Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible materials with significant potential for biotechnological applications. The terpolymer P(3HB-co-3HV-co-4HB) is of interest due to its unique mechanical properties, cell adhesion, and conductivity attributes. While its potential applications, particularly in medical and pharmaceutical arenas, are vast, one of the main challenges in the field has been optimizing its production for increased 4HB content to over 10% for medical application. To address this, an integrated study encompassing thermodynamics and kinetics was conducted. Through these investigations molecular models were developed that elucidate the initiation mechanisms of PHA formation, which have been challenging to detect using traditional physical tracking techniques. Key findings revealed the pronounced reactivity of the 4HB monomer compared to its counterparts such as 3HB and 3HV. Utilizing this knowledge, a feeding strategy was devised, employing carbon sources like glucose, γ- butyrolactone, and propionic acid in an order reflecting monomer reactivity. When implemented in Cupriavidus necator, this strategy resulted in an accumulation of 4HB in the terpolymer, surpassing 50%. Building on the promise of next-generation biotechnology, the archaeon Haloferax mediterranei offers several advantages. A genetically modified strain, Haloferax mediterranei_zxr1.0, was developed to express a 4-hydroxybutyrate-CoA transferase from C.necator N-1, aiming to augment the 4HB content in the synthesized terpolymer. Systematic investigations into wood-derived sugars led to the identification of specific constituents that favorably influenced both PHA accumulation and cellular proliferation. Remarkably, H.mediterranei_zxr1.0 achieved a terpolymer concentration of 0.42 g/L in bioreactor batch fermentations, with 4HB and 3HV constituting 25% and 9%, respectively. Furthermore, in a quest to explore alternative genetic pathways, three H.mediterranei strains, each incorporating a distinct 4HB-CoA transferase/ligase pathway, were examined. Among these, the Hfx_nmar strain displayed optimal growth patterns and terpolymer yields, achieving 0.5 g/L in bioreactor fermentations and a 4HB fraction of up to 40%. Extended experimentation using fed-batch fermentation methods with Hfx_nmar yielded a PHA concentration of 0.7 g/L, while retaining a 4HB fraction of over 50%. This body of research provides a comprehensive roadmap for PHA production, intergrating molecular modeling, genetic engineering, and fermentation techniques. The insights offer ways for the sustainable production of specialized PHAs, paving the way for reduced reliance on petroleum-based materials.
Strategies for bespoke Polyhydroxyalkanoates (PHA) production
Zheng, X. (Author). 1 Aug 2024
Student thesis: Phd