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Bio-derived HIgh Value polymers through novel Enzyme function

Periodic Reporting for period 3 - BHIVE (Bio-derived HIgh Value polymers through novel Enzyme function)

Reporting period: 2018-09-01 to 2020-02-29

"The 21st century has been coined the “Golden Age of Biology”, where society increasingly benefits from system-wide analysis of living systems 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 human 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 have concentrated on lignocellulose bioconversion have predicted 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 most genome sequences encode proteins with entirely no known function. As we enter the post-genomics era, many are recognizing that in order to achieve the transformative potential of “omic” analyses, it will be critically important to address the “elephant in the room”, which is detailed functional characterization of uncharted sequences.

Accordingly, the overall aim of the proposed research program is to mine the uncharted sequence space of genome and metagenome sequences of lignocellulose-active microorganisms, to uncover novel proteins that can be used to synthesize entirely new, high-value biomaterials from plant cell wall polysaccharides. Our approach combines (1) judicious sequence selection through bioinformatics and molecular modelling, (2) high-throughput expression of selected proteins in multiple expression hosts, (3) development of new analytical approaches for functional characterization of targeted proteins, and (4) development of cell-free enzyme systems to create new lignocellulose-derived materials.

1. To date, >70 fungal genes were selected and corresponding proteins were recombinantly expressed in either P. pastoris or A. niger.

2. A manuscript describing time and substrate-dependent transcriptome profiles of a white-rot basidiomycete, which will be used to inform target sequence selections, has been prepared for publication.

3. To date, 33 genes were isolated from metagenome sequences of lignocellulose-active microbial communities, and recombinantly expressed in E. coli.

4. To date, 29 proteins with unknown function have been purified for functional characterization.

5. To date, 14 proteins from selected CAZy families were purified for functional characterization.

6. To date, three new functional assay has been established, including progress towards the 3-D lignocellulose model to test for both substrate preference and accessibility.

7. Crystallization trials were performed for eight purified and active enzymes. Site-specific mutations were constructed in an effort to improve crystal formation, and permit structural characterization of enzyme-substrate complexes.

8. Crystallization trials were performed for six purified and active enzyme with initially unknown function. Site-specific mutations were constructed in an effort to improve crystal formation.

9. Project website has been designed and is being finalized
Our most significant contributions to date are:
1. demonstrating carbohydrate transaminase activity
2. uncovering a novel enzyme family that fills the missing link to full enzymatic conversion of hardwood glucuronoxylans
3. establishing a novel application of oxidoreductases in chemical syntheses
3. establishing novel analytical methods to advance enzyme discovery