Nootkatone, a sesquiterpenoid widely used in the food and cosmetics industries, exhibits diverse biological activities and pharmaceutical prospects. Modification of nootkatone to create new derivatives with desirable activities has attracted significant attention. For this purpose, cytochrome P450 monooxygenases (P450 or CYP) are attractive candidates due to their ability to perform regio- and stereoselective hydroxylation at allylic C-H bonds. In this study, CYP154C4 from Streptomyces cavourensis YBQ59 was cloned and expressed in Escherichia coli. By screening 64 candidate substrates, this P450 was found to catalyze the regio- and stereoselective hydroxylation of nootkatone, yielding a single product, 3β-hydroxynootkatone. Using a whole-cell E. coli system expressing CYP154C4, supported by the heterologous redox partners YkuN from Bacillus subtilis and FdR from E. coli, 3β-hydroxynootkatone was produced on a preparative scale. The structure of this compound was determined by 1H NMR, 13C NMR, NOESY, HMBC, and HSQC. The kinetics of product formation were analyzed using HPLC, and the Km and Kcat values were calculated. Furthermore, structural insights into the selective hydroxylation of nootkatone were elucidated by molecular docking. 3β-Hydroxynootkatone, recently synthesized semi-synthetically from nootkatone, has been reported to exhibit a higher insecticidal activity than its parent compound. Additionally, the functionalization of nootkatone with N-acyl-2-aminothiazole at the C3 and C2 positions, yielding an α-glucosidase inhibitor, has also been previously described. Therefore, 3β-hydroxynootkatone has great potential for further research and for synthesizing new derivatives with valuable biological activities for agricultural and medicinal applications.

Nootkatone, a sesquiterpenoid widely used in the food and cosmetics industries, exhibits diverse biological activities and pharmaceutical prospects. Modification of nootkatone to create new derivatives with desirable activities has attracted significant attention. For this purpose, cytochrome P450 monooxygenases (P450 or CYP) are attractive candidates due to their ability to perform regio- and stereoselective hydroxylation at allylic C-H bonds. In this study, CYP154C4 from Streptomyces cavourensis YBQ59 was cloned and expressed in Escherichia coli. By screening 64 candidate substrates, this P450 was found to catalyze the regio- and stereoselective hydroxylation of nootkatone, yielding a single product, 3(3-hydroxynootkatone. Using a whole-cell E. coli system expressing CYP154C4, supported by the heterologous redox partners YkuN from Bacillus subtilis and FdR from E. coli, 3(3hydroxynootkatone was produced on a preparative scale. The structure of this compound was determined by 1 H NMR, 13 C NMR, NOESY, HMBC, and HSQC. The kinetics of product formation were analyzed using HPLC, and the Km and K cat values were calculated. Furthermore, structural insights into the selective hydroxylation of nootkatone were elucidated by molecular docking. 3(3-Hydroxynootkatone, recently synthesized semi- synthetically from nootkatone, has been reported to exhibit a higher insecticidal activity than its parent compound. Additionally, the functionalization of nootkatone with N-acyl-2-aminothiazole at the C3 and C2 positions, yielding an alpha-glucosidase inhibitor, has also been previously described. Therefore, 3(3-hydroxynootkatone has great potential for further research and for synthesizing new derivatives with valuable biological activities for agricultural and medicinal applications.

Sesquiterpenes are common constituents of essential oil in plants. Their oxygenated derivatives often possess desirable flavor, fragrance, and pharmaceutical properties. Recently, the CYP264B1-based recombinant Escherichia coli whole-cell system has been constructed for the oxidation of sesquiterpenes. However, limiting factors of this system related to the high volatility of substrates and the suitability of the P450 redox partner need to be addressed. In this work, the improvement of the system was implemented with (+)-α-longipinene as a model substrate. By using 2-hydroxypropyl-β-cyclodextrin and an alternative ferredoxin reductase, the conversion of (+)-α-longipinene was improved 77.1%. Applying the optimized conditions, the yields of the main products were 54.2, 34.2, and 47.2 mg L-1, corresponding to efficiencies of 82.1, 51.8, and 71.5% for the conversion of (+)-α-longipinene, (-)-isolongifolene, and α-humulene, respectively, at a 200 mL scale. These products were characterized as 12-hydroxy-α-longipinene, isolongifolene-9-one, and 5-hydroxy-α-humulene, respectively, by nuclear magnetic resonance spectroscopy.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

The inside cover picture shows an E. coli-based whole-cell catalyst that expresses the cytochromes P450 CYP260A1 and CYP264B1 from the myxobacterium Sorangium cellulosum So ce56. Each of the cytochromes P450 is capable of oxidizing plant terpenoids of the eremophilane, humulane, caryophyllane, and cedrane type. Native cytochromes P450 that show such a broad substrate spectrum are valuable as starting materials for developing a toolbox for selective sesquiterpene oxidation. More details can be found in the Full Paper by R. Bernhardt et al. on page 2624 in Issue 18, 2015 (DOI: 10.1002/cbic.201500417).

Sesquiterpenes are natural products derived from the common precursor farnesyl pyrophosphate (FPP) but are highly diverse in structure and function. Cytochrome P450 enzymes (P450s) exhibit the unique ability to introduce molecular oxygen into non-activated C - H bonds. In plant biosynthetic pathways, P450s commonly derivatize sesquiterpene hydrocarbons. However, the potential of bacterial P450s for terpene derivatization is still underinvestigated. This work compares the substrate specificities and regioselectivities of the sesquiterpene hydroxylases CYP260A1 and CYP264B1 from myxobacterium Sorangium cellulosum Soce56. Four tested substrate classes (eremophilanes, humulanes, caryophyllanes, and cedranes) were converted by both P450s. The achievable variety of oxidations is demonstrated on the model substrates (+)-nootkatone and zerumbone. Increasing the number of functionally investigated P450s, this study represents a step towards the selective derivatization of sesquiterpenes.

Terpenoids can be found in almost all forms of life; however, the biosynthesis of bacterial terpenoids has not been intensively studied. This study reports the identification and functional characterization of the gene cluster CYP264B1-geoA from Sorangium cellulosum So ce56. Expression of the enzymes and synthesis of their products for NMR analysis and X-ray diffraction were carried out by employing an Escherichia coli whole-cell conversion system that provides the geoA substrate farnesyl pyrophosphate through simultaneous overexpression of the mevalonate pathway genes. The geoA product was identified as a novel sesquiterpene, and assigned NMR signals unambiguously proved that geoA is an (+)-eremophilene synthase. The very tight binding of (+)-eremophilene (similar to 0.40 mu m), which is also available in S. cellulosum So ce56, and its oxidation by CYP264B1 suggest that the CYP264B1-geoA gene cluster is required for the biosynthesis of (+)-eremophilene derivatives.

Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and β-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and β-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-β-ionone and 13-hydroxy-nootkatone, were confirmed by (1)H and (13)C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the K ( m ) and k (cat) values were calculated. The results of docking α-/β-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations.

In the work presented here, CYP264B1 and the terpene cyclase GeoA of Sorangium cellulosum So ce56 have been characterized. CYP264B1 is able to convert norisoprenoids (a-ionone and b-ionone) and diverse sesquiterpene compounds, including nootkatone. Three products, 3-hydroxy-a-ionone, 3-hydroxy-b-ionone and 13-hydroxy-nootkatone were characterized using HPLC and 1H and 13C NMR. CYP264B1 is the first enzyme reported to be capable to hydroxylate regioselectively both norisoprenoids at the position C-3 as well as nootkatone at the position C-13. The kinetics (Km and Vmax) of the product formation were analyzed by HPLC. The results of docking a-/b-ionone and nootkatone into a homology model of CYP264B1 revealed the structural basis of these selective hydroxylations. In addition, an E. coli whole cell system containing CYP264B1 and its redox partners was created for the biotransformation of CYP264B1 substrates. This system was applied successfully for b-ionone conversion. FPP and GGPP were found to be substrates for GeoA. The sesquiterpene and diterpene products of GeoA are similar to valencene (89%) and neocembrene A (80%), respectively. However, these products are most likely new compounds. In order to characterize them by NMR, a whole cell system based on mevalonate pathwayengineered E. coli was created to faciliate the production of sufficient amounts. The terpene production using this system was investigated, showing that it is possible to obtain the amounts required for NMR analysis if laboratory conditions are optimized. In der vorliegenden Arbeit wurden CYP264B1 und die Terpencyclase GeoA aus So ce56 charakterisiert. CYP264B1 ist in der Lage, Norisoprenoide (a-Ionon und b-Ionon) und diverse Sesquiterpene, inklusive Nootkaton, umzusetzen. Drei Produkte (3-Hydroxy-a-Ionon, 3-Hydroxy-b-Ionone und 13-Hydroxy-Nootkaton) wurden mittels HPLC und 1H und 13C NMR charakterisiert. Damit ist CYP264B1 das erste Enzym, das die Fahigkeit besitzt, regioselektiv Norisoprenoide an Position C-3, sowie Nootkaton an Position C- 13 zu hydroxylieren. Die Kinetik der Produktbildung (Vmax und Km) wurde mittels HPLC analysiert. Durch das Docking von a/b-Ionon und Nootkaton in das Homologiemodell von CYP264B1 konnte die strukturelle Grundlage dieser selektiven Hydroxylierungen aufgeklart werden. Des Weiteren wurde ein E. coli Ganzell- Sumsatzsystem fur die Umsetzung von CYP264B1 Substraten etabliert, das neben CYP264B1 auch seine Redox Partner enthalt. Dieses System wurde erfolgreich fur dieUmsetzung von b-Ionon eingesetzt. FPP und GGPP wurden als Substrate von GeoA identifiziert. Die jeweiligen Sesquiterpen- und Diterpen- Produkte wiesen Ahnlichkeit mit Valencen (89%), bzw. Neocembren (80%) auf. Jedoch handelt es sich bei den gebildeten Produkten hochstwahrscheinlich um neue Verbindungen. Um ausreichende Mengen dieser Verbindungen fur eine NMR Analyse zur Verfugung zu stellen, wurde ein Ganzzell- System aufgebaut, das auf E. coli Zellen die heterolog zusatzliche Proteine des Mevalonat Stoffwechselweges exprimieren, basiert. Durch weitere Optimierung dieses Systems sollte es in Zukunft moglich sein, die fur eine NMR Analyse erforderlichen Produktmengen produzieren.