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.
