Untitled

Abstract

Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur. P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form. The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include beta1,4galactosyltransferase, alpha2,6sialyltransferase, alpha2,3sialyltransferase, alpha1,3fucosyltransferase III and VI and alpha1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.

Exploring the Acceptor Substrate Recognition of the Human β-Galactoside α2,6-Sialyltransferase

“…Human soluble ST6GalI was produced in Pichia pastoris after truncation of the N terminus and the transmembrane fragment as described (29). This deletion was therefore very similar to the FLAG-ST6GalI construct.…”

Enzymatic Biosynthesis of Oligosaccharides and Glycoconjugates

“…Furthermore, their purification procedures are quite complicated with their relative instability, because glycosyltransferases are often membrane-bound or membrane-associated. For these reasons, many efforts have been geared toward the genetic engineering and recombinant sources of glycosyltransferases [33][34][35][36][37]. Historically, most glycosyltransferases investigated have been isolated from mammalian sources reflective of the major focus of glycobiology researchers.…”

Development, Production, and Application of Recombinant Yeast Biocatalysts in Organic Synthesis

“…A variety of plasmid DNA vectors which are capable of transforming auxotrophic yeast strains and which have also bacterial sequences that can be selected for and propagated in E.coli, are suitable for subcloning the glycosyltransferase cDNA and represent important screening tools [43]. The identification of positive transformants of yeast host strains like Pichia pastoris KM71 (arg4his4aox1: ARG4) by direct PCR screening and the inclusion of yeast promoter and terminator sequences for efficient transcription have been used in the construction of recombinant Pichia pastoris strains [44]. It is useful for downstream processing of soluble forms of glycosyltransferases that the vector contains a signal sequence such as the N-terminal signal sequence of Saccharomyces cerevisiae α-factor for the secretion of the expressed protein into the medium.…”

“…Another strategy aims at expressing glycosyltransferases immobilized to the yeast cell wall [45] [46]. In the case of glycosyltransferases, the yeast expression systems have been important for developing a series of soluble functional biocatalysts [44] [47]. For the human β(1→4)-galactosyltransferase gene, the Saccharomyces cerevisiae expression system has been developed.…”

“…The growth of this yeast was controlled with a fedbatch procedure, which demonstrated for the first time that heterologous expression of a gly-cosyltransferase is possible on a large scale [48]. The expression of the human α(1→ 3)-fucosyltransferase VI gene has been developed in the Pichia pastoris system with the alcohol-oxidase (AOX) promoter in the protease-negative KM71-host in shake flasks [44]. The development of efficient fermentation and downstream processing required the development of sensitive and reliable non-radioactive activity assays for in-process control.…”