Synthetic Genetic Elements Enable Rapid Characterization of Inorganic Carbon Uptake Systems in Cupriavidus necator H16

Cupriavidus necator H16
is a facultative
chemolithotroph capable of using CO2 as a carbon source,
making it a promising organism for carbon-negative biomanufacturing
of petroleum-based product alternatives. In contrast to model microbes,
genetic engineering technologies are limited in C.
necator, constraining its utility in basic and applied
research. Here, we developed a genome engineering technology to efficiently
mobilize, integrate, and express synthetic genetic elements (SGEs)
in C. necator. We tested the chromosomal
expression of four inducible promoters to optimize an engineered genetic
landing pad for tunable gene expression. To demonstrate utility, we
employed the SGE system to design, mobilize, and express eight heterologous
inorganic carbon uptake pathways in C. necator. We demonstrated all inorganic carbon uptake systems’ upregulated
intracellular bicarbonate concentrations under heterotrophic conditions.
This work establishes the utility of the SGE strategy for expedited
integration and tunable expression of heterologous pathways, and enhances
intracellular bicarbonate concentrations in C. necator.