This study focuses on the characterization and re-engineering of glucose transport in β-galactosidase (BglD) to enhance its catalytic efficiency. Computational prediction methods were employed to identify key residues constituting access tunnels for lactose and glucose, revealing distinct pockets for both substrates. In silico simulated saturation mutagenesis of residues T215 and T473 led to the identification of eight mutant variants exhibiting potential enhancements in glucose transport. Site-directed mutagenesis at T215 and T473 resulted in mutants with consistently enhanced specific activities, turnover rates, and catalytic efficiencies. These mutants also demonstrated improved galactooligosaccharide (GOS) synthesis, yielding an 8.1–10.6% enhancement over wild-type BglD yield. Structural analysis revealed that the mutants exhibited transformed configurations and localizations of glucose conduits, facilitating expedited glucose release. This study’s findings suggest that the re-engineered mutants offer promising avenues for enhancing BglD’s catalytic efficiency and glucose translocation, thereby improving GOS synthesis. By-product (glucose) re-tunneling is a viable approach for enzyme tunnel engineering and holds significant promise for the molecular evolution of enzymes.

Using Bacillus licheniformis H107 as the initial strain, a novel complementary synbiotics production method was developed through comprehensive metabolic engineering strategies. Key modifications included the systematic analysis and reconstruction of the central carbon metabolism pathway through precise gene editing, targeting the deletion of pflB, alsS, ydaP, and pycA genes while disrupting ganA1 and ganA2 to block galactooligosaccharide (GOS) metabolism. Additionally, heterologous expression of the L-lactate dehydrogenase gene (BcoaLDH) was implemented, resulting in the engineered strain H107-06A. Shake-flask fermentation under anaerobic conditions with 20 g/L glucose yielded L-lactic acid production of 4.45 g/L, representing a 15.3-fold increase compared to the wild type. In a 5 L fermenter using GOS syrup as the carbon source, the engineered system synergistically produced complementary synbiotics, comprising L-lactic acid (42.56 g/L), GOS (141.89 g/L, accounting for 75.09% of total sugars), and viable cells (3.82 g/L). These findings provide a foundation for developing innovative and functional fermentation products.