title: Development of N- and O-linked oligosaccharide engineered Saccharomyces cerevisiae strain.
abstract: Yeast cells have been engineered for the production of glycoproteins as biopharmaceuticals with humanized N-linked oligosaccharides. The suppression of yeast-specific O-mannosylation is important to reduce immune response and to improve heterologous protein productivity in the production of biopharmaceuticals. However, so far, there are few reports of the engineering of both N-linked and O-linked oligosaccharides in yeast cells. In the present study, we describe the generation of a Saccharomyces cerevisiae strain capable of producing a glycoprotein with humanized Man5GlcNAc2N-linked oligosaccharides, an intermediate of mammalian hybrid- and complex-type oligosaccharides, while suppressing O-mannosylation. First, a yeast strain that produces a glycoprotein with Man5GlcNAc2was isolated by introducing msdS encoding α-1,2-mannosidase into a strain synthesizing Man8GlcNAc2N-linked oligosaccharides. Next, to suppress O-mannosylation, an O-mannosyltransferase-deficient strain was generated by disrupting PMT1 and PMT2 Although the relative amount of O-linked oligosaccharides in the disruptant was reduced to approximately 40% of that in wild type cells, this strain exhibited growth defects and decreased protein productivity. To overcome the growth defects, we applied a mutagenesis technique that is based on the disparity theory of evolution. Finally, to improve protein productivity of the growth-recovered strain, vacuolar proteases PEP4 and PRB1 were further disrupted. Thus, by combining genetic engineering and disparity mutagenesis, we generated an Saccharomyces cerevisiae strain whose N- and O-linked oligosaccharide synthetic pathways were engineered to effectively produce the heterologous protein.
authors: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi, Takamatsu, Kagawa 761-0395, Japan abe-abe@aist.go.jp.
authors: Hiroko
authors: H
authors: Abe
authors: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi, Takamatsu, Kagawa 761-0395, Japan.
authors: Kazuya
authors: K
authors: Tomimoto
authors: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi, Takamatsu, Kagawa 761-0395, Japan.
authors: Yasuko
authors: Y
authors: Fujita
authors: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi, Takamatsu, Kagawa 761-0395, Japan.
authors: Tomoko
authors: T
authors: Iwaki
authors: Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors: Yasunori
authors: Y
authors: Chiba
authors: Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors: Ken-Ichi
authors: KI
authors: Nakayama
authors: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi, Takamatsu, Kagawa 761-0395, Japan.
authors: Yoshihiro
authors: Y
authors: Nakajima
mesh_terms: D005818:Genetic Engineering; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides
keywords: N-linked oligosaccharide; O-linked oligosaccharide; S. cerevisiae; disparity mutagenesis; glycan engineering