TY - JOUR

T1 - Whole-Cell Production of Patchouli Oil Sesquiterpenes in Escherichia coli

T2 - Metabolic Engineering and Fermentation Optimization in Solid-Liquid Phase Partitioning Cultivation

AU - Aguilar, Francisco

AU - Ekramzadeh, Kimia

AU - Scheper, Thomas

AU - Beutel, Sascha

N1 - Funding Information: This work was supported by the PINN program from the Ministry of Science, Technology and Telecommunications of Costa Rica (MICITT). We are grateful for the assistance provided for the product identification by Daniel Sandner and Ulrich Krings from the Institute of Food Chemistry at the Leibniz Hannover University. The publication of this article was funded by the Open Access Publishing Fund of Leibniz Universitat Hannover.

PY - 2020/12/22

Y1 - 2020/12/22

N2 - Patchouli oil is a major ingredient in perfumery, granting a dark-woody scent due to its main constituent (-)-patchoulol. The growing demand for patchouli oil has raised interest in the development of a biotechnological process to assure a reliable supply. Herein, we report the production of patchouli oil sesquiterpenes by metabolically engineered Escherichia coli strains, using solid-liquid phase partitioning cultivation. The (-)-patchoulol production was possible using the endogenous methylerythritol phosphate pathway and overexpressing a (-)-patchoulol synthase isoform from Pogostemon cablin but at low titers. To improve the (-)-patchoulol production, the exogenous mevalonate pathway was overexpressed in the multi-plasmid PTS + Mev strain, which increased the (-)-patchoulol titer 5-fold. Fermentation was improved further by evaluating several defined media, and optimizing the pH and temperature of culture broth, enhancing the (-)-patchoulol titer 3-fold. To augment the (-)-patchoulol recovery from fermentation, the solid-liquid phase partitioning cultivation was analyzed by screening polymeric adsorbers, where the Diaion HP20 adsorber demonstrated the highest (-)-patchoulol recovery from all tests. Fermentation was scaled-up to fed-batch bioreactors, reaching a (-)-patchoulol titer of 40.2 mg L-1 and productivity of 20.1 mg L-1 d-1. The terpene profile and aroma produced from the PTS + Mev strain were similar to the patchouli oil, comprising (-)-patchoulol as the main product, and α-bulnesene, trans-β-caryophyllene, β-patchoulene, and guaia-5,11-diene as side products. This investigation represents the first study of (-)-patchoulol production in E. coli by solid-liquid phase partitioning cultivation, which provides new insights for the development of sustainable bioprocesses for the microbial production of fragrant terpenes.

AB - Patchouli oil is a major ingredient in perfumery, granting a dark-woody scent due to its main constituent (-)-patchoulol. The growing demand for patchouli oil has raised interest in the development of a biotechnological process to assure a reliable supply. Herein, we report the production of patchouli oil sesquiterpenes by metabolically engineered Escherichia coli strains, using solid-liquid phase partitioning cultivation. The (-)-patchoulol production was possible using the endogenous methylerythritol phosphate pathway and overexpressing a (-)-patchoulol synthase isoform from Pogostemon cablin but at low titers. To improve the (-)-patchoulol production, the exogenous mevalonate pathway was overexpressed in the multi-plasmid PTS + Mev strain, which increased the (-)-patchoulol titer 5-fold. Fermentation was improved further by evaluating several defined media, and optimizing the pH and temperature of culture broth, enhancing the (-)-patchoulol titer 3-fold. To augment the (-)-patchoulol recovery from fermentation, the solid-liquid phase partitioning cultivation was analyzed by screening polymeric adsorbers, where the Diaion HP20 adsorber demonstrated the highest (-)-patchoulol recovery from all tests. Fermentation was scaled-up to fed-batch bioreactors, reaching a (-)-patchoulol titer of 40.2 mg L-1 and productivity of 20.1 mg L-1 d-1. The terpene profile and aroma produced from the PTS + Mev strain were similar to the patchouli oil, comprising (-)-patchoulol as the main product, and α-bulnesene, trans-β-caryophyllene, β-patchoulene, and guaia-5,11-diene as side products. This investigation represents the first study of (-)-patchoulol production in E. coli by solid-liquid phase partitioning cultivation, which provides new insights for the development of sustainable bioprocesses for the microbial production of fragrant terpenes.

UR - http://www.scopus.com/inward/record.url?scp=85097813150&partnerID=8YFLogxK

U2 - 10.1021/acsomega.0c04590

DO - 10.1021/acsomega.0c04590

M3 - Article

C2 - 33376881

AN - SCOPUS:85097813150

VL - 5

SP - 32436

EP - 32446

JO - ACS Omega

JF - ACS Omega

IS - 50

ER -