Cell regeneration and cyclic catalysis of engineered Kluyveromyces marxianus of a D-psicose-3-epimerase gene from Agrobacterium tumefaciens for D-allulose production.

D-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-1 D-allulose with 750 g L-1 D-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.