Optimization of promoter screening for heterologous expression of carbonic anhydrase and characterization of its enzymatic properties and carbon sequestration performance.
- Author:
Dandan YAO
1
;
Yunhui LI
1
;
Xingjia FU
1
;
Hui WANG
1
;
Yun LIU
1
Author Information
- Publication Type:Journal Article
- Keywords: carbon capture and storage; carbonic anhydrase; catalytic mechanism; heterologous expression; high-throughput promoter screening
- MeSH: Carbonic Anhydrases/biosynthesis*; Promoter Regions, Genetic/genetics*; Escherichia coli/metabolism*; Carbon Sequestration; Carbon Dioxide/metabolism*; Green Fluorescent Proteins/metabolism*
- From: Chinese Journal of Biotechnology 2025;41(4):1588-1604
- CountryChina
- Language:Chinese
- Abstract: In this study, high-throughput promoter screening was employed to optimize the heterologous expression of Mesorhizobium loti carbonic anhydrase (MlCA) in order to reduce the costs associated with carbon capture and storage (CCS). To simplify the complexity of traditional vectors, a fusion protein expression system was constructed using superfolder green fluorescent protein (sfGFP) and MlCA. The synthetic promoter library in Escherichia coli was utilized for efficient one-step screening. Based on fluorescence intensity on agar plates, a total of 143 monoclonal colonies were identified, forming a library with varying expression levels. The top four recombinants with the highest fluorescence intensity were selected, among which MlCA driven by the promoter 342042/+ exhibited the highest enzymatic activity, with a specific activity of the 34.6 Wilbur-Anderson units (WAU)/mg. Optimization experiments revealed that MlCA exhibited the best performance when cultured for 4 days under pH 7.0 and 40 ℃ conditions. The Michaelis constant (Km·hdy) and maximum reaction rate (Vmax·hdy) for CO2 hydration were determined to be 62.46 mmol/L and 0.164 mmol/(s·L), respectively. For esterase hydrolysis, MlCA showed the Km and Vmax of 639.8 mmol/L and 0.035 mmol/(s·L), respectively. MlCA accelerated the CO2 hydration process, promoting CO2 mineralized into CaCO3 within 9 min at low pH and room temperature conditions. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses confirmed that the precipitated product was calcite. This study provides a low-cost and environmentally friendly alternative for future CCS applications.
