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

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. Her...

Full description

Saved in:
Bibliographic Details
Published in:Nature biotechnology 2012-05, Vol.30 (5), p.440-446
Main Authors: JUN FU, XIAOYING BIAN, YOUMING ZHANG, SHENGBAIO HU, HAILONG WANG, FAN HUANG, SEIBERT, Philipp M, PLAZA, Alberto, LIQIU XIA, MÜLLER, Rolf, STEWART, A. Francis
Format: Article
Language:English
Subjects:
Biological and medical sciences
Bioprospecting
Biotechnology
Chemistry, Pharmaceutical - methods
Chromosomes, Artificial, Bacterial
Cloning
Cloning, Molecular - methods
Computational Biology - methods
Deoxyribonucleic acid
Diverse techniques
DNA
DNA, Complementary - metabolism
DNA-Binding Proteins - genetics
Escherichia coli Proteins - genetics
Exodeoxyribonucleases - genetics
Fundamental and applied biological sciences. Psychology
Gene expression
Genes, Bacterial
Genetic aspects
Genetic recombination
Genetic Vectors
Genome
Genomics
Homologous Recombination
Innovations
Metabolites
Methods
Models, Chemical
Models, Genetic
Molecular and cellular biology
Multigene Family
Photorhabdus - metabolism
Photorhabdus luminescens
Sequence Analysis, DNA
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1087-0156
1546-1696
DOI:10.1038/nbt.2183