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.

Implementation of stable and complex biological systems through recombinase-assisted genome engineering

“…The five clones tested were obtained by both plasmid and phage delivery of the integration cassette, demonstrating that the use of either methodology leads to 100% cassette integrity and functionality after selecting for kanamycin resistance and chloramphenicol Figure 1 | Advanced design principle based on the optimal implementation of complex biological systems through RAGE. Top: Construction of biological systems (assembly of genetic parts): A plasmid containing genes responsible for a designed biological function is constructed from either genomic DNA, de novo synthesized DNA and/or DNA fragments through random cloning (library construction), direct cloning or DNA assembly methods [12][13][14][15][16][17] . Middle: Implementation through RAGE (host strain selection and genome engineering): The expression vector is transformed into different heterologous hosts to identify the specific genetic backgrounds yielding the most desirable cellular phenotypes.…”

“…The design of increasingly more complex biological systems has been greatly enhanced by the wide array of tools now available for controlling heterologous protein and pathway expression at both the transcriptional (promoter and messenger RNA) and translational (RBS) levels [9][10][11] . Similarly, the construction of large and complex designs has become an almost routine process through the use of cheap, automated and sophisticated techniques for both de novo DNA synthesis 12,13 and genetic parts assembly [14][15][16][17] . Despite this unprecedented level of genetic control, however, most engineered systems still do not perform as expected when first implemented in a microbial host.…”

“…1. First, the biological system of interest is constructed on a single copy plasmid or bacterial artificial chromosome (BAC) using various cloning or assembly techniques [12][13][14][15][16][17] and then transformed into different hosts for proof-of-concept studies aimed at identifying genetic backgrounds that yield the desired phenotype. (Single copy plasmids or BACs are recommended at this step, as they can maintain large DNA fragments at copy numbers that are representative of genome-based expression.)…”

De novo Biosynthesis of “Non‐Natural” Thaxtomin Phytotoxins

“…However there are many microorganisms that produce useful secondary metabolites, which are not amenable to such genetic manipulation. The rise of synthetic biology has provided access to larger synthetic DNA constructs, rapid DNA capture,7, 8, 9 editing,10, 11 assembly,12, 13 and other advances 14, 15. The prospect of using these new tools to assemble de novo biosynthetic pathways in well‐characterized heterologous host strains16, 17 for diversification and optimization of natural products, derived from less tractable microorganisms, is an attractive goal.…”

The International Conference of Natural Product Biosynthesis (ICNPB, 8th US–Japan seminar on the Biosynthesis of Natural Products)

“…Detection of the compounds from myxobacteria was performed in a high-throughput manner using LC-MS. Construction of the heterologous expression system utilizing linear-linear homologous recombination of several gene clusters was also discussed. 32 Professor Jun Ishikawa (National Institute of Infectious Diseases) presented the construction of a web-based support tool (2nd Find) to find secondary metabolite biosynthetic genes on the basis of Pfam domains. Dr Masaki Fujita (Hokkaido University) described the finding of a bisucaberinproducing clone from the metagenomic library constructed from deep sea samples, 33 and the switching of bisucaberin production to desferrioxamine E production by exchanging an enzyme catalyzing macrocyclization.…”