Engineering Cupriavidus necator for butanediol production
Publication Date
July 18, 2023
Creator
Abstract
The increase in atmospheric greenhouse gases such as carbon dioxide (CO2) is a major factor contributing to global warming. Harnessing CO2-fixing microorganisms to produce platform chemicals, typically synthesised from petrochemicals, can simultaneously reduce greenhouse gas emissions and dependence on non-renewable sources. In this work, the conversion of CO2 to the platform chemical butanediol (BDO) facilitated by the chemolithoautotrophic bacterium Cupriavidus necator H16 has been investigated.
First, C. necator H16 was evaluated for potential deployment as a chassis to produce (R)-1,3-BDO and other BDOs, through consumption and toxicity assays. To aid the engineering of C. necator H16, two inducible gene expression systems, TF/PyhjX from Escherichia coli and AcoR/PacoA system from Acetobacterium woodii were used to develop genetically encoded biosensors. These biosensors were then evaluated for potential use in BDO detection and monitoring.
Next, C. necator H16 was metabolically engineered to produce the optically active (R)- 1,3-BDO. Bioproduction of (R)-1,3-BDO was achieved using two synthetic metabolic pathways consisting of a butanal dehydrogenase, pyruvate decarboxylase, deoxyribose- 5-phosphate aldolase and an aldehyde reductase. Carbon flux towards (R)-1,3-BDO was significantly increased through deletion of the P3HB synthase encoded by phaC1 and the succinyl-CoA synthetase encoded by sucCD. To increase genetic stability, genes encoding (R)-1,3-BDO biosynthesis pathways were integrated into the C. necator H16 genome. Chromosomally integrated strains were then cultivated heterotrophically and autotrophically for bioproduction of (R)-1,3-BDO. This is the first report of production of (R)-1,3-BDO using CO2 as the carbon source.
Finally, C. necator H16 energy metabolism was investigated to ascertain NADPH regeneration mechanism during BDO production from CO2. To elucidate the role of four putative transhydrogenase’s (PntAB1234) and their involvement in NADPH regeneration, a ∆pntAB1234 strain was generated and characterised. A meso-2,3-BDO and (R, R)-2,3-BDO biosynthetic pathways were employed to evaluate transhydrogenase deletion strain ability regenerating NADH and NADPH, respectively. This research shows that through the stepwise engineering, C. necator H16 can be developed as a chassis for platform chemical production demonstrating its potential for biosynthesis of (R)-1,3-BDO from CO2.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
72250
UoN Repository URI
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Gascoyne, JL-Engineering Cupriavidus necator for Butanediol Production_CorrectionsFinal.pdf
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