A Novel Strategy for Highly Efficient Heterologous Expression of Carbonic Anhydrase in Yarrowia lipolytica

Carbonic anhydrases (CAs) efficiently catalyze CO₂ reversible hydration, critical for carbon capture and sequestration (CCS), but naturally low yield limits industrial use. Yarrowia lipolytica, an unconventional yeast, is an ideal heterologous expression host with robust adaptability, post-translational modification capacity, and versatile genetic tools. In this study, 10 α-, β-, and γ-class CAs were successfully expressed in Y. lipolytica, and two top-performing candidates were identified: Methanosarcina mazei γ-CA (MmaCA) and Sulfurihydrogenibium azorense α-CA (SazCA). Their production was further optimized via promoter and gene dosage adjustment, cultural condition optimization and auxiliary protein co-expression. The optimized intracellular MmaCA activity reached 960 U/mL (64.42-fold improvement), and the extracellular SazCA activity peaked at 925 U/mL (70.08-fold enhancement). CO₂ mineralization experiments confirmed both recombinant CAs significantly accelerated CaCO₃ precipitation, demonstrating a promising CCS application potential. To our knowledge, this is the first systematic investigation of CA heterologously expressed in Y. lipolytica, providing a novel strategy for the highly efficient production of CAs to enable their application in industry.