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.

Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae

“…Towards this end, Sso7dstrep and Sso7dhFc were cloned into pct-CT-F2A with the secretory leader sequence SED1SP 41 placed after the F2A peptide (Fig. 7A).…”

Co-fermentation using Recombinant Saccharomyces cerevisiae Yeast Strains Hyper-secreting Different Cellulases for the Production of Cellulosic Bioethanol

“…In addition to the CBHs, we compared the amounts of BGL and EGL secreted into the culture supernatant by the signal peptide MFα, the native signal sequence, and TFPs for SfBGL1 21 and TrEGL2 27, 28 . For EGL activity analysis, the secretion of EGL by the TFP system was analysed by incubating the cell-free culture supernatants of S. cerevisiae harbouring YGaTFPn-EGL with carboxymethylcellulose (CMC) and determining the amount of the reducing sugars formed.…”

Surface Display—An Alternative to Classic Enzyme Immobilization

“…Inokuma et al [53] showed that the replacement of the anchor domain of α-agglutinin with the anchor domain of Sed1 significantly enhanced the activity of surface-displayed β-glucosidase from Aspergillus aculeatus (BGL) and endoglucanase II from Trichoderma reesei (EGII). Further optimization of the promoter and signal peptide of Sed1 additionally increased the display efficiency of both recombinant enzymes [53,54]. Inokuma et al [54] showed that the signal peptide (SP) sequence derived from the S. cerevisiae SED1 resulted in 1.3-and 1.9-fold higher BGL activity in comparison with BGL activity obtained from the constructs bearing SP from R. oryzae glucoamylase (GLUASP) and S. cerevisiae α-mating pheromone (MFα1SP), respectively.…”

“…Further optimization of the promoter and signal peptide of Sed1 additionally increased the display efficiency of both recombinant enzymes [53,54]. Inokuma et al [54] showed that the signal peptide (SP) sequence derived from the S. cerevisiae SED1 resulted in 1.3-and 1.9-fold higher BGL activity in comparison with BGL activity obtained from the constructs bearing SP from R. oryzae glucoamylase (GLUASP) and S. cerevisiae α-mating pheromone (MFα1SP), respectively. Using the Sed1 promoter was also favorable because efficient heterologous protein display was achieved by growth fa or prolonged period without any changes in the carbon source.…”