Fructooligosaccharides Production In Yeast Using Surface-displayed Fructosyltransferase

As functional oligosaccharides,fructooligosaccharides（FOS）are gaining more and more attentions due to their unique properties and great economic potential for food industry.FOS selectively stimulate the growth and activity of health-promoting Bifidobacteria and Lactobacilli in the human intestine,and contribute to the prevention of cardiovascular diseases,colon cancer,and osteoporosis.FOS have been commercially produced either by enzymatic or cell transformation.In this work,we used molecular cloning techniques to obtain a fructosyltransferase gene from Aspergillus oryzae CGMCC3.800 that produced large amount of fructosyltransferases.Using erythritol-producing yeast Yarrowia lipolytica CGMCC7326 as the recipient,the fructosyltransferase was displayed on the cell surface using cell-wall protein Pir1p,which resulted in an engineered strain CGMCC11368 with the ability to transform sucrose to FOS.The engineered strain CGMCC11368 has the highest transfructosylating activity of 0.85±0.04 U/mg dry cell weight,and hydrolytic activity of 0.11±0.02 U/mg dry cell weight,with a ratio of A_T/A_H of 7.7.Through the immunofluorescence labeling of the engineered strain,we confirmed the fusion protein was successfully displayed on the surface of the recombinant yeast.In addition,the optimal temperature for reaction was increased to 60℃,5-10℃ higher than those of free enzymes,which speeded up the reaction rate resulting in enhanced production,and the optimal pH for reaction was 6.0.This newly-developed method was applied to reuse the yeast paste from erythritol fermentaton at least for 10 times.Meanwhile,compared with Aspergillus oryzae CGMCC3.800,the engineered strain CGMCC11368 was found to have higher activity and rate of transformation for FOS production.This engineered strain produced a value-added compound erythritol when glucose was used as a carbon source.At the end of fermentation,yeast paste of engineered strain cells was harvested by centrifugation,and then used as whole-cell catalysts to convert high-concentration of sucrose to FOS.Under the condition of 800 g L^-1 sucrose,5.0 mg/mL cell（dry weight）,pH 6.0,and 60℃,480 g L^-1 FOS was produced within 3 hours,with a yield of 60% of total sucrose and the highest productivity of 160 g L^-1 h^-1 ever reported.Large amount of the by-product glucose was further converted to erythritol by the parental strain CGMCC7326.It appears to be attractive for the large-scale production of FOS using this engineered strain in a low-cost process.