Biosensor-assisted evolution of RhaD for enhancing the biosynthetic yield of d-allulose

d -Allulose, is an improtant rare sugar, having health-promoting properties and applications in the food, pharmaceutical and nutrition sectors. White the aldol reaction-based method shows promise as an alternative to the “Izumori” strategy for ᴅ-allulose bioproduction, its industrial exploitability is hindered by the low product yield in reaction equilibrium and the high costs associated with the use of purified enzymes . To address these limitations, this study explored cell-based biosynthesis of ᴅ-allulose from low-cost glycerol by overexpression the enzymes involved in the ᴅ-allulose aldol condensation (AC) biosynthesis pathway, namely fructose-1-phosphatase (YqaB), alditol oxidase (AldO), and l -rhamnulose-1-phosphate aldolase (RhaD), in Escherichia coli . Results indicated that RhaD is a rate-limiting step of the ᴅ-allulose biosynthetic pathway. Then, biosensor-based high throughput screening was employed for the directed evolution of RhaD. In this way, two RhaD mutants, namely RhaD N156D and RhaD P184S , were successfully screened from 40,129 individual colonies, which exhibited respectively achieved a ᴅ-allulose titer of 3.1 g/L and 2.9 g/L, respectively in shake flasks, representing a more than 20% improvement compared to the wildtype strain. Overall, the ᴅ-allulose biosensor-assisted key enzyme evolution strategy and the high throughput screening method presented in this work provide a paradigm for improving rare sugars’ high-valued productions from cheap substrates.