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RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression

Nature Protocols volume 11, pages 1175–1190 (2016)Cite this article

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Abstract

Full-length RecE and RecT from Rac prophage mediate highly efficient linear–linear homologous recombination that can be used to clone large DNA regions directly from genomic DNA into expression vectors, bypassing library construction and screening. Homologous recombination mediated by Redαβ from lambda phage has been widely used for recombinant DNA engineering. Here we present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes from genomic DNA using one Escherichia coli host that harbors both RecET and Redαβ systems. The pipeline uses standardized cassettes for horizontal gene transfer options, as well as vectors with different replication origins configured to minimize recombineering background through the use of selectively replicating templates or CcdB counterselection. These optimized reagents and protocols facilitate fast acquisition of transgenes from genomic DNA preparations, which are ready for heterologous expression within 1 week.

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Figure 1: Strategy diagram for direct cloning and engineering of natural product biosynthetic gene clusters for heterologous expression.
Figure 2: Addition of homology arms to the three cloning vectors.
Figure 3: Insertion of the conjugation–transposition/site-specific-integration cassettes into the direct cloning vectors.
Figure 4: Retrofitting the cloned gene clusters with a broad-host-range vector.

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References

  1. Van Lanen, S.G. & Shen, B. Microbial genomics for the improvement of natural product discovery. Curr. Opin. Microbiol. 9, 252–260 (2006).

    Article  CAS  Google Scholar 

  2. Nett, M., Ikeda, H. & Moore, B.S. Genomic basis for natural product biosynthetic diversity in the actinomycetes. Nat. Prod. Rep. 26, 1362–1384 (2009).

    Article  CAS  Google Scholar 

  3. Lautru, S., Deeth, R.J., Bailey, L.M. & Challis, G.L. Discovery of a new peptide natural product by Streptomyces coelicolor genome mining. Nat. Chem. Biol. 1, 265–269 (2005).

    Article  CAS  Google Scholar 

  4. Bergmann, S. et al. Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans. Nat. Chem. Biol. 3, 213–217 (2007).

    Article  CAS  Google Scholar 

  5. Wenzel, S.C. & Müller, R. The impact of genomics on the exploitation of the myxobacterial secondary metabolome. Nat. Prod. Rep. 26, 1385–1407 (2009).

    Article  CAS  Google Scholar 

  6. Charlop-Powers, Z., Milshteyn, A. & Brady, S.F. Metagenomic small molecule discovery methods. Curr. Opin. Microbiol. 19, 70–75 (2014).

    Article  CAS  Google Scholar 

  7. Fu, J. et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat. Biotechnol. 30, 440–446 (2012).

    Article  CAS  Google Scholar 

  8. Yamanaka, K. et al. Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc. Natl. Acad. Sci. USA 111, 1957–1962 (2014).

    Article  CAS  Google Scholar 

  9. Tang, Y. et al. Heterologous expression of an orphan NRPS gene cluster from Paenibacillus larvae in Escherichia coli revealed production of sevadicin. J. Biotechnol. 194, 112–114 (2015).

    Article  CAS  Google Scholar 

  10. Yin, W.B. et al. Discovery of cryptic polyketide metabolites from dermatophytes using heterologous expression in Aspergillus nidulans. ACS Synth. Biol. 2, 629–634 (2013).

    Article  CAS  Google Scholar 

  11. Wolpert, M., Heide, L., Kammerer, B. & Gust, B. Assembly and heterologous expression of the coumermycin A1 gene cluster and production of new derivatives by genetic engineering. Chembiochem 9, 603–612 (2008).

    Article  CAS  Google Scholar 

  12. Chai, Y. et al. Heterologous expression and genetic engineering of the tubulysin biosynthetic gene cluster using Red/ET recombineering and inactivation mutagenesis. Chem. Biol. 19, 361–371 (2012).

    Article  CAS  Google Scholar 

  13. Fu, J. et al. Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition. Nucleic Acids Res. 36, e113 (2008).

    Article  Google Scholar 

  14. Perlova, O. et al. Reconstitution of the myxothiazol biosynthetic gene cluster by Red/ET recombination and heterologous expression in Myxococcus xanthus. Appl. Environ. Microbiol. 72, 7485–7494 (2006).

    Article  CAS  Google Scholar 

  15. Wenzel, S.C. et al. Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering. Chem. Biol. 12, 349–356 (2005).

    Article  CAS  Google Scholar 

  16. Wang, H. et al. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 42, e37 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Zhang, Y., Muyrers, J.P., Testa, G. & Stewart, A.F. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18, 1314–1317 (2000).

    Article  CAS  Google Scholar 

  19. Bian, X. et al. Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering. Chembiochem 13, 1946–1952 (2012).

    Article  CAS  Google Scholar 

  20. Bian, X. et al. Heterologous production of glidobactins/luminmycins in Escherichia coli Nissle containing the glidobactin biosynthetic gene cluster from Burkholderia DSM7029. Chembiochem 15, 2221–2224 (2014).

    Article  CAS  Google Scholar 

  21. Bian, X. et al. In vivo evidence for a prodrug activation mechanism during colibactin maturation. Chembiochem 14, 1194–1197 (2013).

    Article  CAS  Google Scholar 

  22. Kouprina, N. & Larionov, V. Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nat. Protoc. 3, 371–377 (2008).

    Article  CAS  Google Scholar 

  23. Shao, Z. & Zhao, H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 37, e16 (2009).

    Article  Google Scholar 

  24. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  25. Muyrers, J.P., Zhang, Y., Testa, G. & Stewart, A.F. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555–1557 (1999).

    Article  CAS  Google Scholar 

  26. Muyrers, J.P. et al. Point mutation of bacterial artificial chromosomes by ET recombination. EMBO Rep. 1, 239–243 (2000).

    Article  CAS  Google Scholar 

  27. Eustaquio, A.S. et al. Heterologous expression of novobiocin and clorobiocin biosynthetic gene clusters. Appl. Environ. Microbiol. 71, 2452–2459 (2005).

    Article  CAS  Google Scholar 

  28. Binz, T.M., Wenzel, S.C., Schnell, H.J., Bechthold, A. & Müller, R. Heterologous expression and genetic engineering of the phenalinolactone biosynthetic gene cluster by using red/ET recombineering. Chembiochem 9, 447–454 (2008).

    Article  CAS  Google Scholar 

  29. Ongley, S.E. et al. High-titer heterologous production in E. coli of lyngbyatoxin, a protein kinase C activator from an uncultured marine cyanobacterium. ACS Chem. Biol. 8, 1888–1893 (2013).

    Article  CAS  Google Scholar 

  30. Tang, L. et al. Elucidating the mechanism of cis double bond formation in epothilone biosynthesis. J. Am. Chem. Soc. 126, 46–47 (2004).

    Article  CAS  Google Scholar 

  31. Bian, X., Plaza, A., Yan, F., Zhang, Y. & Müller, R. Rational and efficient site-directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo. Biotechnol. Bioeng. 112, 1343–1353 (2015).

    Article  CAS  Google Scholar 

  32. Nguyen, K.T. et al. Genetically engineered lipopeptide antibiotics related to A54145 and daptomycin with improved properties. Antimicrob. Agents Chemother. 54, 1404–1413 (2010).

    Article  CAS  Google Scholar 

  33. Nguyen, K.T. et al. Combinatorial biosynthesis of novel antibiotics related to daptomycin. Proc. Natl. Acad. Sci. USA 103, 17462–17467 (2006).

    Article  CAS  Google Scholar 

  34. Alexander, D.C. et al. Development of a genetic system for combinatorial biosynthesis of lipopeptides in Streptomyces fradiae and heterologous expression of the A54145 biosynthesis gene cluster. Appl. Environ. Microbiol. 76, 6877–6887 (2010).

    Article  CAS  Google Scholar 

  35. Yin, J. et al. Direct cloning and heterologous expression of the salinomycin biosynthetic gene cluster from Streptomyces albus DSM41398 in Streptomyces coelicolor A3(2). Sci. Rep. 5, 15081 (2015).

    Article  CAS  Google Scholar 

  36. Wang, J. et al. An improved recombineering approach by adding RecA to lambda Red recombination. Mol. Biotechnol. 32, 43–53 (2006).

    Article  Google Scholar 

  37. Sarov, M. et al. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nat. Methods 3, 839–844 (2006).

    Article  CAS  Google Scholar 

  38. Filutowicz, M., McEachern, M.J. & Helinski, D.R. Positive and negative roles of an initiator protein at an origin of replication. Proc. Natl. Acad. Sci. USA 83, 9645–9649 (1986).

    Article  CAS  Google Scholar 

  39. Sorensen, H.P. & Mortensen, K.K. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J. Biotechnol. 115, 113–128 (2005).

    Article  CAS  Google Scholar 

  40. Li, Y. et al. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis. Sci. Rep. 5, 9383 (2015).

    Article  CAS  Google Scholar 

  41. Frengen, E. et al. A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics 58, 250–253 (1999).

    Article  CAS  Google Scholar 

  42. Mei, J., Benashski, S. & Firshein, W. Interactions of the origin of replication (oriV) and initiation proteins (TrfA) of plasmid RK2 with submembrane domains of Escherichia coli. J. Bacteriol. 177, 6766–6772 (1995).

    Article  CAS  Google Scholar 

  43. Nedelkova, M. et al. Targeted isolation of cloned genomic regions by recombineering for haplotype phasing and isogenic targeting. Nucleic Acids Res. 39, e137 (2011).

    Article  CAS  Google Scholar 

  44. Lynch, M.D. & Gill, R.T. Broad host range vectors for stable genomic library construction. Biotechnol. Bioeng. 94, 151–158 (2006).

    Article  CAS  Google Scholar 

  45. Antoine, R. & Locht, C. Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol. Microbiol. 6, 1785–1799 (1992).

    Article  CAS  Google Scholar 

  46. Bi, C. et al. Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production. Microb. Cell Fact. 12, 107 (2013).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the International ST Cooperation Program of China (ISTCP 2015DFE32850 to J.F.), a Shandong Innovation and Transformation of Achievements Grant (2014ZZCX02601 to Y.Z.) and a Shenzhen Global Experts Innovation and Pioneering Grant (KQCX20120806161031208 to J.F.). Y.Z. is supported by the Recruitment Program of Global Experts in Shandong University. H.W. is supported by the China Postdoctoral Science Foundation (2015T80710) and the Postdoctoral Innovation Program of Shandong Province (201303110). A.F.S. is supported by the TUD Elite University Support the Best program. The authors acknowledge V. Ravichandir's help in proofreading this manuscript.

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Author notes

  1. Hailong Wang and Zhen Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Shandong University–Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China.,

    Hailong Wang, Zhen Li, Ruonan Jia, Yu Hou, Jia Yin, Xiaoying Bian, Aiying Li, Rolf Müller, Jun Fu & Youming Zhang

  2. Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI) and Pharmaceutical Biotechnology, Saarland University, Saarbrücken, Germany

    Rolf Müller

  3. Genomics, Biotechnology Center, Technische Universität Dresden, Dresden, Germany

    A Francis Stewart & Jun Fu

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Contributions

H.W., Z.L., R.M., A.F.S., J.F. and Y.Z. designed the experiments. H.W., Z.L., R.J., Y.H., J.Y., X.B. and A.L. performed the experiments. H.W., A.F.S., J.F. and Y.Z. wrote the manuscript with help from all authors.

Corresponding authors

Correspondence to A Francis Stewart, Jun Fu or Youming Zhang.

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Competing interests

R.M., A.F.S. and Y.Z. are shareholders in Gene Bridges GmbH, which holds exclusive rights to the commercialization of both Redαβ and RecET recombineering technologies.

Integrated supplementary information

Supplementary Figure 1 Derivation of pBAC2015.

(a) pBAC2015 was derived from pBACe3.6 by insertion of oriV and removal of a 30 bp repeat and the loxP site. (b) Comparison of direct cloning with pBACe3.6 and pBAC2015. Homology arms to the 37.5 kb Photorhabdus luminescens plu3535-plu3532 gene cluster were added using the method illustrated in Supplementary Figure 2. Most of the colonies obtained with pBACe3.6 were due to intramolecular recombination through the 30 bp repeat. Its removal in pBAC2015 dramatically reduced the unwanted background so that most colonies contained the intended product.

Supplementary Figure 2 Construction of linear BAC direct cloning vectors.

(a) The cassette containing pBR322, amp and ccdB is amplified by two rounds of PCR to attach homology arms to the gene cluster (ha1 and ha2) and pBAC2015 (ha3 and ha4) respectively. (b) The cassette containing pBR322, amp and ccdB is recombined with pBAC2015 by LLHR in RecET expressing CcdB resistant strain, E. coli GBdir-gyrA462. Because the pBR322-amp-ccdB-rpsL plasmid carries the rpsL gene, in addition to ampicillin and chloramphenicol selection, streptomycin selection can be added to eliminate carryover background from the PCR template. The BAC vector is linearized by BamHI digestion to expose the terminal homology arms.

Supplementary Figure 3 Optimization of R6K plasmids with transposition cassette for different hosts.

(a) pR6K-oriT-tnpA-genta is the same as pR6K-oriT-tnpA-kan except the gentamicin resistance gene replaced the kanamycin resistance gene. (b) pR6K-oriT-tnpA-apra is the same as pR6K-oriT-tnpA-kan except for the inclusion of the apramycin resistance gene and the NcoI site. (c) Exchanging the promoter of tnpA via LLHR. Electroporate the BstZ17I digested pR6K-oriT-TnpA-kan plasmid together with a synthetic oligonucleotide, a synthetic DNA or a PCR product into RecET expressing pir strain, E. coli GBdir-pir116, for LLHR. In case of using synthetic oligonucleotides, the homology arms can be as short as 20 nt to ensure the oligonucleotide is not longer than 140 nt.

Supplementary information

Supplementary Information

Supplementary Figures 1–3, Supplementary Tables 1–3 and Supplementary Notes 1–10 (PDF 764 kb)

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Wang, H., Li, Z., Jia, R. et al. RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. Nat Protoc 11, 1175–1190 (2016). https://doi.org/10.1038/nprot.2016.054

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