Surface Display of Small Peptides on Escherichia coli for Enhanced Calcite Precipitation Rates

Mineralization has emerged as a promising strategy for long-term carbon sequestration. These processes involve carbon dioxide hydration followed by mineral precipitation. We have explored the production of whole-cell biocatalysts engineered with carbonic anhydrase (CA) activity to accelerate the CO₂ hydration reaction. In this study, short polypeptides were displayed on the surface of E. coli cells and whole-cell biocatalysts containing periplasmically expressed CAs in an attempt to enhance calcite mineral formation. It was found that cells coexpressing recombinant periplasmic CA and surface-displayed GPA peptide (PEVPEGAFDTAI) outperformed other peptide-expressing biocatalysts evaluated in terms of the amount of precipitate formed, as well as the overall formation rate of solids. Cells expressing the Cab CA isoform (BLR-pCab) and Cam isoform (BLR-pCam) with the surface-displayed GPA peptide exhibited 36 and 59% improvements in precipitation amounts, as well as 18 and 60% improvements in overall formation rates, respectively, over similar biocatalysts without GPA expression. The biocatalyst with the best performance was BLR-pCam/GPA, which generated 0.15 g of CaCO₃, while BLR cells generated only 0.08 g of CaCO₃ under the same small batch reaction conditions. The BLR-pCam/GPA cells also exhibited the fastest formation rates, achieving the maximum change in solution turbidity after only 2.2 min, as opposed to 6.3 min for BLR cells. These results demonstrate that synthetic biology approaches can be used to create novel biocatalysts with the ability to enhance both catalysis and precipitation activities.