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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.

CYP106A2 is known as a 15β-hydroxylase, but also shows minor 11α-hydroxylase activity for progesterone. 11α-Hydroxyprogesterone is an important pharmaceutical compound with anti-androgenic and blood-pressure-regulating activity. This work therefore focused on directing the regioselectivity of the enzyme towards hydroxylation at position 11 in the C ring of the steroid through a combination of saturation mutagenesis and rational site-directed mutagenesis. With the aid of data from a homology model of CYP106A2 containing docked progesterone, together with site-directed mutagenesis of active-site residues (Lisurek et al. ChemBioChem 2008, 9, 1439-1449), a saturation mutagenesis library at positions A395 and G397 was created. Screening of the library identified the mutants A395I and A395W/G397K as having 11α-hydroxylase activities 8.9 and 11.5 times higher than that of the wild type (WT). In the next step, additional mutations were integrated by a rational site-directed mutagenesis approach to increase the catalytic efficiency. Of the 40 candidates analyzed, the mutants A106T/A395I, A106T/A395I/R409L, and T89N/A395I turned out to display increased 11α-hydroxylase selectivities and activities relative to the WT (14.3-, 12.6-, and 11.8-fold increases in selectivity and 39.3-, 108-, and 24.4- in k(cat)/K(m)). In the last step of the study, the best mutants were applied in a whole-cell biotransformation. In these experiments the production (percentage) of 15β-hydroxyprogesterone decreased from 50.4 % (wild type) to 4.8 % (mutant T89N/A395I), whereas that of 11α-hydroxyprogesterone increased from 27.7 to 80.9 %, thus demonstrating an impressive regioselectivity.

This paper is dedicated to Professor Yoshinori Asakawa for his 65 th birthday. Two recombinant, stereospecific monoterpene synthases, a (-)-limonene synthase (CsTPS1) and a (+)-α-pinene synthase (CsTPS2), encoded by Cannabis sativa L. cv. 'Skunk' trichome mRNA, have been isolated and characterized. Recombinant CsTPS1 showed a K m value of 6.8 μM, a V max of 1.1 x 10-4 µmol/min and V max /K m of 0.016; the pH optimum was determined at pH 6.5, and a temperature optimum at 40°C. Recombinant CsTPS2 showed a K m value of 10.5 µM, a V max of 2.2 x 10-4 µmol/min and V max /K m of 0.021; the pH optimum was determined at pH 7.0, and a temperature optimum at 30°C. Phylogenetic analysis showed that both CsTPSs group within the angiosperms and belong to the Tpsb subgroup of monoterpene synthases. The enzymatic products (-)-limonene and (+)-α-pinene were detected as natural products in C. sativa trichomes.