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References

  1. Metchnikoff E. 1995. Prolongation of Life. William Heinemann, London. UK.
  2. Sanders ME. 1998. Overview of functional foods: emphasis on probiotic bacteria. Int. Dairy J. 8: 341-347.
    CrossRef
  3. Wells JM, Robinson K, Chamberlain LM, Schofield KM, Le Page RW. 1996. Lactic acid bacteria as vaccine delivery vehicles. Antonie Van Leeuwenhoek 70: 317-330.
    Pubmed CrossRef
  4. Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, et al. 2000. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289: 1352-1355.
    Pubmed CrossRef
  5. Le Loir Y, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, Bermudez-Humaran LG, et al. 2005. Protein secretion in Lactococcus lactis: an efficient way to increase the overall heterologous protein production. Microb. Cell Fact. 4: 2.
    Pubmed PMC CrossRef
  6. Yeh CM, Huang XH, Sue CW. 2008. Functional secretion of a type 1 antifreeze protein analogue by optimization of promoter, signal peptide, prosequence, and terminator in Lactococcus lactis. J. Agric. Food Chem. 56: 8442-8450.
    Pubmed CrossRef
  7. Berlec A, Tompa G, Slapar N, Fonovic UP, Rogelj I, Strukelj B. 2008. Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis. Lett. Appl. Microbiol. 46: 227-231.
    Pubmed CrossRef
  8. Morello E, Bermudez-Humaran LG, Llull D, Sole V, Miraglio N, Langella P, Poquet I. 2008. Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion. J. Mol. Microbiol. Biotechnol. 14: 48-58.
    Pubmed CrossRef
  9. Le Loir Y, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, Bermudez-Humaran LG, et al. 2005. Protein secretion in Lactococcus lactis: an efficient way to increase the overall heterologous protein production. Microb. Cell Fact. 4: 2.
    Pubmed PMC CrossRef
  10. Kim JH, Mills DA. 2007. Improvement of a nisin-inducible expression vector for use in lactic acid bacteria. Plasmid. 58: 275-283.
    Pubmed CrossRef
  11. Lan CQ, Oddone G, Mills DA, Block DE. 2006. Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin. Biotechnol. Bioeng. 95: 1070-1080.
    Pubmed CrossRef
  12. Mierau I, Olieman K, Mond J, Smid EJ. 2005. Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications. Microb. Cell Fact. 4: 16.
    Pubmed PMC CrossRef
  13. Oddone GM, Lan CQ, Rawsthorne H, Mills DA, Block DE. 2007. Optimization of fed-batch production of the model recombinant protein GFP in Lactococcus lactis. Biotechnol. Bioeng. 96: 1127-1138.
    Pubmed CrossRef
  14. Rawsthorne H, Turner KN, Mills DA. 2006. Multicopy integration of heterologous genes, using the lactococcal group II intron targeted to bacterial insertion sequences. Appl. Environ. Microbiol. 72: 6088-6093.
    Pubmed PMC CrossRef
  15. Zhang GY, Mills DA, Block DE. 2009. Development of chemically defined media supporting high-cell-density growth of lactococci, enterococci, and streptococci. Appl. Environ. Microbiol. 75: 1080-1087.
    Pubmed PMC CrossRef
  16. Zhang GY, Block DE. 2009. Using highly efficient nonlinear experimental design methods for optimization of Lactococcus lactis fermentation in chemically defined media. Biotechnol. Prog. 25: 1587-1597.
    Pubmed
  17. Samoilis G, Psaroulaki A, Vougas K, Tselentis Y, Tsiotis G. 2007. Analysis of whole cell lysate from the intercellular bacterium Coxiella burnetii using two gel-based protein separation techniques. J. Proteome Res. 6: 3032-3041.
    Pubmed CrossRef
  18. Schmid AK, Lipton MS, Mottaz H, Monroe ME, Smith RD, Lidstrom ME. 2005. Global whole-cell FTICR mass spectrometric proteomics analysis of the heat shock response in the radioresistant bacterium Deinococcus radiodurans. J. Proteome Res. 4: 709-718.
    Pubmed CrossRef
  19. de Godoy LMF, Olsen JV, Cox J, Nielsen ML, Hubner NC, Frohlich F, et al. 2008. Comprehensive mass spectrometry based proteome quantification of haploid versus diploid yeast. Nature 455: 1251-1260.
    Pubmed CrossRef
  20. Jerez CA. 2008. The use of genomics, proteomics and other OMICS technologies for the global understanding of biomining microorganisms. Hydrometallurgy 94: 162-169.
    CrossRef
  21. Shah HN, Keys CJ, Schmid O, Gharbia SE. 2002. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry and proteomics: a new era in anaerobic microbiology. Clin. Infect. Dis. 35: S58-S64.
    Pubmed CrossRef
  22. Blow N. 2008. Mass spectrometry and proteomics: hitting the mark. Nat. Methods 5: 741-747.
    CrossRef
  23. Ahrends R, Pieper S, Kuhn A, Weisshoff H, Hamester M, Lindemann T, et al. 2007. A metal-coded affinity tag approach to quantitative proteomics. Mol. Cell. Proteomics 6: 1907-1916.
    Pubmed CrossRef
  24. Delahunty CM, Yates JR. 2007. MudPIT: multidimensional protein identification technology. Biotechniques 43: 563.
    Pubmed
  25. Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP. 2003. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100: 6940-6945.
    Pubmed PMC CrossRef
  26. Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A, Sevinsky JR, et al. 2005. Comparison of labelfree methods for quantifying human proteins by shotgun proteomics. Mol. Cell. Proteomics 4: 1487-1502.
    Pubmed CrossRef
  27. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1: 376-386.
    Pubmed CrossRef
  28. Putz S, Reinders J, Reinders Y, Sickmann A. 2005. Mass spectrometry-based peptide quantification: applications and limitations. Expert Rev. Proteomics 2: 381-392.
    Pubmed CrossRef
  29. Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, et al. 2001. The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res. 11: 731-753.
    Pubmed PMC CrossRef
  30. Paoletti AC, Parmely TJ, Tomomori-Sato C, Sato S, Zhu DX, Conaway RC, et al. 2006. Quantitative proteomic analysis of distinct mammalian mediator complexes using normalized spectral abundance factors. Proc. Natl. Acad. Sci. USA 103: 18928-18933.
    Pubmed PMC CrossRef
  31. Sardiu ME, Cai Y, Jin JJ, Swanson SK, Conaway RC, Conaway JW, et al. 2008. Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics. Proc. Natl. Acad. Sci. USA 105: 1454-1459.
    Pubmed PMC CrossRef
  32. Zybailov BL, Florens L, Washburn MP. 2007. Quantitative shotgun proteomics using a protease with broad specificity and normalized spectral abundance factors. Mol. Biosyst. 3: 354-360.
    Pubmed CrossRef
  33. Quadri LEN. 2002. Regulation of antimicrobial peptide production by autoinducer-mediated quorum sensing in lactic acid bacteria. Antonie Van Leeuwenhoek 82: 133-145.
    Pubmed CrossRef
  34. Simon D, Chopin A. 1988. Construction of a vector plasmid family and its use for molecular-cloning in Streptococcus lactis. Biochimie 70: 559-566.
    CrossRef
  35. Lechatelier E, Ehrlich SD, Janniere L. 1994. The pAMβ1 CopF repressor regulates plasmid copy number by controlling transcription of the repE gene. Mol. Microbiol. 14: 463-471.
    CrossRef
  36. Wang Z, Xiang L, Shao J, Wegrzyn A, Wegrzyn G. 2006. Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism. Microb. Cell Fact. 5: 34.
    Pubmed PMC CrossRef
  37. Bentley WE, Mirjalili N, Andersen DC, Davis RH, Kompala DS. 1990. Plasmid encoded protein - the principal factor in the metabolic burden associated with recombinant bacteria. Biotechnol. Bioeng. 35: 668-681.
    Pubmed CrossRef
  38. Chapot-Chartier MP, Vinogradov E, Sadovskaya I, Andre G, Mistou MY, Trieu-Cuot P, et al. 2010. Cell surface of Lactococcus lactis is covered by a protective polysaccharide pellicle. J. Biol. Chem. 285: 10464-10471.
    Pubmed PMC CrossRef
  39. Frees D, Ingmer H. 1999. ClpP participates in the degradation of misfolded protein in Lactococcus lactis. Mol. Microbiol. 31: 79-87.
    Pubmed CrossRef
  40. Oddone GM, Mills DA, Block DE. 2009. Dual inducible expression of recombinant GFP and targeted antisense RNA in Lactococcus lactis. Plasmid 62: 108-118.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2017; 27(7): 1345-1358

Published online July 28, 2017 https://doi.org/10.4014/jmb.1703.03065

Copyright © The Korean Society for Microbiology and Biotechnology.

Impact of High-Level Expression of Heterologous Protein on Lactococcus lactis Host

Mina Kim 1, Yerin Jin 2, Hyun-Joo An 2 and Jaehan Kim 1*

1Department of Food and Nutrition,, 2Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea

Received: March 30, 2017; Accepted: April 27, 2017

Abstract

The impact of overproduction of a heterologous protein on the metabolic system of host
Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403
containing two near-identical plasmids that expressed high- and low-level of the green
fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains
via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The
relative amount of each protein in the proteome of both strains was determined by label-free
quantification using the spectral counting method. Although expression level of most proteins
were similar, several significant alterations in metabolic network were identified in the high
GFP-producing strain. These changes include alterations in the pyruvate fermentation
pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for
pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and Nacetylglucosamine
from glucose was suppressed in the high GFP strain. In addition, enzymes
involved in the amino acid synthesis or interconversion pathway were downregulated. The
most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the
expression of stress response and chaperone proteins and increase of caseinolytic peptidase
family proteins. Characterization of these host expression changes witnessed during
overexpression of GFP was might suggested the metabolic requirements and networks that
may limit protein expression, and will aid in the future development of lactococcal hosts to
produce more heterologous protein.

Keywords: Comparative proteomics, green fluorescent protein, label-free quantification, Lactococcus lactis, systems biology, protein overexpression

References

  1. Metchnikoff E. 1995. Prolongation of Life. William Heinemann, London. UK.
  2. Sanders ME. 1998. Overview of functional foods: emphasis on probiotic bacteria. Int. Dairy J. 8: 341-347.
    CrossRef
  3. Wells JM, Robinson K, Chamberlain LM, Schofield KM, Le Page RW. 1996. Lactic acid bacteria as vaccine delivery vehicles. Antonie Van Leeuwenhoek 70: 317-330.
    Pubmed CrossRef
  4. Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, et al. 2000. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289: 1352-1355.
    Pubmed CrossRef
  5. Le Loir Y, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, Bermudez-Humaran LG, et al. 2005. Protein secretion in Lactococcus lactis: an efficient way to increase the overall heterologous protein production. Microb. Cell Fact. 4: 2.
    Pubmed KoreaMed CrossRef
  6. Yeh CM, Huang XH, Sue CW. 2008. Functional secretion of a type 1 antifreeze protein analogue by optimization of promoter, signal peptide, prosequence, and terminator in Lactococcus lactis. J. Agric. Food Chem. 56: 8442-8450.
    Pubmed CrossRef
  7. Berlec A, Tompa G, Slapar N, Fonovic UP, Rogelj I, Strukelj B. 2008. Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis. Lett. Appl. Microbiol. 46: 227-231.
    Pubmed CrossRef
  8. Morello E, Bermudez-Humaran LG, Llull D, Sole V, Miraglio N, Langella P, Poquet I. 2008. Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion. J. Mol. Microbiol. Biotechnol. 14: 48-58.
    Pubmed CrossRef
  9. Le Loir Y, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, Bermudez-Humaran LG, et al. 2005. Protein secretion in Lactococcus lactis: an efficient way to increase the overall heterologous protein production. Microb. Cell Fact. 4: 2.
    Pubmed KoreaMed CrossRef
  10. Kim JH, Mills DA. 2007. Improvement of a nisin-inducible expression vector for use in lactic acid bacteria. Plasmid. 58: 275-283.
    Pubmed CrossRef
  11. Lan CQ, Oddone G, Mills DA, Block DE. 2006. Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin. Biotechnol. Bioeng. 95: 1070-1080.
    Pubmed CrossRef
  12. Mierau I, Olieman K, Mond J, Smid EJ. 2005. Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications. Microb. Cell Fact. 4: 16.
    Pubmed KoreaMed CrossRef
  13. Oddone GM, Lan CQ, Rawsthorne H, Mills DA, Block DE. 2007. Optimization of fed-batch production of the model recombinant protein GFP in Lactococcus lactis. Biotechnol. Bioeng. 96: 1127-1138.
    Pubmed CrossRef
  14. Rawsthorne H, Turner KN, Mills DA. 2006. Multicopy integration of heterologous genes, using the lactococcal group II intron targeted to bacterial insertion sequences. Appl. Environ. Microbiol. 72: 6088-6093.
    Pubmed KoreaMed CrossRef
  15. Zhang GY, Mills DA, Block DE. 2009. Development of chemically defined media supporting high-cell-density growth of lactococci, enterococci, and streptococci. Appl. Environ. Microbiol. 75: 1080-1087.
    Pubmed KoreaMed CrossRef
  16. Zhang GY, Block DE. 2009. Using highly efficient nonlinear experimental design methods for optimization of Lactococcus lactis fermentation in chemically defined media. Biotechnol. Prog. 25: 1587-1597.
    Pubmed
  17. Samoilis G, Psaroulaki A, Vougas K, Tselentis Y, Tsiotis G. 2007. Analysis of whole cell lysate from the intercellular bacterium Coxiella burnetii using two gel-based protein separation techniques. J. Proteome Res. 6: 3032-3041.
    Pubmed CrossRef
  18. Schmid AK, Lipton MS, Mottaz H, Monroe ME, Smith RD, Lidstrom ME. 2005. Global whole-cell FTICR mass spectrometric proteomics analysis of the heat shock response in the radioresistant bacterium Deinococcus radiodurans. J. Proteome Res. 4: 709-718.
    Pubmed CrossRef
  19. de Godoy LMF, Olsen JV, Cox J, Nielsen ML, Hubner NC, Frohlich F, et al. 2008. Comprehensive mass spectrometry based proteome quantification of haploid versus diploid yeast. Nature 455: 1251-1260.
    Pubmed CrossRef
  20. Jerez CA. 2008. The use of genomics, proteomics and other OMICS technologies for the global understanding of biomining microorganisms. Hydrometallurgy 94: 162-169.
    CrossRef
  21. Shah HN, Keys CJ, Schmid O, Gharbia SE. 2002. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry and proteomics: a new era in anaerobic microbiology. Clin. Infect. Dis. 35: S58-S64.
    Pubmed CrossRef
  22. Blow N. 2008. Mass spectrometry and proteomics: hitting the mark. Nat. Methods 5: 741-747.
    CrossRef
  23. Ahrends R, Pieper S, Kuhn A, Weisshoff H, Hamester M, Lindemann T, et al. 2007. A metal-coded affinity tag approach to quantitative proteomics. Mol. Cell. Proteomics 6: 1907-1916.
    Pubmed CrossRef
  24. Delahunty CM, Yates JR. 2007. MudPIT: multidimensional protein identification technology. Biotechniques 43: 563.
    Pubmed
  25. Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP. 2003. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100: 6940-6945.
    Pubmed KoreaMed CrossRef
  26. Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A, Sevinsky JR, et al. 2005. Comparison of labelfree methods for quantifying human proteins by shotgun proteomics. Mol. Cell. Proteomics 4: 1487-1502.
    Pubmed CrossRef
  27. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1: 376-386.
    Pubmed CrossRef
  28. Putz S, Reinders J, Reinders Y, Sickmann A. 2005. Mass spectrometry-based peptide quantification: applications and limitations. Expert Rev. Proteomics 2: 381-392.
    Pubmed CrossRef
  29. Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, et al. 2001. The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res. 11: 731-753.
    Pubmed KoreaMed CrossRef
  30. Paoletti AC, Parmely TJ, Tomomori-Sato C, Sato S, Zhu DX, Conaway RC, et al. 2006. Quantitative proteomic analysis of distinct mammalian mediator complexes using normalized spectral abundance factors. Proc. Natl. Acad. Sci. USA 103: 18928-18933.
    Pubmed KoreaMed CrossRef
  31. Sardiu ME, Cai Y, Jin JJ, Swanson SK, Conaway RC, Conaway JW, et al. 2008. Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics. Proc. Natl. Acad. Sci. USA 105: 1454-1459.
    Pubmed KoreaMed CrossRef
  32. Zybailov BL, Florens L, Washburn MP. 2007. Quantitative shotgun proteomics using a protease with broad specificity and normalized spectral abundance factors. Mol. Biosyst. 3: 354-360.
    Pubmed CrossRef
  33. Quadri LEN. 2002. Regulation of antimicrobial peptide production by autoinducer-mediated quorum sensing in lactic acid bacteria. Antonie Van Leeuwenhoek 82: 133-145.
    Pubmed CrossRef
  34. Simon D, Chopin A. 1988. Construction of a vector plasmid family and its use for molecular-cloning in Streptococcus lactis. Biochimie 70: 559-566.
    CrossRef
  35. Lechatelier E, Ehrlich SD, Janniere L. 1994. The pAMβ1 CopF repressor regulates plasmid copy number by controlling transcription of the repE gene. Mol. Microbiol. 14: 463-471.
    CrossRef
  36. Wang Z, Xiang L, Shao J, Wegrzyn A, Wegrzyn G. 2006. Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism. Microb. Cell Fact. 5: 34.
    Pubmed KoreaMed CrossRef
  37. Bentley WE, Mirjalili N, Andersen DC, Davis RH, Kompala DS. 1990. Plasmid encoded protein - the principal factor in the metabolic burden associated with recombinant bacteria. Biotechnol. Bioeng. 35: 668-681.
    Pubmed CrossRef
  38. Chapot-Chartier MP, Vinogradov E, Sadovskaya I, Andre G, Mistou MY, Trieu-Cuot P, et al. 2010. Cell surface of Lactococcus lactis is covered by a protective polysaccharide pellicle. J. Biol. Chem. 285: 10464-10471.
    Pubmed KoreaMed CrossRef
  39. Frees D, Ingmer H. 1999. ClpP participates in the degradation of misfolded protein in Lactococcus lactis. Mol. Microbiol. 31: 79-87.
    Pubmed CrossRef
  40. Oddone GM, Mills DA, Block DE. 2009. Dual inducible expression of recombinant GFP and targeted antisense RNA in Lactococcus lactis. Plasmid 62: 108-118.
    Pubmed CrossRef