McGregor, Callum
(2021)
Production of 3-hydroxypropionate-containing polymers by engineered C necator H16.
PhD thesis, University of Nottingham.
Abstract
Alternative materials are needed to replace traditional plastics, since their production and recalcitrance in nature have negative effects on the environment. Polyhydroxyalkanoates (PHAs) are bacterially synthesised polymers which are potential alternatives to plastic on account of their availability from renewable sources, plastic-like properties and biodegradability.
Cupriavidus necator H16 is a model organism for PHA production and is also capable of using CO2 as a carbon source. Therefore, an opportunity exists for producing biodegradable plastics and other valuable chemicals from CO2 using engineered strains of C. necator H16. However, the PHA which is produced by C. necator H16, polyhydroxybutyrate (PHB), does not possess properties which are ideal for the manufacture of plastic products.
PHA copolymers, which consist of more than one type of monomer, can often exhibit better properties than PHA homopolymers, such as PHB. Therefore, in this study, the primary goal was to produce the PHA copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate (poly(3HB-co3HP) using engineered C. necator H16. In comparison to PHB, poly(3HBco-3HP) is less crystalline, has a lower melting temperature, and a lower glass transition temperature than PHB, which significantly improves its processability. Through plasmid engineering, medium alteration, and suppression of 3HB-CoA formation, it was possible to produce poly(3HBco-3HP) containing a 3HP molar fraction of between 0 and 91 mol% 3HP using C. necator H16. Previously, the highest 3HP molar fraction reported in a poly(3HB-co-3HP) copolymer produced by an engineered strain of C. necator H16 was 1 mol%.
While PHAs are the most studied biodegradable polymer, a more recent development in the field of biobased materials is the production of polymers containing β-methyl-Δ-valerolactone (βMΔVL). βMΔVL is derived from mevalonate, and can be used in the synthesis of homopolymers or reacted with other compounds to produce copolymers. Mevalonate can be synthesised by engineered C. necator H16, and other mevalonate-derived compounds have been produced from C. necator H16 from CO2. Therefore an opportunity to produce novel biodegradable polymer precursors from CO2 arises. To attempt to stabilize mevalonate biosynthesis in a bioreactor, a plasmid addiction system was also tested. Using a metabolism-based plasmid addiction system it was possible to co-produce approximately 14 g/L mevalonate and over 10 g/L PHB from CO2 in autotrophic batch fermentation. However, during autotrophic continuous fermentation the plasmid addiction system did not support stable mevalonate production.
The results presented in this thesis indicate several directions for future research to continue developing a poly(3HB-co-3HP)-producing strain of C. necator H16. It was also found that C. necator H16 could be engineered to produce significant quantities of mevalonate from CO2, although further work is required to implement a stable plasmid for use in high-density autotrophic cultivation of this organism.
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