Rewiring carbonic anhydrase isoforms for adaptive carbon dioxide capture in Chlamydomonas reinhardtii CC400

Background: Rising CO₂ emissions from fossil fuel combustion are a major driver of climate change, necessitating more sustainable mitigation strategies to reduce atmospheric carbon release. Microalgae offer a viable solution for carbon capture, storage, and utilization (CCUS) due to their photosynthetic ability, efficiently converting CO₂ into biomass. Methods: The carbon capture capability of Chlamydomonas reinhardtii CC400 was engineered by overexpressing carbonic anhydrase (CA) genes from endogenous mitochondrial CAH4 and a transgenic Sulfurihydrogenibium yellowstonense CA (CHJS). Enzymatic and transcriptional analyses were conducted to investigate the role of genetic CA in carbon regulation and rearrangement. The effects of CAH4 and CHJS were evaluated through biomass, protein, starch, and lipid production, along with CO₂ capture efficiency across varying levels to assess strain-specific carbon assimilation trends. Significant findings: Overexpressing intrinsic and extrinsic CAs effectively minimized Rubisco maintenance while promoting biomass accumulation. Notably, both genetic strains displayed distinct metabolic preferences, with starch production reaching 0.64 g/L under mixotrophic conditions in TAP medium. Protein accumulation peaked at 0.38 g/L under autotrophic cultivation in mBG11 medium, highlighting their efficiency in condition-dependent carbon utilization. The modified strains outperformed wild-type CC400 in carbon uptake, with CAH4 achieving a 50 % efficiency increase under direct air capture conditions (0.04 % CO₂). In contrast, CHJS exhibited a moderate enhancement of 10 % and 15 % at CO₂ levels of 1 % and 2 %, respectively. These findings underscore the diverse potential of distinct CAs in modulating carbon flux, optimizing metabolic pathways, and improving carbon fixation under dynamic CO₂ conditions.