Skip to main content
SpringerLink
Log in

Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production

Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

For the efficient production of poly(3-hydroxybutyrate) (PHB) using recombinant Escherichia coli, it is of primal importance to overproduce NADPH, which is necessary for the PHB synthetic pathway. In order to overproduce NADPH in the pentose phosphate (PP) pathway, a recombinant E. coli was constructed in which the phosphoglucose isomerase (pgi) gene was knocked out to force the carbon flow into the PP pathway. The fermentation characteristics of the recombinant E. coli mutant lacking pgi were then investigated to determine the effect of overproduction of NADPH on efficient PHB production. It was found that, compared with the parent strain (E. coli JM109), growth of the E. coli mutant lacking pgi (E. coli DF11) is repressed due to NADPH overproduction in the PP pathway. Furthermore, repressed cell growth can be recovered to some extent by introducing a NADPH-consuming pathway, such as the PHB synthetic pathway. Efficient PHB production using such recombinant E. coli (DF11/pAeKG1) could be attained by appropriately controlling the glucose concentration in the fermentor. Total gene expression was investigated at the protein level by two-dimensional electrophoresis. Out of 22 differentially expressed proteins, 12 were identified with the aid of MALDI-TOF mass spectrometry. Variations in the accumulation of PHB in the recombinant pgi mutant carrying phb (E. coli DF11/pAeKG1) corresponded to the expression of proteins encoded by rpsA, znuA, fabD, potD, fkpA, gapA, ynaF and ibpA. The unfavorable conditions generated by PHB accumulation in the pgi mutant carrying phb resulted in the highest expression of 30S ribosomal protein S1, which ultimately caused a further increase in soluble protein synthesis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price includes VAT (France)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.
Fig. 9.

References

  • Arnold CN, Mcelhanon J, Lee A, Leonhart R, Siegele DA (2001) Global analysis of Escherichia coli gene expression during the acetate-induced acid tolerance response. J Bacteriol 183:2178–2186

    Article  CAS  PubMed  Google Scholar 

  • Bergmeyer HU, (1989) Methods of enzymatic analysis, vol VII, 3rd edn. VCH, Weinheim, Germany

  • Blackstock WP, Weir MP (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol 17:121–127

    CAS  PubMed  Google Scholar 

  • Boonstra B, French CE, Wainwright I, Bruce NC (1999) The udhA gene of Escherichia coli encodes a soluble pyridine nucleotide transhydrogenase. J Bacteriol 181:1030–1034

    CAS  PubMed  Google Scholar 

  • Braunegg G, Sonnteither B, Lafferty RM (1978) A rapid gas chromatography method for the determination of poly-β-hydroxybutyric acid in microbial biomass. Eur J Appl Microbiol Biotechnol 6:29–37

    Google Scholar 

  • Canonaco F, Hess TA, Heri S, Wang T, Szyperski T, Sauer U (2001) Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA. FEMS Microbiol Lett 204:247–252

    Article  CAS  PubMed  Google Scholar 

  • Clarke DM, Loo TW, Gillam S, Bragg PD (1986) Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli. Eur J Biochem 158:647–653

    CAS  PubMed  Google Scholar 

  • Fraenkel DG, Levisohn SR (1967) Glucose and gluconate metabolism in an Escherichia coli mutant lacking phosphoglucose isomerase. J Bacteriol 93:1571–1578

    CAS  PubMed  Google Scholar 

  • Han M-J, Yoon SS, Lee SY (2001) Proteome analysis of metabolically engineered Escherichia coli producing poly (3-hydroxybutyrate). J Bacteriol 183:301–308

    Article  CAS  PubMed  Google Scholar 

  • Hanson RL, Rose C (1980) Effects of insertion mutation in a locus affecting pyridine nucleotide transhydrogenase (pnt::Tn5) on the growth of Escherichia coli. J Bacteriol 141:401–404

    CAS  PubMed  Google Scholar 

  • Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C (1993) Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein databases. Proc Natl Acad Sci USA 90:5011–5015

    Google Scholar 

  • Hochstrasser DE (1998) Proteome in perspective. Clin Chem Lab Med 36:825–836

    PubMed  Google Scholar 

  • Hochstrasser DF, Harrington MG, Hochstrasser AC, Miller MJ, Merril CR (1988) Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal Biochem 173:424–435

    CAS  PubMed  Google Scholar 

  • Kabir MM, Shimizu K (2001) Proteome analysis of a temperature-inducible recombinant Escherichia coli for poly-β-hydroxybutyrate production. J Biosci Bioeng 92:277–284

    Article  CAS  Google Scholar 

  • Kalousek S, Lubitz W (1995) High-level poly(β-hydroxybutyrate) production in recombinant Escherichia coli in sugar free complex medium. Can J Microbiol 41:216–221

    CAS  PubMed  Google Scholar 

  • Kidwell J, Valentin HE, Dennis D (1995) Regulated expression of the Alcaligenes eutrophus pha biosynthesis gene in Escherichia coli. Appl Environ Microbiol 61:1391–1398

    CAS  PubMed  Google Scholar 

  • Kimata K, Tanaka Y, Inada T, Aiba H (2001) Expression of the glucose transporter gene, ptsG, is regulated at the mRNA degradation step in response to glycolytic flux in Escherichia coli. EMBO J 20:3587–3595

    Article  CAS  PubMed  Google Scholar 

  • Lee SY (1994) Suppression of filamentation in recombinant Escherichia coli by amplified ftsZ activity. Biotechnol Lett 16:1247–1252

    CAS  Google Scholar 

  • Lee SY, Chang HN (1995) Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studies. Can J Microbiol 41:207-215

    CAS  PubMed  Google Scholar 

  • Lee SY, Yim KS, Chang HN (1994a) Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) in Escherichia coli. J Biotechnol 32:203–211

    Article  CAS  PubMed  Google Scholar 

  • Lee SY, Lee KM, Chang HN, Steinbüchel A (1994b) Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly(3-hydroxybutyric acid) and morphological changes. Biotechnol Bioeng 44:1337–1347

    CAS  Google Scholar 

  • Link AJ, Robison K, Church GM (1997) Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrophoresis 18:1259–313

    CAS  PubMed  Google Scholar 

  • Lowery OH, Rosbrough JN, Farr AL, Randall RJ (1951) Protein measurement with the folin-phenol regent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  • Missiakas D, Betton J-M, Raina S (1996) New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH. Mol Microbiol 21:871-884

    CAS  PubMed  Google Scholar 

  • O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021

    PubMed  Google Scholar 

  • Pappin DJ, Hojrup P, Bleasby A (1993) Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol 3:327–332

    CAS  Google Scholar 

  • Patterson SD (2000) Mass spectrometry and proteomics. Physiol Genomics 2:59–65

    CAS  PubMed  Google Scholar 

  • Peoples OP, Sinskey AJ (1989) Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). J Biol Chem 264:15298–15303

    CAS  PubMed  Google Scholar 

  • Persidis A (1998) Proteomics. Nat Biotechnol 16:393–394

    CAS  PubMed  Google Scholar 

  • Potapov AP, Subramanian AR (1992). Biochem Int 27:745–753

    CAS  PubMed  Google Scholar 

  • Rosenfeld J, Capdevielle J, Guillemot J, Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after one-or two dimensional gel electrophoresis. Anal Biochem 203:173–179

    CAS  PubMed  Google Scholar 

  • Salas M, Vinuela E, Sols A (1965) Spontaneous and enzymatically catalyzed anomerization of glucose-6-phosphate and anomeric specificity of related enzymes. J Biol Chem 240:561–568

    CAS  Google Scholar 

  • Schubert P, Steinbuchel A, Schlegel HG (1988) Cloning of the Alcaligenes eutrophus poly-β-hydroxybutyrate synthetic pathway and synthesis of PHB in Escherichia coli. J Bacteriol 170:5837–5847

    CAS  PubMed  Google Scholar 

  • Sengupta J, Agrawal RK, Frank J (2001) Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA. Proc Natl Acad Sci USA 98:11991–6

    Article  CAS  PubMed  Google Scholar 

  • Shi H, Nikawa J, Shimizu K (1999) Effect of modifying metabolic network on poly-3-hydroxybutyrate biosynthesis in recombinant Escherichia coli. J Biosci Bioeng 87:666–677

    Article  CAS  Google Scholar 

  • Shi H, Kyuwa K, Takasu M, Shimizu K (2001) Temperature-induced expression of phb genes in Escherichia coli and effect of temperature patterns on the production of poly-3-hydroxybutyrate. J Biosci Bioeng 91:21–26

    Article  CAS  Google Scholar 

  • Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858

    Article  CAS  PubMed  Google Scholar 

  • Sim SJ, Snell KD, Hogan SA, Stubbe J, Rha C, Sinskey AJ (1997) PHA synthesis activity control molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nat Biotechnol 15:63–67

    CAS  PubMed  Google Scholar 

  • Slater SC, Voige WH, Dennis DE (1988) Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-β-hydroxybutyrate biosynthetic pathway. J Bacteriol 170:4431–4436

    CAS  PubMed  Google Scholar 

  • Stebbins CE, Borukhov S, Orlova M, Polyakov A, Goldfarb A, Darst SA (1995) Crystal structure of the greA transcript cleavage factor from Escherichia coli. Nature 373:636–640

    CAS  PubMed  Google Scholar 

  • Tzareva NV, Makhno VI, Boni IV (1994). FEBS Lett 337:189–194

    Article  CAS  PubMed  Google Scholar 

  • Van der Werf MJ, Guettle MV, Jain MK, Zeikus JG (1997) Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch Microbiol 167:332–342

    PubMed  Google Scholar 

  • Wieczorek RA, Pries A, Steinbüchel A, Mayer F (1995) Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligens eutrophus. J Bacteriol 177:2425–2435

    CAS  PubMed  Google Scholar 

  • Wittmann HG (1974) In: Namura M, Tissieres A, Lengyel P (eds) Ribosomes. Cold Spring Harbor Laboratory, Plainview, New York, pp 93–114

Download references

Acknowledgement

This research was supported in part by a grant from New Energy and Industrial Technology Development Organization (NEDO) of the Ministry of Economy, Trade and Industry of Japan (Development of Technological Infrastructure for Industrial Bioprocess Project).

Author information

Authors and Affiliations

  1. Department of Biochemical Engineering and Science, Kyushu Institute of Technology, 820–8502, Iizuka, Fukuoka , Japan

    Md. M. Kabir & K. Shimizu

  2. Institute for Advanced Biosciences, Keio University, 997–0017, Tsuruoka, Yamagata , Japan

    K. Shimizu

Authors
  1. Md. M. Kabir
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K. Shimizu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to K. Shimizu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kabir, M.M., Shimizu, K. Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production. Appl Microbiol Biotechnol 62, 244–255 (2003). https://doi.org/10.1007/s00253-003-1257-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-003-1257-z

Keywords

search

Navigation