High-Level Production of Sesquiterpene Patchoulol in Saccharomyces cerevisiae

Abstract

Patchoulol is a tricyclic sesquiterpene widely used in perfumes and cosmetics. Herein, comprehensive engineering strategies were employed to construct an efficient yeast strain for patchoulol production. First, a platform strain was constructed via pathway modification. Second, three off-pathway genes were deleted, which led to significant physiological changes in yeast. Further, strengthening of the ergosterol pathway, enhancement of the energy supply, and a decrease in intracellular reactive oxygen species were implemented to improve the physiological status of yeast, demonstrating a new promotive relationship between ergosterol biosynthesis and synthesis of patchoulol. Moreover, patchoulol synthase was improved through protein modification and Mg 2+ addition, reaching a final titer of 141.5 mg/L in a shake flask. Finally, a two-stage fermentation with dodecane addition was employed to achieve the highest production (1632.0 mg/L, 87.0 mg/g dry cell weight, 233.1 mg/L/d) ever reported for patchoulol in a 5 L bioreactor. This work lays a foundation for green and efficient patchoulol production.

Enhancement of Patchoulol Production in Escherichia coli via Multiple Engineering Strategies

“…coli 060HA was 24.2-fold higher than the starting strain PTS2 (Figures and S2C). This titer was also 2.4-fold higher than the reported maximum production in the shake-flask experiment . While cultivating in a 5 L bioreactor, a patchoulol titer of 970.1 mg/L with a volumetric productivity of 199 mg/L/d was produced using strain 060HA (Figure ), which were 24.1- and 9.9-fold higher than those of the previous bioreactor experiment using recombinant E.…”

“…Single-site mutations around the active site of PTS were reported recently to limitedly increase patchoulol synthesis in S. cerevisiae . However, post-translational modification in E.…”

“…This titer was also 2.4-fold higher than the reported maximum production in the shake-flask experiment. 12 While cultivating in a 5 L bioreactor, a patchoulol titer of 970.1 mg/L with a volumetric productivity of 199 mg/L/d was produced using strain 060HA (Figure 5), which were 24.1-and 9.9-fold higher than those of the previous bioreactor experiment using recombinant E. coli. 2 Furthermore, strain 060HA has no auxotrophic requirements in mineral salt medium which could save costs in material and purification.…”

“…In addition, patchoulol possesses potential therapeutic functions such as neuroprotective, anti-inflammatory, anticancer, antimicrobial, analgesic, antioxidant, antiplatelet, antithrombotic, antidepressant, and antiemetic activities. − Along with the increase in consumer awareness and the application scope of patchouli oil, the natural production of patchouli oil cannot meet the demand and quality requirements. , From the standpoint of sustainability and cost-effectiveness, terpenoid production using microbial platforms is more advantageous than extraction from plants . Several types of microorganisms have been engineered for patchoulol biosynthesis. ,,− A maximum titer of 1630 mg/L and a recombinant production of patchouli oil (under the brand “Clearwood” by the company Firmenich, SA, Swiss) − were obtained using Saccharomyces cerevisiae as the host strain. However, S.…”

“…7 Several types of microorganisms have been engineered for patchoulol biosynthesis. 2,7,9−11 A maximum titer of 1630 mg/ L 12 and a recombinant production of patchouli oil (under the brand "Clearwood" by the company Firmenich, SA, Swiss) 13−15 were obtained using Saccharomyces cerevisiae as the host strain. However, S. cerevisiae comprises competing pathways for the farnesyl diphosphate (FPP) precursor, such as the ergosterol and carotenoid synthetic pathways, 2 and requires complex medium for synthesis of patchoulol, which increase production impurity and cost.…”