title: Engineering Escherichia coli for high-yield geraniol production with biotransformation of geranyl acetate to geraniol under fed-batch culture.
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.
authors.affiliation: CAS Key Laboratory of Bio-Based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 China ; Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 10090 China ; University of Chinese Academy of Sciences, Beijing, 100049 China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Bio-Based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 China ; University of Chinese Academy of Sciences, Beijing, 100049 China.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.affiliation: CAS Key Laboratory of Bio-Based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 China ; University of Chinese Academy of Sciences, Beijing, 100049 China.
authors.forename: Rubing
authors.initials: R
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Bio-Based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 China.
authors.forename: Tao
authors.initials: T
authors.lastname: Cheng
authors.affiliation: CAS Key Laboratory of Bio-Based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 China.
authors.forename: Yujin
authors.initials: Y
authors.lastname: Cao
authors.affiliation: CAS Key Laboratory of Bio-Based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 China.
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588 USA.
authors.forename: Jiantao
authors.initials: J
authors.lastname: Guo
authors.affiliation: CAS Key Laboratory of Bio-Based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 China.
authors.forename: Huizhou
authors.initials: H
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Bio-Based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 China.
authors.forename: Mo
authors.initials: M
authors.lastname: Xian
keywords: Acetylesterase; Engineered Escherichia coli; Fed-batch fermentation; Geraniol; Geranyl acetate