Yang, Yiheng; (2022) Investigation of plasmid stability in glucose-limited E.coli continuous fermentations for commodity chemicals production. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Commodity chemicals are widely used in various fields ranging from the life science industries to aerospace. The overall market for commodity chemicals continues to grow rapidly owing to economic growth around the world. Currently, production of commodity chemicals is mainly based on petrochemical processes which lead to the consumption of non-renewable feedstocks with associated impacts on the environment. These concerns have led to investigation of more sustainable fermentation-based processes for chemicals production. The economics of chemicals production has rekindled interest in continuous fermentations due to their enhanced productivity over batch-wise processes. However, industrial applications of continuous fermentation have been limited to date due to issues associated with genetic instabilities and currently insufficient productivity due to limited investigation and researches. These issues are addressed in this work, which focuses on citramalate production by metabolically engineered E.coli BW25113 in glucose-limited continuous fermentations. Citramalate is a precursor that can be used in the production of acrylic glasses. Initial experiments focused on glucose-limited batch and fed-batch fermentations to generate benchmark data on citramalate productivity using plasmid-based, inducible expression systems. As a result, the concentrations of biomass and citramalate achieved were 2.4 and 2.2 gL-1 in the batch fermentation and 25 and 31 gL-1 in the fed-batch fermentation. A well-controlled continuous fermentation process was then established for citramalate production in glucose-limited cultures using different plasmid expression systems (pET and pBAD) with different strength constitutive promoters. The combination of the pET plasmid with the constitutive promoter BBaJ23119 demonstrated the highest levels of citramalate synthase expression, and hence citramalate production in continuous cultures. However, a decrease in citramalate production over time was observed which is indicative of culture genetic instability. Improvement in continuous fermentation stability, and maintenance of citramalate production, was achieved by incorporation of the Cer system (plasmid multimer resolution) in the pET plasmid backbone. Analyses indicated that inclusion of the Cer locus could solve issues associated with plasmid segregational instabilities but could not prevent plasmid structural instabilities. The impact of dilution rate, D (0.03, 0.1, 0.17, 0.23 h-1), on citramalate production was also investigated. The results showed that production stability and productivity increased with dilution rate. A relatively high cell density continuous culture (citramalate concentration= 13 gL-1, productivity= 3.1 gL-1h-1) was ultimately established through a combination of process design and media optimization studies. Sequencing of plasmid isolated from the cells during continuous fermentations indicated that citramalate production loss was mainly associated with transposition of host E.coli IS5 and IS1 elements into the plasmids or deletion of gene sequences within the citramalate synthase or promoter genes. Continuous fermentation kinetic models were then established based on parameter estimation from fed-batch cultures taking inhibitory effects on cell growth into consideration. Prediction of continuous fermentation performance using the validated kinetic model indicated that the current system could achieve a maximum citramalate titre of ~40 gL-1 and a productivity of 5.5 gL-1h-1 at specific dilution rates and feed concentrations. Furthermore, citramalate production loss and the take-over of fermentations by non-productive mutants was successfully modelled by assuming a mutant population with increased μmax (1.6 times higher) and decreased Ks (3 times lower) compared to the original productive cells. These model predictions were validated experimentally. This research has provided novel insights into plasmid stability in glucose-limited continuous microbial fermentations. Respectable citramalate concentrations and productivities were achieved but these need to be enhanced further before the process becomes economically viable. Modelling of the fermentation processes has contributed to a more in-depth understanding of the fermentation process dynamics, including the mutant take-over phenomenon arising due to plasmid instability. Overall, the work gives the engineering insights into the technical and commercial feasibility of commodity chemicals production by continuous fermentation.

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