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Research article

References

  1. Lee SY. 1996. Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 49: 1-14.
    Pubmed CrossRef
  2. Madison LL, Huisman GW. 1999. Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev. 63: 21-53.
    Pubmed PMC CrossRef
  3. Steinbüchel A, Valentin HE. 1995. Diversity of bacterial polyhydroxyalkanoic acid. FEMS Microbiol. Lett. 128: 219-228.
    CrossRef
  4. Steinbüchel A, Füchtenbusch B. 1998. Bacterial and other biological systems for polyester production. Trends Biotechnol. 16: 419-427.
    Pubmed CrossRef
  5. Jendrossek D, Handrick R. 2002. Microbial degradation of polyhydroxyalkanoates. Annu. Rev. Microbiol. 56: 403-432.
    Pubmed CrossRef
  6. Kumar A, Gross RA, Jendrossek D. 2000. Poly(3-hydroxybutyrate)depolymerase from Pseudomonas lemoignei: Catalysis of Esterifications in Organic Media. J. Org. Chem. 65: 7800-7806.
    Pubmed CrossRef
  7. Lee SJ, Park JP, Park TJ, Lee SY, Lee S, Park JK. 2005. Selective immobilization of fusion proteins on poly(hydroxyalkanoate) microbeads. Anal. Chem. 77: 5755-5759.
    Pubmed CrossRef
  8. Georgiou G, Stathopoulos C, Daugherty PS, Nayak AR, Iverson BL, Curtiss RI. 1997. Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat. Biotechnol. 15: 29-34.
    Pubmed CrossRef
  9. Chen W, Georgiou G. 2002. Cell-surface display of heterologous proteins: from high-throughput screening to environmental applications. Biotechnol. Bioeng. 79: 496-503.
    Pubmed CrossRef
  10. Lee SY, Choi JH, Xu J. 2003. Microbial cell surface display. Trends Biotechnol. 21: 45-52.
    Pubmed CrossRef
  11. Benhar I. 2001. Biotechnological applications of phage and cell display. Biotechnol. Adv. 19: 1-33.
    Pubmed CrossRef
  12. 2015. Engineering novel and improved biocatalysts by cell surface display. Ing. Eng. Chem. Res. 54: 4021-4031.
    Pubmed PMC CrossRef
  13. Shimazu M, Mulchandani A, Chen W. 2001. Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnol. Prog. 17: 76-80.
    Pubmed CrossRef
  14. Lee SH, Choi J, Han M-J, Choi JH, Lee SY. 2005. Display of lipase on the cell surface of Escherichia coli using OprF as an anchoring motif and its application to enatioselective resolution in organic solvent. Biotechnol. Bioeng. 90: 223-230.
    Pubmed CrossRef
  15. Matsumoto T, Ito M, Fukuda H, Kondo A. 2004. Enantioselective transesterification using lipase-displaying yeast whole-cell biocatalyst. Appl. Microbiol. Biotechnol. 64: 481-485.
    Pubmed CrossRef
  16. Hiraishi T, Yamashita K, Sakono M, Nakanishi J, Tan L-T, Sudesh K, et al. 2012. Display of functionally active PHB depolymerase on Escherichia coli cell surface. Macromol. Biosci. 12: 218-224.
    Pubmed CrossRef
  17. Tan L-T, Hiraishi T, Sudesh K, Maeda M. 2013. Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285. Appl. Microbiol. Biotechnol. 97: 4859-4871.
    Pubmed CrossRef
  18. Saito T, Suzuki K, Yamamoto J, Fukui T, Miwa K, Tomita K, et al. 1989. Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. J. Bacteriol. 171: 184-189.
    Pubmed PMC CrossRef
  19. Lee SH, Choi J, Park SJ, Lee SY, Park BC. 2004. D isplay of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis. Appl. Environ. Microbiol. 70: 5074-5080.
    Pubmed PMC CrossRef
  20. Schumacher SD, Hannemann F, Teese MG, Bernhardt R, Jose J. 2012. Autodisplay of functional CYP106A2 in Escherichia coli. J. Biotechnol. 161: 104-112.
    Pubmed CrossRef
  21. Chen Z, Wang Y, Cheng Y, Wang X, Tong S, Yang H, et al. 2020. Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase. Sci. Total Environ. 709: 136138.
    Pubmed CrossRef
  22. Lee SH, Lee SY, Park B. 2005. Cell surface display of lipase on the Pseudomonas putida using OprF as an anchoring motif and its biocatalytic applications. Appl. Environ. Microbiol. 71: 8581-8586.
    Pubmed PMC CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2020; 30(2): 244-247

Published online February 28, 2020 https://doi.org/10.4014/jmb.2001.01042

Copyright © The Korean Society for Microbiology and Biotechnology.

Cell Surface Display of Poly(3-hydroxybutyrate) Depolymerase and its Application

Seung Hwan Lee 1* and Sang Yup Lee 2

1Department of Biotechnology and Bioengineering, Chonnam National University, Republic of Korea, 2Department of Chemical and Biomolecular Engineering (BK21 Program), Institute of BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea

Received: January 22, 2020; Accepted: February 7, 2020

Abstract

We have expressed extracellular poly(3-hydroxybutyrate) (PHB) depolymerase of Ralstonia pickettii T1 on the Escherichia coli surface using Pseudomonas OprF protein as a fusion partner by C-terminal deletion-fusion strategy. Surface display of depolymerase was confirmed by flow cytometry, immunofluorescence microscopy and whole cell hydrolase activity. For the application, depolymerase was used as an immobilized catalyst of enantioselective hydrolysis reaction for the first time. After 48 h, (R)-methyl mandelate was completely hydrolyzed, and (S)-mandelic acid was produced with over 99% enantiomeric excess. Our findings suggest that surface displayed depolymerase on E. coli can be used as an enantioselective biocatalyst.

Keywords: Cell surface display, depolymerase, immobilization, enantioselective biocatalys, whole cell biocatalyst, OprF

Acknowledgment

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters, that accumulate intracellularly as a carbon and/or energy source in numerous bacteria, to provide renewable resources under nutrient limited conditions. In PHA-accumulating bacteria, many enzymes, including PHA synthase, phasin, epimerase, oligomer hydrolase and depolymerase, play a role in the biosynthesis and degradation of PHAs [1-4]. Of those, PHA depolymerases have drawn much attention for their novel characteristics such as high stability, relatively small molecular weight (< 70 kDa), consisting of only one polypeptide, and strong affinity to hydrophobic materials [5]. Some bacteria secrete depoly-merases to degrade extracellular PHAs and utilize the resulting monomer, 3-hydroxyalkanoic acid, as a nutrient. Several researchers have begun to employ depolymerases as enantioselective catalysts for biotransformation, and selective binding in immunoassays [6, 7].

Thus far, various surface display systems have been reported, with potential applications in the development of vaccines, bioadsorbents, biocatalysts, antibody libraries and biosensors [8-10]. In addition, many researchers have demonstrated the use of enzymes as target proteins for cell surface display due to their unique characteristics, such as chemo-, regio- and enantioselectivity [11,12]. Among the numerous available enzymes only a few have been adapted for cell surface display applications, such as lipase and organophosphorus hydrolase for biocatalysts and bioreme-diation, respectively [13-15]. Therefore, the development of new useful enzymes for display systems is an important research objective. Recently, a Japanese research group successfully applied cell surface display of PHB depolymerase, however, the application was limited to the degradation of the polyester to produce 3-hydroxybutyrate [16, 17]. In this paper, we report the display of the Ralstonia pickettii T1 depolymerase on the surface of E. coli cells using truncated Pseudomonas OprF as an anchoring motif and demonstrate the biocatalytic application of this system to enantioselective hydrolysis of a racemic ester.

To construct pTacOprF164RD and pTacOprF188RD, R. pickettii T1 depolymerase gene was amplified using primers 5’-gctctagagccacggcggggcccggtgc-3’ and 5-cccaagctttcatgg acaattgccgacg-3’, and inserted into the XbaI and HindIII sites of pTacOprF164PL and pTacOprF188PL, respectively. For the immunodetection, 6 histidine residues (underlined) were added to C-terminal of depolymerase gene, which was amplified using primers 5’-gctctagagccacggcggggcccggtgc-3’ and 5’-cccaagctttcaatggtgatgatggtgatgtggacaattgccgacg-3’, and cloned into the XbaI and HindIII sites of pTacOprF164PL and pTacOprF188PL to make pTacOprF164RDH and pTacOprF188RDH, respectively. Primers for the amplification of the R. pickettii T1 PHB depolymerase gene were designed based on the reported sequence (J04223) [18]. Plasmids used in this study are listed in Table 1.

Table 1 . List of plasmids used in this study..

PlasmidRelevant CharacteristicsReference or Source
pTacOprF164PLpTacOprF164 derivative;[14]
P. fluorescens SIK W1 lipase gene
pTacOprF188PLpTacOprF188 derivative;[14]
P. fluorescens SIK W1 lipase gene
pTacOprF188EpTac99A derivative; containing 636 bp fragment of oprF of P. aeruginosa and the stop codon[14]
pTacOprF164RDR. pickettii T1 depolymerase geneThis study
pTacOprF164RDHR. pickettii T1 depolymerase gene with 6 histidine residuesThis study
pTacOprF188RDR. pickettii T1 depolymerase geneThis study
pTacOprF188RDHR. pickettii T1 depolymerase gene with 6 histidine residuesThis study


For detection of displayed depolymerase fused with 6 histidine residues, recombinant cells were labeled with mouse anti-His antibody (Sigma, USA) diluted 1:100, followed by rabbit anti-mouse IgG antibody conjugated fluorescein isothiocyanate (FITC) diluted 1:200. The cells were analyzed using a flow cytometer (FACSCalibur, Becton Dickinson, USA). For the visualization under fluorescence microscope, cells were stained with the mouse anti-His antibody diluted 1:1000 and rabbit anti-mouse IgG conjugated with FITC diluted 1:3000. The samples were visualized by confocal fluorescence microscopy (Zeiss LSM 410, Carl Zeiss, Germany).

After collection by centrifugation at 5,590 ×g and 4°C and washing with distilled water, recombinant cells were further freeze-dried for 48 h with lyophilizer (TFD5505, Ilshin Lab., Korea) for measuring whole cell enzyme activity and application of enantioselective biocatalyst. Hydrolytic activity of genetically immobilized depolymerase was assayed by spectrophotometric method as mentioned elsewhere [14]. In order to observe the enantioselective catalytic property of cell surface displayed depolymerase (Fig. 1), 300 mg of freeze-dried XL10-Gold (pTacO188RD) and 150 mg of racemic methyl mandelate (Sigma) were added to 30 ml of 50 mM Tris buffer (pH 8.0). Reaction mixture was maintained at 250 rpm at 37oC. Quantification of chemicals were determined by HPLC as previously described [19].

Figure 1. Reaction scheme for the enantioselective hydrolysis of racemic methyl mandelic acid.

To date, cell surface display systems have employed many enzymes including lipase, polyethylene terephthalate (PET) degrading enzyme (PETase), cyotochrom P450, amylase, dimeric bovine adrenodoxin and carboxymethylcellulase as target proteins [11, 12, 20, 21]. As we mentioned earlier, PHB depolymerases have ideal characteristics for cell surface display applications. P. aeruginosa PaO1 OprF was selected for depolymerase display as it allows successful and stable display of proteins in an active form on the surface of E. coli and P. putida [14, 22].

First, surface display of the depolymerase was examined by flow cytometer analysis. Fluorescence was analyzed for E. coli (pTacOprF188E) and E. coli (pTacOprF188RDH) incubated with anti-His antibody followed by FITC-conjugated secondary antibody. As shown in Fig. 2A, the mean fluorescence values of XL 10-Gold harboring pTacOprF188RDH was increased when compared to that of XL 10-Gold harboring pTacOprF188E indicating that the fusion protein of truncated OprF (OprFt) and depolymerase, with 6 histidine residues, was expressed outside of the E. coli. In order to confirm surface display of depolymerase, recombinant cells were analyzed using immunofluorescence microscopy. E. coli XL-10 Gold (pTacOprF188RDH) labeled with anti-His antibody followed by binding of FITC-conjugated secondary antibody showed fluorescent spots, which demonstrates depolymerase fused with 6 histidine residues was successfully expressed on the cell surface (Fig.2C). Meanwhile, E. coli XL-10 Gold cells harboring pTacOprF188E showed little fluorescence (Fig. 2B).

Figure 2. Flow cytometry analysis (A) of E. coli XL-10 Gold harboring pTacOprF188E and pTacOprF188RDH. Differential interference micrographs (left) and immunofluorescence micrographs (right) of E. coli XL-10 Gold harboring pTacOprF188E (B) and pTacOprF188RDH (C). Cells were incubated with mouse anti-His probe antibody followed by probing with rabbit anti-mouse IgG-FITC conjugate.

After confirmation of surface display, we further checked whole cell hydrolase activity to ascertain functional expression of depolymerase. After normalizing hydrolase activity for recombinant XL 10-Gold (pTacOprF188RD) obtained with 0.1 mM IPTG induction to a value of 100, the relative activities with 0.01 and 1 mM IPTG were 21.3 and 64.8, respectively. Little hydrolase activity was observed in supernatant fractions. These suggest that OprFt successfully played the role of an anchoring motif for the surface display of depolymerase on the E. coli surface in an active form, without protein secretion and significant cell lysis.

For application in biocatalysts, the enantioselective resolution of racemic methyl mandelate was investigated. The scheme of this reaction is shown in Fig. 1. Time profiles of enantiomeric excess of product during enantioselective resolution are shown in Fig. 3. The conversion of the reaction and enantiomeric excess of the product, (S)-mandelic acid, obtained in 48 h were over 40% and 99%, respectively.

Figure 3. Time profiles for the enantioselective resolution of racemic methyl mandelic acid using cell surface displayed depolymerase. Time profiles of enantiomeric excess (●) of reaction products are shown.

It is known that PHA depolymerase shows enantioselective behavior towards substrate in aqueous solutions, however, the main application of surface displayed depolymerase has been the hydrolysis of polymer or oligomer, composed of monomeric units of 3-hydroxyacid [5, 16, 17]. Here, we have shown for the first time, that cell surface displayed depolymerase can enantioselectively hydrolyze not only polymers or oligomers of PHB, but racemic esters. As shown in Fig. 3, depolymerase displayed on the cell surface exhibited good enzymatic characteristics, such as high enantiomeric excess and conversion as an enantioselective catalyst. It is clear that depolymerase is stably expressed at the outer membrane as an active form, which is very similar to an immobilized form of enzyme without elimination of selectivity and activity. These suggest that E. coli cells displaying depolymerase have potential as industrial catalysts similar to other hydrolases.

In conclusion, we reported the cell surface display of PHB depolymerase on the cell surface of E. coli and demonstrate the application of this system to the enantioselective biotransformation for the first time. Furthermore, improvement of the enzyme characteristics via methods such as immobilization, fusion technique and directed evolution, could result in significant increases in activity, selective binding affinity or other key characteristics of depolymerases. Thus, the results of our efforts can be successfully applied to the various fields including bioremediation, biocatalysis, immunoassay, whole cell microarrays and whole cell biosensors.

Conflict of Interest

This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the MSIT through the NRF of Korea (NRF-2015M1A2A2035814) and Basic Science Research Program through the NRF of Korea funded by the Ministry of Education (NRF-2019R1I1A3A01063492).

Fig 1.

Figure 1.Reaction scheme for the enantioselective hydrolysis of racemic methyl mandelic acid.
Journal of Microbiology and Biotechnology 2020; 30: 244-247https://doi.org/10.4014/jmb.2001.01042

Fig 2.

Figure 2.Flow cytometry analysis (A) of E. coli XL-10 Gold harboring pTacOprF188E and pTacOprF188RDH. Differential interference micrographs (left) and immunofluorescence micrographs (right) of E. coli XL-10 Gold harboring pTacOprF188E (B) and pTacOprF188RDH (C). Cells were incubated with mouse anti-His probe antibody followed by probing with rabbit anti-mouse IgG-FITC conjugate.
Journal of Microbiology and Biotechnology 2020; 30: 244-247https://doi.org/10.4014/jmb.2001.01042

Fig 3.

Figure 3.Time profiles for the enantioselective resolution of racemic methyl mandelic acid using cell surface displayed depolymerase. Time profiles of enantiomeric excess (●) of reaction products are shown.
Journal of Microbiology and Biotechnology 2020; 30: 244-247https://doi.org/10.4014/jmb.2001.01042

Table 1 . List of plasmids used in this study..

PlasmidRelevant CharacteristicsReference or Source
pTacOprF164PLpTacOprF164 derivative;[14]
P. fluorescens SIK W1 lipase gene
pTacOprF188PLpTacOprF188 derivative;[14]
P. fluorescens SIK W1 lipase gene
pTacOprF188EpTac99A derivative; containing 636 bp fragment of oprF of P. aeruginosa and the stop codon[14]
pTacOprF164RDR. pickettii T1 depolymerase geneThis study
pTacOprF164RDHR. pickettii T1 depolymerase gene with 6 histidine residuesThis study
pTacOprF188RDR. pickettii T1 depolymerase geneThis study
pTacOprF188RDHR. pickettii T1 depolymerase gene with 6 histidine residuesThis study

References

  1. Lee SY. 1996. Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 49: 1-14.
    Pubmed CrossRef
  2. Madison LL, Huisman GW. 1999. Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev. 63: 21-53.
    Pubmed KoreaMed CrossRef
  3. Steinbüchel A, Valentin HE. 1995. Diversity of bacterial polyhydroxyalkanoic acid. FEMS Microbiol. Lett. 128: 219-228.
    CrossRef
  4. Steinbüchel A, Füchtenbusch B. 1998. Bacterial and other biological systems for polyester production. Trends Biotechnol. 16: 419-427.
    Pubmed CrossRef
  5. Jendrossek D, Handrick R. 2002. Microbial degradation of polyhydroxyalkanoates. Annu. Rev. Microbiol. 56: 403-432.
    Pubmed CrossRef
  6. Kumar A, Gross RA, Jendrossek D. 2000. Poly(3-hydroxybutyrate)depolymerase from Pseudomonas lemoignei: Catalysis of Esterifications in Organic Media. J. Org. Chem. 65: 7800-7806.
    Pubmed CrossRef
  7. Lee SJ, Park JP, Park TJ, Lee SY, Lee S, Park JK. 2005. Selective immobilization of fusion proteins on poly(hydroxyalkanoate) microbeads. Anal. Chem. 77: 5755-5759.
    Pubmed CrossRef
  8. Georgiou G, Stathopoulos C, Daugherty PS, Nayak AR, Iverson BL, Curtiss RI. 1997. Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat. Biotechnol. 15: 29-34.
    Pubmed CrossRef
  9. Chen W, Georgiou G. 2002. Cell-surface display of heterologous proteins: from high-throughput screening to environmental applications. Biotechnol. Bioeng. 79: 496-503.
    Pubmed CrossRef
  10. Lee SY, Choi JH, Xu J. 2003. Microbial cell surface display. Trends Biotechnol. 21: 45-52.
    Pubmed CrossRef
  11. Benhar I. 2001. Biotechnological applications of phage and cell display. Biotechnol. Adv. 19: 1-33.
    Pubmed CrossRef
  12. 2015. Engineering novel and improved biocatalysts by cell surface display. Ing. Eng. Chem. Res. 54: 4021-4031.
    Pubmed KoreaMed CrossRef
  13. Shimazu M, Mulchandani A, Chen W. 2001. Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnol. Prog. 17: 76-80.
    Pubmed CrossRef
  14. Lee SH, Choi J, Han M-J, Choi JH, Lee SY. 2005. Display of lipase on the cell surface of Escherichia coli using OprF as an anchoring motif and its application to enatioselective resolution in organic solvent. Biotechnol. Bioeng. 90: 223-230.
    Pubmed CrossRef
  15. Matsumoto T, Ito M, Fukuda H, Kondo A. 2004. Enantioselective transesterification using lipase-displaying yeast whole-cell biocatalyst. Appl. Microbiol. Biotechnol. 64: 481-485.
    Pubmed CrossRef
  16. Hiraishi T, Yamashita K, Sakono M, Nakanishi J, Tan L-T, Sudesh K, et al. 2012. Display of functionally active PHB depolymerase on Escherichia coli cell surface. Macromol. Biosci. 12: 218-224.
    Pubmed CrossRef
  17. Tan L-T, Hiraishi T, Sudesh K, Maeda M. 2013. Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285. Appl. Microbiol. Biotechnol. 97: 4859-4871.
    Pubmed CrossRef
  18. Saito T, Suzuki K, Yamamoto J, Fukui T, Miwa K, Tomita K, et al. 1989. Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. J. Bacteriol. 171: 184-189.
    Pubmed KoreaMed CrossRef
  19. Lee SH, Choi J, Park SJ, Lee SY, Park BC. 2004. D isplay of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis. Appl. Environ. Microbiol. 70: 5074-5080.
    Pubmed KoreaMed CrossRef
  20. Schumacher SD, Hannemann F, Teese MG, Bernhardt R, Jose J. 2012. Autodisplay of functional CYP106A2 in Escherichia coli. J. Biotechnol. 161: 104-112.
    Pubmed CrossRef
  21. Chen Z, Wang Y, Cheng Y, Wang X, Tong S, Yang H, et al. 2020. Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase. Sci. Total Environ. 709: 136138.
    Pubmed CrossRef
  22. Lee SH, Lee SY, Park B. 2005. Cell surface display of lipase on the Pseudomonas putida using OprF as an anchoring motif and its biocatalytic applications. Appl. Environ. Microbiol. 71: 8581-8586.
    Pubmed KoreaMed CrossRef