LAUSR

Abstractd-Allulose as a low-energy and special bioactive monosaccharide sugar is essential for human health. In this study, the d-psicose-3-epimerase gene (DPEase) of Agrobacterium tumefaciens was transferred into thermotolerant Kluyveromyces marxianus to decrease the production cost of d-allulose and reduce the number of manufacturing procedures. The cell regeneration of K. marxianus and cyclic catalysis via whole-cell reaction were investigated to achieve the sustainable application of K. marxianus and the consumption of residual d-fructose. Results showed that DPEase, encoding a 33 kDa protein, could be effectively expressed in thermotolerant K. marxianus. The engineered K. marxianus produced 190 g L−1d-allulose with 750 g L−1d-fructose as a substrate at 55 °C within 12 h. Approximately 100 g of residual d-fructose was converted into 34 g of ethanol, and 15 g of the engineered K. marxianus cells was regenerated after fermentation at 37 °C for 21 h. The purity of d-allulose of more than 90% could be obtained without isolating it from d-allulose and d-fructose mixture through residual d-fructose consumption. This study provided a valuable pathway to regenerate engineered K. marxianus cells and achieve cyclic catalysis for d-allulose production.