UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation

Allen, James R; Torres-Acosta, Mario A; Mohan, Naresh; Lye, Gary J; Ward, John M; (2022) Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation. Microbial Cell Factories , 21 (1) , Article 229. 10.1186/s12934-022-01958-3. Green open access

[thumbnail of James Allen Micro Cell Fact.pdf]
Preview
Text
James Allen Micro Cell Fact.pdf - Other

Download (1MB) | Preview

Abstract

Background: The production of chemicals via bio-based routes is held back by limited easy-to-use stabilisation systems. A wide range of plasmid stabilisation mechanisms can be found in the literature, however, how these mechanisms effect genetic stability and how host strains still revert to non-productive variants is poorly understood at the single-cell level. This phenomenon can generate difficulties in production-scale bioreactors as different populations of productive and non-productive cells can arise. To understand how to prevent non-productive strains from arising, it is vital to understand strain behaviour at a single-cell level. The persistence of genes located on plasmid vectors is dependent on numerous factors but can be broadly separated into structural stability and segregational stability. While structural stability refers to the capability of a cell to resist genetic mutations that bring about a loss of gene function in a production pathway, segregational stability refers to the capability of a cell to correctly distribute plasmids into daughter cells to maintain copy number. A lack of segregational stability can rapidly generate plasmid-free variants during replication, which compromises productivity. Results: Citramalate synthase expression was linked in an operon to the expression of a fluorescent reporter to enable rapid screening of the retention of a model chemical synthesis pathway in a continuous fermentation of E. coli. Cells without additional plasmid stabilisation started to lose productivity immediately after entering the continuous phase. Inclusion of a multimer resolution site, cer, enabled a steady-state production period of 58 h before a drop in productivity was detected. Single-cell fluorescence measurements showed that plasmid-free variants arose rapidly without cer stabilisation and that this was likely due to unequal distribution of plasmid into daughter cells during cell division. The addition of cer increased total chemical yield by more than 50%. Conclusions: This study shows the potential remains high for plasmids to be used as pathway vectors in industrial bio-based chemicals production, providing they are correctly stabilised. We demonstrate the need for accessible bacterial ‘toolkits’ to enable rapid production of known, stabilised bacterial production strains to enable continuous fermentation at scale for the chemicals industry.

Type: Article
Title: Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation
Location: England
Open access status: An open access version is available from UCL Discovery
DOI: 10.1186/s12934-022-01958-3
Publisher version: https://doi.org/10.1186/s12934-022-01958-3
Language: English
Additional information: Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Keywords: Science & Technology, Life Sciences & Biomedicine, Biotechnology & Applied Microbiology, Stabilised plasmids, Continuous culture, Cer sequence, Bio-based chemical production, COPY-NUMBER, CITRAMALATE, TIME
UCL classification: UCL
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: https://discovery.ucl.ac.uk/id/eprint/10171807
Downloads since deposit
8Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item