Characterisation and optimisation of Escherichia coli cell factories for large-scale industrial production of human milk oligosaccharides

Fermentation based production has become the alternative process replacing fossil fuel based chemical synthesis and production. Subsequently, bioprocesses have gained significant attention in recent years that can be contributed to rising implementation of circular bioeconomy. The ability of microbes to produce vast range of compounds from pharmaceuticals and fine chemicals to fuels and bulk chemicals is well recognized. To meet economical and production targets in the field of biotechnological production, fermentations processes must be performed on enormous scales. Therefore, there is a huge effort on the development and improvement of microbial platforms for bioprocesses.

The environment of such scales and the natural environment of microorganisms are irreconcilable. Scale-dependent factors arousing from the size of industrial bioreactors can negatively affect strain performance and could potentially lead to economic losses. Yet, microbial cell factories are often tested and characterized in laboratory fermentation setup where the environment is significantly different than in industrial scale bioreactors. To tailor the microbial cell factories for high performance (i.e. high product yields) and robustness in large scale fermenter tanks, comprehensive physiological characterization under industrial conditions and metabolic engineering are required.

This PhD project focused on the development and utilization of a workflow including scale-down fermentations and ‘omics technologies to gain a deeper understanding of the effect of large-scale environment on the physiology of a human milk oligosaccharide (HMO) producing Escherichia coli strains. The project specifically focusing on 2’fucossyllactose (2’FL) production which is the most abundant HMO. Construction of scale-down reactor models mimicking manufacturing scale fermentation and establishment of a pipeline of transcriptomics and proteomics were done within this PhD project

The work presented in this PhD thesis contributes to understanding robustness of a 2’FL producing E. coli strain under industrial scale fermentation conditions and challenging of scale dependent factors. Furthermore, the PhD project had a unique opportunity to characterize scale related aspects in a 450 m³ industrial process and to directly compare scale-down fermentation performance this large-scale process. Characterization included transcriptome and proteome comparison of laboratory-, large-scale and scale-down fermentations. Finally, mutants of the strain characterized in this thesis related to reduced acetic acid production were analysed in high cell density fed-batch culture and overflow triggering stress test.

This PhD project contributes to the understanding of E. coli’s physiology under large-scale fermentation environment and brings us a step forward to improve robustness of industrial strains to improve transition of microbial fermentation processes form small to industrial scale.