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. 2013 Nov 13:12:107.
doi: 10.1186/1475-2859-12-107.

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

Changhao Bi, Peter Su, Jana Müller, Yi-Chun Yeh, Swapnil R Chhabra  1 , Harry R Beller, Steven W Singer, Nathan J Hillson
Affiliations

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

Changhao Bi et al. Microb Cell Fact. .

Abstract

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.

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

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Figures

Figure 1
Figure 1
Plasmid expression vector RFP fluorescence intensities and copy numbers. (A) Induced RFP fluorescence intensity/OD600 (dark bars) and plasmid copy numbers (square dots) for the various origins of replication. (B, C) Induced (dark bars) and uninduced (light bars) RFP fluorescence intensity/OD600 for the various (B) promoters and (C) RBS sequences.
Figure 2
Figure 2
Hydrocarbon titers for the various (A) origins of replication, (B) promoters, and (C) RBS sequences; (dark bars) pentadecane, (light bars) heptadecene, and (grey bars) combined.
Figure 3
Figure 3
Hydrocarbon titers for the two highest producing strains.
Figure 4
Figure 4
RFP expression levels and hydrocarbon production titers. Values normalized to reference plasmids pBADTrfp and pBADTHC. pCM62: pCMTrfp and pCMTHC. PlacUV5: pYIUV5Trfp and pYIUV5THC. pKT: pKTTrfp and pKTTHC. Pxyls: pXylsTrfp and pXylsTHC. nrdDRBS: pBADTnrdDRBSrfp and pBADTnrdDRBSHC. calRBS: pBADTcalRBSrfp and pBADTcalRBSHC. pCM271: pCM271Trfp and pCM271THC. pBADT: pBADTrfp and pBADTHC. pBAD (no T): pBADrfp and pBADHC. pCM271 + calRBS: pCM271TcalRBSrfp and pCM271TcalRBSHC.
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References

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