Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase

Abstract

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.

Whole-Cell Production of Patchouli Oil Sesquiterpenes in Escherichia coli: Metabolic Engineering and Fermentation Optimization in Solid–Liquid Phase Partitioning Cultivation

“… 46 The in vivo optimal pH (found here) is similar to that of the in vitro biotransformation reactions, determined between pH 6.4–6.6. 42 Accordingly, our results are similar to the E. coli strains used for the production of longifolene 47 and sclareol, 48 which used a pH of 7.0 during cultivation. Thereafter, the cultivation conditions of 20 °C and pH 6.75 were used for further evaluations.…”

“…Nevertheless, the lowest temperature tests did not produce the highest (−)-patchoulol titers; possibly, due to the diminished enzyme activity of the (−)-patchoulol synthase. This could be supported because in previous in vitro biotransformation reactions, 42 (−)-patchoulol synthase showed higher activity at higher temperatures, reaching its maximum at 34 °C. Therefore, a balance is required among the production of soluble (−)-patchoulol synthase protein, enzyme activity, and cell growth, as observed in the tests grown at 20 °C, which achieved the highest (−)-patchoulol production.…”

The enzyme mechanism of patchoulol synthase

“…atoms was reported that is explainable by the deprotonation step from E to 6 [10], but contradicts the retainment of this hydrogen as reported by Croteau [9]. Further support for Akhila's mechanism was provided by Ekramzadeh et al, who observed the uptake of two deuterium atoms at C3 and C12 in an incubation of FPP with PTS in deuterium oxide buffer that explain the reprotonations of the neutral intermediates 8 and 6 (Scheme 2C) [13].…”

“…For both experiments a full retainment of labelling was reported for all intermediates until 13, while a loss of tritium was observed for 14 with both substrates. From these experiments it was concluded that the hydrogen H6 must migrate into another position, as realised by the 1,4-hydride shift from C to D. The loss of 3 H in the experiment with [12,[13][14] C,1-3 H]FPP was expected for the aldol reaction of 13, but is more difficult to understand in the experiment with [12,13-14 C,6-3 H]FPP. In this case the loss of 3 H was explained by an exchange against 1 H during catalytic hydrogenation [9].…”

“…Notably, none of the proposed mechanisms in Schemes 1-3 can explain the reported results from all labelling experiments and some of the reported findings are even contradictory. For this reason, we have reinvestigated the enzyme mechanism of PTS in isotopic labelling experiments through methods recently developed in our laboratory that make use of 13 C and 2 H-substituted terpene precursors, and by DFT calculations. The general strategy in these experiments is to use substrates or substrate combinations so that deuterium migrations end at 13 C-labelled carbons, resulting in triplet signals in the 13 C NMR spectra [15,16].…”