Development of the biotechnological production of (+)-zizaene : enzymology, metabolic engineering and in situ product recovery

Abstract

The sesquiterpene (+)-zizaene is the immediate precursor of khusimol, the main compound of the vetiver essential oil from the vetiver grass, which grants its characteristic woody scent. Among its distinct applications, this oil is relevant for the formulation of cosmetics and used in approximately 20% of all men’s perfumery. The traditional supply of the vetiver essential oil had suffered shortages due to natural disasters. Consequently, the biotechnological production of khusimol is an alternative towards a more reliable supply. In this study, we provide new insights towards the microbial production of khusimol by characterizing the zizaene synthase, engineering the metabolic pathway of (+)-zizaene in Escherichia coli and analyzing the in situ recovery of (+)-zizaene from fermentation. In the first chapter, the zizaene synthase, the critical enzyme for khusimol biosynthesis, was characterized. A SUMO-fused zizaene synthase variant was overexpressed in E. coli, and in vitro reactions yielded 90% (+)-zizaene. Furthermore, enzyme characterization comprised enzyme kinetics, optimal reaction conditions, substrate specificity and reaction mechanisms. The in vitro reactions showed high stability through varying pH and temperature values. By in silico docking model, this was explained due to the hydrophobicity of the surrounding loops, which stabilized the closed conformation of the active site. The second chapter addressed the metabolic engineering of the (+)-zizaene biosynthetic pathway in E. coli. A systematic strategy was applied by modulating the substrate FDP and the zizaene synthase to improve the zizaene titers. The optimal (+)-zizaene production was reached by engineering the mevalonate pathway and two copies of the zizaene synthase into a multi-plasmid strain. Optimization of the fermentation conditions such as IPTG, media, pH and temperature improved the production further, achieving a (+)-zizaene titer of 25 mg L‒1. In the third chapter, the in situ recovery of (+)-zizaene from fermentation was analyzed. Initially, liquid-liquid phase partitioning cultivation improved the (+)-zizaene recovery at shake flask scale. Subsequently, solid-liquid phase partitioning cultivation was evaluated by screening polymeric adsorbers, where Diaion HP20 obtained the highest recovery ratio. The bioprocess was scaled up to 2 L fed-batch bioreactors by integrating in situ recovery and fermentation. External and internal (with and without gas stripping) recovery configurations were tested, where the internal configuration obtained the highest (+)-zizaene recovery of all, achieving a (+)-zizaene titer of 211.13 mg L−1 and a productivity of 3.2 mg L−1 h−1.