The project “Targeted Biosynthesis of Pharmaceutical‑Relevant Triterpenoids” (ASPIRANT) was carried out at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) in Münster in cooperation with the Technical University of Munich (TUM) and the companies Vivacell Biotechnology GmbH and Phytowelt GreenTechnologies GmbH. The funding came from the German Federal Ministry of Education and Research (BMBF) under the initiative “Mass‑Customized Bio‑Based Ingredients for a Competitive Bio‑Economy.” The project ran from 1 February 2020 to 31 January 2023, with Dr. Christian Schulze Gronover as project leader. IME was responsible for the design, execution, and reporting of all experimental work, while TUM contributed analytical expertise and the companies supplied industrial partners, substrates, and scale‑up facilities.
The scientific outcome of ASPIRANT is a set of engineered yeast strains capable of producing high‑value triterpenoids at increased scale and purity. Two distinct Saccharomyces cerevisiae strains were developed: Genotype 1, based on a previously published lupeol‑producing strain, and Genotype 2, which carries an additional insertion in the endogenous Galactose10 gene enabling triterpenoid expression in non‑galactose media. These strains were used to upscale lupeol production, yielding more than 10 g of triterpenoid mixtures with a purity above 98 % and a production rate exceeding 100 µg L⁻¹. In addition, three pharmaceutical‑relevant triterpenoids were produced at a scale of 300 mg each, meeting the deliverable requirement of at least 15 compounds above 1 mg and 98 % purity for the first pharmacological screening.
A comprehensive optimisation of cultivation protocols was performed. Three different fermentation protocols were tested, and the most promising one was refined with respect to media composition, aeration, feeding strategy, feeding rate, temperature, and duration. The final protocol delivered the highest titres and was documented for future use. Extraction and downstream processing were systematically evaluated: liquid‑liquid extraction, solid‑phase adsorption, and chromatographic analyses were compared, and a cost‑effective protocol was selected after a critical assessment of technical and economic aspects.
Molecular engineering efforts produced a library of enzymes: ten oxidosqualene cyclases, five triterpenoid‑oxidation enzymes, and five glycosylation enzymes were cloned and expressed in yeast. These enzymes enabled the synthesis of a diverse set of triterpenoid scaffolds. Bioactivity data from the first screening were compiled, allowing the selection of the most promising candidates for further characterization. The optimized yeast strain achieved a production performance of >100 µg L⁻¹ for the target triterpenoids, fulfilling the deliverable of a high‑yielding production strain.
The project also included a workshop (M5) with potential industrial partners to introduce the tailored triterpenoids and discuss commercialization pathways. This event, led by IME, facilitated knowledge transfer and opened avenues for future collaborations. All reports and deliverables were submitted on schedule, and the final project report was completed in accordance with the project plan.
In summary, ASPIRANT delivered engineered yeast strains, optimized fermentation and downstream protocols, a library of triterpenoid‑synthesizing enzymes, and a set of high‑purity pharmaceutical‑relevant triterpenoids ready for further development. The collaboration between academia and industry, supported by BMBF funding, demonstrates the feasibility of converting plant triterpenoid pathways into efficient microbial production platforms.