title: Metabolic engineering of Escherichia coli for high-specificity production of isoprenol and prenol as next generation of biofuels.
abstract: BACKGROUND
  The isopentenols, including isoprenol and prenol, are excellent alternative fuels. However, they are not compounds largely accumulated in natural organism. The need for the next generation of biofuels with better physical and chemical properties impels us to develop biosynthetic routes for the production of isoprenol and prenol from renewable sugar. In this study, we use the heterogenous mevalonate-dependent (MVA) isoprenoid pathway for the synthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) intermediates, and then convert IPP and DMAPP to isoprenol and prenol, respectively.
  
  
  RESULTS
  A mevalonate titer of 1.7 g/L was obtained by constructing an efficient MVA upper pathway in engineered E. coli. Different phosphatases and pyrophosphatases were investigated for their abilities in hydrolyzing the IPP and DMAPP. Consequently, ADP-ribose pyrophosphatase was found to be an efficient IPP and DMAPP hydrolase. Moreover, ADP-ribose pyrophosphatase from Bacillus subtilis (BsNudF) exhibited a equivalent substrate specificity towards IPP and DMAPP, while ADP-ribose pyrophosphatase from E. coli (EcNudF) presented a high substrate preference for DMAPP. Without the expression of any phosphatases or pyrophosphatases, a background level of isopentenols was synthesized. When the endogenous pyrophosphatase genes (EcNudF and yggV) that were capable of enhancing the hydrolyzation of the IPP and DMAPP were knocked out, the background level of isopentenols was still obtained. Maybe the synthesized IPP and DMAPP were hydrolyzed by some unknown hydrolases of E. coli. Finally, 1.3 g/L single isoprenol was obtained by blocking the conversion of IPP to DMAPP and employing the BsNudF, and 0.2 g/L ~80% prenol was produced by employing the EcNudF. A maximal yield of 12% was achieved in both isoprenol and prenol producing strains.
  
  
  CONCLUSIONS
  To the best of our knowledge, this is the first successful report on high-specificity production of isoprenol and prenol by microbial fermentation. Over 1.3 g/L isoprenol achieved in shake-flask experiments represents a quite encouraging titer of higher alcohols. In addition, the substrate specificities of ADP-ribose pyrophosphatases were determined and successfully applied for the high-specificity synthesis of isoprenol and prenol. Altogether, this work presents a promising strategy for high-specificity production of two excellent biofuels, isoprenol and prenol.
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China ; University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Yanning
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: College of Food Science, Sichuan Agricultural University, Yaan, 625014, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.affiliation: College of Food Science, Sichuan Agricultural University, Yaan, 625014, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Li
authors.affiliation: College of Food Science, Sichuan Agricultural University, Yaan, 625014, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Qin
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.
authors.forename: Jianming
authors.initials: J
authors.lastname: Yang
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China ; University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Xinglin
authors.initials: X
authors.lastname: Jiang
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Cheng
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.
authors.forename: Mo
authors.initials: M
authors.lastname: Xian
keywords: Biofuel; Escherichia coli; Isoprenol; Metabolic engineering; Prenol