The patchoulol synthase (PTS) is a multi-product sesquiterpene synthases which is the central enzyme for biosynthesis of patchouli essential oil in the patchouli plant. Sesquiterpene synthases catalyse the formation of various complex carbon backbones difficult to approach by organic synthesis. Here, we report the characterisation of a recombinant patchoulol synthase complementary DNA (cDNA) variant (PTS var. 1), exhibiting significant amino acid exchanges compared to the native PTS. The product spectrum using the natural substrate E,E-farnesyl diphosphate (FDP) as well as terpenoid products resulting from conversions employing alternative substrates was analysed by GC-MS. In respect to a potential use as a biocatalyst, important enzymatic parameters such as the optimal reaction conditions, kinetic behaviour and the product selectivity were studied as well. Adjusting the reaction conditions, an increased patchoulol ratio in the recombinant essential oil was achieved. Nevertheless, the ratio remained lower than in plant-derived patchouli oil. As alternative substrates, several prenyl diposphates were accepted and converted in numerous compounds by the PTS var. 1, revealing its great biocatalytic potential.

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Fusion to rd to expres synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution.

The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5%) and β-caryophyllene (~5.5%) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters K M and k cat were determined using a discontinuous kinetic assay.

Bioproduction of α-humulene in metabolically engineered Escherichia coli and application in zerumbone synthesisZerumbone is a sesquiterpene ketone with potent anti-cancerogenic activities, produced in several ginger species of the Zingiberaceae familiy. We have investigated the biotechnological production of α-humulene, a precursor of zerumbone. By implementing a heterologous mevalonate pathway in combination with the α-humulene synthase expression, we effectively synthesized α-humulene from glucose in Escherichia coli. In this study, we developed a practical and efficient in situ separation method for α-humulene by comparison of extractive and adsorptive strategies. By the in situ adsorption of the product to the hydrophobic resin Amberlite R XAD4 we were able to increase α-humulene yield by 2310% to 60.2 mg/L. Furthermore we present an easy applicable, short subsequent chemical process for the conversion of α-humulene to zerumbone by using transition metal catalysis. To reduce process steps, the chemical reaction was carried out in the same solvent as the eluting solvent that was used to elute α-humulene from the adsorbent resin. By allylic oxidation of α-humulene with manganese II chloride as a catalyst and tert.-butylhydroperoxide as an oxidizing agent we were able to synthetize zerumbone with a selectivity of 51.6%. Product and byproducts of the oxidation reaction were identified by GC-MS.

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Terpene synthases are the key enzymes in terpene biosynthesis that provide a structurally complex and highly diverse product spectrum. A suitable and reliable analytical assay is indispensable to measure terpene synthase activity accurately and precisely. In this study, a malachite green assay (MG) was adapted to rapidly assay terpene synthase activity and was validated in comparison to an already established gas chromatography assay. A linear correlation between both assays was observed. Kinetic properties for the previously described sesquiterpene synthase α‐humulene synthase (HUM) from Zingiber zerumbet Smith were investigated for the bioconversion of the monoterpene precursors geranyl pyrophosphate (2E‐GPP) and neryl pyrophosphate (2Z‐NPP) as well as for the sesquiterpene precursor farnesyl pyrophosphate (2E,6E‐FPP). Also, gas chromatography mass spectrometry (GS‐MS) was carried out to identify the products of the bioconversion of (2E)‐GPP and (2Z)‐NPP.