Efficient microbial glucose-fructose conversion at ambient temperature through adaptive laboratory evolution and application for D-allulose production

Glucose and fructose, the major monosaccharides in nature, are metabolically interconverted in phosphorylated form by glucose 6-phosphate isomerase. An enzyme specifically evolved for interconversion of the non-phosphorylated forms has not been discovered yet. Xylose isomerase catalyzes the conversion as a side reaction, but with very low activity at ambient temperatures. Here, we developed a microbial catalyst enabling the efficient interconversion of glucose and fructose at 30°C. Using adaptive laboratory evolution (ALE) of a selection strain dependent on xylose isomerase activity for growth, we obtained enzyme variants with up to ten-fold improved catalytic efficiency. Fast growth additionally required a mutation in the transporter IolT1, boosting sugar uptake and increasing the intracellular substrate concentration. During engineering of a strain unable to grow on glucose, we identified mutations in the sucrose transporter PtsS, presumably converting it into a transporter for glucose and fructose. Molecular dynamics simulations of the transporter and xylose isomerase variants suggested mechanistic consequences of the mutations. Our novel strains enabled the microbial conversion of glucose to the low-calorie sweetener D-allulose with a yield of 15%.