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Background  The chemoautotrophic bacterium  Ralstonia eutropha  can utilize H 2 /CO 2  for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO 2 , 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.

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