scholarly journals Engineering Pichia pastoris with surface-display minicellulosome for carboxymethyl cellulose hydrolysis and ethanol production

2020 ◽  
Author(s):  
Ce Dong ◽  
Jie Qiao ◽  
Xinping Wang ◽  
Wenli Sun ◽  
Lixia Chen ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Cell Surface ◽  
Ethanol Production ◽  
Carboxymethyl Cellulose ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Cellulose Hydrolysis ◽  
Cell Factory ◽  
Yeast Cell Surface

Abstract Backgrounds: Engineering yeast with cell surface immobilized cellulosome is a promising strategy for consolidated bioprocessing (CBP) to produce bioethanol from the conversion of cellulose. However, previous studies mostly focused on utilization of Saccharomyces cerevisiae , which was able to directly convert phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L. Results: Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosome through in vitro assembly of various recombinant cellulases on the cell surface. For the first time, the yeast can efficiently convert CMC to bioethanol, achieving an impressive ethanol titer of 5.1 g/L. Further, the engineered yeasts were lyophilized to powders that can be utilized as compound cellulases. Conclusions: This research promotes the application of P. pastoris as CBP cell factory in cellulosic ethanol production and provides a promising platform for screening optimal cellulase species and ratios to construct celluosome on the yeast cell surface.

scholarly journals Engineering Pichia pastoris with surface-display minicellulosomes for carboxymethyl cellulose hydrolysis and ethanol production

2020 ◽  
Author(s):  
Ce Dong ◽  
Jie Qiao ◽  
Xinping Wang ◽  
Wenli Sun ◽  
Lixia Chen ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Ethanol Production ◽  
Carboxymethyl Cellulose ◽  
Cellulosic Ethanol ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Cell Factory ◽  
Carbohydrate Binding ◽  
Surface Assembly

Abstract Backgrounds: Engineering yeast as a consolidated bioprocessing (CBP) microorganism by surface assembly of cellulosomes has been aggressively utilized for cellulosic ethanol production. However, most of the previous studies focused on Saccharomyces cerevisiae, achieving efficient conversion of phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L. Results: Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosomes by in vitro assembly of three recombinant cellulases including an endoglucanase (EG), an exoglucanase (CBH) and a β-glucosidase (BGL), as well as a carbohydrate binding module (CBM) on the cell surface. For the first time, the engineered yeasts enable efficient and direct conversion of CMC to bioethanol, observing an impressive ethanol titer of 5.1 g/L. Conclusions: The research promotes the application of P. pastoris as a CBP cell factory in cellulosic ethanol production and provides a promising platform for screening the cellulases from different species to construct surface-assembly celluosome.

scholarly journals Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol

2009 ◽  
Vol 76 (4) ◽  
pp. 1251-1260 ◽  
Author(s):  
Fei Wen ◽  
Jie Sun ◽  
Huimin Zhao
Keyword(s):  
Cell Surface ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Cellulose Hydrolysis ◽  
Cellulase Production ◽  
Single Step ◽  
Yeast Surface Display ◽  
Biofuel Production ◽  
Yeast Cells ◽  
Recombinant Yeast Strain

ABSTRACT By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of Saccharomyces cerevisiae strains displaying a series of uni-, bi-, and trifunctional minicellulosomes. These minicellulosomes consist of (i) a miniscaffoldin containing a cellulose-binding domain and three cohesin modules, which was tethered to the cell surface through the yeast a-agglutinin adhesion receptor, and (ii) up to three types of cellulases, an endoglucanase, a cellobiohydrolase, and a β-glucosidase, each bearing a C-terminal dockerin. Cell surface assembly of the minicellulosomes was dependent on expression of the miniscaffoldin, indicating that formation of the complex was dictated by the high-affinity interactions between cohesins and dockerins. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme synergy and enzyme proximity synergy. More importantly, surface display of the trifunctional minicellulosomes gave yeast cells the ability to simultaneously break down and ferment phosphoric acid-swollen cellulose to ethanol with a titer of ∼1.8 g/liter. To our knowledge, this is the first report of a recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes. The strain reported here represents a useful engineering platform for developing CBP-enabling microorganisms and elucidating principles of cellulosome construction and mode of action.

scholarly journals Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

2013 ◽  
Vol 79 (18) ◽  
pp. 5519-5526 ◽  
Author(s):  
Li Zhang ◽  
Shuli Liang ◽  
Xinying Zhou ◽  
Zi Jin ◽  
Fengchun Jiang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Pichia Pastoris ◽  
Cell Surface ◽  
Signal Sequence ◽  
Recombinant Expression ◽  
Cell Wall Proteins ◽  
Heterologous Proteins ◽  
Content Type ◽  
Yeast Cell Surface

ABSTRACTGlycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different usesin vitro. In the present study, the genome ofPichia pastorisGS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and matureCandida antarcticalipase B (CALB). The expression of fusion proteins on the cell surface ofP. pastorisGS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface ofP. pastorisGS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis ofp-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins inP. pastorisGS115, which can be used to display heterologous proteins on the yeast cell surface.

Construction of a novel system for cell surface display of heterologous proteins on Pichia pastoris

2007 ◽  
Vol 29 (10) ◽  
pp. 1561-1566 ◽  
Author(s):  
Qingjie Wang ◽  
Lei Li ◽  
Min Chen ◽  
Qingsheng Qi ◽  
Peng George Wang
Keyword(s):  
Pichia Pastoris ◽  
Cell Surface ◽  
Surface Display ◽  
Cell Surface Display ◽  
Heterologous Proteins

Yeast cell surface display of xylose isomerase from C. cellulovorans toward efficient xylose fermentation

2012 ◽  
Vol 29 ◽  
pp. S48
Author(s):  
Kouichi Kuroda ◽  
Miki Ota ◽  
Hironobu Morisaka ◽  
Hideo Miyake ◽  
Yutaka Tamaru ◽  
...  
Keyword(s):  
Cell Surface ◽  
Yeast Cell ◽  
Xylose Fermentation ◽  
Surface Display ◽  
Xylose Isomerase ◽  
Cell Surface Display ◽  
Yeast Cell Surface

scholarly journals Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

2011 ◽  
Vol 78 (2) ◽  
pp. 560-567 ◽  
Author(s):  
Denise Knobloch ◽  
Kai Ostermann ◽  
Gerhard Rödel
Keyword(s):  
Cell Surface ◽  
Bacillus Megaterium ◽  
Fusion Proteins ◽  
Shuttle Vector ◽  
Surface Display ◽  
Fluorescent Labeling ◽  
Content Type ◽  
Sporosarcina Ureae

ABSTRACTMonomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report thatBacillus megateriumis an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA fromSporosarcina ureaeATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into theEscherichia coli-Bacillus megateriumshuttle vector pHIS1525. After transformation of the respective plasmids intoBacillus megateriumprotoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemblein vitrointo highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope ofBacillus megaterium, indicating that the cell surface can servein vivoas a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

scholarly journals Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response

2006 ◽  
Vol 72 (11) ◽  
pp. 7140-7147 ◽  
Author(s):  
Frank Breinig ◽  
Björn Diehl ◽  
Sabrina Rau ◽  
Christian Zimmer ◽  
Helmut Schwab ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cell Surface ◽  
Surface Display ◽  
Cell Surface Display ◽  
Constitutive Activation ◽  
Unfolded Protein ◽  
Yeast Cell Surface ◽  
Protein Response

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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