The substrate flexibility of eight purified sesquiterpene cyclases was evaluated using six new heteroatom-modified farnesyl pyrophosphates, and the formation of six new heteroatom-modified macrocyclic and tricyclic sesquiterpenoids is described. GC-O analysis revealed that tricyclic tetrahydrofuran exhibits an ethereal, peppery, and camphor-like olfactoric scent.

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)‐patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH‐value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2‐(N‐morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.

Patchouli oil is a major ingredient in perfumery, granting a dark-woody scent due to its main constituent (−)-patchoulol. The growing demand for patchouli oil has raised interest in the development of a biotechnological process to assure a reliable supply. Herein, we report the production of patchouli oil sesquiterpenes by metabolically engineered Escherichia coli strains, using solid–liquid phase partitioning cultivation. The (−)-patchoulol production was possible using the endogenous methylerythritol phosphate pathway and overexpressing a (−)-patchoulol synthase isoform from Pogostemon cablin but at low titers. To improve the (−)-patchoulol production, the exogenous mevalonate pathway was overexpressed in the multi-plasmid PTS + Mev strain, which increased the (−)-patchoulol titer 5-fold. Fermentation was improved further by evaluating several defined media, and optimizing the pH and temperature of culture broth, enhancing the (−)-patchoulol titer 3-fold. To augment the (−)-patchoulol recovery from fermentation, the solid–liquid phase partitioning cultivation was analyzed by screening polymeric adsorbers, where the Diaion HP20 adsorber demonstrated the highest (−)-patchoulol recovery from all tests. Fermentation was scaled-up to fed-batch bioreactors, reaching a (−)-patchoulol titer of 40.2 mg L –1 and productivity of 20.1 mg L –1 d –1 . The terpene profile and aroma produced from the PTS + Mev strain were similar to the patchouli oil, comprising (−)-patchoulol as the main product, and α-bulnesene, trans-β-caryophyllene, β-patchoulene, and guaia-5,11-diene as side products. This investigation represents the first study of (−)-patchoulol production in E. coli by solid–liquid phase partitioning cultivation, which provides new insights for the development of sustainable bioprocesses for the microbial production of fragrant terpenes.

The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co2+‐immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl2 and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm2 each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (‐)‐patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.

HighlightsBiologically active rhBMP-4 was produced in a prokaryotic host as inclusion bodies.Different refolding recipes were tested for optimal dimerization yield.One-step purification of dimer with cation-exchange membrane chromatography.The product induces trophoblast differentiation in induced pluripotent stem cells.Comparison between commercial rhBMP-4 from cell culture and product from E. coli.