title: Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide.
abstract: Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Kobe, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Inokuma
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Bamba
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Kobe, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ishii
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Kobe, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Hasunuma
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. akondo@kobe-u.ac.jp.|Biomass Engineering Program, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan. akondo@kobe-u.ac.jp.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D002480:Cellulase / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D021381:Protein Transport / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C077394:SED1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D002480:Cellulase
keywords: Pichia pastoris; Saccharomyces cerevisiae; cell surface display; endo-glucanase; secretion signal sequence; β-glucosidase