3 research outputs found
Synthetic Biology Toolkit for Cupriavidus necator H16: An Industrially Relevant Microbe
PhD ThesisCupriavidus necator is a Gram-negative soil bacterium of great biotechnological interest. It
is known as a producer of the bioplastic 3-polyhydroxybutyrate, has been used in
bioremediation efforts, and its lithoautotrophic capabilities raise the possibility that it could
function as a microbial factory upgrading renewable resources to fuels and chemicals.
However, appropriate experimental resources to permit controlled bioengineering and
system optimisations with the bacterium are not well established. In this study, statistical
Design of Experiments (DoE) was used to identify how key media components and their
interactions affect cell growth. The model resulting from this approach is predictive and
was experimentally validated against novel media compositions at different cultivations
scales. Specifically, interaction between histidine and CuSO4 are important for reliable
robust growth prediction. Further, plasmid parts (replication origins, antibiotic cassettes
and reporter proteins) were characterised for improved transformation efficiency,
segregational stability and reporter protein expression in C. necator. Modular minimal
plasmid sets (pCAT) were constructed for C. necator using these well-characterised
biological parts, which were assembled by Golden gate method. The resulting plasmids
were delivered to C. necator via electroporation, and the transformation efficiency
obtained was more than 3000-fold higher in comparison to that obtained with the existing
plasmid, pBHR1. More importantly, the resulting Golden gate restriction-ligation products
can be delivered directly to C. necator via electroporation with high transformation
efficiency and can co-express more than one functional protein carried on a single
plasmid restriction-ligation product. pCAT plasmids can stably propagate for more than six
generation (144 h) without the addition of antibiotics. Furthermore, the application of the
toolkit was demonstrated by engineering C. necator for improved tolerance to ethanol
using directed evolutionary approach. The toolkit established in this study will be crucial in
making future bioengineering applications in C. necator more efficient, controllable and
predictable