Shown here are two photos sets of tomato plant leaves, the top set of leaves have no pseudomonas infection and the bottom set of leaves have pseudomonas infection. [Chi P. Ting]

Bacteria produce small-molecule compounds that play key roles in how the organisms communicate, interact with symbiotic hosts, and compete. Researchers at the University of Illinois at Urbana-Champaign have now discovered a new bacterial pathway for producing natural products that scientists could feasibly exploit for synthetic biology. “The kind of reactions that these enzymes are doing are mind-boggling,” said Wilfred van der Donk, PhD, a Howard Hughes Medical Institute (HHMI) investigator. “When we first saw them, we were scratching our heads…we had to painstakingly prove that the reactions we thought the enzymes were doing, are indeed carried out.”

Van der Donk, who is the Richard E. Heckert endowed chair in chemistry, and his colleagues at Illinois collaborated with HHMI investigator and University of California, Los Angeles, professor of biological chemistry and physiology Tamir Gonen, PhD. The HHMI researchers, including first author Chi Ting, PhD, report on their findings in Science, in a paper titled, “Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products.”

Activity-based screens have traditionally been used to discover new types of bacterial compounds, but scientists can now carry out bacterial genome sequencing to identify biosynthetic gene clusters that encode enzymes which produce a diverse range of natural products. Ting and van der Donk are part of a Carl R. Woese Institute for Genomic Biology team that is harnessing genome mining to discover new microbial products.

“Genome mining allows you to start looking for compounds where you have absolutely no idea what they are going to be,” van der Donk explained. “Many labs in [our team] are trying to find new antibiotics by genome mining…you look for unusual things where we don’t know what is being made, and then you try to make the compound in a friendly organism.”

Proteins and peptides are composed of chains of amino acids, and some bacterial natural products are formed as small peptides that are then modified after translation. Most peptides and proteins are assembled in cells by ribosomes, which link together the correct sequence of amino acids, according to the genetic recipe. These are known as ribosomally synthesized and posttranslationally modified peptides (RiPPs). Other peptide-based natural products are the work of combinations of enzymes that don’t create peptides according to different templates, but instead generate the same amino acid chains and modifications every time, to produce just one product. “In natural product biosynthesis, both pathways are used,” van der Donk said. “… now we stumbled across something that has features from both.”

The researchers made their discovery while examining a cluster of genes in the bacterial plant pathogen Pseudomonas syringae. They found that the cluster included one gene that codes for a peptide made by a ribosome, while another encoded an enzyme that could add another amino acid onto the peptide chain. “In retrospect, it’s just a really clever way of doing things,” van der Donk suggested. “Having an enzyme that can do this to a pre-existing peptide means that now…you can use it as a scaffold and just keep making the natural product time and time again.”

In this novel type of hybrid synthetic process the new amino acid added to the peptide is modified through a series of steps, then broken off, returning the ribosomally created peptide back to its starting step. The process can be likened to a sourdough starter in breadmaking. As long as the starter remains active, it doesn’t need to be recreated from scratch to make a subsequent batch of bread.

The investigators were keen to find out more about the structure of the new natural product, but the nanocrystals were too unstable to analyze by microcrystal electron diffraction (MicroED) microscopy using traditional approaches. They then turned to Gonen’s lab, which had recently applied a cutting-edge approach that uses electron microscopy on flash-frozen microcrystals of purified substances, to the determination of the structures of small molecules. “We then turned to the Falcon III direct electron detector, one of the most sensitive cameras for cryo-EM that was recently demonstrated to be suitable for MicroED data collection and structure determination and that minimizes radiation damage because of its high sensitivity and high frame rate,” the researchers wrote.

“Once you’ve made the natural product, now you need to figure out what it is,” van der Donk noted. “Our collaborators wanted to show the utility of this method for an unknown molecule of natural origins. This was really a win-win situation for both labs. I think the whole natural products community probably will want to start using this technique.”

The researchers say the new molecule isn’t a RiPP—“because it is not ribosomally synthesized, but is made by posttranslational modification reactions—but acknowledge that they don’t yet know the function of the peptide, which contains L-3-thiaglutamate at its C terminus, or whether the unstable compound undergoes further chemical modification. The researches also reason that plants might harness 3-thiaGlu or a derivative in response to infection by P. synringae. “Plants were recently shown to use Glu [glutamate] for a systemic signaling response to pathogens, and it is possible that 3-thiaGlu or a product derived from it interferes with Glu signaling similar to other antimetabolite toxins made by P. syringae that block jasmonate and ethylene signaling pathways,” they stated.

The team separately identified other examples of similar synthetic mechanisms, including the production of an antitumor compound by a soil microorganism. They say they are encouraged by the potential to identify gene clusters that make natural products, and find ways to exploit the newly discovered synthetic pathway. “We’re also excited as to how we might be able to use this for synthetic biology,” van der Donk said. “Because the overhead, the amount of resources that have to go in to make a natural product, is fairly low here. You make this peptide, a few enzymes, and out comes rolling an anti-tumor compound… There’s a lot of interest right now in engineering bacteria to have anticancer activity, and this is relatively low-hanging fruit with respect to making the organism make the molecules for you.”

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