Direct cloning method CAPTUREs novel microbial natural products
Microorganisms possess natural product biosynthetic gene clusters (BGCs) that may harbor unique bioactivities that can be used in drug development and agricultural applications. However, many uncharacterized microbial GBSs remain inaccessible. Researchers at the University of Illinois Urbana-Champaign previously demonstrated a technique using transcription factor decoys to activate large, silent BGCs in bacteria to facilitate natural product discovery.
Now they have developed a direct cloning method that aims to accelerate the large-scale discovery of new natural products. Their results are published in the journal Nature Communications.
Named CAPTURE (Cas12a assisted precise targeted cloning using in vivo Cre-lox recombination), the method allows direct cloning of large genomic fragments, including those with high GC content or sequence repeats. Where existing direct cloning methods fail to efficiently clone BGCs from natural products of this nature, CAPTURE excels.
"With CAPTURE, microbial BGCs from natural products can be directly cloned and heterologously expressed at an unprecedented rate," said Huimin Zhao, study leader and the Steven L. Miller Professor of Chemical and Biomolecular Engineering. Miller Chair, also a member of the Carl R. Woese Institute for Genomic Biology at Illinois. "As a result, CAPTURE enables large-scale cloning of CGBs of natural products from a variety of organisms, which can lead to the discovery of many new natural products."
The researchers first characterized the efficiency and robustness of CAPTURE by cloning 47 BGCs of natural products from Actinomycetes and Bacilli. After demonstrating CAPTURE's nearly 100% efficacy, the researchers cloned and heterologously expressed 43 uncharacterized natural product BGCs from 14 Streptomyces species and three Bacillus species. The compounds produced were purified and determined as 15 novel natural products, including six unprecedented compounds designated as bipentaromycins. Four of the bipentaromycins exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Bacillus anthracis.
"To address the current antimicrobial resistance crisis, new molecules capable of treating drug-resistant infections must be discovered," Zhao said. "The discovery of bipentaromycins not only demonstrates the possibility of discovering new antimicrobials, but also provides an example of how this strategy can be applied for the discovery of unique bioactive compounds for use in drug development and agricultural applications."
The researchers then plan to characterize these compounds for other bioactivities such as anti-cancer, anti-parasitic, and anti-cancer properties. Preliminary results already show anti-cancer properties for some of the compounds.
"Due to its exceptional robustness and efficiency, CAPTURE will likely become the method of choice for direct cloning of large DNA molecules such as natural product GBCs from genomic or metagenomic DNA for various basic and applied biological applications," said Zhao.
