Engineering vesicle trafficking improves the extracellular activity and surface display efficiency of cellulases in Saccharomyces cerevisiae

Abstract

BackgroundCellulase expression via extracellular secretion or surface display in Saccharomyces cerevisiae is 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.ResultsIn 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 in Clostridium thermocellum endoglucanase (CelA)- and Sacchromycopsis fibuligera β-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.ConclusionsWe 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 in S. cerevisiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0738-8) contains supplementary material, which is available to authorized users.

Surface Display—An Alternative to Classic Enzyme Immobilization

“…Tang et al [75] improved the surface display efficiency of recombinant a-agglutinin fusion with Clostridium thermocellum endoglucanase CelA and S. fibuligera β-glucosidase BGL1 by engineering vesicle trafficking. Sec12, Sec13, Erv25, and Bos1 proteins, involved in vesicle trafficking from the ER to the Golgi, and Sso1, Snc2, Sec1, Exo70, Sec4, and Ypt32 proteins, involved in vesicle transport from the Golgi to the plasma membrane, were overexpressed in strains expressing CelA/a-agglutinin and BGL1/a-agglutinin.…”

The Role of Yeast-Surface-Display Techniques in Creating Biocatalysts for Consolidated BioProcessing

“…This was achieved through the utilization of various molecular techniques that lead to the optimization of signal sequences, upregulation of ER folding, modification of vesicle transport, and deletion of host proteinases as discussed in detail by Haan, et al [112]. A recent example of this in relation to the role of surface display for efficient lignocellulose utilization has been demonstrated by Tang, et al [113]. Where the localization and co-display of the C. thermocellum endoglucanase (CelA) along with a S. fibuligera β-glucosidase (BGL) was improved by the over-expression of various vesicle trafficking components.…”

“…Where the localization and co-display of the C. thermocellum endoglucanase (CelA) along with a S. fibuligera β-glucosidase (BGL) was improved by the over-expression of various vesicle trafficking components. Furthermore, their results have shown that there is a protein-specific effect, which limit the vesicle trafficking pathway with respect to the heterologous protein being localized; this provides an insight on how to improve surface display efficiency tailored to the protein of interest [113]. In the semi-rational approach, the genes in the cell wall environment are targeted instead of those in the secretory pathway.…”

Identification of a secretion-enhancing cis regulatory targeting element (SECReTE) involved in mRNA localization and protein synthesis

“…Moreover, S. cerevisiae is a generally recognized as a safe (GRAS) organism, which makes it a favorable for use in the production of biopharmaceuticals 67,68 . Unfortunately, the natural capacity of S. cerevisiae secretory pathway is relatively limited and, thus, mechanisms that improve secreted protein production would be of significant benefit.…”