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

ᴅ-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 RhaDN156D and RhaDP184S, 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.