Experts of the UB and CRAG reconstitute the complete synthesis of carnosic acid in yeast cultures

Moreover, the carnosic acid is the biosynthetic precursor of other bioactive molecules such as the tanshinones from Salvia miltiorriza, molecules of biomedical interest for their high anti-tumor activity.

Synthetic routes in the laboratory to protect plant population

Lots of plants are able to synthesize a wide range of secondary metabolites, which are the source of new molecules with pharmacological activity. The intensive exploitation of these resources can risk natural populations of the producing species, therefore it is important to elucidate and reconstitute the metabolic routes able to produce active compounds of pharmacologic interest without threatening natural species -in other organisms.

With this aim, the new study was carried out in the project Plant terpenoids for human health: a chemical and genomic approach to identify and produce bioactive compounds (TERPMED), funded by the European Union and coordinated by Albert Ferrer, professor at the Department of Biochemistry and Physiology of the University of Barcelona, and member of the Center for Research and Agricultural Genomics (CRAG). This work results from the collaboration of the teams of professors Alain Tissier (Leibniz Institute for Plant Biochemistry, Germany); Albert Ferrer and David Manzano (Faculty of Pharmacy and Food Sciences of the UB and CRAG), and Angelos Kanellis (Aristotile University of Thessaloniki, Greece).

Fighting diseases with natural products

Rosemary and sage are aromatic plants that synthesize carnosic acid and carnosol, two phenolic diterpenes that are research targets due to their antioxidant, antimicrobial, anti-inflammatory and anti-tumor properties, as well as potential therapeutic agents to prevent and treat neurodegenerative diseases.

So far, scientific bibliography had proposed some chemical synthetic pathways of these molecules, processes that are generally long and complex. The new study offers the possibility to explore the uses of yeast culture -as well as other organisms such as plants- as biofactors to produce these two bioactive molecules of high value at an industrial scale. At the moment, carnosic acid and carnosol are mainly used in the fields of nutrition and cosmetics due their high antioxidant and antimicrobial activity. In addition, they have been approved food additives in the European Union, China and Japan.

According to Professor Albert Ferrer, “the work published in Nature Communications also proofs the potential that shows the approach of applied combined biochemistry on yeast to figure out the synthesis of the carnosic acid as a methodology to elucidate synthetic ways and produce other related terpenes of biotechnological interest”.

Working out the synthetic way of carnosic acid

In the new article, the scientific team identified the enzymes that catalyze the conversion of a natural terpenoid (abietatriene) into carnosic acid, and describes the previous reconstitution of the complete synthetic carnosic acid pathway into yeast cells, out of a common structural precursor of the diterpenes, geranylgeranyl diphosphate. In particular, the experts identified the 11-hydroxy ferruginol from Rosemarinus officinalis and Salvia fruticosa (a molecule that catalyzes the sequencing oxidation from abietratriene to ferruginol, and from this molecule to 11-hydrozy ferruginol), and C20-oxidasa, which turns this last intermediary into carnosic acid through three consecutive oxidation reactions.

The team reconstituted the synthetic pathway of the carnosic acid completely, which spontaneously oxidizes to carnosol, through the co-expression in yeast cells of these two enzymes, which are members of the family of cytochrome P450, and from the ones that synthesise the abietatriene precursos out of the geranylgeranyl diphosphate. Throughout a monitored mutagenesis approach, based on molecular modeling, it has been established that the change of only three amino-acids is enough to turn the S. miltiorrhiza ferroginol sintasa enzyme -which can only synthesise ferruginol out of abietratrien- into an equivalent 11-hydroxy ferruginol to the ones in R. officinalis and S. fruticosa.