Public Abstract

DE-SC0019427: Gene regulatory networks enabling fungi to selectively extract sugars from lignocellulose

Award Status: Inactive

Institution: Regents of the University of Minnesota, Minneapolis, MN
UEI: KABJZBBJ4B54
DUNS: 555917996

Most Recent Award Date: 07/13/2021
Number of Support Periods: 3
PM: Adin, Dawn

Current Budget Period: 09/15/2020 - 09/14/2022
Current Project Period: 09/15/2018 - 09/14/2022
PI: Schilling, Jonathan

Supplement Budget Period: N/A

Public Abstract

Fungi dominate the biological decomposition of wood and other lignocellulosic plant tissues in nature. These saprotrophs offer us a proven model for making energy, sustainably, from biomass. They also offer those with commercial interests a range of pathways for unlocking sugars embedded in lignin. Their strategies range from ‘white rot’ mechanisms that remove lignin to gain access to polysaccharides to ‘brown rot’ mechanisms that selectively extract sugars, leaving most lignin behind. This metabolic diversity could be harnessed, industrially, but research has generally been focused more toward white rot delignification pathways. White rot fungi can unsheathe polysaccharides by selectively removing lignin, a capacity that historically attracted interest for the potential to extract intact fibers for papermaking. Modern bioenergy schemes, however, do not aspire for intact fibers - instead, the goal is to depolymerize polysaccharides to release fermentable sugars (saccharification), saving lignin as a co-product, if possible. This is a better fit for the carbohydrate-selective pathways of brown rot fungi, but our grasp of fungal brown rot metabolism lags behind what we know about white rot. The research carried out in this project addresses several key gaps that will advance our capacity to harness brown rot for bioenergy. First, among these gaps, brown rot fungi evolved from white rot ancestors at least six times, converging on faster wood decay mechanisms using reactive oxygen species (ROS) to loosen wood cell walls ahead of saccharification. This pretreatment approach lacks precedent in other organisms, limiting comparative genomics to show only the genes lost in brown rot evolution, not the pathways gained. Second, evolutionary convergence on a similar strategy implies that brown rot pathways were co-opted from white rot ancestors. In making this evolutionary transition, however, brown rot fungi shed >60% of ancestral genes with known lignocellulolytic functions, leaving the novelty of brown rot hidden among functionally ‘gray’ genes. Finally, there is mounting evidence that ROS pretreatments are not the sole upgrade for brown rot. We recently found strong evidence that brown rot carbohydrate-active enzymes (CAZYs) are novel in their own right, adapted for saccharification in oxidative environments after a choreographed delay to avoid oxidative damage. This begs to integrate gene regulation with metabolite feedback, but unfortunately, there has not yet been a strategic systems biology effort to do so. Our collaborative project is aligned to address these gaps, with the goal of producing an integrated regulatory model for brown rot. Our proposed objectives insure stand-alone advances, but will also synergize to push ideas forward in a systems context. Our objective 1 is to identify fungal gene regulation patterns that distinguish brown rot fungi from fungi with other decay modes (e.g., white rot). We plan to compare fungi among relevant lineages but with varied carbohydrate-selectivities. We will culture these strains on solid wood wafers, spatially mapping gene expression and then overlaying fungal/wood metabolite patterns to enable temporally-resolved functional genomics. These maps can isolate patterns unique to brown rot and can target characterization. Our objective 2 focuses on characterization, starting with a short list generated in an earlier transcriptomics study, and progressively adding objective 1 gene targets. We plan to use routine single-/multi-cellular in vitro transformation pipelines, but will complement this with efforts to develop a brown rot transformation system, enabling in vivo manipulations (e.g., Crispr-Cas9). Finally, objective 3 is to use metabolomics to map metabolite-expression feedback over time, providing networks of gene regulation. This approach promises to advance our understanding of this unique brown rot strategy, beyond current ROS-centric models toward a systems view. This project will facilitate a new collaboration that is strategically aligned for systems biology, enabling omics-driven tools for organisms highly relevant to bioenergy. Our proposal is thus well-aligned for this call and with BER’s mission, and our results will have broader scientific impacts in the fields of ecology, evolution, and biogeochemistry. We have also been deliberate in integrating our budgets, to enable the science but also to harness training opportunities for students/postdocs who will ‘bridge’ Universities and National Laboratories. The team at University of Minnesota Drs. Jonathan Schilling, Claudia Schmidt-Dannert, and Jiwei Zhang will collaborate with Dr. David Hibbett (Clark University), Dr. Igor Grigoriev (Lawrence Berkeley National Laboratory - Joint Genome Institute), and Young-Mo Kim (Pacific Northwest National Laboratory - Environmental Molecular Sciences Laboratory).

Fungi dominate the biological decomposition of wood and other lignocellulosic plant tissues in nature. These saprotrophs offer us a proven model for making energy, sustainably, from biomass. They also offer those with commercial interests a range of pathways for unlocking sugars embedded in lignin. Their strategies range from ‘white rot’ mechanisms that remove lignin to gain access to polysaccharides to ‘brown rot’ mechanisms that selectively extract sugars, leaving most lignin behind. This metabolic diversity could be harnessed, industrially, but research has generally been focused more toward white rot delignification pathways. White rot fungi can unsheathe polysaccharides by selectively removing lignin, a capacity that historically attracted interest for the potential to extract intact fibers for papermaking. Modern bioenergy schemes, however, do not aspire for intact fibers - instead, the goal is to depolymerize polysaccharides to release fermentable sugars (saccharification), saving lignin as a co-product, if possible. This is a better fit for the carbohydrate-selective pathways of brown rot fungi, but our grasp of fungal brown rot metabolism lags behind what we know about white rot.

The research carried out in this project addresses several key gaps that will advance our capacity to harness brown rot for bioenergy. First, among these gaps, brown rot fungi evolved from white rot ancestors at least six times, converging on faster wood decay mechanisms using reactive oxygen species (ROS) to loosen wood cell walls ahead of saccharification. This pretreatment approach lacks precedent in other organisms, limiting comparative genomics to show only the genes lost in brown rot evolution, not the pathways gained. Second, evolutionary convergence on a similar strategy implies that brown rot pathways were co-opted from white rot ancestors. In making this evolutionary transition, however, brown rot fungi shed >60% of ancestral genes with known lignocellulolytic functions, leaving the novelty of brown rot hidden among functionally ‘gray’ genes. Finally, there is mounting evidence that ROS pretreatments are not the sole upgrade for brown rot. We recently found strong evidence that brown rot carbohydrate-active enzymes (CAZYs) are novel in their own right, adapted for saccharification in oxidative environments after a choreographed delay to avoid oxidative damage. This begs to integrate gene regulation with metabolite feedback, but unfortunately, there has not yet been a strategic systems biology effort to do so.

Our collaborative project is aligned to address these gaps, with the goal of producing an integrated regulatory model for brown rot. Our proposed objectives insure stand-alone advances, but will also synergize to push ideas forward in a systems context. Our objective 1 is to identify fungal gene regulation patterns that distinguish brown rot fungi from fungi with other decay modes (e.g., white rot). We plan to compare fungi among relevant lineages but with varied carbohydrate-selectivities. We will culture these strains on solid wood wafers, spatially mapping gene expression and then overlaying fungal/wood metabolite patterns to enable temporally-resolved functional genomics. These maps can isolate patterns unique to brown rot and can target characterization. Our objective 2 focuses on characterization, starting with a short list generated in an earlier transcriptomics study, and progressively adding objective 1 gene targets. We plan to use routine single-/multi-cellular in vitro transformation pipelines, but will complement this with efforts to develop a brown rot transformation system, enabling in vivo manipulations (e.g., Crispr-Cas9). Finally, objective 3 is to use metabolomics to map metabolite-expression feedback over time, providing networks of gene regulation. This approach promises to advance our understanding of this unique brown rot strategy, beyond current ROS-centric models toward a systems view.

This project will facilitate a new collaboration that is strategically aligned for systems biology, enabling omics-driven tools for organisms highly relevant to bioenergy. Our proposal is thus well-aligned for this call and with BER’s mission, and our results will have broader scientific impacts in the fields of ecology, evolution, and biogeochemistry. We have also been deliberate in integrating our budgets, to enable the science but also to harness training opportunities for students/postdocs who will ‘bridge’ Universities and National Laboratories. The team at University of Minnesota Drs. Jonathan Schilling, Claudia Schmidt-Dannert, and Jiwei Zhang will collaborate with Dr. David Hibbett (Clark University), Dr. Igor Grigoriev (Lawrence Berkeley National Laboratory - Joint Genome Institute), and Young-Mo Kim (Pacific Northwest National Laboratory - Environmental Molecular Sciences Laboratory).