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

Abstract

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 D-allulose with 750 g L 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.

Review on D-Allulose: In vivo Metabolism, Catalytic Mechanism, Engineering Strain Construction, Bio-Production Technology

“…The exogenous DAEase gene could be effectively expressed in the recombinant S. cerevisiae (Li et al, 2015) and Kluyveromyces marxianus (Yang et al, 2018). The expression vector of pRS424-TEF pr -ss-xy/A carrying A. tumefaciens DAEase could generate a 33 kDa protein in S. cerevisiae AN120 (Li et al, 2015).…”

“…D-Fructose from D-glucose catalyzed by recombinant xylose isomerase is then converted to D-allulose by recombinant DAEase. Yang et al (2018) have provided a valuable pathway to regenerate engineered K. marxianus cells, and thus achieve cyclic catalysis for D-allulose production. The recombinant K. marxianus produced 190 g L −1 D-allulose with 750 g L −1 D-fructose within 12 h. Approximately 100 g residual D-fructose was converted into 34 g ethanol by the engineering strains.…”

“…The recombinant K. marxianus produced 190 g L −1 D-allulose with 750 g L −1 D-fructose within 12 h. Approximately 100 g residual D-fructose was converted into 34 g ethanol by the engineering strains. Besides, the idea of cyclic catalysis for D-allulose production was also provided through a whole-cell reaction (Yang et al, 2018). (Yang et al, 2018), 2018 Bacillus sp., E. coli, and yeast are generally used to construct the recombinant system for DAEase expression.…”

“…Besides, the idea of cyclic catalysis for D-allulose production was also provided through a whole-cell reaction (Yang et al, 2018). (Yang et al, 2018), 2018 Bacillus sp., E. coli, and yeast are generally used to construct the recombinant system for DAEase expression. Different from E. coli, B. subtilis has no outer membrane, and the secreted protein can be released directly to the culture medium.…”

“…Early researchers tend to use E. coli as host bacteria to study the expression and properties of DTEase family enzymes (Ishida et al, 1997a;Mu et al, 2011Mu et al, , 2013. Recently, many researchers have applied B. subtilis and yeast as host bacteria to express DTEase family enzymes for D-allulose production (Chen et al, 2016;Yang et al, 2018). The contents of D-allulose respectively reached 230 g/L (Park et al, 2016b) and 190 g L −1 (Yang et al, 2018) by using recombinant A. tumefaciens DAEase expressed in E. coli and K. marxianus.…”