New Carbohydrate Oxidising Enzymes prepared by directed evolution and their application in synthesis of AminoGlycoside derivatives with antibiotic potential and rare sugars.

Project Information

COxEnzymes - AGs

Grant agreement ID: 101152159

DOI

10.3030/101152159

Project closed

EC signature date 5 March 2024

Start date 1 June 2024

End date 31 May 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 172 618,56

Coordinated by

UNIVERSIDADE NOVA DE LISBOA
Portugal

Periodic Reporting for period 1 - COxEnzymes - AGs (New Carbohydrate Oxidising Enzymes prepared by directed evolution and their application in synthesis of AminoGlycoside derivatives with antibiotic potential and rare sugars.)

Reporting period: 2024-06-01 to 2026-05-31

Summary of the context and overall objectives of the project

Antimicrobial resistance is one of the most pressing global health challenges, creating an urgent need for new strategies to develop improved antibiotics and sustainable manufacturing processes. At the same time, rare sugars are attracting increasing interest because of their potential applications in pharmaceuticals, functional foods and biotechnology. However, both the modification of antibiotics, namely aminoglycosides and the production of rare sugars remain challenging using conventional chemical synthesis, which often requires multiple reaction steps, protecting groups and environmentally demanding processes.

The COxEnzymes-AGs project addressed these challenges by developing sustainable enzyme-based approaches for selectively modifying carbohydrate-containing molecules. The project focused on engineering carbohydrate-oxidising enzymes capable of performing highly selective oxidation reactions under mild conditions using molecular oxygen, providing greener alternatives to conventional chemical methods.

The project pursued two complementary objectives. The first was to develop improved enzymes for the efficient production of rare sugars and their derivatives through chemoenzymatic synthesis. The second was to investigate the potential of engineered enzymes to selectively modify aminoglycoside antibiotics, laying the foundation for the future development of novel antibiotic derivatives.

To achieve these goals, the project combined protein engineering, directed evolution, biocatalysis, carbohydrate chemistry and advanced analytical techniques. The developed enzyme variants were evaluated for their catalytic properties and integrated into sustainable synthetic workflows.

The project contributes to the European Green Deal and Horizon Europe objectives by promoting environmentally friendly chemical manufacturing based on renewable resources and biocatalysis. Its results expand the available toolbox of engineered enzymes and support future innovation in biotechnology, pharmaceutical research and sustainable industrial processes. In addition, the fellowship strengthened international collaboration, knowledge transfer and interdisciplinary training in enzyme engineering and carbohydrate chemistry.

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

The project focused on the engineering and application of carbohydrate-oxidising enzymes for sustainable chemoenzymatic synthesis. Two complementary research directions were pursued: (i) the development of engineered enzymes for the production of rare sugars and carbohydrate derivatives, and (ii) the exploration of enzymatic oxidation of aminoglycoside antibiotics.

During the project, engineered variants of glycoside 3-oxidase (PsG3Ox) and galactose oxidase (GalOx) were evaluated and further characterised. The PsG3Ox platform was successfully applied in the chemoenzymatic synthesis of rare sugars, leading to improved production of D-allose and D-gulose, as well as the preparation of novel allose-containing derivatives for subsequent biological evaluation. The project generated exploitable results that contributed to an ongoing patent application on engineered glycoside 3-oxidases for rare sugar synthesis, on which the fellow is recognised as a co-inventor.

In parallel, a GalOx engineering strategy combining rational design, saturation mutagenesis and directed evolution was established. An engineered GalOx variant exhibiting efficient C6 oxidation of glucose and methyl glucoside was identified and characterised. The regioselectivity of oxidation was confirmed by NMR analysis of the isolated products. The engineered enzyme was subsequently evaluated towards selected aminoglycosides, demonstrating detectable catalytic activity and providing valuable insights for future enzyme engineering.

The project combined enzyme engineering, molecular biology, biocatalysis, organic synthesis and advanced analytical techniques, including enzyme kinetics, chromatography and NMR spectroscopy. Overall, the work expanded the available toolbox of carbohydrate-oxidising enzymes and generated new sustainable chemoenzymatic methodologies with potential applications in biotechnology and pharmaceutical research.

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)

The COxEnzymes-AGs project advanced the field of carbohydrate biocatalysis by expanding the available toolbox of engineered carbohydrate-oxidising enzymes with improved catalytic properties and new synthetic applications. The project demonstrated that enzyme engineering and directed evolution can provide highly selective oxidation catalysts suitable for sustainable chemoenzymatic synthesis under mild reaction conditions.

A major outcome of the project was the development and application of engineered oxidases for the efficient synthesis of rare sugars and their derivatives. The developed biocatalytic methodologies provide environmentally friendly alternatives to conventional multistep chemical synthesis and contribute to improving access to valuable carbohydrate building blocks with potential applications in biotechnology, pharmaceuticals and functional foods.

The project also established proof-of-concept for the enzymatic oxidation of selected aminoglycoside antibiotics. Although further enzyme engineering will be required to improve catalytic efficiency, the obtained results identified promising starting points and provided valuable mechanistic insights that will support the future development of regioselective biocatalytic modification of complex antibiotics.

The fellowship generated results with exploitation potential and contributed to intellectual property protection activities related to engineered glycoside 3-oxidases for rare sugar synthesis. The developed enzyme platforms and synthetic methodologies provide a foundation for future research, technology transfer and potential industrial applications.

Further uptake of the project outcomes will benefit from continued enzyme engineering, process optimisation and scale-up studies, together with biological evaluation of newly synthesised carbohydrate derivatives. These activities will facilitate the translation of the developed technologies towards practical applications in sustainable biotechnology and pharmaceutical research.