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Title: Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi

Abstract

This project aims to investigate the hypothesis that white-rot fungi can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. Evaluating this hypothesis will provide deeper understanding of the role of white-rot fungi in facilitating carbon sequestration in Nature. Additionally, identifying the most promising fungal strains for lignin turnover and catabolism will catalyze future efforts in genetic tool development to enable metabolic engineering in white-rot fungi for lignin bioconversion to bioproducts. Lignin is the second most abundant plant-based biopolymer on Earth and represents up to 40% of the energy density of lignocellulosic biomass. Even though lignin is a massive natural carbon and energy reservoir, only a small group of basidiomycete fungi, namely white-rot fungi (WRF), have evolved the ability to efficiently depolymerize and mineralize lignin to CO2 and H2O. Considerable research efforts have been undertaken to understand how WRF depolymerize lignin but the biochemical reactions that convert lignin into CO2 have been largely neglected. In fact, it is unclear if WRF intracellularly catabolize lignin-derived aromatic compounds to utilize them as a carbon and energy source, or rather if lignin is depolymerized and mineralized extracellularly merely to facilitate access to cellulose and hemicellulose for use asmore » a primary carbon source. To date, we have employed 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to definitively demonstrate that two WRF, Trametes versicolor and Gelatoporia (Ceriporiopsis) subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways [1]. Specifically, 13C-isotopic labeling approaches showed that these WRF utilize poplar-derived aromatic compounds (e.g. 4-hydroxybenzoic acid (4-HBA)) as a carbon source. In silico genome analysis led us to hypothesize a complete catabolic pathway for 4-HBA and identify multiple homologous sequences for enzymes with putative oxidative decarboxylase, hydroxylase, and ring-opening dioxygenase activities, which are among the main biochemical reactions acting on aromatic compounds. Spatial and differential proteomic and metabolomic analyses supported the proposed catabolic pathways and showed alternative catabolic steps in T. versicolor that were not present in G. subvermispora. Based on the in silico, proteomics, and transcriptomics results, we down-selected enzymes for further in vitro characterization, and we have assigned a function to six fungal enzymes (including oxidative decarboxylases, hydroxylases, and ring-opening dioxygenases). Interestingly, even though we selected homologous enzyme pairs from both WRF with similar -omics trends, in a few cases only one of the studied fungi showed activity for the proposed substrate. Based on all the observations from this study [1], we hypothesized that 4-HBA preferentially undergoes oxidative decarboxylation to hydroquinone and subsequent hydroxylation to 1,2,4-benzenetriol in G. subvermispora before ring cleavage, whereas 4-HBA would preferentially undergo hydroxylation to protocatechuate and further oxidative decarboxylation to 1,2,4-benzenetriol in T. versicolor. Examining additional decarboxylases and hydroxylases as well as enzymes from other protein families that can perform the same or similar oxidative reactions, such as cytochromes P450 with aromatic hydroxylation activity, will also be key for elucidating enzyme preferences for specific substrates. Overall, this work forms the foundation of a new research area based on lignin catabolism by WRF, which could be further exploited to convert the undervalued biopolymer lignin into value-added compounds.« less

Authors:
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Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC); USDOE National Renewable Energy Laboratory (NREL), Laboratory Directed Research and Development (LDRD) Program
OSTI Identifier:
1814720
Report Number(s):
NREL/PO-2800-79122
MainId:33348;UUID:9d00be3d-2e26-47f6-af28-dc1b2295abed;MainAdminID:59558
DOE Contract Number:  
DE-AC36-08GO28308
Resource Type:
Conference
Resource Relation:
Conference: Presented at the Genomic Sciences Program Annual Principal Investigator Meeting, 22-24 February 2021
Country of Publication:
United States
Language:
English
Subject:
29 EE - Bioenergy Technologies Office (EE-3B); aromatic catabolic pathways; white-rot fungi

Citation Formats

del Cerro, Carlos, Erickson, Erika, Dong, Tao, Ramirez, Kelsey J., Wong, Allison R., Eder, Elizabeth K., Purvine, Samuel O., Mitchell, Hugh D., Weitz, Karl K., Markillie, Lye Meng, Burnett, Megan C., Hoyt, David W., Chu, Rosalie K., Chen, Jan-Fang, and Salvachua, Davinia. Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi. United States: N. p., 2021. Web.
del Cerro, Carlos, Erickson, Erika, Dong, Tao, Ramirez, Kelsey J., Wong, Allison R., Eder, Elizabeth K., Purvine, Samuel O., Mitchell, Hugh D., Weitz, Karl K., Markillie, Lye Meng, Burnett, Megan C., Hoyt, David W., Chu, Rosalie K., Chen, Jan-Fang, & Salvachua, Davinia. Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi. United States.
del Cerro, Carlos, Erickson, Erika, Dong, Tao, Ramirez, Kelsey J., Wong, Allison R., Eder, Elizabeth K., Purvine, Samuel O., Mitchell, Hugh D., Weitz, Karl K., Markillie, Lye Meng, Burnett, Megan C., Hoyt, David W., Chu, Rosalie K., Chen, Jan-Fang, and Salvachua, Davinia. 2021. "Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi". United States. https://www.osti.gov/servlets/purl/1814720.
@article{osti_1814720,
title = {Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi},
author = {del Cerro, Carlos and Erickson, Erika and Dong, Tao and Ramirez, Kelsey J. and Wong, Allison R. and Eder, Elizabeth K. and Purvine, Samuel O. and Mitchell, Hugh D. and Weitz, Karl K. and Markillie, Lye Meng and Burnett, Megan C. and Hoyt, David W. and Chu, Rosalie K. and Chen, Jan-Fang and Salvachua, Davinia},
abstractNote = {This project aims to investigate the hypothesis that white-rot fungi can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. Evaluating this hypothesis will provide deeper understanding of the role of white-rot fungi in facilitating carbon sequestration in Nature. Additionally, identifying the most promising fungal strains for lignin turnover and catabolism will catalyze future efforts in genetic tool development to enable metabolic engineering in white-rot fungi for lignin bioconversion to bioproducts. Lignin is the second most abundant plant-based biopolymer on Earth and represents up to 40% of the energy density of lignocellulosic biomass. Even though lignin is a massive natural carbon and energy reservoir, only a small group of basidiomycete fungi, namely white-rot fungi (WRF), have evolved the ability to efficiently depolymerize and mineralize lignin to CO2 and H2O. Considerable research efforts have been undertaken to understand how WRF depolymerize lignin but the biochemical reactions that convert lignin into CO2 have been largely neglected. In fact, it is unclear if WRF intracellularly catabolize lignin-derived aromatic compounds to utilize them as a carbon and energy source, or rather if lignin is depolymerized and mineralized extracellularly merely to facilitate access to cellulose and hemicellulose for use as a primary carbon source. To date, we have employed 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to definitively demonstrate that two WRF, Trametes versicolor and Gelatoporia (Ceriporiopsis) subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways [1]. Specifically, 13C-isotopic labeling approaches showed that these WRF utilize poplar-derived aromatic compounds (e.g. 4-hydroxybenzoic acid (4-HBA)) as a carbon source. In silico genome analysis led us to hypothesize a complete catabolic pathway for 4-HBA and identify multiple homologous sequences for enzymes with putative oxidative decarboxylase, hydroxylase, and ring-opening dioxygenase activities, which are among the main biochemical reactions acting on aromatic compounds. Spatial and differential proteomic and metabolomic analyses supported the proposed catabolic pathways and showed alternative catabolic steps in T. versicolor that were not present in G. subvermispora. Based on the in silico, proteomics, and transcriptomics results, we down-selected enzymes for further in vitro characterization, and we have assigned a function to six fungal enzymes (including oxidative decarboxylases, hydroxylases, and ring-opening dioxygenases). Interestingly, even though we selected homologous enzyme pairs from both WRF with similar -omics trends, in a few cases only one of the studied fungi showed activity for the proposed substrate. Based on all the observations from this study [1], we hypothesized that 4-HBA preferentially undergoes oxidative decarboxylation to hydroquinone and subsequent hydroxylation to 1,2,4-benzenetriol in G. subvermispora before ring cleavage, whereas 4-HBA would preferentially undergo hydroxylation to protocatechuate and further oxidative decarboxylation to 1,2,4-benzenetriol in T. versicolor. Examining additional decarboxylases and hydroxylases as well as enzymes from other protein families that can perform the same or similar oxidative reactions, such as cytochromes P450 with aromatic hydroxylation activity, will also be key for elucidating enzyme preferences for specific substrates. Overall, this work forms the foundation of a new research area based on lignin catabolism by WRF, which could be further exploited to convert the undervalued biopolymer lignin into value-added compounds.},
doi = {},
url = {https://www.osti.gov/biblio/1814720}, journal = {},
number = ,
volume = ,
place = {United States},
year = {Wed Aug 18 00:00:00 EDT 2021},
month = {Wed Aug 18 00:00:00 EDT 2021}
}

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