Synthetic Biology Toolkit for Cupriavidus necator H16: An Industrially Relevant Microbe

Abstract

Cupriavidus 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.