Directed evolution of Mesorhizobium loti carbonic anhydrase for carbon dioxide sequestration by MutaT7 and rational codon design

Background: Directed evolution plays a crucial role in accelerating genetic development to obtain superior enzyme variants with high tolerance and activity. Under nature evolution, Mesorhizobium loti carbonic anhydrase (MlCA) stands out as one of the fastest metalloenzymes involved in carbon dioxide capture and sequestration (CCS). However, challenge arises when expressing the recombinant MlCA which affects its total catalytic function. Therefore, we aim to evolve MlCA by enhancing protein production and elevate catalytic efficiency in genetically Escherichia coli. Methods: A growth-dependent CRISPRi-based high-throughput screening platform DEPEND-2.0 with down-regulating essential gene was established. The in vivo target-specific MutaT7 mutator was employed to evolve MlCA and screened the mutants using DEPEND-2.0 based on cell growth. Structure prediction was further applied to analyze the mutant while the degenerated or definitive codons were designed to investigate the codon influence on the mRNA and enzyme production. Significant finding: The DEPEND-2.0 system, utilizing chromosomal dCas9, effectively inhibited cell growth by targeting the promoter region with sgRNA which maintained a repression rate of 65.4% at 4 h using high-copy RSF plasmid. Structural prediction using Alphafold revealed that a mutation introduced by MutaT7 at P105S resulted in a 99% loss of intrinsic activity due to the disruption of hydrogen bonding. Furthermore, synonymous codon designs demonstrated that AT-rich codons at the 5’ terminus of a 30-bp sequence exhibited higher mRNA levels and improved recombinant protein expression. Finally, the MlCA variant with A-rich codons (i.e., MA) effectively facilitated CO₂ biomineralization, displaying high durability with 72% residual activity after five repeated reactions, ultimately accumulating 193 mg of CaCO₃.