Periplasmic carbonic anhydrase CAH1 contributes to high inorganic carbon affinity inChlamydomonas reinhardtii

Carbonic anhydrase (CA), an enzyme conserved across species, is pivotal in the interconversion of inorganic carbon (Ci; CO 2 and HCO 3 − ). Compared to the well-studied intracellular CA, the specific role of extracellular CA in photosynthetic organisms is still not well understood. In the green alga Chlamydomonas reinhardtii , CAH1, located at the periplasmic space, is strongly induced under CO 2 -limiting conditions by the Myb transcription factor LCR1. While it has been observed that the lcr1 mutant shows decreased Ci-affinity, the detailed mechanisms behind this phenomenon are yet to be elucidated. In this study, we aimed to unravel the LCR1-dependent genes essential for maintaining high Ci-affinity. To achieve this, we identified a total of 12 LCR1-dependent inducible genes under CO 2 -limiting conditions, focusing specifically on the most prominent ones - CAH1 , LCI1 , LCI6 , and Cre10.g426800 . We then created mutants of these genes using the CRISPR-Cas9 system, all from the same parental strain, and compared their Ci-affinity. Contrary to earlier findings (Van and Spalding, 1999) that reported no reduction in Ci-affinity in the cah1 mutant, our newly created cah1 -1 mutant exhibited a significant decrease in Ci-affinity under high HCO 3 − /CO 2 -ratio conditions. Additionally, when we treated wild-type cells with a CA inhibitor with low membrane permeability, a similar reduction in Ci-affinity was observed. Moreover, the addition of exogenous CA to the cah1 mutant restored the decreased Ci-affinity. These results, highlighting the crucial function of the periplasmic CAH1 in maintaining high Ci-affinity in Chlamydomonas cells, provide new insights into the functions of periplasmic CA in algal carbon assimilation. One-sentence summary CAH1, a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii , plays a crucial role in maintaining a high affinity for inorganic carbon, particularly under CO 2 -limiting conditions.