Expanding engineering tools for Cupriavidus necator H16 to convert waste stream into useful chemicals via rational design and evolutionary engineering approaches

Cupriavidus necator H16 is a chemolithoautotrophic Gram-negative bacterium that belongs to the class of -proteobacteria. In recent years, it has attracted the biotechnological attention as a cell factory to produce value-added chemicals. Thus, a need for expanding engineering tools for chemical production in C. necator H16 has emerged. In order to use C. necator H16 as a cell factory, mainly two different engineering strategies are reviewed in this work: rational design engineering and evolutionary engineering. Aim: The overall aim of the study is to engineer C. necator H16 with rational design and evolutionary engineering tools to explore the biotechnological potentials of the strain and fine-tune the properties of C. necator H16 for chemical production using waste stream as a feedstock. Thesis content: In Chapter 1, literature review about C. necator H16 was compiled to understand the natural metabolism of the strain and to analyse its different biomanufacturing potentials, as well as engineering strategies used for chemical production in C. necator H16 and other related microorganisms, afterwards, in Chapter 2 the development of an optimised transformation method and a synthetic biology toolbox for C. necator H16 was developed to design robust strains of C. necator H16. Once the synthetic biology toolbox was tailored for C. necator H16, in Chapter 3, the synthetic biology toolbox was tested for the expression of two clusters of genes as well as possibly obtain bioproducts from the expression of these. Then, in Chapter 4, evolutionary engineering tools were optimised to engineer C. necator H16, the evolutionary engineering tool used was directed evolution via random mutagenesis, where an optimised protocol for random mutagenesis with a chemical mutagen for C. necator H16 was optimised and used to try to understand the biotin biosynthesis pathway of C. necator H16, which could furtherly be used for chemical production. In Chapter 5, the knowledge and strategies studied in previous chapters and proven to work in C. necator H16 supported the approaches selected to construct recombinant and evolved strains of C. necator H16 for chemical production (bioplastics) using waste stream (crude glycerol) as a feedstock. Chapter 6 highlights the general discussions, results, future work and perspectives of this PhD work.