High Conversion of Value-Added Lactulose from Lactose-Rich Dairy Waste by In Vivo Cascade Cell Factory

Lactulose, a nondigestible disaccharide, is widely recognized for its significant health-promoting effects. The drawbacks of traditional chemical catalysis, e.g., high environmental pollution and complex separation processes, hinder its sustainable production. In this study, an in vivo cascade system, integrating a cellobiose 2-epimerase (CE) and a mannose isomerase from Marinomonas mediterranea (MmMI), was proposed to produce lactulose from lactose-rich dairy waste. The cascaded system improved the conversion of lactulose up to 90% by eliminating the accumulation of the byproduct epilactose. Moreover, loop-B was revealed to be a crucial structural region for isomerization; upon this new discovery, semirational modification of MmMI by reshaping loop-B resulted in a 17.5% increase in lactulose production. Furthermore, this in vivo cascade cell factory was optimized via ribosome binding site (RBS) engineering. Upon high-density fermentation, a total of 59.3 g/L lactulose was obtained using whey powder as the raw material. This efficient in vivo multienzyme cascade system demonstrates substantial potential for the large-scale production of lactulose.