Functional Reconstitution of the Bacterial CO2 Concentrating Mechanism

Bacterial autotrophs often rely on biophysical CO₂ concentrating mechanisms (CCMs) to assimilate carbon. It is postulated that principles - and perhaps even components – of these CCMs could be used to improve CO₂ assimilation in plants. Surprisingly, a systematic ‘parts list’ of the CCM remains unknown. To this end, we have carried out a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the É£-proteobacterium H. neapolitanus. Screening revealed that the CCM comprises at least 17 and likely no more than 25 genes, most of which are encoded in 3 operons. Found within these genes is a new class of C_i­ pump which we term DAB, for ‘DABs accumulate bicarbonate,’ that is widespread amongst prokaryotes and readily expressed in a heterologous fashion. Informed by this information, we have also performed a functional reconstitution of the bacterial CCM in vivo. Using a novel strain of Rubisco-dependent E. coli, we have assessed the importance of known and poorly characterized activities of the CCM for achieving efficient CO₂ assimilation and, ultimately, have engineered a strain which is capable of growth due to efficient CO₂ fixation directly from ambient air.