Enhancing Lactulose Production by An Engineered via Enzymatic Modification and Compartmentalization.

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 CE (DithCE) 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 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.