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Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to Escherichia coli Genome

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Abstract

Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets (FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P trc and P araB ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.

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References

  1. Makrides, S. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiological Reviews, 60, 512–538.

    CAS  Google Scholar 

  2. Zhang, K., Li, H., Cho, K. M., & Liao, J. C. (2010). Expanding metabolism for biosynthesis of nonnatural alcohols. Proceedings of the National Academy of Sciences of the United States of America, 105, 20653–202658.

    Article  Google Scholar 

  3. Prather, K. L. J., & Martin, C. H. (2008). De novo biosynthetic pathways: Rational design of microbial chemical factories. Current Opinion in Biotechnology, 19, 468–474.

    Article  Google Scholar 

  4. Minami, H., Kim, J. S., Ikezawa, N., Takemura, T., Katayama, T., Kumagi, H., et al. (2008). Microbial production of plant benzylisoquinoline alkaloids. Proceedings of the National Academy of Sciences of the United States of America, 105, 7393–7398.

    Article  CAS  Google Scholar 

  5. Wróbel, B., & Wegrzyn, G. (1998). Replication regulation of ColE1-like plasmids in amino acid-starved Escherichia coli. Plasmid, 39, 48–62.

    Article  Google Scholar 

  6. Balbás, P., & Gosset, G. (2001). Chromosomal editing in E. coli: Vectors for DNA integration and excision. Molecular Biotechnology, 19, 1–12.

    Article  Google Scholar 

  7. Palomares, L. A., Estrada-Mondaca, S., & Ramírez, O. T. (2004). Production of recombinant proteins: Challenges and solutions. Methods in Molecular Biology, 267, 15–52.

    CAS  Google Scholar 

  8. Gutterson, N. I., & Koshland, D. E., Jr. (1983). Replacement and amplification of bacterial genes with sequences altered in vitro. Proceedings of the National Academy of Sciences of the United States of America, 80, 4894–4898.

    Article  CAS  Google Scholar 

  9. Peredelchuk, M. Y., & Bennett, G. N. (1997). A method for construction of E. coli strains with multiple DNA insertions in the chromosome. Gene, 187, 231–238.

    Article  CAS  Google Scholar 

  10. El Karoui, M., Dabert, P., Gruss, A., & Amundsen, S. K. (1999). Gene replacement with linear DNA in electroporated wild-type Escherichia coli. Nucleic Acids Research, 27, 1296–1299.

    Article  CAS  Google Scholar 

  11. Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P., & Kushner, S. (1989). New method for generating deletions and gene replacements in Escherichia coli. Journal of Bacteriology, 177, 4617–4622.

    Google Scholar 

  12. Shevell, D. E., Abou-Zamzam, A. M., Demple, B., & Walker, G. C. (1988). Construction of an Escherichia coli K-12 ada deletion by gene replacement in a recD strain reveals a second methyltransferase that repairs alkylated DNA. Journal of Bacteriology, 170, 3294–3296.

    CAS  Google Scholar 

  13. Huang, L. C., Wood, E. A., & Cox, M. M. (1997). Convenient and reversible site-specific targeting of exogenous DNA into a bacterial chromosome by use of the FLP recombinase: The FLIRT system. Journal of Bacteriology, 179, 6076–6083.

    CAS  Google Scholar 

  14. Pósfai, G., Kolisnychenko, V., Bereczki, Z., & Blattner, F. R. (1999). Markerless gene replacement in Escherichia coli stimilated by double-stranded break in the chromosome. Nucleic Acids Research, 27, 4409–4415.

    Article  Google Scholar 

  15. Zhang, Y., Buchholz, F., Muyrers, J. P., & Stewart, A. F. (1998). A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics, 20, 123–128.

    Article  CAS  Google Scholar 

  16. Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 97, 6640–6645.

    Article  CAS  Google Scholar 

  17. Murphy, K. C. (1998). Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. Journal of Bacteriology, 180, 2063–2071.

    CAS  Google Scholar 

  18. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., et al. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Molecular Systems Biology, 2, 1–11.

    Article  Google Scholar 

  19. Jasin, M., & Schimmel, P. (1984). Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments. Journal of Bacteriology, 159, 783–786.

    CAS  Google Scholar 

  20. Lee, D. J., Bingle, L. E., Heurlier, K., Pallen, M. J., Penn, C. W., Busby, S. J., et al. (2009). Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains. BMC Microbiol, 9, 252.

    Article  Google Scholar 

  21. Amann, E., Ochs, B., & Abel, K. J. (1988). Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene, 69, 301–315.

    Article  CAS  Google Scholar 

  22. Chou, C. P., Yu, C.-C., Tseng, J.-H., Lin, M.-I., & Lin, H.-K. (1999). Genetic manipulation to identify limiting steps and develop strategies for high-level expression of penicillin acylase in Escherichia coli. Biotechnology and Bioengineering, 63, 263–272.

    Article  CAS  Google Scholar 

  23. Perez–Perez, J., & Gutierrez, J. (1995). An arabinose-inducible expression vector, pAR3, compatible with ColE1-derived plasmids. Gene, 158, 141–142.

    Article  Google Scholar 

  24. Narayanan, N., & Chou, C. P. (2009). Alleviation of proteolytic sensitivity to enhance recombinant lipase production in Escherichia coli. Applied and Environmental Microbiology, 75, 5424–5427.

    Article  CAS  Google Scholar 

  25. Don, R., Cox, P., Wainwright, B., Baker, K., & Mattick, J. (1991). ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Research, 19, 4008.

    Article  CAS  Google Scholar 

  26. Meevootisom, V., Somsuk, P., Prachaktam, R., & Flegel, T. W. (1983). Simple screening method for isolation of penicillin acylase-producing bacteria. Applied and Environmental Microbiology, 46, 1227–1229.

    CAS  Google Scholar 

  27. Narayanan, N., Hsieh, M.-Y., Xu, Y., & Chou, C. P. (2006). Arabinose-induction of lac-derived promoter systems for penicillin acylase production in Escherichia coli. Biotechnology Progress, 22, 617–625.

    Article  CAS  Google Scholar 

  28. Vorderwulbecke, T., Kieslich, K., & Erdmann, H. (1992). Comparison of lipases by different assays. Amsterdam, PAYS-BAS: Elsevier.

    Google Scholar 

  29. Diges, C. M., & Uhlenbeck, O. C. (2001). Escherichia coli DbpA is an RNA helicase that requires hairpin 92 of 23S rRNA. EMBO Journal, 20, 5503–5512.

    Article  CAS  Google Scholar 

  30. Hudson, J. M., & Fried, M. G. (1990). Co-operative interactions between the catabolite gene activator protein and the lac repressor at the lactose promoter. Journal of Molecular Biology, 214, 381–396.

    Article  CAS  Google Scholar 

  31. Torres, B., Porras, G., Garcia, J. L., & Diaz, E. (2003). Regulation of the mhp cluster responsible for 3-(3-hydroxyphenyl)propionic acid degradation in Escherichia coli. Journal of Biological Chemistry, 278, 27575–27585.

    Article  CAS  Google Scholar 

  32. Narayanan, N., Khan, M., & Chou, C. P. (2010). Enhancing functional expression of heterologous lipase B in Escherichia coli by extracellular secretion. Journal of Industrial Microbiology and Biotechnology, 37, 349–361.

    Article  CAS  Google Scholar 

  33. Fernandez, L. A., Sola, I., Enjuanes, L., & Lorenzo, V. D. (2000). Specific secretion of active single-chain Fv antibodies into the supernatants of Escherichia coli cultures by use of the hemolysin system. Applied and Environmental Microbiology, 66, 5024–5029.

    Article  CAS  Google Scholar 

  34. Woodstock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., Decruz, E., & Noyerweidner, M. (1989). Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Research, 17, 3469–3478.

    Article  Google Scholar 

  35. Boyer, H. W., & Roulland-Dussoix, D. (1969). A complementation analysis of the restriction and modification of DNA in Escherichia coli. Journal of Molecular Biology, 41, 459–472.

    Article  CAS  Google Scholar 

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Acknowledgments

This study was supported in part by the Natural Sciences and Engineering Research Council (NSERC) and the Canada Research Chair (CRC) program of Canada.

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Authors and Affiliations

  1. Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada

    Karan Sukhija, Michael Pyne, Saad Ali, Valerie Orr, Murray Moo-Young & C. Perry Chou

  2. Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy, Isfahan University of Medical Sciences, 81745-359, Isfahan, Iran

    Daryoush Abedi

Authors
  1. Karan Sukhija
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  2. Michael Pyne
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  3. Saad Ali
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  7. C. Perry Chou
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Correspondence to C. Perry Chou.

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Sukhija, K., Pyne, M., Ali, S. et al. Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to Escherichia coli Genome. Mol Biotechnol 51, 109–118 (2012). https://doi.org/10.1007/s12033-011-9442-2

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