Functional plasticity of HCO3- uptake and CO2 fixation in Cupriavidus necator H16

Uptake and fixation of CO2 are central to strategies for CO2-based biomanufacturing. Cupriavidus necator H16 has emerged as a promising industrial host for this purpose. Despite its prominence, the ability to engineer C. necator inorganic carbon uptake and fixation is underexplored. Here, we test the role of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of one of the four carbonic anhydrases in C. necator, {beta}-carbonic anhydrase can, had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in saturating CO2 in batch culture. Several heterologous strains with Halothiobacillus neopolitanus DAB2 (hnDAB2) expressed from the chromosome in combination with diverse rubisco homologs grew in CO2 equally or better than the wild-type strain. Our experiments suggest that the primary role of Can carbonic anhydrase during autotrophic growth is for bicarbonate accumulation to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3- uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/593039v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@83c601org.highwire.dtl.DTLVardef@13ca34forg.highwire.dtl.DTLVardef@5b7f8org.highwire.dtl.DTLVardef@1710e48_HPS_FORMAT_FIGEXP M_FIG C_FIG