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Optimisation of enzyme cascades for chiral amino alcohol synthesis in aid of host cell integration using a statistical experimental design approach

Villegas-Torres, MF; Ward, JM; Baganz, F; (2018) Optimisation of enzyme cascades for chiral amino alcohol synthesis in aid of host cell integration using a statistical experimental design approach. Journal of Biotechnology , 281 pp. 150-160. 10.1016/j.jbiotec.2018.07.014. Green open access

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Abstract

Chiral amino alcohols are compounds of pharmaceutical interest as they are building blocks of sphingolipids, antibiotics, and antiviral glycosidase inhibitors. Due to the challenges of chemical synthesis we recently developed two TK-TAm reaction cascades using natural and low cost feedstocks as substrates: a recycling cascade comprising of 2 enzymes and a sequential 3-step enzyme cascade yielding 30% and 1% conversion, respectively. In order to improve the conversion yield and aid the future host strain engineering for whole cell biocatalysis, we used a combination of microscale experiments and statistical experimental design. For this we implemented a full factorial design to optimise pH, temperature and buffer type, followed by the application of Response Surface Methodology for the optimisation of substrates and enzymes concentrations. Using purified enzymes we achieved 60% conversion for the recycling cascade and 3-fold improvement using the sequential pathway. Based on the results, limiting steps and individual requirements for host cell metabolic integration were identified expanding the understanding of the cascades without implementing extensive optimisation modelling. Therefore, the approach described here is well suited for optimising reaction conditions as well as defining the relative enzyme expression levels required for construction of microbial cell factories.

Type: Article
Title: Optimisation of enzyme cascades for chiral amino alcohol synthesis in aid of host cell integration using a statistical experimental design approach
Location: Netherlands
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.jbiotec.2018.07.014
Publisher version: https://doi.org/10.1016/j.jbiotec.2018.07.014
Language: English
Additional information: This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
Keywords: Recycling cascade, chiral amino alcohol, sequential pathway, statistical experimental design
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: http://discovery.ucl.ac.uk/id/eprint/10053233
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