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The sesquiterpene (+)-zizaene is the direct precursor of khusimol, the main fragrant compound of the vetiver essential oil from Chrysopogon zizanioides and used in nearly 20% of men’s fine perfumery. The biotechnological production of such fragrant sesquiterpenes is a promising alternative towards sustainability; nevertheless, product recovery from fermentation is one of the main constraints. In an effort to improve the (+)-zizaene recovery from a metabolically-engineered Escherichia coli, we developed an integrated bioprocess by coupling fermentation and (+)-zizaene recovery using adsorber extractants. Initially, (+)-zizaene volatilization was confirmed from cultivations with no extractants but application of liquid–liquid phase partitioning cultivation (LLPPC) improved (+)-zizaene recovery nearly 4-fold. Furthermore, solid–liquid phase partitioning cultivation (SLPPC) was evaluated by screening polymeric adsorbers, where Diaion HP20 reached the highest recovery. Bioprocess was scaled up to 2 L bioreactors and in situ recovery configurations integrated to fermentation were evaluated. External recovery configuration was performed with an expanded bed adsorption column and improved (+)-zizaene titers 2.5-fold higher than LLPPC. Moreover, internal recovery configuration (IRC) further enhanced the (+)-zizaene titers 2.2-fold, whereas adsorption velocity was determined as critical parameter for recovery efficiency. Consequently, IRC improved the (+)-zizaene titer 8.4-fold and productivity 3-fold from our last report, achieving a (+)-zizaene titer of 211.13 mg L⁻¹ and productivity of 3.2 mg L⁻¹ h⁻¹. This study provides further knowledge for integration of terpene bioprocesses by in situ product recovery, which could be applied for many terpene studies towards the industrialization of fragrant molecules. Full article

The vetiver essential oil from Chrysopogon zizanioides contains fragrant sesquiterpenes used widely in the formulation of nearly 20% of men’s cosmetics. The growing demand and issues in the supply have raised interest in the microbial production of the sesquiterpene khusimol, the main compound of the vetiver essential oil due to its woody smell. In this study, we engineered the biosynthetic pathway for the production of (+)-zizaene, the immediate precursor of khusimol. A systematic approach of metabolic engineering in Escherichia coli was applied to modulate the critical bottlenecks of the metabolic flux towards (+)-zizaene. Initially, production of (+)-zizaene was possible with the endogenous methylerythritol phosphate pathway and the codon-optimized zizaene synthase (ZS). Raising the precursor E,E-farnesyl diphosphate supply through the mevalonate pathway improved the (+)-zizaene titers 2.7-fold, although a limitation of the ZS supply was observed. To increase the ZS supply, distinct promoters were tested for the expression of the ZS gene, which augmented 7.2-fold in the (+)-zizaene titers. Final metabolic enhancement for the ZS supply by using a multi-plasmid strain harboring multiple copies of the ZS gene improved the (+)-zizaene titers 1.3-fold. The optimization of the fermentation conditions increased the (+)-zizaene titers 2.2-fold, achieving the highest (+)-zizaene titer of 25.09 mg L⁻¹. This study provides an alternative strategy to enhance the terpene synthase supply for the engineering of isoprenoids. Moreover, it demonstrates the development of a novel microbial platform for the sustainable production of fragrant molecules for the cosmetic industry. Full article