title: Engineering vesicle trafficking improves the extracellular activity and surface display efficiency of cellulases inSaccharomyces cerevisiae.
abstract: BACKGROUND
  Cellulase expression via extracellular secretion or surface display inSaccharomyces cerevisiaeis one of the most frequently used strategies for a consolidated bioprocess (CBP) of cellulosic ethanol production. However, the inefficiency of the yeast secretory pathway often results in low production of heterologous proteins, which largely limits cellulase secretion or display.
  
  
  RESULTS
  In this study, the components of the vesicle trafficking from the endoplasmic reticulum (ER) to the Golgi and from the Golgi to the plasma membrane, involved in vesicle budding, tethering and fusion, were over-expressed inClostridium thermocellumendoglucanase (CelA)- andSacchromycopsis fibuligeraβ-glucosidase (BGL1)-secreting or -displaying strains. Engineering the targeted components in the ER to Golgi vesicle trafficking, including Sec12p, Sec13p, Erv25p and Bos1p, enhanced the extracellular activity of CelA. However, only Sec13p over-expression increased BGL1 secretion. By contrast, over-expression of the components in the Golgi to plasma membrane vesicle trafficking, including Sso1p, Snc2p, Sec1p, Exo70p, Ypt32p and Sec4p, showed better performance in increasing BGL1 secretion compared to CelA secretion, and the over-expression of these components all increased BGL1 extracellular activity. These results revealed that various cellulases showed different limitations in protein transport, and engineering vesicle trafficking has protein-specific effects. Importantly, we found that engineering the above vesicle trafficking components, particularly from the ER to the Golgi, also improved the display efficiency of CelA and BGL1 when a-agglutinin was used as surface display system. Further analyses illustrated that the display efficiency of a-agglutinin was increased by engineering vesicle trafficking, and the trend was consistent with displayed CelA and BGL1. These results indicated that fusion with a-agglutinin may affect the proteins' properties and alter the rate-limiting step in the vesicle trafficking.
  
  
  CONCLUSIONS
  We have demonstrated, for the first time, engineering vesicle trafficking from the ER to the Golgi and from the Golgi to the plasma membrane can enhance the protein display efficiency. We also found that different heterologous proteins had specific limitations in vesicle trafficking pathway and that engineering the vesicle trafficking resulted in a protein-specific effect. These results provide a new strategy to improve the extracellular secretion and surface display of cellulases inS. cerevisiae.
authors.affiliation: The School of Life Science, State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100 China.
authors.forename: Hongting
authors.initials: H
authors.lastname: Tang
authors.affiliation: The School of Life Science, State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100 China.
authors.forename: Meihui
authors.initials: M
authors.lastname: Song
authors.affiliation: The School of Life Science, State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100 China.
authors.forename: Yao
authors.initials: Y
authors.lastname: He
authors.affiliation: The School of Life Science, State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100 China.
authors.forename: Jiajing
authors.initials: J
authors.lastname: Wang
authors.affiliation: The School of Life Science, State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100 China.
authors.forename: Shenghuan
authors.initials: S
authors.lastname: Wang
authors.affiliation: The School of Life Science, State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100 China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Shen
authors.affiliation: The School of Life Science, State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100 China.
authors.forename: Jin
authors.initials: J
authors.lastname: Hou
authors.affiliation: The School of Life Science, State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100 China.|Shandong Provincial Key Laboratory of Microbial Engineering, Qi Lu University of Technology, Jinan, 250353 China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Bao
keywords: Cellulases; Saccharomyces cerevisiae; Secretory pathway; Surface display protein; Vesicle trafficking