A combinatorial assembly strategy to optimise biodegradation pathway performance in yeast

Combinatorial assembly methods, enhanced by automation, aim to facilitate the optimisation of a wide range of biosynthetic and biodegradative pathways to obtain a maximal product yield by varying the rate of expression of genes. The aim of this thesis was to develop a combinatorial strategy that allows the variation of expression of metabolic pathways in yeast. Initially, two yeast species, Pichia pastoris and Saccharomyces cerevisiae, were selected and compared to determine what the better suited host would be for metabolic pathway expression. The secretion of a fluorescent reporter (ymNeonGreen) was used as marker and P. pastoris was found to be a more suitable host for extracellular secretion of recombinant proteins. This PhD work was an iCASE collaboration with the biologics company Ingenza, Edinburgh, Scotland, which provided seven novel cellulase sequences that could be optimised for efficient cellulose degradation. The individual heterologous extracellular secretion of these cellulases was carried out in P. pastoris and the cellulases that were not expressed were not selected for further optimisation. To be able to co-express these cellulases and create a cellulose degradation pathway in P. pastoris, a plasmid utilising marker recycling via cre-recombinases was selected. However, unwanted recombination of this plasmid was occurring in E. coli prior to P. pastoris transformation, preventing high throughput use of this plasmid. Therefore, modifications were made to the plasmid that allowed the development of a high-throughput integration method. A combinatorial strategy was implemented in this method with the use of promoters with different expression strength. This strategy was used to create a cellulose degrading P. pastoris strain. The same method was utilised for the integration of ferulic acid degradation pathway in P. pastoris, validating this method as an efficient high-throughput method to integrate and express metabolic pathway products in P. pastoris