Unveiling a classical mutant in the context of the GH3 β-glucosidase family in Neurospora crassa

Zhang, Yuxin; Nada, Basant; Baker, Scott E.; Evans, James E.; Tian, Chaoguang; Benz, J. Philipp; Tamayo, Elisabeth

doi:10.1186/s13568-023-01658-0

Unveiling a classical mutant in the context of the GH₃ β-glucosidase family in Neurospora crassa

Journal Article · Fri Jan 05 00:00:00 EST 2024 · AMB Express

DOI:https://doi.org/10.1186/s13568-023-01658-0· OSTI ID:2282268

Zhang, Yuxin ^[1]; Nada, Basant ^[1]; Baker, Scott E. ^[2]; Evans, James E. ^[3]; Tian, Chaoguang ^[4]; Benz, J. Philipp ^[1]; ^[1]

Technical University of Munich (Germany)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Chinese Academy of Sciences (CAS), Tianjin (China); National Technology Innovation Center of Synthetic Biology, Tianjin (China)

Classical fungal mutant strains obtained by mutagenesis have helped to elucidate fundamental metabolic pathways in the past. In the filamentous fungus Neurospora crassa, the gluc-1 strain was isolated long ago and characterized by its low level of β-glucosidase activity, which is essential for the degradation of cellulose, the most abundant biopolymer on Earth and the main polymeric component of the plant cell wall. Based on genomic resequencing, we hypothesized that the causative mutation resides in the β-glucosidase gene gh3-3 (bgl6, NCU08755). In this work, growth patterns, enzymatic activities and sugar utilization rates were analyzed in several mutant and overexpression strains related to gluc-1 and gh3-3. In addition, different mutants affected in the degradation and transport of cellobiose were analyzed. While overexpression of gh3-3 led to the recovery of β-glucosidase activity in the gluc-1 mutant, as well as normal utilization of cellobiose, the full gene deletion strain Δgh3-3 was found to behave differently than gluc-1 with lower secreted β-glucosidase activity, indicating a dominant role of the amino acid substitution in the point mutated gh3-3 gene of gluc-1. Our results furthermore confirm that GH3-3 is the major extracellular β-glucosidase in N. crassa and demonstrate that the two cellodextrin transporters CDT-1 and CDT-2 are essential for growth on cellobiose when the three main N. crassa β-glucosidases are absent. Overall, these findings provide valuable insight into the mechanisms of cellulose utilization in filamentous fungi, being an essential step in the efficient production of biorefinable sugars from agricultural and forestry plant biomass.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 2282268

Report Number(s):: PNNL-SA--194079

Journal Information:: AMB Express, Journal Name: AMB Express Journal Issue: 1 Vol. 14; ISSN 2191-0855

Publisher:: SpringerCopyright Statement

Country of Publication:: United States

Language:: English

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Unveiling a classical mutant in the context of the GH₃ β-glucosidase family in Neurospora crassa