Results from Matteo Vajente (ESR 01)

Climate change is an urgent and collective challenge, and several solutions are necessary to reduce CO₂ emissions or to increase carbon capture and utilization from the atmosphere. Nature has been evolving CO₂ utilization pathways for billions of years, and biotechnology could be used to exploit them and transform CO₂ into useful molecules for human applications. Cupriavidus necator H16 is one of the most studied autotrophic microorganisms, able to convert H₂, CO₂ and O₂ into biomass and bioplastic. It has been engineered for the production of several compounds, but the slow and cumbersome engineering process limits synthetic biology applications. In his project, Matteo developed an easy-to-use inducible expression plasmid compatible with C. necator as well as other organisms, which allow for quick cloning and expression of genes of interest using Golden Gate assembly. The transformation bottlenecks have also been investigated and characterized, and it is now possible to reliably transform expression plasmids and even suicide plasmids by electroporation. This considerably shortens the engineering time and allows quicker testing and screening of potential strains. While more work is ongoing towards the development of genetic tools for C. necator, the current results can be found in a recently published study (DOI: 10.1021/acssynbio.4c00380). The pMVRha plasmid is also available on Addgene (#229553). This study highlights the steps that can be undertaken to increase electroporation efficiency in other non-model bacteria, using a combination of bioinformatic prediction and experimental confirmation via ad-hoc plasmids.