This research, published in the Proceedings of the National Academy of Sciences (PNAS), details a method that uses enzymes to ‘fold’ individual molecules to create many different variations each with unique chemical properties.
Triterpenes, a large group of plant compounds, are already used in the food and nutrition industry with uses as a foaming agent and emulsifier in food and beverages, and glycyrrhizin (from liquorice), a sweetener around 50 times sweeter than sugar.
The researchers believe this technique will eventually be able to create triterpenes that can be added to (or bred into) food. One example could be a tailored triterpene specifically created to maintain good intestinal flora health and depressing pathogenic microbes.
“For these types of applications it will be critical to compare the effects of the food matrix with and without the triterpene components in order to carry out a rigorous analysis of the effects of these compounds,” said Professor Anne Osbourn, lead author and director of the Norwich Research Park Industrial Biotechnology Alliance.
Osbourn and her US collaborators detail a technique that uses a mutated version of the enzyme SAD1 from the oat plant. This enzyme is a triterpene synthase enzyme responsible for a crucial step in triterpene construction.
Normal triterpenes contain a pentacyclic scaffold (5 carbon rings). This is then further modified by other enzymes to produce hundreds of different triterpene compounds.
With one of the mutated enzymes, triterpenes with tetracyclic scaffolds (four carbon rings) are formed resulting in different chemical properties.
Osbourn’s team, based at the John Innes Centre in Norwich, also discovered that the same mutation in the same gene from a different plant, Arabidopsis thaliana, which gave identical results.
The researchers believe this ‘molecular switch’ from pentacyclic to tetracyclic triterpene production is a feature conserved between different plant species.
“This was an exciting discovery,” said Osbourn, “because we realised that we could not only modify the enzyme to produce different triterpene scaffolds, but we could also modify the building block to make different more highly oxygenated scaffolds.”
The team then placed the mutant SAD1 gene into yeast to see if large quantities of triterpenes could be made. Here, they found that the SAD1 enzyme preferred dioxidosqualene (DOS) as a substrate rather than 2,3-oxidosqualene (OS).
This preference is important for the generation of pentacyclic rather than tetracyclic products and may be a “substrate specificity switch” in certain forms of the triterpene synthase enzyme.
While the discovery is exciting and signals a new direction, Osborn is well aware of the challenges of customising triterpenes.
“We now need to build a systematic framework in order to be able to deepen our understanding of the amount of triterpene diversity that is out there in nature (currently around 30,000 triterpenes so far),” she said.
“We also need to enhance our ability to modify different triterpene scaffolds in as many positions as possible to really advance our designer tool kit, and build our capacity to make and test the suites of structural analogs that we make.”
Triterpenes are already used for wide range of applications in the food and nutrition industry. Saponin-containing plant extracts (e.g. from yucca and quillaja) are used for a wide variety of purposes including as foaming agents and emulsifiers in food and beverages. Another type of triterpene, ginsenosides are found in ginseng.
The triterpene-rich plants eaten by the Masai tribe have been linked with low levels of cholesterol and heart disease despite their high meat diet.
Source: Proceedings of the National Academy of Sciences.
Published online ahead of print, doi: 10.1073/pnas.1605509113
“A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases.”
Authors: Anne Osbourn et al.