Display of lipase on the cell surface of <i>Escherichia coli</i> using OprF as an anchor and its application to enantioselective resolution in organic solvent

Lee, Seung Hwan; Choi, Jong‐il; Han, Mee‐Jung; Choi, Jong Hyun; Lee, Sang Yup

doi:10.1002/bit.20399

Cited by 42 publications

(46 citation statements)

References 41 publications

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“…To date, the expression systems for display of heterologous proteins on the surface of microorganisms have attained considerable progress in various applications in biotechnology and microbiology, for example whole-cell biocatalysis, vaccines vehicles, peptide library screening, biosensors, environmental bioadsorbents, and antibody production (Lee et al, 2003;Richins et al, 1997;Samuelson et al, 2002;Ståhl and Uhlén, 1997;Wernérus and Ståhl, 2004). In display systems for gram-negative bacteria, various anchoring motifs have been employed including outer membrane proteins (Francisco et al, 1992;Lee et al, 2005), lipoproteins, and ice nucleation protein (INP) (Jung et al, 1998a,b), poly-g-glutamate (PGA) synthetase complex (Narita et al, 2006). In fact, INP is a more promising display system for displaying enzymes (Jung et al, 1998a,b;Kim et al, 2000;Li et al, 2004;Shimazu et al, 2001aShimazu et al, ,b, 2003Wang et al, 2002).…”

Section: Introductionmentioning

confidence: 97%

Surface display of transglucosidase onEscherichia coli by using the ice nucleation protein ofXanthomonas campestris and its application in glucosylation of hydroquinone

Giridhar

2006

Biotechnol. Bioeng.

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A surface anchoring motif using the ice nucleation protein (INP) of Xanthomonas campestris pv. campestris BCRC 12,846 for display of transglucosidase has been developed. The transglucosidase gene from Xanthomonas campestris pv. campestris BCRC 12,608 was fused to the truncated ina gene. This truncated INP consisting of N- and C-terminal domains (INPNC) was able to direct the expressed transglucosidase fusion protein to the cell surface of E. coli with apparent high enzymatic activity. The localization of the truncated INPNC-transglucosidase fusion protein was examined by Western blot analysis and immunofluorescence labeling, and by whole-cell enzyme activity in the glucosylation of hydroquinone. The glucosylation reaction was carried out at 40 degrees C for 1 h, which gave 23 g/L of alpha-arbutin, and the molar conversion based on the amount of hydroquinone reached 83%. The use of whole-cells of the wild type strain resulted in an alpha-arbutin concentration of 4 g/L and a molar conversion of 16% only under the same conditions. The results suggested that E. coli displaying transglucosidase using truncated INPNC as an anchoring motif can be employed as a whole-cell biocatalyst in glucosylation.

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Section: Introductionmentioning

confidence: 97%

Surface display of transglucosidase onEscherichia coli by using the ice nucleation protein ofXanthomonas campestris and its application in glucosylation of hydroquinone

Giridhar

2006

Biotechnol. Bioeng.

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“…It was estimated that 1,534 lipase molecules were displayed per cell of P. putida harboring pMO188PL. This value is quite high compared with the value for E. coli cells displaying lipase using OmpC or OprF as an anchoring motif (14,15). It was also found that induction with a low IPTG FIG.…”

Section: Discussionmentioning

confidence: 89%

“…This property should be useful for enantioselective biocatalysis in an organic solvent. Lipases have been used as target proteins for several cell surface display systems (8,12,14,15,19,25). Of these, only yeast and E. coli have been employed for biocatalysis in an organic solvent at a moderate temperature.…”

Section: Discussionmentioning

confidence: 99%

Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

Lee¹,

Lee

Park

2005

Appl Environ Microbiol

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We developed a new cell surface display system in Pseudomonas putida KT2442 using OprF, an outer membrane protein of Pseudomonas aeruginosa, as an anchoring motif in a C-terminal deletion-fusion strategy. The Pseudomonas fluorescens SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of P. putida KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47°C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47°C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (R)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in P. putida KT2442 and consequently for various biocatalytic applications.

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“…Cell surface display allows peptides and proteins to be displayed on the surfaces of microbial cells by fusing them with the anchoring motif through recombinant DNA techniques. A wide range of applications of the display technology include bioadsorption (15)(16)(17)(18)(19)(20), whole-cell biocatalyst (21)(22)(23)(24)(25)(26)(27), peptide library screening (28,29), live vaccine development (30)(31)(32), antibody production (33), and biosensors (34,35). Although cell attachment through cell surface display is not a well-known application, cell attachment through displaying peptides with binding affinity has been reported on chitin, cellulose, and gold substrates.…”

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confidence: 99%

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

Park

Choi

Kim

et al. 2014

Appl Environ Microbiol

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c Mussels 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 on Escherichia coli surfaces 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 engineered E. coli exhibited 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 sticky E. coli is that no chemistry for cell attachment are necessary, and the ability of spontaneous E. coli attachment allows one to immobilize the cells on challenging material surfaces such as synthetic polymers. Therefore, we envision that mussel-inspired catecholamine yielded sticky E. coli that 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. Microbial cell attachment is a crucial process in the overall efficiency of a variety of systems that exploits the whole microbial cell, such as biocatalysts (1-9) or biosensors (10). Existing methods of attaching microbial cells to surfaces utilize various chemistries, such as adsorption (11), covalent attachment (12, 13), and entrapment within matrices (13, 14). However, adsorption often results in the detachment of bacteria due to the weak bond with the substrates, and covalent coupling requires crosslinking agents which results in the loss of cell activity and viability. The entrapment process requires harsh conditions in some cases, which jeopardizes cellular activity and viability. Another important issue is that the types of materials that the conventional methods can be applied to are largely limited due to the available surface chemistry. Therefore, cell attachment to variety of material surfaces without surface modification or harsh chemical treatment remains a great challenge. The facile cell attachment would circumvent the complex chemical modifications and reduce the loss of cellular activity and viability of the whole cell.As an emerging method for cell attachment, engineered cells expressing binding affinity tags on the cell surfaces through display technology have been utilized, although the approach is not prevalent. Cell surface display allows peptides and proteins to be displayed on the surfaces of mi...

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Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent

Cited by 42 publications

References 41 publications

Surface display of transglucosidase onEscherichia coli by using the ice nucleation protein ofXanthomonas campestris and its application in glucosylation of hydroquinone

Surface display of transglucosidase onEscherichia coli by using the ice nucleation protein ofXanthomonas campestris and its application in glucosylation of hydroquinone

Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

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

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