Heterologous Expression and Functional Verification of Extracellular Carbonic Anhydrases in Bacillus safensis yw6 from Mariana Trench.

The exploration and exploitation of deep-sea microbial resources is of great scientific value for understanding biological evolution under extreme conditions. Deep-sea microorganisms are critical in the ocean carbon cycle, and marine heterotrophic microorganisms secrete extracellular carbonic anhydrase (CA) to fix inorganic carbon, an important process in climate regulation. Extracellular CA provides a green method for fixing carbon dioxide into stable minerals containing Ca2+. However, studies on extracellular CA in deep-sea microorganisms are limited. In this study, Bacillus safensis yw6 was isolated from Mariana Trench sediments and three candidate extracellular CA genes (β-ca1, β-ca2, and γ-ca) were identified by whole genome sequencing. Bioinformatics analyses showed that these CAs have different structural compositions, with the β-CA having α-helix and random coiling, whereas the γ-CA has more random coiling and stretched strands. Heterologous expression in E. coli BL21 (DE3) showed that β-CA2 had the highest enzyme activity, followed by γ-CA and β-CA1. Field emission scanning electron microscopy (FESEM) observations showed that the engineered strains with β-ca2 genes produced deposits that were like those from natural sources. This finding not only provides new perspectives for the utilization of deep-sea microbial resources, but also provides an important scientific basis for the molecular mechanisms of extracellular CAs of deep-sea microbes.