Efficient production of D-tagatose through cell membrane permeabilization modification and multi-enzymes...

D-tagatose, a rare sugar with 92 % of sucrose's sweetness but only one-third of its calories, represents a promising low-calorie sweetener and functional food ingredient. However, microbial production of D-tagatose from lactose is often limited by inefficient substrate transport across cell membranes and suboptimal enzyme catalytic efficiency. In this study, we deployed a combination of cell membrane engineering and spatial enzyme organization to improve D-tagatose biosynthesis in Escherichia coli. First, we improved lactose uptake efficiency through deletion of the Braun lipoprotein (lpp) gene and overexpression of lactose permease (lacY), reducing fermentation time by one-third while maintaining comparable yields. Second, we addressed cofactor imbalance by chromosomally integrating the membrane-bound transhydrogenase gene pntAB, which further increased D-tagatose titer by 1.20-fold. Finally, we employed a RGG-RGG motif-mediated liquid-liquid phase separation strategy to compartmentalize the key pathway enzymes-xylitol reductase (SgXR) and galactitol dehydrogenase (RlGDH)-into membraneless organelles. Upon optimizing promoter regulation and induction timing, the engineered strain BLPBT04 achieved a D-tagatose titer of 10.01 g/L with a conversion yield of 95.2 % of the theoretical maximum (0.50 g/g lactose) and a volumetric productivity of 0.209 g/L/h in shake-flask fermentation, representing a 4.35-fold improvement over the parental strain. This work demonstrates that combining cell membrane engineering with enzyme compartmentalization via phase separation provides a powerful approach for enhancing rare sugars production in microbial cell factories.