Engineering Escherichia coli for high-yield geraniol production with biotransformation of geranyl acetate to geraniol under fed-batch culture

Wei Liu, Xin Xu, Rubing Zhang, Tao Cheng, Yujin Cao, Xiaoxiao Li, Jiantao Guo, Huizhou Liu, Mo Xian

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Background: Geraniol is an acyclic monoterpene alcohol, which exhibits good prospect as a gasoline alternative. Geraniol is naturally encountered in plants at low concentrations and an attractive target for microbial engineering. Geraniol has been heterologously produced in Escherichia coli, but the low titer hinders its industrial applications. Moreover, bioconversion of geraniol by E. coli remains largely unknown. Results: Recombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase, and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6 ± 3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol production to 78.8 mg/L. To further improve the production yield, the fermentation conditions were optimized. Firstly, 81.4 % of volatile geraniol was lost during the first 5 h of fermentation in a solvent-free system. Hence, isopropyl myristate was added to the culture medium to form an aqueous-organic two-phase culture system, which effectively prevented volatilization of geraniol. Secondly, most of geraniol was eventually biotransformed into geranyl acetate by E. coli, thus decreasing geraniol production. For the first time, we revealed the role of acetylesterase (Aes, EC 3.1.1.6) from E. coli in hydrolyzing geranyl acetate to geraniol, and production of geraniol was successfully increased to 2.0 g/L under controlled fermentation conditions. Conclusions: An efficient geraniol production platform was established by overexpressing several key pathway proteins in engineered E. coli strain combined with a controlled fermentation system. About 2.0 g/L geraniol was obtained using our controllable aqueous-organic two-phase fermentation system, which is the highest yield to date. In addition, the interconversion between geraniol and geranyl acetate by E. coli was first elucidated. This study provided a new and promising strategy for geraniol biosynthesis, which laid a basis for large-scale industrial application.

Original languageEnglish (US)
Article number466
JournalBiotechnology for Biofuels
Volume9
Issue number1
DOIs
StatePublished - Mar 11 2016

Fingerprint

Batch Cell Culture Techniques
biotransformation
Biotransformation
Escherichia coli
fermentation
acetate
Fermentation
engineering
Industrial applications
Monoterpenes
monoterpene
Production platforms
Bioconversion
volatilization
Biosynthesis
geranyl acetate
geraniol
alcohol
Vaporization
Gasoline

Keywords

  • Acetylesterase
  • Engineered Escherichia coli
  • Fed-batch fermentation
  • Geraniol
  • Geranyl acetate

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Renewable Energy, Sustainability and the Environment
  • Energy(all)
  • Management, Monitoring, Policy and Law

Cite this

Engineering Escherichia coli for high-yield geraniol production with biotransformation of geranyl acetate to geraniol under fed-batch culture. / Liu, Wei; Xu, Xin; Zhang, Rubing; Cheng, Tao; Cao, Yujin; Li, Xiaoxiao; Guo, Jiantao; Liu, Huizhou; Xian, Mo.

In: Biotechnology for Biofuels, Vol. 9, No. 1, 466, 11.03.2016.

Research output: Contribution to journalArticle

Liu, Wei ; Xu, Xin ; Zhang, Rubing ; Cheng, Tao ; Cao, Yujin ; Li, Xiaoxiao ; Guo, Jiantao ; Liu, Huizhou ; Xian, Mo. / Engineering Escherichia coli for high-yield geraniol production with biotransformation of geranyl acetate to geraniol under fed-batch culture. In: Biotechnology for Biofuels. 2016 ; Vol. 9, No. 1.
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AU - Liu, Wei

AU - Xu, Xin

AU - Zhang, Rubing

AU - Cheng, Tao

AU - Cao, Yujin

AU - Li, Xiaoxiao

AU - Guo, Jiantao

AU - Liu, Huizhou

AU - Xian, Mo

PY - 2016/3/11

Y1 - 2016/3/11

N2 - Background: Geraniol is an acyclic monoterpene alcohol, which exhibits good prospect as a gasoline alternative. Geraniol is naturally encountered in plants at low concentrations and an attractive target for microbial engineering. Geraniol has been heterologously produced in Escherichia coli, but the low titer hinders its industrial applications. Moreover, bioconversion of geraniol by E. coli remains largely unknown. Results: Recombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase, and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6 ± 3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol production to 78.8 mg/L. To further improve the production yield, the fermentation conditions were optimized. Firstly, 81.4 % of volatile geraniol was lost during the first 5 h of fermentation in a solvent-free system. Hence, isopropyl myristate was added to the culture medium to form an aqueous-organic two-phase culture system, which effectively prevented volatilization of geraniol. Secondly, most of geraniol was eventually biotransformed into geranyl acetate by E. coli, thus decreasing geraniol production. For the first time, we revealed the role of acetylesterase (Aes, EC 3.1.1.6) from E. coli in hydrolyzing geranyl acetate to geraniol, and production of geraniol was successfully increased to 2.0 g/L under controlled fermentation conditions. Conclusions: An efficient geraniol production platform was established by overexpressing several key pathway proteins in engineered E. coli strain combined with a controlled fermentation system. About 2.0 g/L geraniol was obtained using our controllable aqueous-organic two-phase fermentation system, which is the highest yield to date. In addition, the interconversion between geraniol and geranyl acetate by E. coli was first elucidated. This study provided a new and promising strategy for geraniol biosynthesis, which laid a basis for large-scale industrial application.

AB - Background: Geraniol is an acyclic monoterpene alcohol, which exhibits good prospect as a gasoline alternative. Geraniol is naturally encountered in plants at low concentrations and an attractive target for microbial engineering. Geraniol has been heterologously produced in Escherichia coli, but the low titer hinders its industrial applications. Moreover, bioconversion of geraniol by E. coli remains largely unknown. Results: Recombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase, and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6 ± 3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol production to 78.8 mg/L. To further improve the production yield, the fermentation conditions were optimized. Firstly, 81.4 % of volatile geraniol was lost during the first 5 h of fermentation in a solvent-free system. Hence, isopropyl myristate was added to the culture medium to form an aqueous-organic two-phase culture system, which effectively prevented volatilization of geraniol. Secondly, most of geraniol was eventually biotransformed into geranyl acetate by E. coli, thus decreasing geraniol production. For the first time, we revealed the role of acetylesterase (Aes, EC 3.1.1.6) from E. coli in hydrolyzing geranyl acetate to geraniol, and production of geraniol was successfully increased to 2.0 g/L under controlled fermentation conditions. Conclusions: An efficient geraniol production platform was established by overexpressing several key pathway proteins in engineered E. coli strain combined with a controlled fermentation system. About 2.0 g/L geraniol was obtained using our controllable aqueous-organic two-phase fermentation system, which is the highest yield to date. In addition, the interconversion between geraniol and geranyl acetate by E. coli was first elucidated. This study provided a new and promising strategy for geraniol biosynthesis, which laid a basis for large-scale industrial application.

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KW - Engineered Escherichia coli

KW - Fed-batch fermentation

KW - Geraniol

KW - Geranyl acetate

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