scholarly journals Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes

2002 ◽  
Vol 68 (10) ◽  
pp. 5136-5141 ◽  
Author(s):  
Yasuya Fujita ◽  
Shouji Takahashi ◽  
Mitsuyoshi Ueda ◽  
Atsuo Tanaka ◽  
Hirofumi Okada ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Yeast Strain ◽  
Surface Engineering ◽  
Synthetic Medium ◽  
Hydrolytic Activity ◽  
Yeast Cells ◽  
Cellulolytic Enzymes ◽  
Efficient Production ◽  
Engineering System

ABSTRACT For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on α-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His6) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-α-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley β-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and β-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing β-glucan as the sole carbon source and could directly ferment 45 g of β-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.

scholarly journals Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme

2004 ◽  
Vol 70 (2) ◽  
pp. 1207-1212 ◽  
Author(s):  
Yasuya Fujita ◽  
Junji Ito ◽  
Mitsuyoshi Ueda ◽  
Hideki Fukuda ◽  
Akihiko Kondo
Keyword(s):  
Cell Surface ◽  
Yeast Strain ◽  
Cellulose Degradation ◽  
Surface Display ◽  
Hydrolytic Activity ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Enzyme ◽  
Amorphous Cellulose ◽  
Endoglucanase Ii ◽  
Cellobiohydrolase Ii

ABSTRACT A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.

scholarly journals Construction of a Xylan-Fermenting Yeast Strain through Codisplay of Xylanolytic Enzymes on the Surface of Xylose-Utilizing Saccharomyces cerevisiae Cells

2004 ◽  
Vol 70 (9) ◽  
pp. 5407-5414 ◽  
Author(s):  
Satoshi Katahira ◽  
Yasuya Fujita ◽  
Atsuko Mizuike ◽  
Hideki Fukuda ◽  
Akihiko Kondo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Yeast Strain ◽  
High Performance ◽  
Surface Engineering ◽  
Xylose Reductase ◽  
Pichia Stipitis ◽  
Direct Conversion ◽  
Engineering System ◽  
Xylose Utilization

ABSTRACT Hemicellulose is one of the major forms of biomass in lignocellulose, and its essential component is xylan. We used a cell surface engineering system based on α-agglutinin to construct a Saccharomyces cerevisiae yeast strain codisplaying two types of xylan-degrading enzymes, namely, xylanase II (XYNII) from Trichoderma reesei QM9414 and β-xylosidase (XylA) from Aspergillus oryzae NiaD300, on the cell surface. In a high-performance liquid chromatography analysis, xylose was detected as the main product of the yeast strain codisplaying XYNII and XylA, while xylobiose and xylotriose were detected as the main products of a yeast strain displaying XYNII on the cell surface. These results indicate that xylan is sequentially hydrolyzed to xylose by the codisplayed XYNII and XylA. In a further step toward achieving the simultaneous saccharification and fermentation of xylan, a xylan-utilizing S. cerevisiae strain was constructed by codisplaying XYNII and XylA and introducing genes for xylose utilization, namely, those encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. After 62 h of fermentation, 7.1 g of ethanol per liter was directly produced from birchwood xylan, and the yield in terms of grams of ethanol per gram of carbohydrate consumed was 0.30 g/g. These results demonstrate that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S. cerevisiae strain.

scholarly journals Cell Surface Engineering of a β-Galactosidase for Galactooligosaccharide Synthesis

2009 ◽  
Vol 75 (18) ◽  
pp. 5938-5942 ◽  
Author(s):  
Yumei Li ◽  
Lili Lu ◽  
Hongmei Wang ◽  
Xiaodong Xu ◽  
Min Xiao
Keyword(s):  
Cell Surface ◽  
Surface Engineering ◽  
Yeast Cells ◽  
Penicillium Expansum ◽  
Yeast Nitrogen Base ◽  
Gene Encoding ◽  
Nitrogen Base ◽  
Reading Frame ◽  
Acid Protein ◽  
Casamino Acids

ABSTRACT A novel gene encoding transglycosylating β-galactosidase (BGase) was cloned from Penicillium expansum F3. The sequence contained a 3,036-bp open reading frame encoding a 1,011-amino-acid protein. This gene was subsequently expressed on the cell surface of Saccharomyces cerevisiae EBY-100 by galactose induction. The BGase-anchored yeast could directly utilize lactose to produce galactooligosaccharide (GOS), as well as the by-products glucose and a small quantity of galactose. The glucose was consumed by the yeast, and the galactose was used for BGase expression, thus greatly facilitating GOS synthesis. The GOS yield reached 43.64% when the recombinant yeast was cultivated in yeast nitrogen base-Casamino Acids medium containing 100 g/liter initial lactose at 25°C for 5 days. The yeast cells were harvested and recycled for the next batch of GOS synthesis. During sequential operations, both oligosaccharide synthesis and BGase expression were maintained at high levels with GOS yields of over 40%, and approximately 8 U/ml of BGase was detected in each batch.

scholarly journals Display of α-Amylase on the Surface of Lactobacillus casei Cells by Use of the PgsA Anchor Protein, and Production of Lactic Acid from Starch

2006 ◽  
Vol 72 (1) ◽  
pp. 269-275 ◽  
Author(s):  
Junya Narita ◽  
Kenji Okano ◽  
Tomoe Kitao ◽  
Saori Ishida ◽  
Tomomitsu Sewaki ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Cell Surface ◽  
Fusion Protein ◽  
Lactobacillus Casei ◽  
Surface Display ◽  
Flow Cytometric Analysis ◽  
Soluble Starch ◽  
Engineering System ◽  
Acid Yield ◽  
Anchor Protein

ABSTRACT We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying α-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.

Construction of a Cell Surface Engineered Yeast Aims to Selectively Recover Molybdenum, a Rare Metal

2017 ◽  
Vol 262 ◽  
pp. 421-424 ◽  
Author(s):  
Mei Fang Chien ◽  
Naoya Ikeda ◽  
Kengo Kubota ◽  
Chihiro Inoue
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Rare Metal ◽  
Yeast Cells ◽  
Functional Domain ◽  
Rare Metals ◽  
Extraction Technology ◽  
Engineered Yeast ◽  
Immunofluorescence Labeling

The depletion of rare metals is an issue of major concern since rare metals are limited in the abundance but essential for high technology industry. However, the present rare metal recovery technical by chemical methods has high environmental impact, poor selectivity, and is too expensive to be practical. To resolve these problems, this study aimed to create a rare metal recover system using yeast, and molybdenum was selected as the first target. A molybdenum binding protein, ModE, which was derived from Escherichia coli was selected. A fusion gene was generated by linking partial modE with a secretion signal and a domain of α-agglutinin to display the ModE on the surface of yeast cells. The expression of fusion protein on the cell surface was detected by immunofluorescence labeling. As for the recovery experiment, the engineered yeast cells were incubated in 10 mM of sodium molybdate solution for 2 h, and the recovery of molybdenum ion was measured by ICP-AES. The results of fluorescence micrographs showed that the designed fusion protein was successfully expressed on yeast cell surface. According to the results of ICP-AES, the cell surface engineered yeast adsorbed molybdenum and the cells after 72~84 h incubation gave the best adsorption. Besides, the results suggested that the optimization of each functional domain in the fusion protein was important. The selectivity and the lower limit of recoverable concentration are under investigation, while this study provides a preliminary result of bio-extraction technology using cell surface engineered yeast.

Molecular Breeding of Polysaccharide-Utilizing Yeast Cells by Cell Surface Engineering

1998 ◽  
Vol 864 (1 ENZYME ENGINE) ◽  
pp. 528-537 ◽  
Author(s):  
MITSUYOSHI UEDA ◽  
TOSHIYUKI MURAI ◽  
YUMI SHIBASAKI ◽  
NAOMI KAMASAWA ◽  
MASAKO OSUMI ◽  
...  
Keyword(s):  
Cell Surface ◽  
Molecular Breeding ◽  
Surface Engineering ◽  
Yeast Cells ◽  
Cell Surface Engineering

Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

1977 ◽  
Vol 35 ◽  
pp. 596-597
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Synthetic Medium ◽  
Freeze Fracture ◽  
Translational Diffusion ◽  
Yeast Cells ◽  
Distilled Water ◽  
Intramembrane Particles ◽  
The Matrix ◽  
Water Cell

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

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