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.

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 (∼0.40 μM), 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.

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 So ce56. 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.

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, 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.

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.

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