V. natriegens genome-scale metabolic model development and its application for the production of polyhydroxybutyrate
File(s)
Author(s)
Coppens, Lucas
Type
Thesis
Abstract
The widespread adoption of microbes for the bioproduction of commodity chemicals as a sustainable alternative to petrochemical synthesis is hindered by low productivities.
Vibrio natriegens, the fastest growing organism known, is gaining interest to address these challenges through its potential as a high-volumetric productivity platform. This thesis aims to further establish V. natriegens’s position as a burgeoning biomanufac- turing host through the development of systems and synthetic biology tools and their application for the production of biopolymer polyhydroxybutyrate (PHB).
This thesis starts with the development of the first genome-scale metabolic model for V. natriegens, iLC858, which is validated by comparing experimental data to model
predictions. iLC858 is subsequently used for a comparison to E. coli metabolism. An analysis of the V. natriegens respiratory system leads to the identification of a sodium-dependent oxaloacetate decarboxylase as a central player in V. natriegens’s metabolism. Finally, iLC858 is used for the development of a resource balance analysis model, allowing to study metabolic resource tradeoffs.
The second part of this thesis explores the application of the iLC858 model for metabolic engineering of V. natriegens to improve PHB production. A novel two-stage bioproduction promoter is identified in V. natriegens to drive expression of the PHB operon. Subsequently, a combinatorial library of 16 metabolic engineering targets suggested by iLC858 is subjected to fluorescence-activated cell sorting (FACS) screening, and a strain is identified that shows improved PHB production owing to knockouts of citrate synthase and phosphate acetyltransferase.
This thesis provides systems biology models that can be used for further studies into the biology of V. natriegens as well as to produce metabolic engineering strategies for strain design. The combination of systems biology with high-throughput combinatorial engineering methods such as MuGENT and FACS further paves the way for the accelerated development of V. natriegens as a biomanufacturing platform.
Vibrio natriegens, the fastest growing organism known, is gaining interest to address these challenges through its potential as a high-volumetric productivity platform. This thesis aims to further establish V. natriegens’s position as a burgeoning biomanufac- turing host through the development of systems and synthetic biology tools and their application for the production of biopolymer polyhydroxybutyrate (PHB).
This thesis starts with the development of the first genome-scale metabolic model for V. natriegens, iLC858, which is validated by comparing experimental data to model
predictions. iLC858 is subsequently used for a comparison to E. coli metabolism. An analysis of the V. natriegens respiratory system leads to the identification of a sodium-dependent oxaloacetate decarboxylase as a central player in V. natriegens’s metabolism. Finally, iLC858 is used for the development of a resource balance analysis model, allowing to study metabolic resource tradeoffs.
The second part of this thesis explores the application of the iLC858 model for metabolic engineering of V. natriegens to improve PHB production. A novel two-stage bioproduction promoter is identified in V. natriegens to drive expression of the PHB operon. Subsequently, a combinatorial library of 16 metabolic engineering targets suggested by iLC858 is subjected to fluorescence-activated cell sorting (FACS) screening, and a strain is identified that shows improved PHB production owing to knockouts of citrate synthase and phosphate acetyltransferase.
This thesis provides systems biology models that can be used for further studies into the biology of V. natriegens as well as to produce metabolic engineering strategies for strain design. The combination of systems biology with high-throughput combinatorial engineering methods such as MuGENT and FACS further paves the way for the accelerated development of V. natriegens as a biomanufacturing platform.
Version
Open Access
Date Issued
2024-11-28
Date Awarded
2025-07-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
Ledesma-Amaro, Rodrigo
Publisher Department
Department of Bioengineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)