Metabolic engineering of Cupriavidus necator H16 for sustainable biomanufacturing

Thesis

Uncoupling fuel and chemical production from the consumption of fossil resources is a fundamental challenge for sustainable development. In industrial biomanufacturing, this challenge is most often addressed by using heterotrophic microbes to convert plant biomass into value-added compounds. To improve the sustainability of industrial bioprocesses, a shift to alternative feedstocks is required. This includes the direct utilisation of excess atmospheric carbon dioxide (CO2), as well as other one carbon (C1) or two-carbon (C2) compounds which can be derived from abiotic CO2 reduction or alternative waste streams. As these feedstocks are broadly inaccessible to common industrial microbes, their bioconversion requires engineering specialised production strains.

The facultative chemolithoautotroph Cupriavidus necator H16 (C. necator) holds demonstrable potential as a host strain for sustainable biomanufacturing, owing to its malleable metabolism and natural ability to utilise a wide range of feedstocks. To realise its potential, tailored molecular tools and extensive biological insights are needed. Addressing these requirements is the overarching aim of this thesis. A novel electroporation protocol was optimised, allowing for more rapid and efficient transformation of wildtype C. necator than previously attainable. This advancement, which accelerates the prototyping of synthetic DNA constructs, enabled the development of an efficient genome editing tool. The method uses homologous recombination and CRISPR-Cas counterselection to streamline the genomic manipulation of C. necator, facilitating the installation of new or enhanced cellular functions. During the development of this genome editing tool, the performance of several different genetic regulatory modules was characterised. These DNA parts are applicable in other biotechnological contexts, enabling the implementation of more complex genetic circuitry in C. necator. As an example, the implementation of a genetic logic gate was demonstrated.

Strategies to improve growth and production from C1 and C2 substrates were also investigated. An evolutionary engineering approach was implemented to improve the growth rate of C. necator on formate, leading to the identification of evolved isolates with enhanced phenotypic traits. Genotypic analysis of these isolates revealed likely causative mutations, which could be harnessed for further metabolic engineering. Finally, the bioconversion of acetate to ethanol was investigated. Ethanologenic C. necator strains were constructed, recording improvements in titer, rate and yield over previous reports. Taken together, the findings documented in this thesis provide valuable tools, methods and insights relevant to C. necator engineering, thereby contributing to its development as a platform strain for sustainable biomanufacturing.

Authors: Della Valle, S

• DOI: 10.5287/ora-bmv7gpjbo
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: synthetic biology; biotechnology; biomanufacturing; genome editing
• Subjects: Biotechnology; Gene editing; Synthetic biology