Functional plasticity of HCO3-uptake and CO2fixation inCupriavidus necatorH16

Abstract

AbstractUptake and fixation of CO2are central to strategies for CO2-based biomanufacturing.Cupriavidus necatorH16 has emerged as a promising industrial host for this purpose. Despite its prominence, the ability to engineerC. necatorinorganic carbon uptake and fixation is underexplored. Here, we test the role of endogenous and heterologous genes onC. necatorinorganic carbon metabolism. Deletion of one of the four carbonic anhydrases inC. necator, β-carbonic anhydrasecan, had the most deleterious effect onC. necatorautotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters fromCyanobacteriaand chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT)C. necatorin saturating CO2in batch culture. Several heterologous strains withHalothiobacillus neopolitanusDAB2 (hnDAB2) expressed from the chromosome in combination with diverse rubisco homologs grew in CO2equally 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 CO2fixation inC. necator, providing new pathways for CO2-based biomanufacturing.Graphical abstract