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


RESULTS
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 P(BAD), T7, P(xyls/PM), P(lacUV5), 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.


CONCLUSIONS
We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.
Changhao
C
Bi
Peter
P
Su
Jana
J
Müller
Yi-Chun
YC
Yeh
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. swap.chhabra@gmail.com.
Swapnil R
SR
Chhabra
Harry R
HR
Beller
Steven W
SW
Singer
Nathan J
NJ
Hillson
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D056804:Biofuels / Q000382:microbiology*; D015964:Gene Expression Regulation, Bacterial; D006838:Hydrocarbons / Q000378:metabolism*; D060847:Metabolic Engineering
D001426:Bacterial Proteins; D056804:Biofuels; D006838:Hydrocarbons