Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.626536
Title: Design and characterization of novel biocatalytic cascades for chiral amino alcohol synthesis
Author: Villegas Torres, M. F.
ISNI:       0000 0004 5362 1839
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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Abstract:
Chiral amino alcohols are building blocks of several pharmaceuticals such as antibiotics, and retrovirals. De novo biocatalytic strategies have been developed for their production as chemical synthesis presents several challenges in establishing chiral centres. One of the successful approaches previously implemented in the Biochemical Engineering Department is the coupling of a transketolase with a transaminase step. This scheme depends on the use of hydroxypyruvate (HPA) and methylbenzylamine (MBA), which are expensive non-natural substrates limiting both the application of the pathway on an industrial scale, and its integration with the host metabolism. Therefore, this thesis examines the ability of two novel biocatalytic cascades to synthesize chiral amino alcohols from natural cheaper compounds, and the issues faced when attempting to integrate the pathways with Escherichia coli metabolism. Two different biocatalytic strategies were studied: a recycling cascade and a sequential pathway. The former, directly aminates erythrulose using serine as amino donor for the simultaneous synthesis of 2-amino-1,3,4-butanetriol (ABT) and HPA. The latter, involves three enzymatic steps carried out by two transaminases and one transketolase using serine, pyruvate and glycolaldehyde (GA) as substrates. Both coupled reactions depend on a transaminase able to use serine as amino donor. As a result, this work initiates with screening studies to identify the ideal candidates from a library of 100 transaminases. The selected enzymes were then characterized based on their main kinetic constants, KM and kcat, and their amino acceptors profiles when using serine as amino donor. From the results, two transaminases were selected for the proof of concept of the cascade reactions in a purified system. Due to the low bioconversions achieved, the work progressed towards their optimization implementing Response Surface Methodology (RSM) using a Central Composite Design (CCD). Finally, the optimized cascades were then evaluated in a whole cell environment using E. coli resting cells, exposing the challenges to be faced during their future metabolic integration. This study finalizes with a first attempt to design and build a more suitable expression system required for future work. Within this work, three new transaminases were described and characterized: two ω-transaminases isolated from Rhodobacter sphaeroides with broad amino acceptors profile using serine as amino donor; and a serine: pyruvate transaminase isolated from Deinococcus geothermalis with high substrates specificity. Both cascades were successfully tested, where the recycling system gave 30-times higher yields (30% conversion), and was more advantageous for the integration in comparison to the three-step sequential pathway. After the optimization, the yields of the recycling cascade and the sequential pathway were 2-fold and 3-fold improved, respectively. Finally based on the preliminary whole cell study of the recycling cascade and the first two steps of the sequential pathway, it became clear that the main challenges for the future metabolic integration are HPA consumption by the host, and the differential effect of the expression of different recombinant enzymes on the E. coli host cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.626536  DOI: Not available
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