Sibley, Michael; (2018) A Cell Engineering Approach to Enzyme-Based Fed-Batch Fermentation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

One of the major disadvantages of batch fermentation is the difficulty in achieving high cell densities; in E.coli K12, much of this is attributed to the production of acetate via a phenomenon known as overflow metabolism. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch mechanisms require components which become problematic when applying them to smaller scale systems such as shake flasks. As a result, a number of slow release carbon techniques have been developed; one of which uses the enzymatic degradation of starch to slowly release glucose into the culture medium following the addition of an amylolytic enzyme. This reduces acetate production due to the low initial glucose concentration, leading to an increased cell density, and an increased product yield. To date, these amylolytic enzymes have been added to the culture exogenously, whereas this project aims to employ a cell engineering approach to design and build a self-secreting amylolytic chassis capable of enzyme-based fed-batch fermentation. The study explores the use of starch as an alternative carbon source, and describes the ability of a highly active amylolytic E. coli strain secreting S. thermoviolaceus α-amylase to degrade and utilise starch as a sole carbon source. Bacterial exo-acting amylolytic enzymes have been identified and cloned into E. coli for characterisation studies, with enhanced secretion of the novel C. violaceum glucoamylase using the DsbA signal peptide resulting in direct conversion of starch to glucose within the media. Further investigations reveal expression can be negatively regulated using a glucose sensitive promoter, providing a basis for self-regulation. Lastly, vectors have been constructed to simultaneously express the C. violaceum glucoamylase and a target recombinant protein (eGFP), resulting in higher biomass and increased recombinant protein expression when grown on starch compared to an equivalent amount of glucose, the first demonstration of a cell engineered approach to enzyme-based fed-batch fermentation.

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