Periodic Reporting for period 2 - BioUPGRADE (Biocatalytic upgrading of natural biopolymers for reassembly as multipurpose materials)
Reporting period: 2022-05-01 to 2023-10-31
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 resource. 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 combines computational biology, genomics and material sciences to realize the potential of biotechnology in sustainable manufacturing of renewable products. Our focus in on biocatalyst (enzymes) that transform major sources of renewable fibre into of high-value textiles and packaging materials, conductive inks for bioelectronics, and customized hydrogels for health and personal care products.
Our specific project objectives are 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.
The BioUPGRADE action combines computational biology, genomics and material sciences to realize the potential of biotechnology in sustainable manufacturing of renewable products. Our focus in on biocatalyst (enzymes) that transform major sources of renewable fibre into of high-value textiles and packaging materials, conductive inks for bioelectronics, and customized hydrogels for health and personal care products.
Our specific project objectives are 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.
We have now completed the design, production and testing of ancestral expansins, as well as an ancestral endoglucanase and ancestral lytic polysaccharide monooxygenase. In general, we have demonstrated that ancestral version of enzymes and proteins display properties that are not present in their modern counterparts and that represent an advantage for biotechnological applications. Considerable efforts were dedicated to scaling-up protein production. Prioritized microbial expansins were produced using 5 L to 200 L bioreactor systems, producing sufficient quantities of microbial expansin for initial for application trials in cellulose fibre processing. In addition to bioreactor productions, a new expression system was established based on Bacillus subtilis. In this method, the recombinant protein is fused to a signal peptide, driving the recombinant protein to be exported into the extracellular environment. This approach allows for high yields of recombinant protein expression, lacking the need for downstream purification steps.
Numerous complementary biophysical methods are now established to investigate the function of diverse microbial expansin. For example, we developed complementary microscopy techniques (i.e. fluorescence microscopy and TEM) to visualize expansin localization in plant cell walls. We also established methods that use small angle neutron scattering (BioSANS) and small angle x-ray scattering (SAXS) to characterize cellulose networks after expansin treatment. Furthermore, quartz-crystal microbalance with dissipation (QCM-D) was used to compare the potential of different microbial expansins to bind cellulosic materials and alter their network structure.
In addition to studies and applications of microbial expansins, we validated the activity of a carbohydrate oxidase and transaminase cascade towards diverse hemicelluloses. These studies established methods to isolate and characterize hemicelluloses from different wood and agricultural sources, including substrates from our industry partners.
Using the characterized microbial expansins, along with engineered enzymes and carbohydrate oxidase-transaminase cascade, we have now 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.
Several mobility activities have taken place across our BioUPGRADE network, including two visits from trainees at KTH to Aalto and visits from trainees at UPM to Aalto and KTH. More than six cross-network collaborations and co-publications were also initiated, including enzymes for nanocellulose preparation (bioGUNE + Aalto), expansins for fibre fibrillation (UPM, Aalto, bioGUNE), and the production of carbohydrate oxidases and transaminases for hemicellulose valorization (KTH, Aalto). In addition to scientific publications, our research activities were further disseminated over the reporting period through eleven international conferences.
Numerous complementary biophysical methods are now established to investigate the function of diverse microbial expansin. For example, we developed complementary microscopy techniques (i.e. fluorescence microscopy and TEM) to visualize expansin localization in plant cell walls. We also established methods that use small angle neutron scattering (BioSANS) and small angle x-ray scattering (SAXS) to characterize cellulose networks after expansin treatment. Furthermore, quartz-crystal microbalance with dissipation (QCM-D) was used to compare the potential of different microbial expansins to bind cellulosic materials and alter their network structure.
In addition to studies and applications of microbial expansins, we validated the activity of a carbohydrate oxidase and transaminase cascade towards diverse hemicelluloses. These studies established methods to isolate and characterize hemicelluloses from different wood and agricultural sources, including substrates from our industry partners.
Using the characterized microbial expansins, along with engineered enzymes and carbohydrate oxidase-transaminase cascade, we have now 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.
Several mobility activities have taken place across our BioUPGRADE network, including two visits from trainees at KTH to Aalto and visits from trainees at UPM to Aalto and KTH. More than six cross-network collaborations and co-publications were also initiated, including enzymes for nanocellulose preparation (bioGUNE + Aalto), expansins for fibre fibrillation (UPM, Aalto, bioGUNE), and the production of carbohydrate oxidases and transaminases for hemicellulose valorization (KTH, Aalto). In addition to scientific publications, our research activities were further disseminated over the reporting period through eleven international conferences.
Progress reached through BioUPGRADE goes beyond the state of the art by creating enzymes and enzyme systems that upgrade rather than degrade major biomass structures, including cellulose, hemicelluloses and chitin. Specific demonstrations include new enzyme systems, improved through ancestral sequence resurrection, for the preparation of nanocelluloses and nanochitins; and cell-free enzyme cascade to chemically functionalize underused hemicelluloses. Our studies of enzymes and non-lytic expansin-related proteins that act on bio-fibres and bio-polymers are also uncovering evolved signatures for surface-acting biocatalysts, which can guide protein design and advance the application of cell-free enzyme systems in biomanufacturing platforms.
Expected outcomes until the end of the project include:
1) Decipher evolved signatures for surface-acting biocatalysts
2) Deepen our understanding of sequence and structural determinants of substrate preference among microbial expansin-related proteins, which represent untapped biocatalysts for bio-based material manufacturing
3) Establish at least three engineered enzyme systems that transform renewable biomass to demonstrator applications in conductive inks, packaging, and personal care products.
Our team created a series of project videos in an effort to increase public access to our research breakthroughs. We are also organizing a symposium at an international conference in an effort to widen our industry partnerships.
Expected outcomes until the end of the project include:
1) Decipher evolved signatures for surface-acting biocatalysts
2) Deepen our understanding of sequence and structural determinants of substrate preference among microbial expansin-related proteins, which represent untapped biocatalysts for bio-based material manufacturing
3) Establish at least three engineered enzyme systems that transform renewable biomass to demonstrator applications in conductive inks, packaging, and personal care products.
Our team created a series of project videos in an effort to increase public access to our research breakthroughs. We are also organizing a symposium at an international conference in an effort to widen our industry partnerships.