启动子筛选优化碳酸酐酶异源表达及其酶学性质与固碳性能

Optimization of promoter screening for heterologous expression of carbonic anhydrase and characterization of its enzymatic properties and carbon sequestration performance

摘要:

本研究通过高通量启动子筛选，优化了百脉根生根瘤菌碳酸酐酶(Mesorhizobium loti carbonic anhydrase,MlCA)的异源表达，以降低碳捕获与储存(carbon capture and storage,CCS)成本。为简化传统载体的复杂性，构建了超级折叠绿色荧光蛋白(superfolder green fluorescent protein,sfGFP)与MlCA的融合蛋白表达系统，并利用大肠杆菌(Escherichia coli)合成启动子库进行一次性高效筛选。通过检测琼脂平板上菌落的荧光强度筛选出143个单克隆，形成不同表达水平的文库。筛选出荧光强度最高的前4个重组子。使用启动子342042/+的MlCA表现出最高酶活性，比活性达到34.6 Wilbur-Anderson units (WAU)/mg。优化实验表明，MlCA在pH 7.0、40℃条件下培养4 d表现最佳，其CO₂水合反应的米氏常数(K_m·hdy)和最大反应速率(V_max·hdy)分别为62.46 mmol/L和0.164 mmol/(s·L)，酯酶水解K_m和V_max分别为639.8 mmol/L和0.035 mmol/(s·L)。MlCA可在9 min内、较低pH值及室温条件下，促进CO₂矿化为CaCO₃。扫描电子显微镜(scanning electron microscopy,SEM)分析和X射线衍射(X-ray diffraction,XRD)分析证实沉淀物为方解石。本研究为CCS提供了一种低成本、环保的替代方案。

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 (K_m·hdy) and maximum reaction rate (V_max·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 K_mand V_maxof 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.