Optimization of Cupriavidus necator growth for carbon dioxide bioconversion in polyhydroxybutyrate through genetic engineering and application of nutritional stress

The escalating levels of carbon dioxide emissions into the atmosphere represent one of the foremost concerns of our era. A promising solution for mitigating this issue lies in the bioconversion of carbon dioxide into sustainable materials. Biopolymers, exemplified by polyhydroxyalkanoates, can be synthesized through bioprocesses involving microorganisms. These biopolymers, characterized by their biocompatibility and biodegradability, offer a potential resolution to the pervasive problem of plastic pollution. Among the polyhydroxyalkanoates, polyhydroxybutyrate (PHB) stands out as a well-known member with the capacity to substitute petroleum-based plastics. Microorganisms, such as Cupriavidus necator, play a crucial role in the production of these biopolymers, concurrently contributing to Carbon Capture, Utilization, and Storage through carbon dioxide fixation. The primary objective of this study was to augment polyhydroxybutyrate production and carbon fixation in C. necator through a dual approach involving genetic engineering and the application of nutritional stresses, including nitrogen and phosphate starvation, along with oxygen limitation. Employing molecular biology techniques such as transformation and cloning, engineered strains were developed to overexpress specific genes associated with autotrophic metabolism, aiming to enhance carbon dioxide uptake rates. Three mutants and a plasmid control were constructed, with one overexpressing a Calvin cycle enzyme displaying reduced growth on elevated levels of carbon dioxide. Instead, nutritional stresses did not significantly impact C. necator growth and gas consumption but elicited an augmented production of polyhydroxybutyrate. Notably, nitrogen starvation resulted in a total PHB accumulation of 55% on dry cell weight. Overall, these findings underscore the effectiveness of nutrient stresses in enhancing polyhydroxybutyrate content in C. necator. However, the ambitious goal of elevating the carbon dioxide uptake rate necessitates further engineering endeavors and metabolic analyses as promising avenues for exploration.