Skip to main content
SpringerLink
Log in

Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences

  • Applied genetics and molecular biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~10−8/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10−8/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

Similar content being viewed by others

References

  • Arbabi-Ghahroudi M, Tanha J, MacKenzie R (2005) Prokaryotic expression of antibodies. Cancer Metastasis Rev 24(4):501–519. doi:10.1007/s10555-005-6193-1

    Article  PubMed  Google Scholar 

  • Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22(11):1399–1408. doi:10.1038/nbt1029

    Article  CAS  PubMed  Google Scholar 

  • Barbotin JN (1994) Immobilization of recombinant bacteria. A strategy to improve plasmid stability. Ann N Y Acad Sci 721:303–309

    Article  CAS  PubMed  Google Scholar 

  • Bell CE (2005) Structure and mechanism of Escherichia coli RecA ATPase. Mol Microbiol 58(2):358–366. doi:10.1111/j.1365-2958.2005.04876.x

    Article  CAS  PubMed  Google Scholar 

  • 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(7):668–681. doi:10.1002/bit.260350704

    Article  CAS  PubMed  Google Scholar 

  • Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997) The complete genome sequence of Escherichia coli K-12. Science 277(5331):1453–1462

    Article  CAS  PubMed  Google Scholar 

  • Bower DM, Prather KL (2009) Engineering of bacterial strains and vectors for the production of plasmid DNA. Appl Microbiol Biotechnol 82(5):805–813. doi:10.1007/s00253-009-1889-8

    Article  CAS  PubMed  Google Scholar 

  • Charlesworth B, Sniegowski P, Stephan W (1994) The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371(6494):215–220

    Article  CAS  PubMed  Google Scholar 

  • Csorgo B, Feher T, Timar E, Blattner FR, Posfai G (2012) Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs. Microb Cell Factories 11:11. doi:10.1186/1475-2859-11-11

    Article  CAS  Google Scholar 

  • Durfee T, Nelson R, Baldwin S, Plunkett G 3rd, Burland V, Mau B, Petrosino JF, Qin X, Muzny DM, Ayele M, Gibbs RA, Csorgo B, Posfai G, Weinstock GM, Blattner FR (2008) The complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse. J Bacteriol 190(7):2597–2606. doi:10.1128/JB.01695-07

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Friehs K (2004) Plasmid copy number and plasmid stability. Adv Biochem Eng Biotechnol 86:47–82

    CAS  PubMed  Google Scholar 

  • Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, Smith HO (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319(5867):1215–1220. doi:10.1126/science.1151721

    Article  CAS  PubMed  Google Scholar 

  • Gillet R, Felden B (2001) Emerging views on tmRNA-mediated protein tagging and ribosome rescue. Mol Microbiol 42(4):879–885

    Article  CAS  PubMed  Google Scholar 

  • Glick BR (1995) Metabolic load and heterologous gene expression. Biotechnol Adv 13(2):247–261

    Article  CAS  PubMed  Google Scholar 

  • Ikeda M (2003) Amino acid production processes. Adv Biochem Eng Biotechnol 79:1–35

    CAS  PubMed  Google Scholar 

  • Jain C (2002) Degradation of mRNA in Escherichia coli. IUBMB Life 54(6):315–321. doi:10.1080/15216540216036

    Article  CAS  PubMed  Google Scholar 

  • Jana S, Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67(3):289–298. doi:10.1007/s00253-004-1814-0

    Article  CAS  PubMed  Google Scholar 

  • Jang SA, Sung BH, Cho JH, Kim SC (2009) Direct expression of antimicrobial peptides in an intact form by a translationally coupled two-cistron expression system. Appl Environ Microbiol 75(12):3980–3986. doi:10.1128/AEM.02753-08

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Keasling JD (1999) Gene-expression tools for the metabolic engineering of bacteria. Trends Biotechnol 17(11):452–460

    Article  CAS  PubMed  Google Scholar 

  • Keseler IM, Mackie A, Peralta-Gil M, Santos-Zavaleta A, Gama-Castro S, Bonavides-Martinez C, Fulcher C, Huerta AM, Kothari A, Krummenacker M, Latendresse M, Muniz-Rascado L, Ong Q, Paley S, Schroder I, Shearer AG, Subhraveti P, Travers M, Weerasinghe D, Weiss V, Collado-Vides J, Gunsalus RP, Paulsen I, Karp PD (2013) EcoCyc: fusing model organism databases with systems biology. Nucleic Acids Res 41(Database issue):D605–D612. doi:10.1093/nar/gks1027

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kim MH, Billiar TR, Seol DW (2004) The secretable form of trimeric TRAIL, a potent inducer of apoptosis. Biochem Biophys Res Commun 321(4):930–935. doi:10.1016/j.bbrc.2004.07.046

    Article  CAS  PubMed  Google Scholar 

  • Kwok R (2010) Five hard truths for synthetic biology. Nature 463(7279):288–290. doi:10.1038/463288a

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  CAS  PubMed  Google Scholar 

  • Lee SY (1996) High cell-density culture of Escherichia coli. Trends Biotechnol 14(3):98–105. doi:10.1016/0167-7799(96)80930-9

    Article  CAS  PubMed  Google Scholar 

  • Lee C, Kim J, Shin SG, Hwang S (2006) Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol 123(3):273–280. doi:10.1016/j.jbiotec.2005.11.014

    Article  CAS  PubMed  Google Scholar 

  • Lee JH, Sung BH, Kim MS, Blattner FR, Yoon BH, Kim JH, Kim SC (2009) Metabolic engineering of a reduced-genome strain of Escherichia coli for l-threonine production. Microb Cell Factories 8:2. doi:10.1186/1475-2859-8-2

    Article  Google Scholar 

  • Lesic B, Zouine M, Ducos-Galand M, Huon C, Rosso ML, Prevost MC, Mazel D, Carniel E (2012) A natural system of chromosome transfer in Yersinia pseudotuberculosis. PLoS Genet 8(3):e1002529. doi:10.1371/journal.pgen.1002529

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25(4):402–408. doi:10.1006/meth.2001.1262

    Article  CAS  PubMed  Google Scholar 

  • Luo Q, Shen YL, Wei DZ, Cao W (2006) Optimization of culture on the overproduction of TRAIL in high-cell-density culture by recombinant Escherichia coli. Appl Microbiol Biotechnol 71(2):184–191. doi:10.1007/s00253-005-0131-6

    Article  CAS  PubMed  Google Scholar 

  • Morales-Soto N, Forst SA (2011) The xnp1 P2-like tail synthesis gene cluster encodes xenorhabdicin and is required for interspecies competition. J Bacteriol 193(14):3624–3632. doi:10.1128/JB.00092-11

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nakamura K, Inouye M (1981) Inactivation of the Serratia marcescens gene for the lipoprotein in Escherichia coli by insertion sequences, IS1 and IS5; sequence analysis of junction points. Mol Gen Genet 183(1):107–114

    Article  CAS  PubMed  Google Scholar 

  • Nesbeth DN, Perez-Pardo MA, Ali S, Ward J, Keshavarz-Moore E (2012) Growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain. Biotechnol Bioeng 109(2):517–527. doi:10.1002/bit.23316

    Article  CAS  PubMed  Google Scholar 

  • O'Kennedy RD, Baldwin C, Keshavarz-Moore E (2000) Effects of growth medium selection on plasmid DNA production and initial processing steps. J Biotechnol 76(2–3):175–183

    Article  PubMed  Google Scholar 

  • Parkhill J, Wren BW, Thomson NR, Titball RW, Holden MT, Prentice MB, Sebaihia M, James KD, Churcher C, Mungall KL, Baker S, Basham D, Bentley SD, Brooks K, Cerdeno-Tarraga AM, Chillingworth T, Cronin A, Davies RM, Davis P, Dougan G, Feltwell T, Hamlin N, Holroyd S, Jagels K, Karlyshev AV, Leather S, Moule S, Oyston PC, Quail M, Rutherford K, Simmonds M, Skelton J, Stevens K, Whitehead S, Barrell BG (2001) Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413(6855):523–527. doi:10.1038/35097083

    Article  CAS  PubMed  Google Scholar 

  • Peternel S, Komel R (2011) Active protein aggregates produced in Escherichia coli. Int J Mol Sci 12(11):8275–8287. doi:10.3390/ijms12118275

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pfleger BF, Pitera DJ, Smolke CD, Keasling JD (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol 24(8):1027–1032. doi:10.1038/nbt1226

    Article  CAS  PubMed  Google Scholar 

  • Posfai G, Plunkett G 3rd, Feher T, Frisch D, Keil GM, Umenhoffer K, Kolisnychenko V, Stahl B, Sharma SS, de Arruda M, Burland V, Harcum SW, Blattner FR (2006) Emergent properties of reduced-genome Escherichia coli. Science 312(5776):1044–1046. doi:10.1126/science.1126439

    Article  CAS  PubMed  Google Scholar 

  • Rawat P, Kumar S, Pental D, Burma PK (2009) Inactivation of a transgene due to transposition of insertion sequence (IS136) of Agrobacterium tumefaciens. J Biosci 34(2):199–202. doi:10.1007/s12038-009-0023-5

    Article  CAS  PubMed  Google Scholar 

  • Riesenberg D, Menzel K, Schulz V, Schumann K, Veith G, Zuber G, Knorre WA (1990) High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1. Appl Microbiol Biotechnol 34(1):77–82

    CAS  PubMed  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Schneider D, Lenski RE (2004) Dynamics of insertion sequence elements during experimental evolution of bacteria. Res Microbiol 155(5):319–327. doi:10.1016/j.resmic.2003.12.008

    Article  CAS  PubMed  Google Scholar 

  • Schneider D, Duperchy E, Coursange E, Lenski RE, Blot M (2000) Long-term experimental evolution in Escherichia coli: IX. Characterization of insertion sequence-mediated mutations and rearrangements. Genetics 156(2):477–488

    CAS  PubMed Central  PubMed  Google Scholar 

  • Serres MH, Goswami S, Riley M (2004) GenProtEC: an updated and improved analysis of functions of Escherichia coli K-12 proteins. Nucleic Acids Res 32(Database issue):D300–D302. doi:10.1093/nar/gkh087

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Siguier P, Varani A, Perochon J, Chandler M (2012) Exploring bacterial insertion sequences with ISfinder: objectives, uses, and future developments. Methods Mol Biol 859:91–103. doi:10.1007/978-1-61779-603-6_5

    Article  CAS  PubMed  Google Scholar 

  • Solyga A, Bartosik D (2004) Entrapment vectors—how to capture a functional transposable element. Pol J Microbiol 53(3):139–144

    CAS  PubMed  Google Scholar 

  • Sprenger M (2011) ECDC and the Escherichia coli outbreak in Germany. Lancet 377(9784):2180. doi:10.1016/S0140-6736(11)60963-X

    Article  PubMed  Google Scholar 

  • Stroeher UH, Jedani KE, Dredge BK, Morona R, Brown MH, Karageorgos LE, Albert MJ, Manning PA (1995) Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139. Proc Natl Acad Sci U S A 92(22):10374–10378

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tobes R, Pareja E (2006) Bacterial repetitive extragenic palindromic sequences are DNA targets for Insertion Sequence elements. BMC Genomics 7:62. doi:10.1186/1471-2164-7-62

    Article  PubMed Central  PubMed  Google Scholar 

  • Umenhoffer K, Feher T, Baliko G, Ayaydin F, Posfai J, Blattner FR, Posfai G (2010) Reduced evolvability of Escherichia coli MDS42, an IS-less cellular chassis for molecular and synthetic biology applications. Microb Cell Factories 9:38. doi:10.1186/1475-2859-9-38

    Article  Google Scholar 

  • Vallejo LF, Brokelmann M, Marten S, Trappe S, Cabrera-Crespo J, Hoffmann A, Gross G, Weich HA, Rinas U (2002) Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli. J Biotechnol 94(2):185–194

    Article  CAS  PubMed  Google Scholar 

  • Wang ZJ, Le GW, Shi YH, Wegrzyn G (2001) Medium design for plasmid DNA production based on stoichiometric model. Process Biochem 36(11):1085–1093. doi:10.1016/S0032-9592(01)00149-2

    Article  CAS  Google Scholar 

  • Win MN, Smolke CD (2007) A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. Proc Natl Acad Sci U S A 104(36):14283–14288. doi:10.1073/pnas.0703961104

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA (1988) Novel regulators of bone formation: molecular clones and activities. Science 242(4885):1528–1534

    Article  CAS  PubMed  Google Scholar 

  • Yamazaki Y, Niki H, Kato J (2008) Profiling of Escherichia coli Chromosome database. Methods Mol Biol 416:385–389. doi:10.1007/978-1-59745-321-9_26

    Article  CAS  PubMed  Google Scholar 

  • Yau SY, Keshavarz-Moore E, Ward J (2008) Host strain influences on supercoiled plasmid DNA production in Escherichia coli: implications for efficient design of large-scale processes. Biotechnol Bioeng 101(3):529–544. doi:10.1002/bit.21915

    Article  CAS  PubMed  Google Scholar 

  • Yu Y, Lee C, Kim J, Hwang S (2005) Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89(6):670–679. doi:10.1002/bit.20347

    Article  CAS  PubMed  Google Scholar 

  • Yu BJ, Kang KH, Lee JH, Sung BH, Kim MS, Kim SC (2008) Rapid and efficient construction of markerless deletions in the Escherichia coli genome. Nucleic Acids Res 36(14):e84. doi:10.1093/nar/gkn359

    Article  PubMed Central  PubMed  Google Scholar 

  • Zhou J, Rudd KE (2013) EcoGene 3.0. Nucleic Acids Res 41(Database issue):D613–D624. doi:10.1093/nar/gks1235

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (2011–0031955) and the Next-Generation BioGreen 21 Program (SSAC; PJ008110). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Department of Biological Sciences, KAIST, Daejeon, 305-701, South Korea

    Myung Keun Park, Sang Hee Lee, Kyung Seok Yang, Suk-Chae Jung & Sun Chang Kim

  2. KAIST Institute for Biocentury, KAIST, Daejeon, 305-701, South Korea

    Jun Hyoung Lee

Authors
  1. Myung Keun Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sang Hee Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kyung Seok Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Suk-Chae Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jun Hyoung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sun Chang Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jun Hyoung Lee or Sun Chang Kim.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 56 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Park, M.K., Lee, S.H., Yang, K.S. et al. Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences. Appl Microbiol Biotechnol 98, 6701–6713 (2014). https://doi.org/10.1007/s00253-014-5739-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-5739-y

Keywords

Search

Navigation