Efficient cell surface display of Lip2 lipase using C-domains of glycosylphosphatidylinositol-anchored cell wall proteins of Yarrowia lipolytica

ABSTRACT Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

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An Alternative Approach to Synthesizing Galactooligosaccharides by Cell-Surface Display of β-Galactosidase on Yarrowia lipolytica

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.5b06138 ◽

2016 ◽

Vol 64 (19) ◽

pp. 3819-3827 ◽

Cited By ~ 15

Author(s):

Jin An ◽

Lebin Zhang ◽

Lijuan Li ◽

Dawen Liu ◽

Huiling Cheng ◽

...

Keyword(s):

Cell Surface ◽

Yarrowia Lipolytica ◽

Surface Display ◽

Cell Surface Display ◽

Alternative Approach

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Cell Wall Anchoring of a Bacterial Chitosanase in Lactobacillus plantarum Using a Food-Grade Expression System and Two Versions of an LP × TG Anchor

International Journal of Molecular Sciences ◽

10.3390/ijms21113773 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3773 ◽

Cited By ~ 1

Author(s):

Mai-Lan Pham ◽

Anh-Minh Tran ◽

Geir Mathiesen ◽

Hoang-Minh Nguyen ◽

Thu-Ha Nguyen

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Lactobacillus Plantarum ◽

Expression System ◽

Surface Display ◽

Cell Surface Display ◽

Expression Vectors ◽

Selection Marker ◽

Food Grade ◽

Cell Factories

Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the alr (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in Lactobacillus plantarum using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from Bacillus subtilis 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in L. plantarum WCFS1. The expression and cell-surface display efficiency driven by the food-grade alr-based system were compared with those obtained from the erm-based pSIP system in terms of enzyme activities and their localisation on L. plantarum cells. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest enzymatic activity of CsnA-displaying cells was obtained from the strain carrying the alr-based expression plasmid with short cell wall anchor S. However, the attachment of chitosanase on L. plantarum cells via the long anchor L was shown to be more stable compared with the short anchor after several repeated reaction cycles. CsnA displayed on L. plantarum cells is catalytically active and can convert chitosan into chito-oligosaccharides, of which chitobiose and chitotriose are the main products.

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Determination of the Domain of the Lactobacillus delbrueckii subsp. bulgaricus Cell Surface Proteinase PrtB Involved in Attachment to the Cell Wall after Heterologous Expression of the prtB Gene in Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.69.6.3377-3384.2003 ◽

2003 ◽

Vol 69 (6) ◽

pp. 3377-3384 ◽

Cited By ~ 19

Author(s):

Jacques-Edouard Germond ◽

MichÃ¯Â¿Â½le Delley ◽

Christophe Gilbert ◽

DaniÃ¯Â¿Â½le Atlan

Keyword(s):

Amino Acids ◽

Cell Wall ◽

Cell Surface ◽

Lactococcus Lactis ◽

Culture Medium ◽

Surface Display ◽

Cell Surface Display ◽

Lactobacillus Delbrueckii ◽

Heterologous Proteins

ABSTRACT Belonging to the subtilase family, the cell surface proteinase (CSP) PrtB of Lactobacillus delbrueckii subsp. bulgaricus differs from other CSPs synthesized by lactic acid bacteria. Expression of the prtB gene under its own promoter was shown to complement the proteinase-deficient strain MG1363 (PrtP− PrtM−) of Lactococcus lactis subsp. cremoris. Surprisingly, the maturation process of PrtB, unlike that of lactococcal CSP PrtPs, does not require a specific PrtM-like chaperone. The carboxy end of PrtB was previously shown to be different from the consensus anchoring region of other CSPs and exhibits an imperfect duplication of 59 amino acids with a high lysine content. By using a deletion strategy, the removal of the last 99 amino acids, including the degenerated anchoring signal (LPKKT), was found to be sufficient to release a part of the truncated PrtB into the culture medium and led to an increase in PrtB activity. This truncated PrtB is still active and enables L. lactis MG1363 to grow in milk supplemented with glucose. By contrast, deletion of the last 806 amino acids of PrtB led to the secretion of an inactive proteinase. Thus, the utmost carboxy end of PrtB is involved in attachment to the bacterial cell wall. Proteinase PrtB constitutes a powerful tool for cell surface display of heterologous proteins like antigens.

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