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Development of a broad-host synthetic biology toolbox for ralstonia eutropha and its application to engineering hydrocarbon biofuel production

Changhao Bi12, Peter Su13, Jana Müller1, Yi-Chun Yeh14, Swapnil R Chhabra1*, Harry R Beller5, Steven W Singer5 and Nathan J Hillson1*

Author Affiliations

1 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

2 Present address: Tianjin Institute of Biotechnology, Chinese Academy of Sciences, Tianjin, China

3 Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA 94720, USA

4 National Taiwan Normal University, Taipei, Taiwan

5 Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

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Microbial Cell Factories 2013, 12:107  doi:10.1186/1475-2859-12-107

Published: 13 November 2013



The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors.


We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5’ mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts.


We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.

Broad-host; Synthetic biology; Ralstonia eutropha; Hydrocarbon; Chemolithoautotroph