TY - JOUR
T1 - Development of a broad-host synthetic biology toolbox for ralstonia eutropha and its application to engineering hydrocarbon biofuel production
AU - Bi, Changhao
AU - Su, Peter
AU - Müller, Jana
AU - Yeh, Yi Chun
AU - Chhabra, Swapnil R.
AU - Beller, Harry R.
AU - Singer, Steven W.
AU - Hillson, Nathan J.
N1 - Funding Information:
This work was funded by the Department of Energy, Advanced Research Projects Agency-Energy (ARPA-E) Electrofuels Program, under contract DE-0000206-1577 to Lawrence Berkeley National Laboratory. This work was performed at the Joint BioEnergy Institute, which is funded by the Department of Energy, Office of Science, Office of Biological and Environmental Research under contract DE-AC02-5CH11231 to Lawrence Berkeley National Laboratory. We thank Vivek Mutalik for providing access to the Guava easyCyte Flow cytometry system. We thank Jonathan Vroom for assistance in constructing trfA mutations and the T7 stem-loop. We thank Yung Hsu Tang for assistance in constructing and testing pKTTrfp, pCMTrfp, pCM271Trfp, and pCM271TcalRBSrfp.
PY - 2013/11/13
Y1 - 2013/11/13
N2 - 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 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.Conclusion: We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.
AB - 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 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.Conclusion: We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.
KW - Broad-host
KW - Chemolithoautotroph
KW - Hydrocarbon
KW - Ralstonia eutropha
KW - Synthetic biology
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U2 - 10.1186/1475-2859-12-107
DO - 10.1186/1475-2859-12-107
M3 - Article
C2 - 24219429
AN - SCOPUS:84887388915
SN - 1475-2859
VL - 12
JO - Microbial Cell Factories
JF - Microbial Cell Factories
IS - 1
M1 - 107
ER -