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Title: Prototyping in Escherichia coli of stable and effective metabolic pathways for Synechocystis sp. PCC 6803
Author: Taylor, George
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Engineering well-functioning metabolic pathways remains a challenge, largely because designing a metabolic pathway-encoding construct to express proteins at intended levels is limited and often leads to an imbalance of the pathway’s protein concentration profile and therefore a poor performance. Whilst combinatorial and iterative approaches have proven effective for optimising the pathway balance in model organisms, implementing these approaches has typically proven arduous and time-consuming for many other organisms with phenotypes of industrial relevance. Such organisms are often considered as non-model because they are difficult to engineer, often lacking the tractability, fast-growth rates and high transformation efficiencies of model organisms. This project aims to implement rapid prototyping of metabolic pathway-encoding constructs in the tractable organism, Escherichia coli, before transferring the construct for expression in the non-model organism, the cyanobacterium Synechocystis sp. PCC 6803. Synechocystis is a photoautotroph that offers the potential to sustainably produce products from carbon dioxide. However, its slow growth rate, genetic instability and lack of genetic tools make it difficult to engineer. To test the rapid prototyping of metabolic pathway-encoding constructs, genetic parts and a design framework were required. For this, libraries of synthetic promoters and synthetic RBSs were developed, from which portable promoters and RBSs were identified that offered predictive protein expression between the two organisms. A novel multi-part, modular DNA assembly method was developed, termed Start-Stop Assembly, to serve as a framework. Using these tools, variations of the lycopene pathway were compared using current state-of-the-art and novel rapid prototyping designs. In this instance, rapid prototyping did not allow the performance of a pathway in E. coli to be used as a prediction of its performance in Synechocystis, and therefore it offered no benefit to the pathways design. Implementing a combinatorial approach did however improve the proportion of effective and stable pathways in Synechocystis, which led to lycopene being directly produced from carbon dioxide.
Supervisor: Heap, John Sponsor: Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral