Unmuting Large Silent Genes Lets Bacteria Produce New Molecules, Potential Drug Candidates.
Illinois researchers developed a technique to unmute silent genes in Streptomyces bacteria using decoy DNA (Deoxyribonucleic acid is a molecule composed of two chains that coil around each other to form a double helix carrying the genetic instructions used in the growth, development, functioning, and reproduction of all known living organisms and many viruses) fragments to lure away repressors. Pictured from left: postdoctoral researcher X professor Y and postdoctoral researcher Z. By enticing away the repressors dampening unexpressed, silent genes in Streptomyces bacteria researchers at the Georgian Technical University have unlocked several large gene clusters for new natural products.
Since many antibiotics, anti-cancer agents and other drugs have been derived from genes readily expressed in Streptomyces the researchers hope that unsilencing genes that have not previously been expressed in the lab will yield additional candidates in the search for new antimicrobial drugs says study leader and chemical and biomolecular engineering professor Y.
“There are so many undiscovered natural products lying unexpressed in genomes. We think of them as the dark matter of the cell” Y said. “Anti-microbial resistance has become a global challenge so clearly there’s an urgent need for tools to aid the discovery of novel natural products. In this work we found new compounds by activating silent gene clusters that have not been explored before”.
The researchers previously demonstrated a technique to activate small silent gene clusters using CRISPR (CRISPR is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments from viruses that have previously infected the prokaryote and are used to detect and destroy DNA from similar viruses during subsequent infections) technology. However large silent gene clusters have remained difficult to unmute. Those larger genes are of great interest to Y’s group since a number of them have sequences similar to regions that code for existing classes of antibiotics such as tetracycline.
To unlock the large gene clusters of greatest interest Y’s group created clones of the DNA (Deoxyribonucleic acid is a molecule composed of two chains that coil around each other to form a double helix carrying the genetic instructions used in the growth, development, functioning, and reproduction of all known living organisms and many viruses) fragments they wanted to express and injected them into the bacteria in hopes of luring away the repressor molecules that were preventing gene expression. They called these clones transcription factor decoys. “Others have used this similar kind of decoys for therapeutic applications in mammalian cells but we show here for the first time that it can be used for drug discovery by activating silent genes in bacteria” said Y.
To prove that the molecules they coded for were being expressed, researchers tested the decoy method first on two known gene clusters that synthesize natural products. Next they created decoys for eight silent gene clusters that had been previously unexplored. In bacteria injected with the decoys the targeted silent genes were expressed and the researchers harvested new products.
“We saw that the method works well for these large clusters that are hard to target by other methods” Y said. “It also has the advantage that it does not disturb the genome; it’s just pulling away the repressors. Then the genes are expressed naturally from the native DNA (Deoxyribonucleic acid is a molecule composed of two chains that coil around each other to form a double helix carrying the genetic instructions used in the growth, development, functioning, and reproduction of all known living organisms and many viruses)”.
In the search for drug candidates each product needs to be isolated and then studied to determine what it does. Of the eight new molecules produced the researchers purified and determined the structure of two molecules and described one in detail in the study – a type of oxazole a class of molecules often used in drugs. The researchers plan next to characterize the rest of the eight compounds and run various assays to find out whether they have any anti-microbial, anti-fungal, anti-cancer or other biological activities.
Y’s group also plans to apply the decoy technique to explore more silent biosynthetic gene clusters of interest in Streptomyces and in other bacteria and fungi to find more undiscovered natural products. Other research groups are welcome to use the technique for gene clusters they are exploring Y said.
“The principle is the same, assuming that gene expression is repressed by transcription factors and we just need to release that expression by using decoy DNA (Deoxyribonucleic acid is a molecule composed of two chains that coil around each other to form a double helix carrying the genetic instructions used in the growth, development, functioning and reproduction of all known living organisms and many viruses) fragments” Y said.