报告题目：GT(Sialyltransferase and fucosyltransferase) mutagenesis for the synthesis of sialyloligosaccharides from Milk
报 告 人：Byung-Gee Kim, Professor
Seoul National University
联 系 人：杨广宇 021-34207248
Oligosachharides in human milk have various useful biological functions for infant and applications in industry. Among them, sialyllactose(3’/6‘-SL) and fucosyllactose(2‘/3-FL) draw our atttention. To produce these oligosaccharides in large quantity, economic supply of CMP-Neu5Ac, highly active sialyltransferase(ST) and fucosyltransferase(FT) are keys to the success of the process development. Most sialylated oligosaccharides consist of Neu5Ac attached to galactose by an α2,3- or α2,6- linkage. In this research, α2,3-ST and α2,6-ST were engineered by hybrid approach to improve production of siayllactose. Hybrid approach is combined with rational design and directed evolution. This method can reduce library size by selecting target region such as substrate binding pocket and functional residues based on alanine scanning and computational mutation analysis. Saturation mutagenesis was done for selected residues to find best hits.
First, multifunctional α2,3 ST from Pasteurella multocida was engineered by this approach. We selected non-conserved residues located in substrates binding site by alignment with STs in GT80 family. And we applied alanine scanning for the selected residues. Mutants which show neutral activity were selected for saturation mutagenesis, and a single mutant interacting with lactose showed 168% of increased specific activity. Also, specific activity of a combination mutant was increased 200% compared to wild-type. In addition, α2,6 side reaction was reduced significantly for R313 mutants.
Second, α2,6 ST from Photobacterium damselae showed low activity and protein expression level. Therfore, it was engineered to increase catalytic activity. Substrate binding sites were predicted through homology modelling and functional residues were selected by the same method. Several mutants which show higher activity than wild type were screened by color assay method. Among them, single mutant interacting with CMP showed 4-5 times higher activity than wild type. Thus, α2,3- and α2,6-ST mutants obtained by hybrid approach will be an efficient tool for the improvement of production of sialyllactose.
In addition, Fucosyltransferase is used for the synthesis of fucosyllactose(2‘/3-FL) and sialyl-LewisX derivatives, and the same hybrid approach was undertaken. The major problem of FTs was their inclusion body formation and low level of soluble expression. How we have overcome this problem will be discussed.