Expressing Xylanases in Escherichia Coli by Cell Surface Display

2013 ◽  
Vol 634-638 ◽  
pp. 965-969
Author(s):  
Mei Na Zhao ◽  
Zongbao Zheng ◽  
Tao Chen
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Cell Surface Display ◽  
Whole Cell ◽  
E Coli ◽  
Metabolic Burden ◽  
Whole Cell Biocatalyst ◽  
Anchor Proteins

In this research, xylan was utilized by a recombinant whole cell biocatalyst, which was developed by expressing three xylanases — β-xylosidase, endoxylanase, and α-arabinofuranosidase — on the surface of the E. coli BL21 (DE3). The xylanases were displayed on the surface of the cells by fusing with anchor proteins, Blc. The assimilation of xylan by cell surface display was the first step in the consolidated bioprocessing (CBP). This result shows that the engineering strains could be endowed with the ability to assimilate xylan. The co-display engineering strains utilized xylan and expressed less metabolic burden than the engineering strains secreting extracellular xylanases.

scholarly journals Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity

2008 ◽  
Vol 74 (24) ◽  
pp. 7733-7739 ◽  
Author(s):  
Chao Yang ◽  
Yaran Zhu ◽  
Jijian Yang ◽  
Zheng Liu ◽  
Chuanling Qiao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Ice Nucleation Protein ◽  
Whole Cell ◽  
E Coli ◽  
Dual Functional ◽  
Whole Cell Biocatalyst ◽  
Mineralizing Activity ◽  
Green Fluorescent

ABSTRACT Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of Escherichia coli by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered E. coli and a natural p-nitrophenol (PNP) degrader, Ochrobactrum sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites.

scholarly journals Enhanced Display of Lipase on the Escherichia coli Cell Surface, Based on Transcriptome Analysis

2009 ◽  
Vol 76 (3) ◽  
pp. 971-973 ◽  
Author(s):  
Jong Hwan Baek ◽  
Mee-Jung Han ◽  
Seung Hwan Lee ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Transcriptome Analysis ◽  
Surface Display ◽  
Cell Surface Display ◽  
Display System ◽  
E Coli ◽  
A Cell ◽  
Anchoring Motif ◽  
Cell Surface Display System

ABSTRACT A cell surface display system was developed using Escherichia coli OmpC as an anchoring motif. The fused Pseudomonas fluorescens SIK W1 lipase was successfully displayed on the surface of E. coli cells, and the lipase activity could be enhanced by the coexpression of the gadBC genes identified by transcriptome analysis.

scholarly journals Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis

2004 ◽  
Vol 70 (9) ◽  
pp. 5074-5080 ◽  
Author(s):  
Seung Hwan Lee ◽  
Jong-Il Choi ◽  
Si Jae Park ◽  
Sang Yup Lee ◽  
Byoung Chul Park
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Kinetic Resolution ◽  
Surface Display ◽  
Active Form ◽  
Enantiomeric Excess ◽  
Fatty Acid Transport ◽  
Whole Cell ◽  
E Coli ◽  
Anchoring Motif

ABSTRACT We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50°C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70°C, and retained over 90% of the full activity after incubation at 50°C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.

scholarly journals Cell Surface Display of Poliovirus Receptor on Escherichia coli, a Novel Method for Concentrating Viral Particles in Water

2011 ◽  
Vol 77 (15) ◽  
pp. 5141-5148 ◽  
Author(s):  
Morteza Abbaszadegan ◽  
Absar Alum ◽  
Hamed Abbaszadegan ◽  
Valerie Stout
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Water Samples ◽  
Stop Codon ◽  
Surface Display ◽  
Cell Surface Display ◽  
Bacterial Cells ◽  
Content Type ◽  
E Coli ◽  
Poliovirus Receptor

ABSTRACTThe lack of efficient methods for concentrating viruses in water samples leads to underreporting of viral contamination in source water. A novel strategy for viral concentration was developed using the expression of target virus receptors on bacterial cells. Poliovirus type 1, the most studied enterovirus, was used as a surrogate for enteric viruses. The human poliovirus receptor (hPVR) gene was expressed on the surface ofEscherichia colicells by using the ice nucleation protein (INP) gene. ThehPVRgene was ligated to the 3′ end of theINPgene after the removal of the stop codon. The resulting open reading frame (ORF) was used for the projection of hPVR onto the outer membrane ofE. coli. Gene expression was tested by SDS-PAGE, Western blot, and dot blot analyses, and virion capture ability was confirmed by transmission electron microscopy. The application of engineeredE. colicells for capturing viruses in 1-liter samples of source and drinking water resulted in 75 to 99% procedural recovery efficiency. Cell surface display of viral receptors on bacterial cells opens a new prospect for an efficient and inexpensive alternative tool for capturing and concentrating waterborne viruses in water samples.

Cell Surface Display of Yarrowia lipolytica Lipase Lip2p Using the Cell Wall Protein YlPir1p, Its Characterization, and Application as a Whole-Cell Biocatalyst

2015 ◽  
Vol 175 (8) ◽  
pp. 3888-3900 ◽  
Author(s):  
Evgeniya Y. Yuzbasheva ◽  
Tigran V. Yuzbashev ◽  
Natalia I. Perkovskaya ◽  
Elizaveta B. Mostova ◽  
Tatiana V. Vybornaya ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Yarrowia Lipolytica ◽  
Surface Display ◽  
Cell Surface Display ◽  
Cell Wall Protein ◽  
Whole Cell ◽  
Whole Cell Biocatalyst

Research on Construction of a Whole-Cell Biocatalyst for Xylan Degradation

2011 ◽  
Vol 347-353 ◽  
pp. 2599-2603
Author(s):  
Jian Zhang Lu ◽  
Mei Lin Cui ◽  
Shan Shan Du ◽  
Lu Yang ◽  
Qin Guo ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Display ◽  
Cell Surface Display ◽  
Whole Cell ◽  
Secretion Signal ◽  
Gal1 Promoter ◽  
Dry Cell Weight ◽  
Whole Cell Biocatalyst ◽  
Genes Encoding ◽  
Secretion Expression

Endo-1,4-β-xylanase (E.C.3.2.1.8) is a family of glycoside hydrolase. It is capable of hydrolyzing the backbone of substituted xylan polymers into fragments of random size. Due to this ability, xylanase can serve the degradation of lignocellulose, and facilitate the application of xylan. Cell-surface display of enzymes is one of the most attractive applications in yeast. It is a promising utilization in constructing the whole-cell biocatalyst of xylanase. For this purpose, a cDNA sequence of endo-1,4-β-xylanase B (XylB) from Aspergillus niger BCC14405 was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae. The genes encoding galactokinase (GAL1) promoter, α-mating factor 1 (MFα1) pre-pro secretion signal, fully codon-optimized XylB, the 320 amino acids of C terminal of α-agglutinin, alcohol dehydrogenase (ADH1) terminator and kanMX cassette were amplified and cloned into YEplac181 to construct a cell-surface display vector called pGMAAK-XylB with α-agglutinin as an anchor. Then pGMAAK-XylB was transformed into S. cerevisiae. The results show XylB was immobilized and actively expressed on S. cerevisiae. Meanwhile, a secretion expression plasmid was also constructed using the above elements except α-agglutinin as a control strain in the study of characteristic of XylB. After an induction of 48 h by 2% galactose, the activity of displayed XylB reached 63 U/g dry-cell weight. The optimal pH of displayed XylB has changed from 5 to 6 and the optimal temperature has changed from 50 °C to 60 °C, comparing to the recombinant secretion XylB.

scholarly journals Preparation of Sticky Escherichia coli through Surface Display of an Adhesive Catecholamine Moiety

2013 ◽  
Vol 80 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Joseph P. Park ◽  
Min-Jung Choi ◽  
Se Hun Kim ◽  
Seung Hwan Lee ◽  
Haeshin Lee
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Communication Systems ◽  
Cell Attachment ◽  
Surface Display ◽  
Content Type ◽  
E Coli ◽  
Material Surfaces ◽  
Mussel Adhesive ◽  
Adhesive Proteins

ABSTRACTMussels attach to virtually all types of inorganic and organic surfaces in aqueous environments, and catecholamines composed of 3,4-dihydroxy-l-phenylalanine (DOPA), lysine, and histidine in mussel adhesive proteins play a key role in the robust adhesion. DOPA is an unusual catecholic amino acid, and its side chain is called catechol. In this study, we displayed the adhesive moiety of DOPA-histidine onEscherichia colisurfaces using outer membrane protein W as an anchoring motif for the first time. Localization of catecholamines on the cell surface was confirmed by Western blot and immunofluorescence microscopy. Furthermore, cell-to-cell cohesion (i.e., cellular aggregation) induced by the displayed catecholamine and synthesis of gold nanoparticles on the cell surface support functional display of adhesive catecholamines. The engineeredE. coliexhibited significant adhesion onto various material surfaces, including silica and glass microparticles, gold, titanium, silicon, poly(ethylene terephthalate), poly(urethane), and poly(dimethylsiloxane). The uniqueness of this approach utilizing the engineered stickyE. coliis that no chemistry for cell attachment are necessary, and the ability of spontaneousE. coliattachment allows one to immobilize the cells on challenging material surfaces such as synthetic polymers. Therefore, we envision that mussel-inspired catecholamine yielded stickyE. colithat can be used as a new type of engineered microbe for various emerging fields, such as whole living cell attachment on versatile material surfaces, cell-to-cell communication systems, and many others.

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