Enhancing Lactulose Production by An Engineered Escherichia coli via Enzymatic Modification and Compartmentalization

作者：Meihereayi Mutailifu, Nuo-Qiao Lin, Fei Liu, Jian-Zhong Liu · 发表于：Journal of Agricultural and Food Chemistry · 年份：2025 · DOI：10.1021/acs.jafc.5c04270 · 被引用次数：3 · 研究领域：Enzyme Production and Characterization、Enzyme Catalysis and Immobilization、Enzyme Structure and Function

Lactulose, a high-value lactose derivative with extensive pharmaceutical and nutritional applications, continues to face production challenges in enzymatic synthesis using cellobiose 2-epimerase (CE). To address this limitation, we present an integrated biocatalytic strategy combining enzyme engineering, spatial compartmentalization, and process optimization. Through targeted artificial DNA replisome (TADR)-directed evolution and rational design with UniKP computational prediction, we developed a quadruple mutant Dictyoglomus thermophilum CE (DithCE MUT ) with enhanced catalytic efficiency. Systematic evaluation of four compartmentalization approaches revealed that vesicle-nucleating peptide 6 (VNp6)-mediated spatial organization increased lactulose production by 2.3-fold compared to no-compartmentalization enzyme systems. We further established three streamlined bioconversion platforms (whole-cell, crude enzyme and heat-treated enzyme biocatalysis), with the heat-treated enzyme from VNp6-compartmentalized DithCE MUT achieving record productivity: 320.49 ± 4.81 g/L lactulose with a yield of 94.5% in the presence of borate. These strategies of enzymatic modification and compartmentalization are powerful tools for synthetic biomanufacturers.