Abstract
BackgroundThe 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.ResultsWe 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.ConclusionWe constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.
Highlights
The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions
We have initiated the development of a synthetic biology toolbox to enable complex metabolic engineering applications in R. eutropha H16
We investigated the impact of the toolbox on hydrocarbon production titer in R. eutropha, and compared the resulting titers with corresponding rfp levels across the various expression configurations
Summary
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. Chemoautotrophic “Knallgas” bacteria can utilize H2/ CO2 for growth under aerobic conditions, and have great potential to directly produce liquid fuels from CO2 and/or syngas [1,2]. While R. eutropha has great potential to be engineered to produce desired compounds (beyond PHB) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. This work develops and demonstrates the engineering utility of a plasmid-based toolbox for R. eutropha
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