Gude, Christian
(2021)
Metabolic engineering of Cupriavidus necator H16 for the production of C5 platform chemicals.
PhD thesis, University of Nottingham.
Abstract
Raising demand for plastics, especially in developing countries, poses a challenge for suppliers to satisfy the need in a sustainable way. Developing carbon-neutral C5 platform chemicals for the production of easily recyclable biopolymers is an integral part of this mission.
In this work, Cupriavidus necator H16 was metabolic engineered to produce the C5 precursors 5-aminovaleric acid and 5-hydroxyvaleric acid for plastic production from industrial waste gases. C. necator H16 was selected as host organism for metabolic engineering because of its ability to grow lithoautotrophically, using CO2 waste gas as carbon source, ammonia as nitrogen source and hydrogen as energy source.
The novel pathway designs for these C5 platform chemicals were first tested in background hosts such as the C. necator H16 wildtype and engineered deletion mutant strains. Once all pathways were designed, tested and chassis strains engineered, optimisations were made. Synthetic biology tools such as the use of synthetic ribosomal binding sites, use of the promoters Ptrp and Ptrc and feedback-resistant enzyme mutants were introduced to the pathways
The production of 5-hydroxyvaleric acid (5-OHV), a precursor for biodegradable polyesters was investigated, using a novel reverse beta-oxidation chain elongation pathway from β-alanine. An engineered C. necator H16 ∆phaCAB ∆mmsA1 ∆mmsA2 ∆mmsA3 strain expressing the genes ydfGEC, bapatCV, prpEEC, bktBCN, phaBCN, crt3CN and terTD produced 5-OHV with a yield of 17.1 mg L-1 from 20 mM β-alanine.
The production of 5-aminovaleric acid (5-AV) via L-lysine was investigated. 5-AV is a precursor for the industrial production of the polyamide nylon-5. Feedback-resistant variants of the lysine biosynthesis pathway enzymes aspartate kinase (LysC) and DHDPS (DapA) were introduced. Furthermore, a by-pass pathway using DAPDH (Ddh) enhanced L-lysine biosynthesis. The enzymes DavB and DavA, encoding a lysine monooxygenase and a δ-aminovaleramidase, completed the pathway from L-lysine to 5-AV. Lastly, disruption of the gene gabT, encoding a GABA aminotransferase with activity towards 5-AV, eliminated 5-AV degradation. As a result of the above modifications, this work showed the first to date production of 5-AV from C. necator H16 central metabolism with a yield of 10.5 mg L-1 from 10 g L-1 mM sodium gluconate.
Furthermore, a novel transcription-factor based biosensor design with fluorescent output for the detection of 5-AV and quantities >50 mM was investigated. This work also demonstrates a second biosensor design with the ability to detect δ-valerolactam in a graded manner for quantities >2 mM.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Kovacs, Katalin
Minton, Nigel
Conradie, Alex |
| Keywords: |
Biopolymers; Platform chemicals; Metabolic engineering; Cupriavidus necator H16 |
| Subjects: |
T Technology > TP Chemical technology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
65718 |
| Depositing User: |
Gude, Christian
|
| Date Deposited: |
10 Jul 2023 09:53 |
| Last Modified: |
10 Jul 2023 09:53 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/65718 |
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