High Conversion of Value-Added Lactulose from Lactose-Rich Dairy Waste by <i>In Vivo</i> 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 <i>in vivo</i> cascade system, integrating a cellobiose 2-epimerase (CE) and a mannose isomerase from <i>Marinomonas mediterranea</i> (<i>Mm</i>MI), 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 <i>Mm</i>MI by reshaping loop-B resulted in a 17.5% increase in lactulose production. Furthermore, this <i>in vivo</i> 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 <i>in vivo</i> multienzyme cascade system demonstrates substantial potential for the large-scale production of lactulose.