Expression, purification and activity assay of a patchoulol synthase cDNA variant fused to thioredoxin in Escherichia coli

Characterisation of a Recombinant Patchoulol Synthase Variant for Biocatalytic Production of Terpenes

“…To maintain reliable and high yield in production of the PTS in soluble form, the PTS var. 1 gene was previously optimised to the codon usage of E. coli [10]. The PTS var.…”

“…The sequence of these reactions and the formation of particular intermediates and products is guided by the chemical appearance and the plasticity of the active site pocket which act as a template for the stereo electrical driven reactions [9]. With no crystal structure of the PTS being published so far, the only structural insights available in literature are based on in silico modelling [10,11]. As in other sesquiterpene synthases, the DDXXD motif binds cofactor Mg 2+ and ensures the right orientation of the FDP molecule in the active site pocket [12].…”

“…The enzyme was produced heterologously in Escherichia coli applying a solubility enhancing strategy by using a codon optimised PTS gene in order to increase the yield of active PTS var. 1 [10]. In combination with a short organic synthesis of the prenyl diphosphate substrates using a modified method described by Keller et al [18], sesquiterpene synthases are promising tools for in vitro biocatalysis to build up challenging sesquiterpene carbon backbones, including precursors for pharmaceutical active molecules such as artemisinin and zerumbone [19].…”

“…The patchoulol synthase variant used in this work demonstrates several amino acid mutations within and around the NSE/DTE motif [10]. The position and chemical properties of the amino acids within the active site of the enzyme are crucial for the particular constitution of the final sesquiterpene at the end of the biocatalytic process.…”

“…1 was characterised in terms of its product spectrum and selectivity, optimal reaction conditions, kinetic properties and substrate scope. The enzyme consists of 553 amino acids with a theoretical weight of 64.2 kDA and a pI of 5.2 [10,23].…”