重组大肠杆菌全细胞一步法催化乳糖合成d-塔格糖

One-step whole-cell synthesis of d-tagatose from lactose catalyzed by recombinant Escherichia coli

摘要:

d-塔格糖是一种功能性稀有糖，因其具有热量低、降低血糖、抗龋齿和改善肠道菌群等特性而受到广泛关注。现有的生产d-塔格糖的方法仍存在生产效率低、成本高等问题。为了解决这一问题，本研究通过在大肠杆菌中构建双酶高效表达系统，实现了全细胞高效催化乳糖合成d-塔格糖。l-阿拉伯糖异构酶(l-arabinose isomerase, l-AI)是生物合成法中催化半乳糖异构化生成d-塔格糖的关键酶。本研究首先通过对不同来源的l-AI进行筛选，发现来源于发酵乳杆菌(Lactobacillus fermentum) CGMCC 2921的l-AI具有较好的催化能力。通过对LfAI进行理性设计，获得最优突变体D390V/V468L，在最适40 ℃条件下，其半衰期延长至72 h，酶活提高了36.68%。随后，以pET28a为载体，通过启动子优化，将最优突变体基因(LfaraA^D390V/V468L)和大肠杆菌来源的β-半乳糖苷酶基因(EclacZ)在大肠杆菌(Escherichia coli) BL21(DE3)中串联表达，建立双酶偶联全细胞体系。最后，通过优化全细胞催化体系，在5 L发酵罐的最优条件(pH 7.0、温度50 ℃、2.5 mmol/L Mn²⁺)下，以500 g/L乳糖为底物转化48 h获得115.21 g/L d-塔格糖，转化率为23.09%。本研究利用重组全细胞催化法以乳糖为底物合成d-塔格糖，展现出了显著的工业应用潜力和 价值。

Abstract:

d-tagatose, a functional rare sugar, has garnered increasing attention because of its low calories, blood glucose-lowering, anti-caries, and intestinal flora-improving properties. The existing methods of producing d-tagatose still have problems of low production efficiency and high costs. In this study, we achieved whole-cell catalytic synthesis of d-tagatose from lactose by constructing a dual-enzyme efficient expression system in Escherichia coli. l-arabinose isomerase (l-AI) is a key enzyme for the isomerization of galactose to d-tagatose in biosynthesis. In this study, we screened the l-AIs from different sources and found that the l-AI from Lactobacillus fermentum CGMCC 2921 had better catalytic ability. The optimal mutant D390V/V468L was obtained by rational design of LfAI. Its half-life was extended to 72 h, and the enzyme activity was increased by 36.68% under the optimum temperature of 40 ℃. Then, with pET28a as a vector, the optimal mutant gene (LfaraA^D390V/V468L) and the β-galactosidase gene from E. coli (EclacZ) were co-expressed in E. coli BL21(DE3) through promoter optimization. Finally, after optimization of the catalytic system, 115.21 g/L d-tagatose was obtained after fermentation under the optimal conditions (pH 7.0, 50 ℃, and 2.5 mmol/L Mn²⁺) in a 5 L fermenter with 500 g/L lactose as the substrate for 48 h, with a conversion rate of 23.09%. This study has a good industrial application value in the one-step whole-cell catalytic synthesis of d-tagatose from lactose.