechanistic understanding of the metabolites. The presented method is the first MS-based method to screen CYP BM3 mutant libraries for diversity in combination with a chemometrical approach to interpret results and visualize differences between the tested mutants.Graphical abstractGeneral worklfow in screening mutant enzyme libraries for catalytic efficiency and diversityElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-9241-x) contains supplementary material, which is available to authorized users.

Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants

A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3–mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small library of novel CYP BM3 mutants in flow-injection analysis mode in the presence of the organic modifiers methanol, acetonitrile, and isopropanol. Mutants with enhanced tolerability toward all three solvents were identified, and the EAD setup was adapted to facilitate CYP BM3 activity screening against a gradient of an organic modifier to study the behavior of the small library of CYP BM3 mutants in more detail. The simple methodology used in this study was shown to be a very powerful tool to screen for novel CYP BM3 mutants with increased tolerability toward organic solvents.

Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

In the present study, the whole-cell biotransformation using strains expressing CYP BM3 mutants has been evaluated for the stereoselective hydroxylation of the steroid norethisterone (NET), a widely used contraceptive. First, an in vitro CYP BM3 mutant library screen was performed to identify mutants with high activity, as well asstereoselectivity. Subsequently, two different whole-cell setups (resting and growing cells) were tested in two different host organisms (Escherichia coli and Saccharomyces cerevisiae) expressing CYP BM3. It was found that resting E. coli whole cells produced the highest amounts of products and therefore this biocatalytic setup was further optimized for application in a laboratory-scale fermentor. In the optimized fermentor setup, high product yields (0.3g/L 15β-OH-NET and 0.16g/L 16β-OH-NET) were achieved while it was also shown that the regio- and stereoselectivities of the steroid hydroxylations, as determined during the in vitro library screen, were preserved in the whole-cell system. The combination of a mutant CYP BM3 library and the optimized whole-cell oxidation system represents a promising and cost-effective alternative to a wide range of in vitro biosynthetic routes.

Biosynthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme.

In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the steroid with >95% regioselectivity to form 16-β-OH norandrostenedione, as confirmed by NMR analysis. Whole cells of R. erythropolis RG9 expressing P450 BM3 M02 enzyme also converted norandrostenedione into the 16-β-hydroxylated product, resulting in the formation of about 0.35g/L. Whole cells of strain RG9 itself did not convert norandrostenedione, indicating that metabolite formation is P450 BM3 M02 enzyme mediated. This study shows that R. erythropolis is a novel and interesting host for the heterologous expression of highly selective and active P450 BM3 M02 enzyme variants able to perform steroid bioconversions.

Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants

Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy.We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity.To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3′-OH-methyl-meclofenamic acid (1a), 5-OH-meclofenamic acid (1b) and 4′-OH-meclofenamic acid (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of 1 respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000–6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4′ position of all three drugs tested with high regioselectivity. Reference metabolites produced by P450 BM3 mutants allowed the characterisation of activity and regioselectivity of metabolism of all three NSAIDs by thirteen recombinant human P450s. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.

Combination of biotransformation by P450 BM3 mutants with on-line post-column bioaffinity and mass spectrometric profiling as a novel strategy to diversify and characterize p38α kinase inhibitors

Mutants of bacterial cytochrome P450 BM3 from Bacillus megaterium have gained increasing interest to support drug metabolism studies by producing human relevant biotransformation products and/or to support drug discovery by producing libraries of new chemical entities based on initial lead compounds. In this study, a library of 33 P450 BM3 mutants was used to diversify TAK-715, a representative (highly lipophilic) inhibitor of p38α mitogen-activated protein kinase (p38α). To simultaneously determine the individual bioaffinity and identity of the different products, an analytical high-resolution screening approach was used, based on post-column on-line bioaffinity profiling with parallel mass spectrometric identification. The screening of the product mixtures produced demonstrated that introducing mutations in the active site of the P450 BM3 resulted in different product profiles. Several P450 BM3 mutants were mimicking the metabolic profile obtained by human liver microsomes. The major biotransformation products of TAK-715 could be produced with the most active P450 BM3 mutant in sufficient amounts to enable further structure elucidation by 1H-NMR and quantification of their p38α affinity.

Hydroxylation of non-substituted polycyclic aromatic hydrocarbons by cytochrome P450 BM3 engineered by directed evolution

Chrysene and pyrene are known toxic compounds recalcitrant to biodegradation. Here directed evolution allowed us to identify two new mutants of cytochrome P450 BM3 that are able to hydroxylate both compounds. Random mutagenesis has been used to generate libraries of mutants of P450 BM3 active toward polycyclic aromatic hydrocarbons (PAHs) PAHs. After two rounds of error-prone PCR and backcross with parental DNA, three mutants were identified for improved activity toward pyrene and for the first time a new activity toward chrysene in comparison to the wild type enzyme. The mutants show higher affinity and coupling efficiency for chrysene with faster rates of product formation compared to the wild type. Furthermore, the mutants are able to hydroxylate chrysene in different positions, producing four metabolites, 1-, 3-, 4-, and 6-hydroxychrysene, and to hydroxylate pyrene to 1-hydroxypyrene.The majority of the mutation sites are found to be far from the active site, demonstrating the power of directed evolution in identifying mutations difficult to predict with a rational design approach.The different product profiles obtained for the different P450 BM3 mutants indicate that substrate orientation in the catalytic pocket of the protein can be modified by protein engineering. The mutants can be used for metabolic engineering for safe and cost-effective sustainable production of hydroxylated PAHs for industrial purposes as well as for the assessment of their carcinogenic activity in mammals.

Regio‐ and Stereoselective Hydroxylation of Optically Active α‐Ionone Enantiomers by Engineered Cytochrome P450 BM3 Mutants

AbstractThe selective hydroxylation of an unactivated CH bond is a crucial step in the synthesis of fine chemicals such as hydroxylated terpenoids. In the present study, the ability of 40 cytochrome P450 BM3 mutants to perform the regio‐ and stereoselective hydroxylation of α‐ionone has been investigated. Based on their activity and selectivity to produce 3‐hydroxy‐α‐ionone from racemic α‐ionone, 6 BM3 mutants were selected. Out of these, 3 mutants (M01 A82W, M11 A82W and M11 V87I) showed high selectivity for trans‐3‐hydroxy‐α‐ionone formation while 3 other mutants (M11 L437N, M11 L437S and M11 L437T) formed almost equal amounts of both cis‐3‐hydroxy‐ and trans‐3‐hydroxy‐α‐ionone. Incubation with individual enantiomers showed that M11 L437N, M11 L437S and M11 L437T exhibited opposite stereoselectivity producing (3S,6S)‐hydroxy‐α‐ionone with the (6S)‐enantiomer and (3S,6R)‐hydroxy‐α‐ionone with the (6R)‐enantiomer. Thus for the first time, BM3 mutants that can selectively produce diastereomers of 3‐hydroxy‐α‐ionone (>90% de), with high turnover numbers and minimal secondary metabolism, have been identified. Docking studies have been performed to rationalize the basis of the experimentally observed selectivity. In conclusion, engineered P450 BM3s are promising biocatalysts for regio‐ and stereoselective production of hydroxylated α‐ionones for industrial applications.