Biocatalytic upgrading of natural biopolymers for reassembly as multipurpose materials

Project Information

BioUPGRADE

Grant agreement ID: 964764

DOI

10.3030/964764

Project closed

EC signature date 19 January 2021

Start date 1 May 2021

End date 31 October 2025

Funded under

• EXCELLENT SCIENCE - Future and Emerging Technologies (FET)

Total cost

€ 2 992 140,00

EU contribution

€ 2 992 140,00

Coordinated by

AALTO KORKEAKOULUSAATIO SR
Finland

Periodic Reporting for period 3 - BioUPGRADE (Biocatalytic upgrading of natural biopolymers for reassembly as multipurpose materials)

Reporting period: 2023-11-01 to 2025-10-31

Summary of the context and overall objectives of the project

Countries world-wide have expressed commitment to reduce greenhouse gas (GHG) emissions from today’s more than 60 GtCO2e yr-1 to 25–30 GtCO2e yr−1 by 2030 and net zero by 2050. Achieving this goal will require a multipronged approach that includes establishing sustainable bio-based economies characterized by reduced reliance on fossil resources. Biological systems naturally recycle GHGs while generating an enormous array of chemicals and materials. Biomanufacturing harnesses this capacity by deploying microorganisms and enzymes that convert renewable bioresources to desired fuels, platform chemicals and materials, including bioderived alternatives to plastics. By 2030, its estimated that the biotechnologies that catalyze these transformations will be producing over 35% of the chemicals and materials we use every day.

The BioUPGRADE action combined computational biology, functional genomics and material sciences to establish biocatalysts in bio-based materials manufacturing. Our goal has been to develop biotechnologies that tailor the chemical and/or physical properties of major structural polysaccharides (e.g. cellulose, hemicellulose, chitin) for application in sustainable textiles, conductive inks for packaging, and customized hydrogels for health and personal care products.

Our specific project objectives were to 1) design biocatalysts that introduce new chemical or physical functionality to structural polysaccharides from plant and fungal sources, 2)establish application-driven functional screens for biocatalysts in materials engineering, and 3) develop procedures for the controlled assembly of tailored bio-fibres for multipurpose bio-based materials.

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

We successfully applied computational methods to identify molecular signatures of biocatalysts that act on high molecular weight substrates; in particular, structural polysaccharides. Approaches included comparative genomics, ancestral sequence resurrection (ASR), structure prediction using deep learning models such as Alphafold2 and molecular dynamics simulations to select and design (i) microbial expansin-related proteins that disrupt non-covalent associations within cellulose or chitin fibres, and (ii) carbohydrate-active enzymes with ability to selectively oxidize and aminate structural polysaccharides.

Prioritized proteins were recombinantly produced in a bacterial or eukaryotic host and then functionally characterized using complementary, application-driven functional screens that uncovered biocatalysts best suited for materials engineering. Targeted biocatalyst activities included (i) microbial expansins that alter the rheology, porosity, and/or surface accessibility of cellulose and chitin, (ii) carbohydrate oxidoreductases that introduce carbonyls at specific positions within cellulose, hemicellulose, and/or chitin substrates, and (iii) carbohydrate-active transaminases that accept high molecular weight, oxidized carbohydrates. Functional screens of microbial expansins were established to measure changes to biofibre organisation using rheometry, BioSANS, SAXS, zeta potential measurements, microscopy, and quartz crystal microbalance with dissipation (QCM-D). Functional screens for carbohydrate oxidoreductases and carbohydrate transaminases were based on fluorometric and colorimetric detection of reaction products, and designed to accommodate 96-well plate formats for rapid screening. Detailed characterizations then used NMR to evaluate the regioselectivity of carbohydrate oxidoreductases, and mass spectrometry to determine the degree of oxidation and amination of tested substrates.

Best performing biocatalysts were then scaled-up using bioreactor units with capacities up to 200L. Using the characterized microbial expansins, along with engineered enzymes and carbohydrate oxidase-transaminase cascade, we demonstrated biocatalytic pathways to conductive inks and chemically functionalized hemicelluloses relevant to hydrogel formulations. We also demonstrated the feasibility of expansin-related proteins to enable non-lytic disruption of cellulose networks relevant to film and packaging applications. The BioUPGRADE project exceeded its dissemination KPIs with over 17 peer-reviewed open-access publications; over 60 outreach events and conferences engaging more than 10,000 stakeholders globally; and exploitation activities delivering four European patents filed and one licensed.

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 novelty of the BioUPGRADE project has been reflected in the following three ways:
1) Transition from a discovery-based approach to biocatalyst development, to a design-based approach aided by increased computational capacity to extract information from large biological datasets
2) Transition from the deconstruction-first paradigm that has dominated biocatalyst applications in biomass processing, to a bio-customize framework that leverages the carbon, energy and function captured in native biomass structures
3) Focus on surface-acting enzymes that fine-tune the chemical, physical and morphological properties of natural polymers and fibres for their customized applications in value-added, renewable materials.

Progress achieved through BioUPGRADE went beyond the state of the art by creating enzymes and multi-enzyme systems that upgrade rather than degrade major biomass structures, including cellulose, hemicelluloses and chitin. Specific demonstrations included new expansin-related proteins, oxidative enzymes and transaminases, improved through ancestral sequence resurrection, for the preparation of nanocelluloses and nanochitins used to make conductive inks for packaging; absorbent cellulosic materials relevant to textile applications; and aminated plant polysaccharides relevant to personal care products for vegan markets. Our studies also uncovered evolved signatures for surface-acting biocatalysts, which can guide protein design and advance the application of biotechnology in bio-based materials manufacturing.

Key outcomes of the BioUPGRADE project included:
1) Deciphered evolved signatures for surface-acting enzymes
2) Development of application-driven functional screens for enzymes that introduce new physical and chemical functionality into natural polymers and fibres
3) Application of four novel and scalable enzyme technologies that transform renewable biomass to demonstrator applications in conductive inks, packaging, and personal care products

Through instructional videos, training in science communication, organizing an international symposium, and our technology dissemination and exploitation activities, the BioUPGRADE project increased public awareness of the role biotechnology plays in defossilizing consumer products and widened its industry partnerships to accelerate research translation to practice.

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