Elucidation of the biosynthesis pathway of the glaucoma drug pilocarpine in Pilocarpus plants

Project Information

PiloSyn

Grant agreement ID: 101066903

DOI

10.3030/101066903

Project closed

EC signature date 29 May 2022

Start date 1 May 2023

End date 30 April 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 214 934,40

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - PiloSyn (Elucidation of the biosynthesis pathway of the glaucoma drug pilocarpine in Pilocarpus plants)

Reporting period: 2023-05-01 to 2025-04-30

Summary of the context and overall objectives of the project

The medical drug pilocarpine is one of the treatments for glaucoma or dry mouth. Pilocarpine is an alkaloid made by Pilocarpus shrubs naturally grown in the Amazon rainforest. As it is not cost-effective to chemically synthesize pilocarpine, commercial demands are met by extracting it from its natural source, the leaves of Pilocarpus plants. Since cultivated Pilocarpus plants have lower pilocarpine content than wild plants, leaves of wild-grown Pilocarpus are preferred for production. Excessive leaf harvesting of wild populations has led to the official listing of several Pilocarpus species as threatened. Synthetic biology offers a sustainable approach for the production of pilocarpine through its synthesis in heterologous hosts, such as microbes. However, to realize this vision, a detailed knowledge about the biosynthetic pathway of pilocarpine in Pilocarpus plants is needed. The aim of PiloSyn is to understand how Pilocarpus plants make pilocarpine, thus laying the foundation for a more sustainable pilocarpine production.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

PiloSyn had access to Pilocarpus microphyllus seeds harvested in the Amazon rainforest. Seed germination was challenging, but it was possible to grow a few dozen P. microphyllus plants under controlled conditions. PiloSyn analyzed the content of pilocarpine and related alkaloids in several P. microphyllus tissues of different ages. The analysis revealed that the plants only start to produce pilocarpine at a certain age. Furthermore, feeding studies with isotopically labeled compounds identified the biosynthetic tissue responsible for pilocarpine biosynthesis of related alkaloids. These results provide a roadmap for future RNAseq analysis aimed at identifying enzymes involved in pilocarpine biosynthesis.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

PiloSyn provided valuable insights into how P. microphyllus plants make pilocarpine by identifying the relevant biosynthetic tissues and discovering that the plants only start to produce pilocarpine at a certain age. This lays the foundation for further studies on pilocarpine biosynthesis, especially via tissue- and age-specific RNASeq analysis. Beyond pilocarpine research, PiloSyn, obtained comprehensive metabolite datasets from different P. microphyllus tissues and ages. As the plant tissue is not widely accessible, the data sets are of scientific value. The scientific community can use the datasets to search for new molecules, which can be evaluated as e.g new active pharmaceutical ingredients (API).