title: Protein engineering of α2,3/2,6-sialyltransferase to improve the yield and productivity of in vitro sialyllactose synthesis.
abstract: In the large-quantity production of α2,3- and α2,6-sialyllactose (Neu5Ac(α2,3)Galβ1,4Glc (3'-SL) and Neu5Ac(α2,6)Galβ1,4Glc (6'-SL)) using sialyltransferases (STs), there are major hurdles to overcome for further improvement in yield and productivity of the enzyme reactions. Specifically, Pasteurella multocida α2,3-sialyltransferase (α2,3PST) forms a by-product to a certain extent, owing to its multifunctional activity at pH below 7.0, and Photobacterium damselae α2,6-sialyltransferase (α2,6PdST) shows relatively low ST activity. In this study, α2,3PST and α2,6PdST were successfully engineered using a hybrid approach that combines rational design with site-saturation mutagenesis. Narrowly focused on the substrate-binding pocket of the STs, putative functional residues were selected by multiple sequence alignment and alanine scanning, and subsequently subjected to site-saturation mutagenesis. In the case of α2,3PST, R313N single mutation improved its activity slightly (by a factor of 1.5), and further improvement was obtained by making the double mutants (R313N/T265S and R313H/T265S) resulting in an overall 2-fold improvement in its specific α2,3 ST activity, which is mainly caused by the increase in kcat. It was revealed that the R313 mutations to N, D, Y, H or T greatly reduced the α2,6 ST side-reaction activity of α2,3PST at below pH 7.0. In the case of α2,6PdST, single-mutation L433S/T and double-mutation I411T/L433T exhibited 3- and 5-fold enhancement of the α2,6 ST-specific activity compared with the wild-type, respectively, via increase in kcat values. Our results show a very good model system for enhancing ST activity and demonstrate that the generated mutants could be used efficiently for the mass production of 3'-SL and 6'-SL with enhanced productivity and yield.
authors.affiliation: Interdisciplinary Program for Biochemical Engineering and Biotechnology, Institute of Molecular Biology and Genetics.
authors.forename: Yun Hee
authors.initials: YH
authors.lastname: Choi
authors.forename: Jong Hoon
authors.initials: JH
authors.lastname: Kim
authors.forename: Joon Ho
authors.initials: JH
authors.lastname: Park
authors.forename: Nahum
authors.initials: N
authors.lastname: Lee
authors.forename: Dae-Hee
authors.initials: DH
authors.lastname: Kim
authors.forename: Kyoung-Soon
authors.initials: KS
authors.lastname: Jang
authors.forename: Il-Hyang
authors.initials: IH
authors.lastname: Park
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
mesh_terms: D003001:Cloning, Molecular; D007785:Lactose / Q000031:analogs & derivatives* / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D030781:Organisms, Genetically Modified; D016979:Pasteurella multocida / Q000235:genetics / Q000378:metabolism; D010776:Photobacterium / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017422:Sequence Analysis, DNA; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: C403777:6'-sialyllactose; D050505:Mutant Proteins; D012799:Sialyltransferases; D007785:Lactose; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
keywords: hybrid approach; protein engineering; sialyllactose; sialyltransferase