The SONATA BIS project focuses on discovering and understanding a new class of biocatalysts capable of performing precise structural modifications of pentacyclic triterpenes (PTs), natural compounds with confirmed biological activities, including anticancer, anti-inflammatory, metabolic, and cardioprotective effects.

A key component of the project is the development and full characterization of a new enzyme variant, AcmB2, which, for the first time, enables enzymatic dehydrogenation at the C1 position in compounds whose stereochemistry has previously hindered biotransformation. This reaction leads to the formation of 1-dehydro-3-keto triterpenes, molecules with unique biological properties that have been virtually inaccessible through chemical synthesis.

The SONATA BIS project is carried out in collaboration with leading research institutions in Poland and worldwide, including The University of Queensland (Australia), Forschungszentrum Jülich (Germany), Cracow University of Technology (Poland), Silesian University of Technology (Poland), University of Silesia in Katowice (Poland), Malopolska Centre of Biotechnology (MCB, Poland) and Natchem s.c.

The 1-dehydro-3-keto triterpenes obtained in this project may form the basis for new therapeutic directions, including:

• anticancer applications (mitochondrial apoptosis, EMT modulation, PI3K/Akt and NF-κB regulation),

• anti-inflammatory and antioxidant activity,

• metabolic regulation (analogous to activated steroid forms),

• dermatological and cosmetic applications (bioactive ingredients with documented functionality).

• The newly developed biocatalysts and reaction cascades may be used in the future for:

• sustainable API synthesis,

• bioproduction of natural metabolites,

• commercialization of triterpenes in pharmaceutical, medical, and cosmetic markets.

Pentacyclic triterpenes represent one of the richest families of natural bioactive compounds, yet their bioavailability and biological activity depend on subtle structural modifications. One of the most desirable but technically challenging reactions is A-ring dehydrogenation, which significantly enhances anticancer and metabolic activity (analogous to structural activation in steroids, e.g., prednisone vs. cortisone).

Until now, there has been a lack of:

• enzymes capable of oxidizing compounds with bulky substituents at C4,

• tools allowing controlled access to 1-dehydro triterpenes,

• mechanistic knowledge describing the biological potential of these compounds.

The SONATA BIS project fills this gap, opening the way to a new generation of bioactive PT derivatives.

The project integrates advanced experimental and computational methods that enable both enzyme engineering and mechanistic understanding of catalytic transformations:

• protein engineering – targeted mutagenesis, construction of AcmB and AcmB2 variants, and evaluation of how individual mutations alter catalytic activity,

• molecular modelling and MD simulations – analysis of active-site dynamics, transition-state energetics, and individual steps of the C1 dehydrogenation reaction,

• bioinformatic mining of the KstD family – identification of evolutionarily distant enzyme variants with potentially unique substrate specificities,

• enzymatic reactions in model, semi-preparative, and preparative scales – enabling isolation of triterpenes in quantities required for biological testing.

The newly discovered AcmB2 enzyme exhibits previously unreported activity toward dipterocarpol and triterpenes bearing bulky C4 substituents. Comparative studies with AcmB revealed significant differences in active-site geometry, pocket flexibility, and FAD coordination—all determining catalytic performance. These insights enable the design of enzymes with predictable specificity tailored to the synthesis of targeted triterpene derivatives.

This project establishes a novel enzymatic route to structurally complex, pharmacophoric molecules whose chemical synthesis is expensive, multistep, or impossible by conventional methods. Combining enzyme engineering with experimental validation creates a platform extendable to other classes of natural products.

The project is funded under the NCN SONATA BIS call, grant no. 2024/54/E/ST4/00364:
“Enzymatic preparation of novel dehydrogenated pentacyclic triterpenes of pharmacological significance.”