Investigating the potential of division of labour in synthetic bacterial communities for bioproduction
File(s)
Author(s)
Mehta, Harman
Type
Thesis
Abstract
With recent advances in bioengineering, the ability to engineer organisms to perform desired functions has expanded significantly. However, adding new functions to a cell can increase metabolic burden and induce genetic instability, making engineered cells more prone to negative selection. This limitation of isogenic monocultures can be mitigated by designing synthetic microbial cocultures with division of labour, in which metabolic pathways are distributed across multiple organisms to reduce the load on any single cell. Such an approach can lead to higher product titres by freeing more cellular resources for the target pathway and enables the biosynthesis of complex compounds requiring multi-step pathways that are difficult to achieve in monocultures. Maintaining stable cocultures over long periods requires a control system to prevent one population from dominating the other.
In this thesis, the potential of division of labour for the bioproduction of high-value compounds using synthetic bacterial communities was explored. Two coculture systems were constructed: an Escherichia coli coculture producing violacein and a Pseudomonas putida coculture producing limonene and perillyl alcohol, with subpopulations expressing different segments of their respective metabolic pathways. For violacein production, several pathway-splitting strategies were tested by partitioning the pathway at different intermediates, and culture conditions were optimised to maximise production. Coculture performance exceeded that of monocultures when three conditions were met: efficient exchange of intermediates, similar growth rates between subpopulations, and balanced initial inoculation ratios. For P. putida, two strains were engineered—one producing limonene and the other converting it to perillyl alcohol. These were optimised for D- and L-enantiomers of both compounds, and cocultures consistently outperformed monocultures. Finally, a control system was proposed for two-species microbial communities, decoupling growth and ratiometric control. Two quorum-sensing systems were characterised for communication, and three bacteriocins were evaluated for growth regulation, culminating in an integrated control circuit design.
In this thesis, the potential of division of labour for the bioproduction of high-value compounds using synthetic bacterial communities was explored. Two coculture systems were constructed: an Escherichia coli coculture producing violacein and a Pseudomonas putida coculture producing limonene and perillyl alcohol, with subpopulations expressing different segments of their respective metabolic pathways. For violacein production, several pathway-splitting strategies were tested by partitioning the pathway at different intermediates, and culture conditions were optimised to maximise production. Coculture performance exceeded that of monocultures when three conditions were met: efficient exchange of intermediates, similar growth rates between subpopulations, and balanced initial inoculation ratios. For P. putida, two strains were engineered—one producing limonene and the other converting it to perillyl alcohol. These were optimised for D- and L-enantiomers of both compounds, and cocultures consistently outperformed monocultures. Finally, a control system was proposed for two-species microbial communities, decoupling growth and ratiometric control. Two quorum-sensing systems were characterised for communication, and three bacteriocins were evaluated for growth regulation, culminating in an integrated control circuit design.
Version
Open Access
Date Issued
2025-05-19
Date Awarded
01/11/2025
License URL
Advisor
Ledesma-Amaro, Rodrigo
Jimenez, Jose
Stan, Guy-Bart
Publisher Department
Department of Bioengineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)