Periodic Reporting for period 4 - ABIONYS (Artificial Enzyme Modules as Tools in a Tailor-made Biosynthesis)
Periodo di rendicontazione: 2025-02-01 al 2025-10-31
Chemical production has long been deeply intertwined with fossil resources. A vast proportion of the materials and compounds that make up our everyday lives, from pharmaceuticals to plastics, have historically been derived from crude oil and natural gas. The consequences of this dependency are well known: greenhouse gas emissions, environmental pollution, and health risks affecting communities worldwide. Transforming the chemical industry towards sustainable, renewable alternatives is one of the defining challenges of our era. The ABIONYS project contributed to this transformation over five years by developing a new generation of biological tools for chemical production. The central idea: instead of relying on harsh conditions and hazardous reagents, why not harness the molecular machines that living cells already use? These machines called enzymes are proteins acting as highly precise catalysts, driving reactions under mild, environmentally friendly conditions. They are naturally produced by bacteria and yeasts, making them inherently renewable.
The challenge is that nature's enzyme toolkit was not designed with industrial chemistry in mind. Natural enzymes evolved for cellular survival, not for the reactions chemists need to synthesise valuable compounds. ABIONYS aimed to go beyond what nature provides: discovering, designing, and deploying enzymes capable of reactions entirely new to biology.
Teaching Enzymes New Tricks: Peroxidases and laccases, normally involved in breaking down plant material, were repurposed to form carbon-nitrogen bonds critical in pharmaceutical synthesis, and the C–N bond forming methodology was directly empolyed on existing drug molecules to open new avenues for drug diversification. Moreover, lytic polysaccharide monooxygenases (LPMOs), natural biomass-decomposers, were recruited for carbene transfer reactions, and a novel class of miniaturised microenzymes was developed using AlphaFold as a design tool, capable of halogenation reactions essential in producing biologically active compounds.
Enzymatic Assembly Lines: ABIONYS assembled cascades of multiple enzymes converting simple starting materials into complex bioactive molecules. In one-pot approaches, a series of enzymes performed multiple steps in a single reaction vessel without isolation of intermediates, dramatically reducing waste and energy consumption. Seven biocatalytic synthesis routes were implemented, providing access to bioactive natural products such as tenuipesone A/B, angiolactone B, and cephalotaxine; with yields of up to 72% over five consecutive enzymatic steps.
Programming Microbes as Living Factories: The ultimate vision was to transfer successful enzymatic reactions into living microorganisms by programming bacteria or yeasts to perform the desired chemistry from within. A proof-of-concept study showed bacteria could be engineered to co-express multiple enzymes and convert biorefinery waste (furans) into value-added compounds. Further attempts to implement more exotic enzymatic reactions inside living cells proved highly challenging, and this objective remains the subject of future research.
Over five years, ABIONYS delivered new enzymatic tools, elegant multi-enzyme cascades, and a portfolio of high-quality publications in leading international chemistry journals, substantially advancing the field and demonstrating that enzymes, with the right ingenuity, can become versatile tools for a greener chemical future.
The challenge is that nature's enzyme toolkit was not designed with industrial chemistry in mind. Natural enzymes evolved for cellular survival, not for the reactions chemists need to synthesise valuable compounds. ABIONYS aimed to go beyond what nature provides: discovering, designing, and deploying enzymes capable of reactions entirely new to biology.
Teaching Enzymes New Tricks: Peroxidases and laccases, normally involved in breaking down plant material, were repurposed to form carbon-nitrogen bonds critical in pharmaceutical synthesis, and the C–N bond forming methodology was directly empolyed on existing drug molecules to open new avenues for drug diversification. Moreover, lytic polysaccharide monooxygenases (LPMOs), natural biomass-decomposers, were recruited for carbene transfer reactions, and a novel class of miniaturised microenzymes was developed using AlphaFold as a design tool, capable of halogenation reactions essential in producing biologically active compounds.
Enzymatic Assembly Lines: ABIONYS assembled cascades of multiple enzymes converting simple starting materials into complex bioactive molecules. In one-pot approaches, a series of enzymes performed multiple steps in a single reaction vessel without isolation of intermediates, dramatically reducing waste and energy consumption. Seven biocatalytic synthesis routes were implemented, providing access to bioactive natural products such as tenuipesone A/B, angiolactone B, and cephalotaxine; with yields of up to 72% over five consecutive enzymatic steps.
Programming Microbes as Living Factories: The ultimate vision was to transfer successful enzymatic reactions into living microorganisms by programming bacteria or yeasts to perform the desired chemistry from within. A proof-of-concept study showed bacteria could be engineered to co-express multiple enzymes and convert biorefinery waste (furans) into value-added compounds. Further attempts to implement more exotic enzymatic reactions inside living cells proved highly challenging, and this objective remains the subject of future research.
Over five years, ABIONYS delivered new enzymatic tools, elegant multi-enzyme cascades, and a portfolio of high-quality publications in leading international chemistry journals, substantially advancing the field and demonstrating that enzymes, with the right ingenuity, can become versatile tools for a greener chemical future.
ABIONYS addressed its key challenges through three individual yet interconnected work packages.
The Tools Work Package developed a range of new-to-nature biocatalytic modules to broaden the enzymatic reaction portfolio and make enzymes a more attractive, renewable and sustainable catalyst in chemical production. Since November 2020, the team delivered a series of new methodologies based on non-natural transformations with high relevance in modern organic synthesis. New enzymatic modules were developed for ene reactions and cycloadditions forming heterocycles (Angew. Chem. Int. Ed. 2023, e202213671; Green Chem. 2023, 3166-3174). Oxidative C–N bond formation mediated by peroxidases and laccases was extended to the direct late-stage functionalization of hydroxamic acid-based pharmaceuticals, enabling rapid diversification of bioactive compounds (ChemRxiv, 10.26434/chemrxiv.10001704/v1). A borrowing hydrogen biocatalysis strategy was implemented for the asymmetric ring contraction of 2-hydroxypyranones, a transformation with broad utility in the synthesis of chiral building blocks (Chem. Sci. 2025, d5sc02591e). Additional tools based on the bromocyclization of allenols by chloroperoxidase (ChemistryOpen 2021, open.202100236) and multienzymatic synthesis of gamma-lactam building blocks (Eur. J. Org. Chem. 2023, ejoc.202300288) further expanded the biocatalytic repertoire. In a significant advance towards the end of the project, ABIONYS also developed a novel class of miniaturised microenzymes using AlphaFold as a design platform. By repurposing catalytically inactive carbohydrate-binding modules into novel metal-binding protein scaffolds, the team created unprecedented small enzymes capable of copper-dependent carbene transfer catalysis and vanadium-dependent haloperoxidase activity (ChemRxiv, 10.26434/chemrxiv-2025-spvxf) bridging synthetic chemistry, structural biology, and AI-guided protein design.
The Applications Work Package utilised these enzymatic tools to construct highly complex reaction cascades targeting bioactive natural products. Furans derived from wood biorefinery side streams served as a key renewable feedstock. The intrinsic mutual tolerance of enzymes allows multiple chemical transformations to be performed in a single reaction vessel, eliminating isolation and purification steps and thereby saving time, energy, and chemical waste. This strategy was showcased in the total synthesis of Angiopterlactone B, a highly complex tricyclic plant metabolite (Angew. Chem. Int. Ed. 2023, e202301178), and extended through biocatalytic rearrangement of furans to spirolactones (ACS Catal. 2023, acscatal.3c00132) and O-heterocycle synthesis via bifunctional lipase-metal biohybrids (ChemCatChem 2022, cctc.202200362). The asymmetric one-pot synthesis of Tenuipesone A/B (ChemistryEurope 2026, ceur.202500440) brought the total to seven biocatalytic natural product syntheses: crassalactone, cavernosine, osmundalin, tenuipesone A/B, angiolactone B, lanceolactone, and cephalotaxine. Yields of 72% over five consecutive enzymatic steps were achieved in selected cases – reflecting both the efficiency and selectivity of the assembled cascades. Choline oxidase-based peroxidase cascade systems were also developed as supporting tools (ChemCatChem 2024, cctc.202401216).
The Cell Factories Work Package worked towards creating tailor-made microbial producers capable of performing non-natural chemical transformations from within the cell. A first breakthrough was achieved when parts of the furan valorization cascade were successfully transferred into a genetically modified bacterium (ChemSusChem 2023, e202201790). Further attempts to implement the newer enzymatic tools, particularly those based on carbene transfer and new-to-nature oxidative chemistry, inside living cells encountered significant challenges, and this work package remained only partially fulfilled by the end of the project.
Across all three work packages, ABIONYS generated 13 publications in high-impact journals including Angewandte Chemie, Chemical Science, ACS Catalysis, & Green Chemistry, alongside 3 preprints currently under review. The project also opened new and sometimes unexpected research directions, including AI-guided protein design for de novo biocatalyst development and preliminary findings on copper-dependent enzymes in combating mycobacterial infections – both anticipated to generate follow-up research activities in the coming years.
The Tools Work Package developed a range of new-to-nature biocatalytic modules to broaden the enzymatic reaction portfolio and make enzymes a more attractive, renewable and sustainable catalyst in chemical production. Since November 2020, the team delivered a series of new methodologies based on non-natural transformations with high relevance in modern organic synthesis. New enzymatic modules were developed for ene reactions and cycloadditions forming heterocycles (Angew. Chem. Int. Ed. 2023, e202213671; Green Chem. 2023, 3166-3174). Oxidative C–N bond formation mediated by peroxidases and laccases was extended to the direct late-stage functionalization of hydroxamic acid-based pharmaceuticals, enabling rapid diversification of bioactive compounds (ChemRxiv, 10.26434/chemrxiv.10001704/v1). A borrowing hydrogen biocatalysis strategy was implemented for the asymmetric ring contraction of 2-hydroxypyranones, a transformation with broad utility in the synthesis of chiral building blocks (Chem. Sci. 2025, d5sc02591e). Additional tools based on the bromocyclization of allenols by chloroperoxidase (ChemistryOpen 2021, open.202100236) and multienzymatic synthesis of gamma-lactam building blocks (Eur. J. Org. Chem. 2023, ejoc.202300288) further expanded the biocatalytic repertoire. In a significant advance towards the end of the project, ABIONYS also developed a novel class of miniaturised microenzymes using AlphaFold as a design platform. By repurposing catalytically inactive carbohydrate-binding modules into novel metal-binding protein scaffolds, the team created unprecedented small enzymes capable of copper-dependent carbene transfer catalysis and vanadium-dependent haloperoxidase activity (ChemRxiv, 10.26434/chemrxiv-2025-spvxf) bridging synthetic chemistry, structural biology, and AI-guided protein design.
The Applications Work Package utilised these enzymatic tools to construct highly complex reaction cascades targeting bioactive natural products. Furans derived from wood biorefinery side streams served as a key renewable feedstock. The intrinsic mutual tolerance of enzymes allows multiple chemical transformations to be performed in a single reaction vessel, eliminating isolation and purification steps and thereby saving time, energy, and chemical waste. This strategy was showcased in the total synthesis of Angiopterlactone B, a highly complex tricyclic plant metabolite (Angew. Chem. Int. Ed. 2023, e202301178), and extended through biocatalytic rearrangement of furans to spirolactones (ACS Catal. 2023, acscatal.3c00132) and O-heterocycle synthesis via bifunctional lipase-metal biohybrids (ChemCatChem 2022, cctc.202200362). The asymmetric one-pot synthesis of Tenuipesone A/B (ChemistryEurope 2026, ceur.202500440) brought the total to seven biocatalytic natural product syntheses: crassalactone, cavernosine, osmundalin, tenuipesone A/B, angiolactone B, lanceolactone, and cephalotaxine. Yields of 72% over five consecutive enzymatic steps were achieved in selected cases – reflecting both the efficiency and selectivity of the assembled cascades. Choline oxidase-based peroxidase cascade systems were also developed as supporting tools (ChemCatChem 2024, cctc.202401216).
The Cell Factories Work Package worked towards creating tailor-made microbial producers capable of performing non-natural chemical transformations from within the cell. A first breakthrough was achieved when parts of the furan valorization cascade were successfully transferred into a genetically modified bacterium (ChemSusChem 2023, e202201790). Further attempts to implement the newer enzymatic tools, particularly those based on carbene transfer and new-to-nature oxidative chemistry, inside living cells encountered significant challenges, and this work package remained only partially fulfilled by the end of the project.
Across all three work packages, ABIONYS generated 13 publications in high-impact journals including Angewandte Chemie, Chemical Science, ACS Catalysis, & Green Chemistry, alongside 3 preprints currently under review. The project also opened new and sometimes unexpected research directions, including AI-guided protein design for de novo biocatalyst development and preliminary findings on copper-dependent enzymes in combating mycobacterial infections – both anticipated to generate follow-up research activities in the coming years.
ABIONYS pushed the boundaries of biocatalysis by delivering enzymatic tools and strategies well beyond what was previously possible. Enzymatic carbon-nitrogen bond formation via peroxidases and laccases, carbene transfer by LPMOs, and AI-designed miniaturised metal-binding enzymes all lacked established precedent, each representing a genuinely new reaction type added to the biocatalytic repertoire. In multi-step synthesis, the project moved well beyond single-enzyme transformations, demonstrating integrated one-pot cascades capable of assembling highly complex natural products from simple renewable feedstocks at a level of efficiency and selectivity previously unattainable without whole-cell fermentation or lengthy chemical routes. The use of AlphaFold to repurpose non-catalytic protein scaffolds into functional metalloenzymes represents a frontier advance in AI-guided biocatalyst design with broad future implications. Together, these achievements mark a genuine step change in what enzyme-based synthesis can accomplish.