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Applied Microbiology and Biotechnology

, Volume 103, Issue 17, pp 7177–7189 | Cite as

Construction of a novel Escherichia coli expression system: relocation of lpxA from chromosome to a constitutive expression vector

  • Lei Zhao
  • Xiaoqing Hu
  • Ye Li
  • Zhen Wang
  • Xiaoyuan WangEmail author
Methods and protocols
  • 112 Downloads

Abstract

The selective marker in the plasmid-based expression system is usually a gene that encodes an antibiotic-resistant protein; therefore, the antibiotic has to add to maintain the plasmid when growing the bacteria. This antibiotic addition would lead to increase of production cost and the environment contamination. In this study, a novel Escherichia coli expression system, the lpxA deletion mutant harboring an lpxA-carrying vector, was developed. To develop this system, three plasmids pCas9Cre, pTF-A-UD, and pRSFCmlpxA were constructed. The plasmid pCas9Cre produces enzymes Cas9, λ-Red, and Cre and can be cured by growing at 42 °C; pTF-A-UD contains several DNA fragments required for deleting the chromosomal lpxA and can be cured by adding isopropyl-D-thiogalactopyranoside; pRSFCmlpxA contains the lpxA mutant lpxA123 and CamR. When E. coli were transformed with these three plasmids, the chromosomal lpxA and the CamR in pRSFCmlpxA can be efficiently removed, resulting in an E. coli lpxA mutant harboring pRSFlpxA. The lpxA is essential for the growth of E. coli; its relocation from chromosome to a constitutive expression vector is an ideal strategy to maintain the vector without antibiotic addition. The lpxA123 in pRSFlpxA can complement the deletion of the chromosomal lpxA and provide a strong selective pressure to maintain the plasmid pRSFlpxA. This study provides an experimental evidence that this novel expression system is convenient and efficient to use and can be used to improve l-threonine biosynthesis in the wild type E. coli MG1655 and an l-threonine producing E. coli TWF006.

Keywords

Escherichia coli lpxA Expression system lpxA-carrying vector Cas9 l-threonine production 

Notes

Funding information

This study was supported by the National Key R&D Program of China (2017YFC1600102), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-10), and the Collaborative Innovation Center of Jiangsu Modern Industrial Fermentation.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Compliance with ethics requirements

This article does not contain any studies with human participants or animals.

References

  1. Aiello AE, Larson E (2003) Antibacterial cleaning and hygiene products as an emerging risk factor for antibiotic resistance in the community. Lancet Infect Dis 3(8):501–506Google Scholar
  2. Ali SA, Chew YW (2015) FabV/triclosan is an antibiotic-free and cost-effective selection system for efficient maintenance of high and medium-copy number plasmids in Escherichia coli. PLoS One 10(6):e0129547.  https://doi.org/10.1371/journal.pone.0129547 Google Scholar
  3. Ali SA, Chew YW, Omar TC, Azman N (2015) Use of FabV-triclosan plasmid selection system for efficient expression and production of recombinant proteins in Escherichia coli. PLoS One 10(12):e0144189.  https://doi.org/10.1371/journal.pone.0144189 Google Scholar
  4. Arakawa H, Lodygin D, Buerstedde JM (2001) Mutant loxP vectors for selectable marker recycle and conditional knock-outs. BMC Biotechnol 1(1):7Google Scholar
  5. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2(2006):0008.  https://doi.org/10.1038/msb4100050 Google Scholar
  6. Benson RE, Gottlin EB, Christensen DJ, Hamilton PT (2003) Intracellular expression of peptide fusions for demonstration of protein essentiality in bacteria. Antimicrob Agents Chemother 47(9):2875–2881.  https://doi.org/10.1128/aac.47.9.2875-2881.2003 Google Scholar
  7. Cadot (2010) Expression system for the antibiotic-free production of polypeptides. United States Patent US2010/0248306A1Google Scholar
  8. Cooper TF, Heinemann JA (2000) Postsegregational killing does not increase plasmid stability but acts to mediate the exclusion of competing plasmids. Proc Natl Acad Sci U S A 97(23):12643–12648Google Scholar
  9. Cranenburgh RM, Hanak JAJ, Williams SG, Sherratt DJ (2001) Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration. Nucleic Acids Res 29:e26Google Scholar
  10. Crowell DN, Anderson MS, Raetz CR (1986) Molecular cloning of the genes for lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase in Escherichia coli. J Bacteriol 168(1):152–159Google Scholar
  11. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97(12):6640–6645Google Scholar
  12. Davison J (1999) Genetic exchange between bacteria in the environment. Plasmid 42(2):73–91Google Scholar
  13. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471(7340):602–607Google Scholar
  14. Dong WR, Xiang LX, Shao JZ (2010) Novel antibiotic-free plasmid selection system based on complementation of host auxotrophy in the NAD de novo synthesis pathway. Appl Environ Microbiol 76(7):2295–2303.  https://doi.org/10.1128/AEM.02462-09 Google Scholar
  15. Enyeart PJ, Chirieleison SM, Mai ND, Perutka J, Quandt EM, Yao J, Whitt JT, Keatinge-Clay AT, Lambowitz AM, Ellington AD (2013) Generalized bacterial genome editing using mobile group II introns and Cre-lox. Mol Syst Biol 9(1):685Google Scholar
  16. Fujishima H, Nishimura A, Wachi M, Takagi H, Hirasawa T, Teraoka H, Nishimori K, Kawabata T, Nishikawa K, Nagai K (2002) kdsA mutations affect FtsZ-ring formation in Escherichia coli K-12. Microbiology 148(1):103–112Google Scholar
  17. Galan JE, Curtiss R (1991) Distribution of the invA. -B, -C, and -D genes of Salmonella typhimurium among other Salmonella serovars: InvA mutants of Salmonella typhi are deficient for entry into mammalian cells. Infect Immun 59(9):2901–2908Google Scholar
  18. Galloway SM, Raetz CR (1990) A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J Biol Chem 265(11):6394–6402Google Scholar
  19. Gopaul DN, Guo F, Van Duyne GD (2014) Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J 17(14):4175–4187Google Scholar
  20. Hagg P, de Pohl JW, Abdulkarim F, Isaksson LA (2004) A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli. J Biotechnol 111(1):17–30.  https://doi.org/10.1016/j.jbiotec.2004.03.010 Google Scholar
  21. Han Y, Li Y, Chen J, Tan Y, Guan F, Wang X (2013) Construction of monophosphoryl lipid A producing Escherichia coli mutants and comparison of immuno-stimulatory activities of their lipopolysaccharides. Mar Drugs 11(2):363–376.  https://doi.org/10.3390/md11020363 Google Scholar
  22. Hu J, Li Y, Zhang H, Tan Y, Wang X (2014) Construction of a novel expression system for use in Corynebacterium glutamicum. Plasmid 75:18–26.  https://doi.org/10.1016/j.plasmid.2014.07.005 Google Scholar
  23. Jawale CV, Lee JH (2013) Development of a biosafety enhanced and immunogenic Salmonella Enteritidis ghost using an antibiotic resistance gene free plasmid carrying a bacteriophage lysis system. PLoS One 8(10):11.  https://doi.org/10.1371/journal.pone.0078193 Google Scholar
  24. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3):233–239.  https://doi.org/10.1038/nbt.2508 Google Scholar
  25. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81(7):2506–2514.  https://doi.org/10.1128/AEM.04023-14 Google Scholar
  26. Junjie Y, Bingbing S, He H, Yu J, Liuyang D, Biao C, Chongmao X, Xin W, Jinle L, Weihong J (2014) High-efficiency scarless genetic modification in Escherichia coli by using lambda red recombination and I-SceI cleavage. Appl Environ Microbiol 80(13):3826–3834Google Scholar
  27. Karberg M, Guo H, Zhong J, Coon R, Perutka J, Lambowitz AM (2001) Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat Biotechnol 19(12):1162–1167Google Scholar
  28. Koros A, Varga Z, Molnar-Perl I (2008) Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography. J Chromatogr A 1203(2):146–152.  https://doi.org/10.1016/j.chroma.2008.07.035 Google Scholar
  29. Kroll J, Klinter S, Schneider C, Voss I, Steinbuchel A (2010) Plasmid addiction systems: perspectives and applications in biotechnology. Microb Biotechnol 3(6):634–657.  https://doi.org/10.1111/j.1751-7915.2010.00170.x Google Scholar
  30. Krute CN, Krausz KL, Markiewicz MA, Joyner JA, Pokhrel S, Hall PR, Bose JL (2016) Generation of a stable plasmid for in vitro and in vivo studies of Staphylococcus species. Appl Environ Microbiol 82(23):6859–6869.  https://doi.org/10.1128/AEM.02370-16 Google Scholar
  31. 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.  https://doi.org/10.1186/1475-2859-8-2 Google Scholar
  32. Liang A, Riaz H, Dong F, Luo X, Yu X, Han Y, Chong Z, Han L, Guo A, Yang L (2014) Evaluation of efficacy, biodistribution and safety of antibiotic-free plasmid encoding somatostatin genes delivered by attenuated Salmonella enterica serovar Choleraesuis. Vaccine 32(12):1368–1374.  https://doi.org/10.1016/j.vaccine.2014.01.026 Google Scholar
  33. Luke J, Carnes AE, Hodgson CP, Williams JA (2009) Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system. Vaccine 27(46):6454–6459Google Scholar
  34. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-Guided human genome engineering via Cas9. Science 339(6121):823–826.  https://doi.org/10.1126/science.1232033 Google Scholar
  35. Nilsson J, Skogman SG (1986) Stabilization of Escherichia coli tryptophan-production vectors in continuous cultures: a comparison of three different systems. Bio/Technology 4:901–903Google Scholar
  36. Nina C, Court DL (2003) Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants. Proc Natl Acad Sci U S A 100(26):15748–15753Google Scholar
  37. Ogura T, Hiraga S (1983) Mini-F plasmid genes that couple host cell division to plasmid proliferation. Proc Natl Acad Sci U S A 80:4784–4788Google Scholar
  38. Oliveira PH, Mairhofer J (2013) Marker-free plasmids for biotechnological applications - implications and perspectives. Trends Biotechnol 31(9):539–547.  https://doi.org/10.1016/j.tibtech.2013.06.001 Google Scholar
  39. Peubez I, Chaudet N, Mignon C, Hild G, Husson S, Courtois V, De Luca K, Speck D, Sodoyer R (2010) Antibiotic-free selection in E. coli: new considerations for optimal design and improved production. Microb Cell Factories 9:65Google Scholar
  40. Pósfai G, Kolisnychenko V, Bereczki Z, Blattner FR (1999) Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucleic Acids Res 27(22):4409–4415Google Scholar
  41. Sánchez-Pascuala A, De LV, Nikel PI (2017) Refactoring the Embden-Meyerhof-Parnas pathway as a whole of portable GlucoBricks for implantation of glycolytic modules in Gram-negative bacteria. ACS Synth Biol 6(5):793–805Google Scholar
  42. Selvamani RSV, Telaar M, Friehs K, Flaschel E (2014) Antibiotic-free segregational plasmid stabilization in Escherichia coli owing to the knockout of triosephosphate isomerase (tpiA). Microb Cell Factories 13:58Google Scholar
  43. Shan G, Good L (2008) Plasmid selection in Escherichia coli using an endogenous essential gene marker. BMC Biotechnol 8(1):1–9Google Scholar
  44. Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4(2):206–223Google Scholar
  45. Tacket CO, Kelly SM, Schodel F, Losonsky G, Nataro JP, Edelman R, Levine MM, Curtiss R (1997) Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system. Infect Immun 65(8):3381–3385Google Scholar
  46. Vidal L, Pinsach J, Striedner G, Caminal G, Ferrer P (2008) Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli. J Biotechnol 134(1-2):127–136.  https://doi.org/10.1016/j.jbiotec.2008.01.011 Google Scholar
  47. Vuorio R, Vaara M (2010) Comparison of the phenotypes of the lpxA and lpxD mutants of Escherichia coli. FEMS Microbiol Lett 134(2-3):227–232Google Scholar
  48. Wang X, Quinn PJ (2010) Lipopolysaccharide: biosynthetic pathway and structure modification. Prog Lipid Res 49:97–107Google Scholar
  49. Wang X, Quinn PJ, Yan A (2015) Kdo2-lipid A: structural diversity and impact on immunopharmacology. Biol Rev Camb Philos Soc 90:408–427Google Scholar
  50. Zhao H, Fang Y, Wang X, Zhao L, Wang J, Li Y (2018) Increasing L-threonine production in Escherichia coli by engineering the glyoxylate shunt and the L-threonine biosynthesis pathway. Appl Microbiol Biotechnol 102:5505–5518.  https://doi.org/10.1007/s00253-018-9024-3 Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Lei Zhao
    • 1
    • 2
  • Xiaoqing Hu
    • 1
    • 2
  • Ye Li
    • 1
    • 2
  • Zhen Wang
    • 1
    • 2
  • Xiaoyuan Wang
    • 1
    • 2
    • 3
    Email author
  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.International Joint Laboratory on Food SafetyJiangnan UniversityWuxiChina
  3. 3.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina

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