Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

ABSTRACT The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa , we identified two genes ( qc - 1 and qc - 2 ) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc - 1 and qc - 2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc - 1 Δqc - 2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability. IMPORTANCE Pyroglutamate modification is the post-translational conversion of N-terminal glutamine or glutamate into a cyclized amino acid derivative. This modification is well studied in animal systems but poorly explored in fungal systems. In Neurospora crassa , we show that this modification takes place in the ER and is catalyzed by two well-conserved enzymes, ubiquitously conserved throughout the fungal kingdom. We demonstrate that the modification is important for the structural stability and aminopeptidase resistance of CBH-1 and GH5-1, two important cellulase enzymes utilized in industrial plant cell wall deconstruction. Many additional fungal proteins predicted in the genome of N. crassa and other filamentous fungi are predicted to carry an N-terminal pyroglutamate modification. Pyroglutamate addition may also be a useful way to stabilize secreted proteins and peptides, which can be easily produced in fungal production systems.

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Network reconstruction and systems analysis of plant cell wall deconstruction byneurospora crassa

10.1101/133165 ◽

2017 ◽

Author(s):

Areejit Samal ◽

James P. Craig ◽

Samuel T. Coradetti ◽

J. Philipp Benz ◽

James A. Eddy ◽

...

Keyword(s):

Cell Wall ◽

Neurospora Crassa ◽

Filamentous Fungi ◽

Plant Cell ◽

Plant Cell Wall ◽

Plant Biomass ◽

Degradation Pathway ◽

Heterogeneous Data ◽

Data Types ◽

Cell Wall Polysaccharides

AbstractPlant biomass degradation by fungal derived enzymes is rapidly expanding in economic importance as a clean and efficient source for biofuels. The ability to rationally engineer filamentous fungi would facilitate biotechnological applications for degradation of plant cell wall polysaccharides. However, incomplete knowledge of biomolecular networks responsible for plant cell wall deconstruction impedes experimental efforts in this direction. To expand this knowledge base, a detailed network of reactions important for deconstruction of plant cell wall polysaccharides into simple sugars was constructed for the filamentous fungusNeurospora crassa. To reconstruct this network, information was integrated from five heterogeneous data types: functional genomics, transcriptomics, proteomics, genetics, and biochemical characterizations. The combined information was encapsulated into a feature matrix and the evidence weighed to assign annotation confidence scores for each gene within the network. Comparative analyses of RNA-seq and ChIP-seq data shed light on the regulation of the plant cell wall degradation network (PCWDN), leading to a novel hypothesis for degradation of the hemicellulose mannan. The transcription factor CLR-2 was subsequently experimentally shown to play a key role in the mannan degradation pathway ofNeurospora crassa. Our network serves as a scaffold for integration of diverse experimental data, leading to elucidation of regulatory design principles for plant cell wall deconstruction by filamentous fungi, and guiding efforts to rationally engineer industrially relevant hyper-production strains.

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DNA nanostructures coordinate gene silencing in mature plants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818290116 ◽

2019 ◽

Vol 116 (15) ◽

pp. 7543-7548 ◽

Cited By ~ 34

Author(s):

Huan Zhang ◽

Gozde S. Demirer ◽

Honglu Zhang ◽

Tianzheng Ye ◽

Natalie S. Goh ◽

...

Keyword(s):

Cell Wall ◽

Gene Silencing ◽

Plant Cell ◽

Mammalian Cells ◽

Plant Cell Wall ◽

Plant Cells ◽

Design Parameters ◽

Dna Nanostructures ◽

Dna Nanostructure ◽

The Impact

Delivery of biomolecules to plants relies onAgrobacteriuminfection or biolistic particle delivery, the former of which is amenable only to DNA delivery. The difficulty in delivering functional biomolecules such as RNA to plant cells is due to the plant cell wall, which is absent in mammalian cells and poses the dominant physical barrier to biomolecule delivery in plants. DNA nanostructure-mediated biomolecule delivery is an effective strategy to deliver cargoes across the lipid bilayer of mammalian cells; however, nanoparticle-mediated delivery without external mechanical aid remains unexplored for biomolecule delivery across the cell wall in plants. Herein, we report a systematic assessment of different DNA nanostructures for their ability to internalize into cells of mature plants, deliver siRNAs, and effectively silence a constitutively expressed gene inNicotiana benthamianaleaves. We show that nanostructure internalization into plant cells and corresponding gene silencing efficiency depends on the DNA nanostructure size, shape, compactness, stiffness, and location of the siRNA attachment locus on the nanostructure. We further confirm that the internalization efficiency of DNA nanostructures correlates with their respective gene silencing efficiencies but that the endogenous gene silencing pathway depends on the siRNA attachment locus. Our work establishes the feasibility of biomolecule delivery to plants with DNA nanostructures and both details the design parameters of importance for plant cell internalization and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.

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Studies on the application of plant cell wall degrading enzymes from Aspergillus terreus and Neurospora crassa

Biotechnology Letters ◽

10.1007/bf00132232 ◽

1983 ◽

Vol 5 (7) ◽

pp. 481-486 ◽

Cited By ~ 2

Author(s):

Anil Sharma ◽

Richard Joseph

Keyword(s):

Cell Wall ◽

Neurospora Crassa ◽

Plant Cell ◽

Aspergillus Terreus ◽

Plant Cell Wall ◽

Cell Wall Degrading Enzymes ◽

Degrading Enzymes

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Direct Target Network of the Neurospora crassa Plant Cell Wall Deconstruction Regulators CLR-1, CLR-2, and XLR-1

mBio ◽

10.1128/mbio.01452-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 53

Author(s):

James P. Craig ◽

Samuel T. Coradetti ◽

Trevor L. Starr ◽

N. Louise Glass

Keyword(s):

Gene Expression ◽

Cell Wall ◽

Transcription Factors ◽

Neurospora Crassa ◽

Plant Cell ◽

Plant Cell Wall ◽

Nutrient Sensing ◽

Wall Material ◽

Content Type ◽

Genes Encoding

ABSTRACTFungal deconstruction of the plant cell requires a complex orchestration of a wide array of intracellular and extracellular enzymes. InNeurospora crassa, CLR-1, CLR-2, and XLR-1 have been identified as key transcription factors regulating plant cell wall degradation in response to soluble sugars. The XLR-1 regulon was defined using a constitutively active mutant allele, resulting in hemicellulase gene expression and secretion under noninducing conditions. To define genes directly regulated by CLR-1, CLR-2, and XLR-1, we performed chromatin immunoprecipitation and next-generation sequencing (ChIPseq) on epitope-tagged constructs of these three transcription factors. WhenN. crassais exposed to plant cell wall material, CLR-1, CLR-2, and XLR-1 individually bind to the promoters of the most strongly induced genes in their respective regulons. These include promoters of genes encoding cellulases for CLR-1 and CLR-2 (CLR-1/CLR-2) and promoters of genes encoding hemicellulases for XLR-1. CLR-1 bound to its regulon under noninducing conditions; however, this binding alone did not translate into gene expression and enzyme secretion. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest paralog inSaccharomyces cerevisiae, Gal4p. Coimmunoprecipitation studies showed that CLR-1 and CLR-2 act in a homocomplex but not as a CLR-1/CLR-2 heterocomplex.IMPORTANCEUnderstanding fungal regulation of complex plant cell wall deconstruction pathways in response to multiple environmental signals via interconnected transcriptional circuits provides insight into fungus/plant interactions and eukaryotic nutrient sensing. Coordinated optimization of these regulatory networks is likely required for optimal microbial enzyme production.

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