Microorganisms have natural product biosynthetic gene clusters (BGCs) that may contain unique bioactivities for drug development and agricultural applications. However, many uncharacterized microbial BGCs remain inaccessible. Researchers at the University of Illinois' Urbana-Champaign previously demonstrated a technique that uses transcription factor bait to activate large, silent BGCs in bacteria to aid in natural product discovery.
Now they have developed a direct cloning method that aims to accelerate the discovery of novel natural products on a large scale. Their results are reported in the journal Communication with nature.
The process, named Cas12a, supported precisely targeted cloning using in vivo Cre-lox recombination (CAPTURE) and enables the direct cloning of large genomic fragments, including those with high GC content or sequence repeats. When existing direct cloning methods cannot effectively clone such natural product BGCs, CAPTURE is excellent.
"CAPTURE enables microbial natural product BGCs to be cloned directly and heterologously expressed at an unprecedented rate," said study director and Steven L. Miller professor of chemical and biomolecular engineering, Huimin Zhao, also a member of the Carl R. Woese Institute for Genome Biology in Illinois . "As a result, CAPTURE enables the large-scale cloning of natural product BGCs from various organisms, which can lead to the discovery of numerous new natural products."
The researchers first characterized the efficiency and robustness of CAPTURE by cloning 47 natural product BGCs from both actinomycetes and bacilli. After the almost 100% efficiency of CAPTURE, 43 uncharacterized natural product BGCs from 14 Streptomyces and three Bacillus species were cloned and heterologously expressed by researchers. The compounds made were purified and identified as 15 new natural products, including six unprecedented compounds called bipentaromycins. Four of the bipentaromycins showed antimicrobial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Bacillus anthracis.
"To tackle the current antibiotic resistance crisis, novel molecules must be discovered that are capable of treating drug-resistant infections," said Zhao. "The discovery of bipentaromycins not only demonstrates the possibility of discovering novel antimicrobial agents, it also provides an example of how this strategy can be applied to the discovery of unique bioactive compounds for use in drug development and agricultural applications."
The researchers next plan to characterize these compounds for other bioactivities such as anti-cancer, anti-parasite and anti-cancer properties. Preliminary results already show anti-cancer properties for some of the compounds.
"Because of its exceptional robustness and efficiency, CAPTURE is likely to be the method of choice for the direct cloning of large DNA molecules such as natural product BGCs from genomic or metagenomic DNA for various basic and applied biological applications," said Zhao.
Source of the story:
materials provided by Carl R. Woese Institute of Genome Biology, University of Illinois at Urbana-Champaign. Originally written by Alisa King-Klemperer. Note: the content can be edited by style and length.