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Combinatorial Evolution of DNA with RECODE

  • Zhen KangEmail author
  • Wenwen Ding
  • Peng Jin
  • Guocheng Du
  • Jian Chen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1772)

Abstract

In past decades, DNA engineering protocols have led to the rapid development of synthetic biology. To engineer the natural proteins, many directed evolution methods based on molecular biology have been presented for generating genetic diversity or obtaining specific properties. Here, we provide a simple (PCR operation), efficient (larger amount of products), and powerful (multiple point mutations, deletions, insertions, and combinatorial multipoint mutagenesis) RECODE method, which is capable of reediting the target DNA flexibly to restructure regulatory regions and remodel enzymes by using the combined function of the thermostable DNA polymerase and DNA ligase in one pot. RECODE is expected to be an applicable choice to create diverse mutant libraries for rapid evolution and optimization of enzymes and synthetic pathways.

Keywords

Combinatorial multiple mutagenesis Single-stranded DNA oligonucleotides Directed evolution Pathway optimization Synthetic biology 

Notes

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (31670092), the Fundamental Research Funds for the Central Universities (JUSRP51707A), and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT_15R26).

References

  1. 1.
    Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485:185–194CrossRefPubMedGoogle Scholar
  2. 2.
    Schoemaker HE, Mink D, Wubbolts MG (2003) Dispelling the myths-biocatalysis in industrial synthesis. Science 299:1694–1697CrossRefPubMedGoogle Scholar
  3. 3.
    Lu TK, Khalil AS, Collins JJ (2009) Next-generation synthetic gene networks. Nat Biotechnol 27:1139–1150CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Carr PA, Church GM (2009) Genome engineering. Nat Biotechnol 27:1151–1162CrossRefPubMedGoogle Scholar
  5. 5.
    Dalby PA (2011) Strategy and success for the directed evolution of enzymes. Curr Opin Struct Biol 21:473–480CrossRefPubMedGoogle Scholar
  6. 6.
    Johannes TW, Zhao HM (2006) Directed evolution of enzymes and biosynthetic pathways. Curr Opin Microbiol 9:261–267CrossRefPubMedGoogle Scholar
  7. 7.
    Romero PA, Arnold FH (2009) Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Biol 10:866–876CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Stemmer WPC (1994) Rapid evolution of a protein in-vitro by DNA shuffling. Nature 370:389–391CrossRefPubMedGoogle Scholar
  9. 9.
    Zhao HM, Giver L, Shao ZX, Affholter JA, Arnold FH (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16:258–261CrossRefPubMedGoogle Scholar
  10. 10.
    Shao ZX, Zhao HM, Giver L, Arnold FH (1998) Random-priming in vitro recombination: an effective tool for directed evolution. Nucleic Acids Res 26:681–683CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Coco WM, Levinson WE, Crist MJ, Hektor HJ, Darzins A, Pienkos PT, Squires CH, Monticello DJ (2001) DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat Biotechnol 19:354–359CrossRefPubMedGoogle Scholar
  12. 12.
    Ness JE, Kim S, Gottman A, Pak R, Krebber A, Borchert TV, Govindarajan S, Mundorff EC, Minshull J (2002) Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Nat Biotechnol 20:1251–1255CrossRefPubMedGoogle Scholar
  13. 13.
    Ostermeier M, Shim JH, Benkovic SJ (1999) A combinatorial approach to hybrid enzymes independent of DNA homology. Nat Biotechnol 17:1205–1209CrossRefPubMedGoogle Scholar
  14. 14.
    Sieber V, Martinez CA, Arnold FH (2001) Libraries of hybrid proteins from distantly related sequences. Nat Biotechnol 19:456–460CrossRefPubMedGoogle Scholar
  15. 15.
    Herman A, Tawfik DS (2007) Incorporating synthetic oligonucleotides via gene reassembiv (ISOR): a versatile tool for generating targeted libraries. Protein Eng Des Sel 20:219–226CrossRefPubMedGoogle Scholar
  16. 16.
    Stemmer WPC (1994) DNA shuffling by random fragmentation and reassembly in-vitro recombination for molecular evolution. Proc Natl Acad Sci U S A 91:10747–10751CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Crameri A, Stemmer WPC (1995) Combinatorial multiple cassette mutagenesis creates all the permutations of mutant and wild-type sequences. Biotechniques 18:194–196Google Scholar
  18. 18.
    Reetz MT, Wilensek S, Zha DX, Jaeger KE (2001) Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis. Angew Chem Int Ed Engl 40:3589–3591CrossRefGoogle Scholar
  19. 19.
    Bloom JD (2014) An experimentally determined evolutionary model dramatically improves phylogenetic fit. Mol Biol Evol 31:1956–1978CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Hidalgo A, Schliessmann A, Molina R, Hermoso J, Bornscheuer UT (2008) A one-pot, simple methodology for cassette randomisation and recombination for focused directed evolution. Protein Eng Des Sel 21:567–576CrossRefPubMedGoogle Scholar
  21. 21.
    Reetz MT, Carballeira JD (2007) Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2:891–903CrossRefPubMedGoogle Scholar
  22. 22.
    Jain PC, Varadarajan R (2014) A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library. Anal Biochem 449:90–98CrossRefPubMedGoogle Scholar
  23. 23.
    Seyfang A, Jin JHQ (2004) Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round. Anal Biochem 324:285–291CrossRefPubMedGoogle Scholar
  24. 24.
    Young L, Dong QH (2003) TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening. Nucleic Acids Res 31:e11CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Sawano A, Miyawaki A (2000) Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res 28:E78CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Packer MS, Liu DR (2015) Methods for the directed evolution of proteins. Nat Rev Genet 16:379–394CrossRefPubMedGoogle Scholar
  27. 27.
    Coussement P, Maertens J, Beauprez J, Van Bellegem W, De Mey M (2014) One step DNA assembly for combinatorial metabolic engineering. Metab Eng 23:70–77CrossRefPubMedGoogle Scholar
  28. 28.
    Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Firnberg E, Ostermeier M (2012) PFunkel: efficient, expansive, user-defined mutagenesis. PLoS One 7:e52031CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2005) Tuning genetic control through promoter engineering. Proc Natl Acad Sci U S A 102:12678–12683CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27:946–950CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KLJ, Keasling JD (2009) Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27:753–759CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Pfleger BF, Pitera DJ, D Smolke C, Keasling JD (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol 24:1027–1032CrossRefPubMedGoogle Scholar
  34. 34.
    Zhang F, Carothers JM, Keasling JD (2012) Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat Biotechnol 30:354–359CrossRefPubMedGoogle Scholar
  35. 35.
    Ajikumar PK, Xiao W-H, Tyo KEJ, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G (2010) Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science 330:70–74CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Xu P, Gu Q, Wang W, Wong L, Bower AGW, Collins CH, Koffas MAG (2013) Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat Commun 4:1409CrossRefPubMedGoogle Scholar
  37. 37.
    Du J, Yuan Y, Si T, Lian J, Zhao H (2012) Customized optimization of metabolic pathways by combinatorial transcriptional engineering. Nucleic Acids Res 40:e142CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY (2013) Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat Biotechnol 31:170–174CrossRefGoogle Scholar
  39. 39.
    Kang Z, Wang X, Li Y, Wang Q, Qi Q (2012) Small RNA RyhB as a potential tool used for metabolic engineering in Escherichia coli. Biotechnol Lett 34:527–531CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Lee SY, Kim HU (2015) Systems strategies for developing industrial microbial strains. Nat Biotechnol 33:1061–1072CrossRefPubMedGoogle Scholar
  41. 41.
    Dai Z, Nielsen J (2015) Advancing metabolic engineering through systems biology of industrial microorganisms. Curr Opin Biotechnol 36:8–15CrossRefPubMedGoogle Scholar
  42. 42.
    Jin P, Kang Z, Zhang J, Zhang L, Du G, Chen J (2016) Combinatorial evolution of enzymes and synthetic pathways using one-otep PCR. ACS Synth Biol 5:259–268CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Zhen Kang
    • 1
    • 2
    • 3
    Email author
  • Wenwen Ding
    • 1
  • Peng Jin
    • 1
  • Guocheng Du
    • 1
    • 2
  • Jian Chen
    • 1
    • 2
  1. 1.The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina
  2. 2.Synergetic Innovation Center of Food Safety and NutritionJiangnan UniversityWuxiChina
  3. 3.The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina

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