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Bio-derived HIgh Value polymers through novel Enzyme function

Periodic Reporting for period 4 - BHIVE (Bio-derived HIgh Value polymers through novel Enzyme function)

Reporting period: 2020-03-01 to 2020-12-31

"The 21st century has been coined the “Golden Age of Biology”, where society increasingly benefits from system-wide analysis of living organisms enabled by the emergence of “omics” technologies (i.e. large-scale DNA, RNA and protein sequencing platforms), along with improved and automated systems for genetic engineering, and increased computational power for data storage and analysis. At the same time, it is clear that this century will be judged by how well we respond to urgent calls for more sustainable lifestyles and enterprise.

Plants synthesize the most abundant source of renewable biopolymers on Earth, which include cellulose, hemicelluloses and lignin (together called ""lignocellulose""). Genomics analyses that concentrate on lignocellulose bioconversion have uncovered the critical importance of microbial enzymes to creating novel and valuable materials from lignocellulose, which would increase environmental health while creating new opportunities for forest, agriculture, and biotechnology sectors.

Notably, however, a minimum of 30-40% of predicted gene sequences encode proteins with entirely unknown function. As we enter the post-genomics era, many are recognizing that to achieve the transformative potential of “omic” analyses, it will be critically important to address the “elephant in the room”: the detailed functional characterization of uncharted protein families.

Through the BHIVE project, we combined genomics approaches, including co-expression and metagenome analyses, with the creation of novel and application-driven functional screens, to uncover entirely new biocatalysts with potential to sustainably upgrade renewable bioresources into sought-after chemicals and materials that reduce our reliance on fossil fuels. Key achievements of the BHIVE project include 1) the discovery of new enzyme families that selectively introduce specific functional groups into natural biopolymers, advance their application in sustainable textiles and packaging materials; 2) the biophysical characterization of unclassified proteins with ability to alter the assembly and architecture of cellulosic materials; and 3) the establishment of new functional screens to identify non-catalytic proteins that impact fibre porosity.

In line with the overarching goal of the ERC Consolidator to “continue to develop a successful career in Europe”, international collaborations forged through the BHIVE project led to a successful FET Open initiative in 2020, which will maximize the impact of BHIVE research and ensure European strength in biotechnology to sustainably upgrade nature’s main structural biopolymers into high-value and multipurpose materials."
1. Over 70 fungal genes were selected and corresponding proteins were recombinantly expressed in either Pichia pastoris or Aspergillus niger. A manuscript describing time and substrate-dependent transcriptome profiles of a white-rot basidiomycete has also been published.

2. Over 30 genes were isolated from metagenome sequences of lignocellulose-active microbial communities, and recombinantly expressed in Escherichia coli.

3. Over 40 bacterial and fungal proteins with unknown function were recombinantly expressed purified for functional characterization. This analysis led to the discovery of previously unknown carbohydrate transaminase activity, new carbohydrate esterase families, and unclassified microbial expansin related proteins that impact the assembly of cellulosic materials. Four articles describing this work have been published; one article in now in review, and four additional manuscripts are in preparation for publication in 2021.

4. Over 35 proteins from the selected CAZy families were purified for functional characterization. We demonstrated the application of carbohydrate oxidoreductases from families AA3, AA5, and AA7 for the synthesis of a) bio-based crosslinkers, b) “bio-lego” building blocks for hemicellulose reassembly, and c) biocatalytic cascades to aminated carbohydrates.

5. Newly discovered glycoside hydrolases from families GH62 and GH115 were successfully applied in pathways that upgrade xylan recovered from underused biomass fractions created at biofuel plants and pulp mills. Specific applications included: a) production of glucaric acid, b) deconstruction of lignin-carbohydrate complexes, and c) recovery of intact xylans from effluent streams for use in bio-based packaging and food additives.

6.We developed a 3-D lignocellulose model (“PACER”: a novel 3D Plant cell wall model for the Analysis of non-Catalytic and Enzymatic Responses) to test for both substrate preference and accessibility.

7. We established methods for functional enzyme screens using surface analysis techniques (ToF SIMS, XPS, fluorescence microscopy).

8. More than 25 recombinantly produced proteins were functionally chararacterized in detail. Furthermore, crystallization trials were performed for 7 purified CAZymes, including trials to obtain enzyme-substrate complexes. Three structures were solved by the end of the project; structural characterization of the additional targets will continue through other funds.

9. Detailed functional analyses were performed for more than 16 proteins with initially unknown function. Site-specific mutations were constructed in an effort to improve crystal formation. By the end of the project, crystals were generated for 4 proteins; their structural characterization will continue through other funds.

10. Functional analyses were established to permit functional characterization through surface analysis techniques (e.g. ToF SIMS, XPS) and novel measures of protein mobility and accessibility to composite substrates (the PACER assay). These new methods established through BHIVE were successfully used to uncover novel proteins for lignocellulose processing and upgrading into high value and sustainable materials, including carbohydrate transaminases, new carbohydrate esterase families, and microbial expansin related proteins (e.g. loosenins, ceratoplatanins).

Key achievements of the BHIVE project include 1) the discovery of new enzyme families that chemically functionalize renewable bioresources for broader use in sustainable textiles and packaging materials, 2) the biophysical characterization of unclassified proteins with ability to alter the assembly and architecture of cellulosic materials, 3) the establishment of novel functional screens to identify non-catalytic proteins that impact fibre porosity. Through the BHIVE project, we also leveraged genomics technologies, including co-expression and metagenome analyses, to prioritize protein target selections for production and functional characterization. In addition to the 21 publications listed on the Project Continuous Report, three manuscripts are under review and four manuscripts are being prepared for submission.
Our most significant contributions include:
1. the discovery of carbohydrate transaminase activity
2. the discovery of a novel enzyme family that fills the missing link to full enzymatic conversion of major xylan sources
3. the establishment of novel biocatalytic pathways to telechelic molecules from underused, renewable polysaccharides
4. the functional characterization of microbial expansin related proteins with potential to modify cellulose and chitin assembly
5. the invention of new functional screens to identify uncharacterized proteins with potential value in bio-based material construction
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