Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces

Abstract

Several strategies have been used to clone large DNA fragments directly from bacterial genome. Most of these approaches are based on different site-specific recombination systems consisting of a specialized recombinase and its target sites. In this study, a novel strategy based on phage ϕBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters. This method has been proved successful for the cloning of actinorhodin gene cluster from Streptomyces coelicolor M145, napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 at a frequency higher than 80%. Furthermore, the system could be used to increase the titer of antibiotics as we demonstrated with actinorhodin and daptomycin, and it will be broadly applicable in many Streptomyces.

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.…”

Engineering microbial hosts for production of bacterial natural products

“…Target genomic fragments up to 157 kb are subsequently captured into a multi-copy plasmid in vivo via site-specific recombination mediated by the phiBT1 integrase with more than 80% efficiency. 55 Given that native BGCs may not be expressed even when transferred to another host, 238 it is noteworthy that these direct capture methods are compatible with downstream cluster refactoring involving λ RED recombineering in E. coli or HR in yeast. In yeast, TAR has been further adapted for simultaneous capture and refactoring of BGCs by insertion of multiple promoter cassettes for expression in an S. albus host, demonstrating streamlined and scalable workflow for heterologous production of microbial natural products.…”

“…Expression of phiBT1 integrase leads to excision of target fragment into a plasmid backbone that is propagated in vivo . 55 …”

New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters

“…Using this method, ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens were cloned into expression vectors and two of them were expressed in a heterologous host which led to the identification of luminmycin A and luminmide A/B 27 . More recently, another strategy based on phage φBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters 28 . Using this strategy, the large actinorhodin gene cluster from Streptomyces coelicolor M145 and the napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 were successfully cloned with a success rate higher than 80% 28 .…”

“…More recently, another strategy based on phage φBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters 28 . Using this strategy, the large actinorhodin gene cluster from Streptomyces coelicolor M145 and the napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 were successfully cloned with a success rate higher than 80% 28 . Similarly, using the recombination-based cloning strategy, fungal heterologous expression vectors that encode the cryptic clusters from Dermatophytes were constructed in yeast and integrated into Aspergillus nidulans , which led to the production of neosartoricin B 29 .…”