Reasearch Awards nomination

Email updates

Keep up to date with the latest news and content from Biotechnology for Biofuels and BioMed Central.

Open Access Highly Accessed Research

Metabolic engineering of Escherichia coli for high-specificity production of isoprenol and prenol as next generation of biofuels

Yanning Zheng12, Qiang Liu3, Lingling Li3, Wen Qin3, Jianming Yang1, Haibo Zhang1, Xinglin Jiang12, Tao Cheng1, Wei Liu1, Xin Xu1 and Mo Xian1*

Author Affiliations

1 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

2 University of Chinese Academy of Sciences, Beijing, 100049, China

3 College of Food Science, Sichuan Agricultural University, Yaan, 625014, China

For all author emails, please log on.

Biotechnology for Biofuels 2013, 6:57  doi:10.1186/1754-6834-6-57

Published: 24 April 2013

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.

Keywords:
Isoprenol; Prenol; Metabolic engineering; Escherichia coli; Biofuel