Metabolic Engineering of and Process Optimisation with Cupriavidus necator H16 for Polyhydroxybutyrate and Lactic Acid Production from CO2-Derived Formic Acid

Climate change, driven by greenhouse gas emissions, and the increasing demand for sustainably produced goods and materials are two of the greatest current global challenges. Both issues are intrinsically linked since conventional production processes and reliance on fossil fuel-derived materials are key drivers of the human-accelerated impacts of climate change. Thus, it has become imperative to find solutions to solve not only both issues individually but ones that can tackle them simultaneously. In this respect, the concept of the circular bioeconomy in which materials are bio-based and fully recyclable is becoming an important framework in policies, industries and research. One aspect of this concept is the utilisation of greenhouse gases as a feedstock to produce sustainable materials and thereby create recycling loops. Autotrophic (micro-) organisms can be ideal catalysts to tap into one-carbon (C1) gasses like CO2 as a resource to create biomass, small molecules or even bio-based polymers. One such organism is the bacterium Cupriavidus necator (C. necator), which, in contrast to phototrophic organisms such as plants or microalgae, uses molecular hydrogen (H2) as an energy source to capture CO2. The organism is well known for its natural ability to store carbon in the biodegradable polymer poly[R-(–)-3-hydroxybutyrate] (PHB) but has also been engineered to produce a multitude of other products of interest. While the bacterium can use CO2 and H2 directly as the respective carbon and energy source, working with these gasses can be technologically challenging. An alternative can be found in formic acid, a C1 molecule that can be synthesised directly from the chemical reaction of CO2 and H2.
In this work, formic acid was used to improve carbon fixation in the strain C. necator H16 through adaptive laboratory evolution, leading to a strain (C. necator ALE26) with an almost two-times higher maximum growth rate on the substrate than the wildtype strain. The improved strain was then employed in process optimisation to accumulate PHB in a continuous bioprocess with formate derived from atmospheric CO2 as the sole carbon source with productivities of up to 8.67 ± 1.14 mg/L/h. Finally, as an additional product of interest, lactate production was attempted with the organism. While minor amounts of the desired molecule could be generated in a biotransformation with resting cells that overexpressed heterologous lactate dehydrogenases, genetic engineering for a reliably lactate-producing strain was unsuccessful in this work.