15-04: Synthetic Biology Tool Box for the Metabolic Engineering of Ralstonia eutropha

Synthetic Biology Tool Box for the Metabolic Engineering of Ralstonia eutropha

The bacterium Ralstonia eutropha has great potential to autotrophically produce bio-based chemicals and fuels from H₂ and CO₂. We have developed a synthetic biology tool box that enables the controlled expression of multi-gene metabolic pathways in R. eutropha. Broad host-range plasmid vectors, designed for transconjucation and replication within R. eutropha, were constructed with several alternate origins of replication, including pCM62 (IncP), pBBR1 (IncQ), and pKT (ATCC 37294). Mutations were made within the pCM62 origin to increase copy number and hence gene expression. An array of multi-strength chemically inducible promoters incuding P_BAD, PT7, Pxyls/pm, and PLacUV5Y, along with a set of PCbbR promoters, induced under autotrophic growth conditions, were engineered for the tool box. Ribosomal-binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational design tool, along with a mRNA 5’ UTR stem-loop structure, were tested for downstream gene expression impacts. The toolbox was then employed to optimize hydrocarbon production. Four sets of plasmids were constructed to assess the relationship between genetic parts and hydrocarbon yield. Preliminary results show that the tool box has enabled a 20-fold improvement in hydrocarbon yield, suggesting the great utility of the platform for the metabolic engineering of R. eutropha.