Cell Surface-displaying The ?-Galactosidase On Yarrowia Lipolytica:A New Approach To Synthesizing Galactooligosaccharides

With the development of molecular biology and cell surface display system,yeast surface display technology has been widely used for the research of protein and enzyme engineering.Y.lipolytica is a common host for the expression of heterologous proteins,more than 40 kinds of heterologous proteins have been expressed in this host.GOS which is usually considered as human prebiotic was produced by?-galactosidase catalyzing transglycosylation reaction of lactose,including enzymatic synthesis,fermentation,immo-bilized enzymes and transformation,etc.But among all these methods,some are expensive,some are time-consuming,so finding a new methods that save cost and time is expected.This article presents a new strategy to synthesize GOS by an erythritol-producing yeast Y.lipolytica using surface display technology.?-galactosidase from A.oryzae was effectively immobilized on cell sur-face of Y.lipolytica start strain CGMCC7326,which resulted in an engineered strain CGMCC11369.The yeast surface display expression cassette with hp4d or FBA_INN promoter was used to transform Y.lipolytica CGMCC7326,activity detection obtained yeast transformants H4 and F1which have higher activity with 0.53±0.03 U/mg?dwc?and 0.50±0.03U/mg?dwc?,respectively.Enzyme activity assay showed both promoter for the expression of?-galactosidase were no significant difference,so H4 transformant?A_T/A_H=3.7?which has higher glycosyltransferase activity was selected for the research.This engineered strain with surface-displayed?-galactosidase can efficiently synthesize GOS from lactose.An amount of 160 g/L GOS was produced within six hours in a solution of 500 g/L lactose and 5 mg/mL cell?dry weight?at pH 5.5 and 60?,with the highest yield of 51%of consumed lactose monohydrate.This newly-developed method was app-lied with waste yeast paste from the erythritol industry at least 10 times.In addition,the optimal reaction temperature increased to 60?,about10²0?higher than that of free enzymes,which is helpful to enhance the reaction rate.It appeared to be an attractive approach for the industrial production of GOS in a low-cost proceed.