Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting

Abstract

Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.

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Figure 1: Full-length RecE mediates recombination between two substrates.
Figure 2: Full-length RecE plus RecT is required for enhanced LLHR.
Figure 3: LCHR and LLHR are mechanistically different.
Figure 4: Evaluation of different configurations of inducible recombinase expression.
Figure 5: Expression of directly cloned PKS-NRPS biosynthetic gene clusters.
Figure 6: A two-step, double recombination 'fishing' strategy enhances the identification of desired products.

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Acknowledgements

Research work in R&D in Gene Bridges (Y.Z.) was partially funded by the Bundesministerium für Bildung und Forschung (MiPro). This work was supported by funding to A.F.S. from the EU 6th and 7th Framework projects, EUCOMM and EUCOMMTOOLS. Research in the laboratory of R.M. was funded by the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung. X.B. is supported by China Scholarship Council. The authors thank J. Herrmann in the laboratory of R.M. for cytotoxic activity assay.

Author information

J.F., A.F.S. and Y.Z. discovered the LLHR activity of full-length RecE. J.F., X.B., S.H., H.W., F.H., P.M.S., L.X. and Y.Z. made the DNA constructs and E. coli strains. X.B., A.P., R.M. and Y.Z. analyzed the secondary metabolites. J.F., X.B.,L.X., R.M., A.F.S. and Y.Z. designed the experiments and wrote the paper.

Correspondence to Rolf Müller or A Francis Stewart or Youming Zhang.

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Competing interests

R.M., A.F.S. and Y.Z. are shareholders of the company Gene Bridges that holds the exclusive rights to Red and RecET recombineering methodologies.

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Supplementary Tables 1–7, Supplementary Results and Supplementary Figures 1–13 (PDF 4526 kb)

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Fu, J., Bian, X., Hu, S. et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat Biotechnol 30, 440–446 (2012). https://doi.org/10.1038/nbt.2183

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