Engineering the genetic components of a whole‐cell catalyst for improved enzymatic CO₂ capture and utilization

Abstract

Carbonic anhydrase (CA) is a diffusion‐limited enzyme that rapidly catalyzes the hydration of carbon dioxide (CO2). CA has been proposed as an eco‐friendly yet powerful catalyst for CO2 capture and utilization. A bacterial whole‐cell biocatalyst equipped with periplasmic CA provides an option for a cost‐effective CO2‐capturing system. However, further utilization of the previously constructed periplasmic system has been limited by its relatively low activity and stability. Herein, we engineered three genetic components of the periplasmic system for the construction of a highly efficient whole‐cell catalyst: a CA‐coding gene, a signal sequence, and a ribosome‐binding site (RBS). A stable and halotolerant CA (hmCA) from the marine bacterium Hydrogenovibrio marinus was employed to improve both the activity and stability of the system. The improved secretion and folding of hmCA and increased membrane permeability were achieved by translocation via the Sec‐dependent pathway. The engineering of RBS strength further enhanced whole‐cell activity by improving both the secretion and folding of hmCA. The newly engineered biocatalyst displayed 5.7‐fold higher activity and 780‐fold higher stability at 60°C compared with those of the previously constructed periplasmic system, providing new opportunities for applications in CO2 capture and utilization.

An Intrinsically Disordered Peptide Tag that Confers an Unusual Solubility to Aggregation-Prone Proteins

“…CA activity was measured by a colorimetric CO 2 hydration assay (38, 39). The purified enzyme in 20 mM phosphate buffer (pH 7.5) was diluted to a concentration of 1 μM.…”

Characterization and High-Level Periplasmic Expression of Thermostable α-Carbonic Anhydrase from Thermosulfurimonas Dismutans in Escherichia Coli for CO2 Capture and Utilization

“…The variant tdCA S82Y did not show any difference in both activity and stability compared to the wild-type tdCA (Figure 5b), implying that the mutation S82Y is neutral for the characteristics of tdCA. Next, we tried to express and translocate tdCA into the periplasm of E. coli via the Sec-dependent pathway, which is a better pathway than the Twin-arginine translocation pathway for CA secretion [33], by genetically fusing PelB signal peptide (SP PelB ) to the N terminus of tdCA. This design was originally intended to construct a whole-cell biocatalyst with periplasmic tdCA (see below).…”

“…The whole-cell catalyst with periplasmic taCA was not considered for further testing because the expression level of periplasmic taCA was too low to be compared with that of periplasmic tdCA (data not shown). Instead, the highly active periplasmic whole-cell biocatalyst (RBS 2 SP PelB ::hmCA) previously constructed by using a halophilic CA (hmCA) from Hydrogenovibrio marinus and by engineering the ribosome binding site (RBS) was used for the comparison [33]. The strains with periplasmic tdCA showed much higher whole-cell activities than that of the RBS 2 SP PelB ::hmCA strain ( Figure 6).…”

“…The time (t 0 ) for the uncatalyzed reaction was also measured by adding a corresponding blank buffer instead of an enzyme sample. The enzyme unit (U) was calculated, as (t 0 − t) / (t × 5) as previously described [33].…”

“…The whole-cell activity was measured as previously described [33]. After cultivation, cells were harvested and resuspended in phosphate-buffered saline (PBS; 8 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na 2 HPO 4 , and 0.24 g/l KH 2 PO 4 ) at a cell concentration of 8-14 OD 600 .…”