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Title: An evaluation of the process engineering options for a multi step biotransformation
Author: Vanier, Fiona Olivia
ISNI:       0000 0001 3542 6421
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2000
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The majority of commercially used biotransformations tend to be single step involving the conversion of a substrate to a structurally similar compound. The exploitation of an existing host native pathway such that a novel compound is synthesised on the addition of a foreign gene is also possible. The cloning of a section of a foreign metabolic pathway in such a way that the enzymes synthesise a specific product on the addition of a substrate is a powerful technique that has been seldom investigated from a process design perspective. The aim of this work was to evaluate the processing options necessary to carry out a multi step biotransformation, undertake the decision-making stage of process design by following guidelines defined in a feasibility study, and put into practice the design of the bioprocess necessary to over accumulate a specific product using a model system. This research was based on the concept that a properly defined structured approach could be used to develop a particular biotransformation, and this approach could facilitate the development of any multi step biotransformation. The cloning of a whole section of a foreign metabolic pathway was possible due to the advent of metabolic pathway engineering using recombinant DNA technology. The distinguishing feature of this bioprocess when compared to other common biotransformations is the existence of pathway intermediates and pathway enzymes not naturally found in the host. The metabolic pathway used as the model system was the TOL meta-cleavage pathway, and the genes which convert benzoate to 2-hydroxymuconic semialdehyde in a linear three-step portion of the pathway were ligated into a host vector and used to transform an E. coli host strain JM107. This thesis describes the design of a fermentation protocol resulting in a biomass concentration of 18 - 22 g.L-1 and an investigation into the effect of induction on foreign gene expression including a long term induction experiment over a 20 hour period. A substrate feeding strategy was also designed and product yields as high as 17% during multi step biotransformations were compared to a single step counterpart with a yield of 22%. A method for the removal of the product using an Amberlite resin in a bisulphite form was proposed, and the resin was experimentally assessed for its use in this model system.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Fermentation protocol; Enzyme synthesis