Spotlight on CYP4B1.

The mammalian cytochrome P450 monooxygenase CYP4B1 can bioactivate a wide range of xenobiotics, such as its defining/hallmark substrate 4-ipomeanol leading to tissue-specific toxicities. Similar to other members of the CYP4 family, CYP4B1 has the ability to hydroxylate fatty acids and fatty alcohols. Structural insights into the enigmatic role of CYP4B1 with functions in both, xenobiotic and endobiotic metabolism, as well as its unusual heme-binding characteristics are now possible by the recently solved crystal structures of native rabbit CYP4B1 and the p.E310A variant. Importantly, CYP4B1 does not play a major role in hepatic P450-catalyzed phase I drug metabolism due to its predominant extra-hepatic expression, mainly in the lung. In addition, no catalytic activity of human CYP4B1 has been observed owing to a unique substitution of an evolutionary strongly conserved proline 427 to serine. Nevertheless, association of CYP4B1 expression patterns with various cancers and potential roles in cancer development have been reported for the human enzyme. This review will summarize the current status of CYP4B1 research with a spotlight on its roles in the metabolism of endogenous and exogenous compounds, structural properties, and cancer association, as well as its potential application in suicide gene approaches for targeted cancer therapy.

Use of Copper as a Trigger for the in Vivo Activity of E. coli Laccase CueO: A Simple Tool for Biosynthetic Purposes.

Laccases are multi-copper oxidases that catalyze the oxidation of various electron-rich substrates with concomitant reduction of molecular oxygen to water. The multi-copper oxidase/laccase CueO of Escherichia coli is responsible for the oxidation of Cu+ to the less harmful Cu2+ in the periplasm. CueO has a relatively broad substrate spectrum as laccase, and its activity is enhanced by copper excess. The aim of this study was to trigger CueO activity in vivo for the use in biocatalysis. The addition of 5 mM CuSO4 was proven effective in triggering CueO activity at need with minor toxic effects on E. coli cells. Cu-treated E. coli cells were able to convert several phenolic compounds to the corresponding dimers. Finally, the endogenous CueO activity was applied to a four-step cascade, in which coniferyl alcohol was converted to the valuable plant lignan (-)-matairesinol.

Synthesis of (-)-deoxypodophyllotoxin and (-)-epipodophyllotoxin via a multi-enzyme cascade in E. coli.

BACKGROUND: The aryltetralin lignan (-)-podophyllotoxin is a potent antiviral and anti-neoplastic compound that is mainly found in Podophyllum plant species. Over the years, the commercial demand for this compound rose notably because of the high clinical importance of its semi-synthetic chemotherapeutic derivatives etoposide and teniposide. To satisfy this demand, (-)-podophyllotoxin is conventionally isolated from the roots and rhizomes of Sinopodophyllum hexandrum, which can only grow in few regions and is now endangered by overexploitation and environmental damage. For these reasons, targeting the biosynthesis of (-)-podophyllotoxin precursors or analogues is fundamental for the development of novel, more sustainable supply routes. RESULTS: We recently established a four-step multi-enzyme cascade to convert (+)-pinoresinol into (-)-matairesinol in E. coli. Herein, a five-step multi-enzyme biotransformation of (-)-matairesinol to (-)-deoxypodophyllotoxin was proven effective with 98 % yield at a concentration of 78 mg/L. Furthermore, the extension of this cascade to a sixth step leading to (-)-epipodophyllotoxin was evaluated. To this end, seven enzymes were combined in the reconstituted pathway involving inter alia three plant cytochrome P450 monooxygenases, with two of them being functionally expressed in E. coli for the first time. CONCLUSIONS: Both, (-)-deoxypodophyllotoxin and (-)-epipodophyllotoxin, are direct precursors to etoposide and teniposide. Thus, the reconstitution of biosynthetic reactions of Sinopodophyllum hexandrum as an effective multi-enzyme cascade in E. coli represents a solid step forward towards a more sustainable production of these essential pharmaceuticals.

Novel insights into oxidation of fatty acids and fatty alcohols by cytochrome P450 monooxygenase CYP4B1.

CYP4B1 is an enigmatic mammalian cytochrome P450 monooxygenase acting at the interface between xenobiotic and endobiotic metabolism. A prominent CYP4B1 substrate is the furan pro-toxin 4-ipomeanol (IPO). Our recent investigation on metabolism of IPO related compounds that maintain the furan functionality of IPO while replacing its alcohol group with alkyl chains of varying structure and length revealed that, in addition to cytotoxic reactive metabolite formation (resulting from furan activation) non-cytotoxic ω-hydroxylation at the alkyl chain can also occur. We hypothesized that substrate reorientations may happen in the active site of CYP4B1. These findings prompted us to re-investigate oxidation of unsaturated fatty acids and fatty alcohols with C9-C16 carbon chain length by CYP4B1. Strikingly, we found that besides the previously reported ω- and ω-1-hydroxylations, CYP4B1 is also capable of α-, ß-, γ-, and δ-fatty acid hydroxylation. In contrast, fatty alcohols of the same chain length are exclusively hydroxylated at ω, ω-1, and ω-2 positions. Docking results for the corresponding CYP4B1-substrate complexes revealed that fatty acids can adopt U-shaped bonding conformations, such that carbon atoms in both arms may approach the heme-iron. Quantum chemical estimates of activation energies of the hydrogen radical abstraction by the reactive compound 1 as well as electron densities of the substrate orbitals led to the conclusion that fatty acid and fatty alcohol oxidations by CYP4B1 are kinetically controlled reactions.

Structure-Activity Relationships for CYP4B1 Bioactivation of 4-Ipomeanol Congeners: Direct Correlation between Cytotoxicity and Trapped Reactive Intermediates.

Cytochrome P450 4B1 (CYP4B1) has been explored as a candidate enzyme in suicide gene systems for its ability to bioactivate the natural product 4-ipomeanol (IPO) to a reactive species that causes cytotoxicity. However, metabolic limitations of IPO necessitate discovery of new "pro-toxicant" substrates for CYP4B1. In the present study, we examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells expressing CYP4B1. This compound series maintains the furan warhead of IPO while replacing its alcohol group with alkyl chains of varying length (C1-C8). Compounds with C3-C6 carbon chain lengths showed similar potency to IPO (LD50 ≈ 5 µM). Short chain analogs (<3 carbons) and long chain analogs (>6 carbons) exhibited reduced toxicity, resulting in a parabolic relationship between alkyl chain length and cytotoxicity. A similar parabolic relationship was observed between alkyl chain length and reactive intermediate formation upon trapping of the putative enedial as a stable pyrrole adduct in incubations with purified recombinant rabbit CYP4B1 and common physiological nucleophiles. These parabolic relationships reflect the lower affinity of shorter chain compounds for CYP4B1 and increased ω-hydroxylation of the longer chain compounds by the enzyme. Furthermore, modest time-dependent inhibition of CYP4B1 by N-pentyl-3-furancarboxamide was completely abolished when trapping agents were added, demonstrating escape of reactive intermediates from the enzyme after bioactivation. An insulated CYP4B1 active site may explain the rarely observed direct correlation between adduct formation and cell toxicity reported here.

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1.

Oxidative biocatalytic reactions performed by cytochrome P450 enzymes (P450s) are of high interest for the chemical and pharmaceutical industries. CYP267B1 is a P450 enzyme from myxobacterium Sorangium cellulosum So ce56 displaying a broad substrate scope. In this work, a search for new substrates was performed, combined with product characterization and a structural analysis of substrate-bound complexes using X-ray crystallography and computational docking. The results demonstrate the ability of CYP267B1 to perform in-chain hydroxylations of medium-chain saturated fatty acids (decanoic acid, dodecanoic acid and tetradecanoic acid) and a regioselective hydroxylation of flavanone. The fatty acids are mono-hydroxylated at different in-chain positions, with decanoic acid displaying the highest regioselectivity towards ω-3 hydroxylation. Flavanone is preferably oxidized to 3-hydroxyflavanone. High-resolution crystal structures of CYP267B1 revealed a very spacious active site pocket, similarly to other P450s capable of converting macrocyclic compounds. The pocket becomes more constricted near to the heme and is closed off from solvent by residues of the F and G helices and the B-C loop. The crystal structure of the tetradecanoic acid-bound complex displays the fatty acid bound near to the heme, but in a nonproductive conformation. Molecular docking allowed modeling of the productive binding modes for the four investigated fatty acids and flavanone, as well as of two substrates identified in a previous study (diclofenac and ibuprofen), explaining the observed product profiles. The obtained structures of CYP267B1 thus serve as a valuable prediction tool for substrate hydroxylations by this highly versatile enzyme and will encourage future selectivity changes by rational protein engineering.

A novel cytochrome P450 mono-oxygenase from Streptomyces platensis resembles activities of human drug metabolizing P450s.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.

Three-steps in one-pot: whole-cell biocatalytic synthesis of enantiopure (+)- and (-)-pinoresinol via kinetic resolution.

BACKGROUND: Pinoresinol is a high-value plant-derived lignan with multiple health supporting effects. Enantiomerically pure pinoresinol can be isolated from natural sources, but with low efficiency. Most chemical and biocatalytic approaches that have been described for the synthesis of pinoresinol furnish the racemic mixture. In this study we devised a three-step biocatalytic cascade for the production of enantiomerically pure pinoresinol from the cheap compound eugenol. Two consecutive oxidations of eugenol through vanillyl-alcohol oxidase and laccase are followed by kinetic resolution of racemic pinoresinol by enantiospecific pinoresinol reductases. RESULTS: The addition of the enantiospecific pinoresinol reductase from Arabidopsis thaliana for kinetic resolution of (±)-pinoresinol to an in vitro cascade involving the vanillyl-alcohol oxidase from Penicillium simplicissimum and the bacterial laccase CgL1 from Corynebacterium glutamicum resulted in increasing ee values for (+)-pinoresinol; however, an ee value of 34% was achieved in the best case. The ee value could be increased up to ≥ 99% by applying Escherichia coli-based whole-cell biocatalysts. The optimized process operated in a one-pot "two-cell" sequential mode and yielded 876 µM (+)-pinoresinol with an ee value of 98%. Switching the reductase to the enantiospecific pinoresinol lariciresinol reductase from Forsythia intermedia enabled the production of 610 µM (-)-pinoresinol with an ee value of 97%. CONCLUSION: A new approach for the synthesis of enantiomerically pure (+)- and (-)-pinoresinol is described that combines three biotransformation steps in one pot. By switching the reductase in the last step, the whole-cell biocatalysts can be directed to produce either (+)- or (-)-pinoresinol. The products of the reductases' activity, (-)-lariciresinol and (-)-secoisolariciresinol, are valuable precursors that can also be applied for the synthesis of further lignans.

In situ formation of H2O2 for P450 peroxygenases.

An in situ H2O2 generation approach to promote P450 peroxygenases catalysis was developed through the use of the nicotinamide cofactor analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) and flavin mononucleotide (FMN). Final productivity could be enhanced due to higher enzyme stability at low H2O2 concentrations. The H2O2 generation represented the rate-limiting step, however it could be easily controlled by varying both FMN and BNAH concentrations. Further characterization can result in an optimized ratio of FMN/BNAH/O2/biocatalyst enabling high reaction rates while minimizing H2O2-related inactivation of the enzyme.

Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s.

Candida apicola belongs to a group of yeasts producing surface-active glycolipids consisting of sophorose and long-chain (ω)- or (ω-1)-hydroxy fatty acids. Hydroxylation of the fatty acids in this strain is likely catalyzed by cytochrome P450 monooxygenases (P450), which require reducing equivalents delivered via a cytochrome P450-diflavin reductase (CPR). We herein report cloning and characterization of the cpr gene from C. apicola ATCC 96134. The gene encoding a protein of 687 amino acids was cloned in Escherichia coli and the enzyme was expressed in functional form after truncation of its N-terminal putative membrane anchor. The truncated recombinant protein showed cytochrome c reducing activity (K (M) of 13.8 µM and k (cat) of 1,915 per minute). Furthermore, we herein demonstrate to our best knowledge for the first time the use of a eukaryotic CPR to transfer electrons to bacterial P450s (namely CYP109B1 and CYP154E1). Cloning and characterization of this CPR therefore is not only an important step in the study of the P450 systems of C. apicola, but also provides a versatile redox partner for the characterization of other bacterial P450s with appealing biotechnological potential. The GenBank accession number of the sequence described in this article is JQ015264.

Light-driven biocatalysis with cytochrome P450 peroxygenases.

The cytochrome P450 peroxygenases P450(Bsß) (CYP152A1) from Bacillus subtilis and P450(Cla) (CYP152A2) from Clostridium acetobutylicum belong to a unique group of P450s with high synthetic potential. They consume hydrogen peroxide via the peroxide shunt and therefore do not require additional electron transfer proteins for biocatalytic activity. Their high synthetic potential is, however, impaired by their rather poor operational stability in the presence of hydrogen peroxide. Herein, we report the use of a light-driven approach utilizing light-excited flavins (riboflavin, flavin mononucleotide, or flavin adenine dinucleotide) and the electron donor ethylenediaminetetraacetate as the electron source for the in situ generation of hydrogen peroxide. This approach represents a simple and easily applicable way to promote oxyfunctionalization reactions catalyzed by P450 peroxygenases and is useful for biocatalysis with these enzymes.

Novel family members of CYP109 from Sorangium cellulosum So ce56 exhibit characteristic biochemical and biophysical properties.

The members of the CYP109 family (CYP109C1, CYP109C2, and CYP109D1) from Sorangium cellulosum So ce56 are among the 21 P450 enzymes, of which only CYP109D1 and CYP264B1 have so far been functionally characterized. Here, we attempted to characterize two other P450s (CYP109C1 and CYP109C2) for the first time and compare their biochemical, biophysical, and functional properties to those of the fatty acid hydroxylating CYP109D1. Considering the physiological importance of fatty acids, we investigated saturated fatty acid binding and conversion for all members of the CYP109 family. The interaction between the CYP109 members and different autologous/heterologous redox partners was compared using Biacore measurements in which only CYP109D1 and bovine adrenodoxin (Adx) formed a complex. Surprisingly, this interaction was similarly efficient as the interaction of Adx with its mammalian redox partners. The in vitro reconstitution assays showed no activity when using CYP109C1, although substrate binding was demonstrated; also, there was subterminal hydroxylation of saturated fatty acids, when using CYP109C2 and CYP109D1, where CYP109D1 was a much more efficient fatty acid hydroxylase. Interestingly, the hydroxylation position moved inside the fatty acid chain when using long-chain fatty acids, thus producing possible precursors for physiologically important products.

Expression, purification and characterization of two Clostridium acetobutylicum flavodoxins: potential electron transfer partners for CYP152A2.

Two flavodoxin genes from Clostridium acetobutylicum, CacFld1 (CAC0587) and CacFld2 (CAC3417), were expressed in Escherichia coli and investigated for their ability to support activity of CYP152A2, a fatty acid hydroxylase from C. acetobutylicum. E. coli flavodoxin reductase (FdR) was used as a redox partner, since flavodoxin reductase CacFdR (CAC0196) from C. acetobutylicum could not be purified in a functional form. CacFld1 was shown to accept electrons from FdR and transfer them to CYP152A2. Since H2O2 was generated by uncoupling at different stages of the reconstituted electron transfer chain, catalase was used as H2O2 scavenger in order to exclude peroxygenation by CYP152A2. The reconstituted P450 system with CacFld1 and FdR oxidized myristic acid with a K(M) of 137 µM and a k(cat) of 36 min⁻¹. Furthermore, the hydroxylase activity of CYP152A2 towards myristic acid with CacFld1 was 17-fold higher than without CacFld1. Along with CYP152A2 and a physiological flavodoxin reductase, CacFld1 is therefore likely to be involved in oxygen detoxification in C. acetobutylicum. Flavodoxin CacFld2 did not accept electrons from NADPH-reduced FdR, though it cannot be excluded as a candidate redox partner for CYP152A2 in the presence of an appropriate physiological reductase.

A targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway.

Microbial synthesis of monolignols and lignans from simple substrates is a promising alternative to plant extraction. Bottlenecks and byproduct formation during heterologous production require targeted metabolomics tools for pathway optimization. In contrast to available fractional methods, we established a comprehensive targeted metabolomics method. It enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. Several cell disruption methods were compared. Hot water extraction was best suited regarding monolignol and lignan stability as well as extraction efficacy. The method was applied to compare enzymes for alleviating bottlenecks during heterologous monolignol and lignan production in E. coli. Variants of tyrosine ammonia-lyase had a considerable influence on titers of subsequent metabolites. The choice of multicopper oxidase greatly affected the accumulation of lignans. Metabolite titers were monitored during batch fermentation of either monolignol or lignan-producing recombinant E. coli strains, demonstrating the dynamic accumulation of metabolites. The new method enables efficient time-resolved targeted metabolomics of monolignol- and lignan-producing E. coli. It facilitates bottleneck identification and byproduct quantification, making it a valuable tool for further pathway engineering studies. This method will benefit the bioprocess development of biotransformation or fermentation approaches for microbial lignan production.

Characterization of the versatile monooxygenase CYP109B1 from Bacillus subtilis.

The oxidizing activity of CYP109B1 from Bacillus subtilis was reconstituted in vitro with various artificial redox proteins including putidaredoxin reductase and putidaredoxin from Pseudomonas putida, truncated bovine adrenodoxin reductase and adrenodoxin, flavodoxin reductase and flavodoxin from Escherichia coli, and two flavodoxins from B. subtilis (YkuN and YkuP). Binding and oxidation of a broad range of chemically different substrates (fatty acids, n-alkanes, primary n-alcohols, terpenoids like (+)-valencene, alpha- and beta-ionone, and the steroid testosterone) were investigated. CYP109B1was found to oxidize saturated fatty acids (conversion up to 99%) and their methyl and ethyl esters (conversion up to 80%) at subterminal positions with a preference for the carbon atoms C11 and C12 counted from the carboxyl group. For the hydroxylation of primary n-alcohols, the omega(-2) position was preferred. n-Alkanes were not accepted as substrates by CYP109B1. Regioselective hydroxylation of terpenoids alpha-ionone (approximately 70% conversion) and beta-ionone (approximately 91% conversion) yielded the allylic alcohols 3-hydroxy-alpha-ionone and 4-hydroxy-beta-ionone, respectively. Furthermore, indole was demonstrated to inhibit fatty acid oxidation.

Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56.

Sesquiterpenes are particularly interesting as flavorings and fragrances or as pharmaceuticals. Regio- or stereoselective functionalizations of terpenes are one of the main goals of synthetic organic chemistry, which are possible through radical reactions but are not selective enough to introduce the desired chiral alcohol function into those compounds. Cytochrome P450 monooxygenases are versatile biocatalysts and are capable of performing selective oxidations of organic molecules. We were able to demonstrate that CYP109D1 from Sorangium cellulosum So ce56 functions as a biocatalyst for the highly regioselective hydroxylation of norisoprenoids, alpha- and beta-ionone, which are important aroma compounds of floral scents. The substrates alpha- and beta-ionone were regioselectively hydroxylated to 3-hydroxy-alpha-ionone and 4-hydroxy-beta-ionone, respectively, which was confirmed by (1)H NMR and (13)C NMR. The results of docking alpha- and beta-ionone into a homology model of CYP109D1 gave a rational explanation for the regio-selectivity of the hydroxylation. Kinetic studies revealed that alpha- and beta-ionone can be hydroxylated with nearly identical V (max) and K (m) values. This is the first comprehensive investigation of the regioselective hydroxylation of norisoprenoids by CYP109D1.

Assembly of Plant Enzymes in E. coli for the Production of the Valuable (-)-Podophyllotoxin Precursor (-)-Pluviatolide.

Lignans are plant secondary metabolites with a wide range of reported health-promoting bioactivities. Traditional routes toward these natural products involve, among others, the extraction from plant sources and chemical synthesis. However, the availability of the sources and the complex chemical structures of lignans often limit the feasibility of these approaches. In this work, we introduce a newly assembled biosynthetic route in E. coli for the efficient conversion of the common higher-lignan precursor (+)-pinoresinol to the noncommercially available (-)-pluviatolide via three intermediates. (-)-Pluviatolide is considered a crossroad compound in lignan biosynthesis, because the methylenedioxy bridge in its structure, resulting from the oxidation of (-)-matairesinol, channels the biosynthetic pathway toward the microtubule depolymerizer (-)-podophyllotoxin. This oxidation reaction is catalyzed with high regio- and enantioselectivity by a cytochrome P450 monooxygenase from Sinopodophyllum hexandrum (CYP719A23), which was expressed and optimized regarding redox partners in E. coli. Pinoresinol-lariciresinol reductase from Forsythia intermedia (FiPLR), secoisolariciresinol dehydrogenase from Podophyllum pleianthum (PpSDH), and CYP719A23 were coexpressed together with a suitable NADPH-dependent reductase to ensure P450 activity, allowing for four sequential biotransformations without intermediate isolation. By using an E. coli strain coexpressing the enzymes originating from four plants, (+)-pinoresinol was efficiently converted, allowing the isolation of enantiopure (-)-pluviatolide at a concentration of 137 mg/L (ee ≥99% with 76% isolated yield).

Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system.

BACKGROUND: (+)-Nootkatone (4) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (1) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (4) from (+)-valencene (1) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (1) at allylic C2-position to produce (+)-nootkatone (4) via cis- (2) or trans-nootkatol (3). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida in Escherichia coli. RESULTS: Addressing the whole-cell system, the cytochrome CYP109B1 from Bacillus subtilis was found to catalyze the oxidation of (+)-valencene (1) yielding nootkatol (2 and 3) and (+)-nootkatone (4). However, when the in vivo biooxidation of (+)-valencene (1) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents - isooctane, n-octane, dodecane or hexadecane - were set up, resulting in accumulation of nootkatol (2 and 3) and (+)-nootkatone (4) of up to 97% of the total product. The best productivity of 120 mg l-1 of desired products was achieved within 8 h in the system comprising 10% dodecane. CONCLUSION: This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (4), as it is safe and can easily be controlled and scaled up.

Cytochrome P450 Monooxygenases in Biotechnology and Synthetic Biology.

Cytochromes P450 (P450 or CYP) are heme-containing enzymes that catalyze the introduction of one atom of molecular oxygen into nonactivated C-H bonds, often in a regio- and stereoselective manner. This ability, combined with a tremendous number of accepted substrates, makes P450s powerful biocatalysts. Sixty years after their discovery, P450 systems are recognized as essential bio-bricks in synthetic biology approaches to enable production of high-value complex molecules in recombinant hosts. Recent impressive results in protein engineering led to P450s with tailored properties that are even able to catalyze abiotic reactions. The introduction of P450s in artificial multi-enzymatic cascades reactions and chemo-enzymatic processes offers exciting future perspectives to access novel compounds that cannot be synthesized by nature or by chemical routes.

Engineering of versatile redox partner fusions that support monooxygenase activity of functionally diverse cytochrome P450s.

Most bacterial cytochrome P450 monooxygenases (P450s or CYPs) require two redox partner proteins for activity. To reduce complexity of the redox chain, the Bacillus subtilis flavodoxin YkuN (Y) was fused to the Escherichia coli flavodoxin reductase Fpr (R), and activity was tuned by placing flexible (GGGGS)n or rigid ([E/L]PPPP)n linkers (n = 1-5) in between. P-linker constructs typically outperformed their G-linker counterparts, with superior performance of YR-P5, which carries linker ([E/L]PPPP)5. Molecular dynamics simulations demonstrated that ([E/L]PPPP)n linkers are intrinsically rigid, whereas (GGGGS)n linkers are highly flexible and biochemical experiments suggest a higher degree of separation between the fusion partners in case of long rigid P-linkers. The catalytic properties of the individual redox partners were best preserved in the YR-P5 construct. In comparison to the separate redox partners, YR-P5 exhibited attenuated rates of NADPH oxidation and heme iron (III) reduction, while coupling efficiency was improved (28% vs. 49% coupling with B. subtilis CYP109B1, and 44% vs. 50% with Thermobifida fusca CYP154E1). In addition, YR-P5 supported monooxygenase activity of the CYP106A2 from Bacillus megaterium and bovine CYP21A2. The versatile YR-P5 may serve as a non-physiological electron transfer system for exploitation of the catalytic potential of other P450s.