The use of microbial platforms for the production of commercially valuable compounds has grown over the last few decades as concern over the sustainability of other production methods, such as extraction and chemical synthesis, has been come into question. Advancements in the understanding of microbial metabolism and regulation with the aid of -omics research, and a steady increase in the number of available genetic tools, has allowed model organisms such as the bacteria Escherichia coli and the yeast Saccharomyces cerevisiae to be engineered for high titre production of a number of value products. The economical production of these compounds on an industrial scale can, however, be hampered by the toxicity of a given end-product upon accumulation in culture. The mode of action by which these products interfere with cellular homeostasis is often multifaceted, making it difficult to address. As such, considerable research has gone into developing strategies to minimize this microbial toxicity in platform hosts; including engineering strains for increased tolerance, and the development of fermentation strategies for the in situ removal of toxic end-products. These efforts have had mixed success, with the microbial titres of a number of value compounds continuing to be limited due low tolerance thresholds of the host. In this work, an alternative strategy for minimizing the toxicity of value alcohols in E. coli was investigated. This strategy involved the in vivo sequestration of an endogenously produced alcohol into a more neutral short/medium chain ester via enzyme mediated esterification by an alcohol acyl transferase (AAT). The rationale behind this strategy being that the incorporation of a toxic alcohol into a less toxic ester molecule may facilitate higher product accumulation in culture, as the ester end-product would be minimally inhibitory. Further, the alcohol component of the ester could be easily recovered downstream through hydrolysis, while the recovered acid component could be recycled as substrate for this strategy in future cultures. In this work, the feasibility of this detoxification strategy was first validated and then applied to two commercially valuable alcohols in E. coli. The first alcohol to which this strategy was applied was the proposed gasoline alternative short chain alcohol, butanol, which is detrimental to culture health when present at concentrations above 1.5% (v/v); and the second alcohol trialled was the common flavour and fragrance constituent monoterpene alcohol, geraniol, which is inhibitory when present above 0.05% (v/v) in culture.
|Date of Award||21 Mar 2018|
|Supervisor||David Leak (Supervisor) & Albert Bolhuis (Supervisor)|