Biomass Depolymerization projects

Systems Biology Analyses of Plant Cell Wall Deconstruction by the Model Filamentous Fungus, Neurospora Crassa

This project is working to define how a filamentous fungus remodels both its internal and extracellular metabolism to efficiently deconstruct plant cell wall material. Neurospora crassa, a filamentous fungus found on burnt grasses, sugarcane stalks and sugarcane bagasse in nature, has been used in the laboratory as a model organism for 80 years; its genome has been sequenced, and many biochemical, molecular, genetic and cell biological tools are available for this organism. The team has characterized the transcriptional response when N. crassa grows on plant cell wall material and are characterizing regulatory processes associated with sensing plant cell wall material and induction of enzyme gene expression and secretion. It is developing high-throughput methodology and cell biological approaches to enable defining mechanistic aspects and function of proteins encoded by genes, that when mutated, decrease or increase hydrolytic enzyme production.

project Highlights

2014 Highlights

Our first area of focus is an analysis of the transcriptional regulatory network associated with plant cell wall deconstruction; during 2014 we further characterized four essential transcription factors required for induction of cellulase or hemicellulase genes (CLR-1, CLR-2, XLR-1, VIB-1, COL-26) and embarked on forward and reverse genetic approaches to define regulatory mechanisms of cellulose/plant cell wall sensing as well as mechanisms associated with carbon catabolite repression. The second focus is the dissection of the pathway to hyper-secretion of lignocellulolytic enzymes, using biochemical, genetic and live cell imaging tools that we are developing for N. crassa. During 2014, we characterized a number of CAZY signal peptides and developed a quantitative measure for protein translocation. We identified new mutants that led to a hyper-secretion phenotype. We also developed new tools that allow us to regulate the production of secretory proteins. We began developing CRISPR-Cas9 system for filamentous fungi and explored a number of thermophilic species for production of biomass-degrading enzymes. The ultimate objective is to be able to rationally engineer hyper-secretion strains in any cellulolytic filamentous ascomycete species as well as engineer fermentation organisms to more efficiently secrete plant cell wall degrading enzymes, enabling expense reduction in production of biofuels from lignocellulosic material.

2013 Highlights

This program uses a systems biology approach to deciphering the mechanism of plant cell wall deconstruction by the filamentous fungus Neurospora crassa. Our first focus is an analysis of the transcriptional regulatory network associated with plant cell wall deconstruction; during 2013 we characterized four essential transcription factors required for induction of cellulase or hemicellulase genes. The second area of focus is the dissection of the pathway to hyper-secretion of lignocellulolytic enzymes, using biochemical, genetic and live cell imaging tools that we are developing for N. crassa. During 2013, we elucidated components of the secretory pathway important for cellulase secretion. Furthermore, we developed a plant biomass deconstruction metabolic model for N. crassa that will ultimately lead to predictions regarding function of proteins associated with plant biomass deconstruction. The ultimate objective is to be able to rationally engineer hyper-secretion strains in any cellulolytic filamentous ascomycete species as well as engineer fermentation organisms to more efficiently secrete plant cell wall degrading enzymes, enabling expense reduction in production of biofuels from lignocellulosic material.

2012 Highlights

In the wild, the filamentous fungus Neurospora crassa degrades plant material killed by fire. In the laboratory, N. crassa is a model organism with a plethora of genetic, biochemical, genomic and cell biological tools. We utilize N. crassa as model organism to decipher mechanisms of plant cell wall deconstruction by fungi. The goal of this program is to apply systems biology approaches to elucidate the full genomic and proteomic repertoire that N. crassa uses for the deconstruction of plant biomass.

 

During the past year, we have determined how N. crassa senses plant cell wall material and have identified transcription factors that directly regulate genes encoding enzymes involved in plant cell wall deconstruction. Importantly, we can now control the production of plant cell wall degrading enzymes by regulation of a cellulose-specific transcription factor. Comparative analyses of enzyme cocktails in filamentous fungi enabled by our comparative transcriptomic and proteomic approaches in N. crassa and Aspergillus nidulans will advance efforts to define tailored enzyme mixtures for maximal efficiency on different lignocellulosic feedstocks for biofuels and production of value- added chemicals. Our ultimate objective is to be able to rationally engineer hyper-secretion fungal strains that produce lignocellulolytic enzymes under regulated conditions. In addition, we are involved in transferring filamentous fungal functionalities in plant cell wall deconstruction to fermentation organisms (yeast or bacteria) in an effort to increase biofuel production via more efficient utilization of lignocellulosic biomass.

2011 Highlights

Glass’ lab has characterized the transcriptional response when N. crassa grows on plant cell wall material and is characterizing regulatory processes associated with sensing plant cell wall material and induction of enzyme gene expression and secretion.

 

Publications

Published in 2014

 

A Systems-Level Analysis of Polysaccharide-Elicited Responses in Neurospora crassa Reveals the Importance of Pectin for Efficient Plant Cell Wall Degradation, J. P. Benz, B. H. Chau, D. Zheng, S. Bauer, N. L. Glass and C. R. Somerville, Molecular Microbiology, V. 91, pp. 275-99, 2014. 

 

Engineering the Filamentous Fungus Neurospora crassa for Lipid Production from Lignocellulosic Biomass, C. M. Roche, N. L. Glass, H. W. Blanch and D. S. Clark, Biotechnology and Bioengineering, V. 111, pp. 1097-1107, April 3, 2014. 

 

The Proteome and Phosphoproteome of Neurospora crassa in Response to Cellulose, Sucrose and Carbon Starvation, Y. Xiong, S. T. Coradetti, X. Li, M. A. Gritsenko, T. Clauss, V. Petyuk, D. Camp, R. Smith, J. H. D. Cate, F. Yang and N. L. Glass, Fungal Genetics and Biology, doi: 10.1016/j.fgb.2014.05.005, 2014. 

 

Plant Cell Wall-Degrading Enzymes and their Secretion in Plant-Pathogenic Fungi, C. P. Kubicek, T. L. Starr and N. L. Glass, Annual Review of Phytopathology, V. 52, pp. 427-451, June 6, 2014. 

 

VIB1, a Link Between Glucose Signaling and Carbon Catabolite Repression, is Essential for Plant Cell Wall Degradation by Neurospora crassa, Y. Xiong, J. Sun and N. L. Glass, PLoS Genetics, doi: 10.1371/journal.pgen.1004500, August 21, 2014. 

Published in 2013

 

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa, E. A. Znameroski, X. Li, J. C. Tsai, N. L. Glass and J. H. D. Cate, Journal of Biological Chemistry, V. 289, pp. 2610-2619, December 2014. 

 

Using a Model Filamentous Fungus to Unravel Mechanisms of Lignocellulose Deconstruction, E. A. Znameroski, N. L. Glass, Biotechnology for Biofuels 6(1), 6, doi: Artn 6 Doi 10.1186/1754-6834-6-6, January 22, 2013.

 

Analysis of Conserved Cellulase Transcriptional Regulator Reveals Inducer-Independent Production of Cellulolytic Enzymes in Neurospora Crassa, Samuel T. Coradetti, Yi Xiong, N. Louise Glass, Microbiology Open 2, pp. 595-609, doi: 10.1002/mbo3.94, May 2013.

 

Plant Cell Wall Deconstruction by Ascomycete Fungi, N. L. Glass, M. Schmoll, J. H. Cate, S. Coradetti, Annual Review of Microbiology 67, pp. 477-498,  doi: 10.1146/annurev-micro-092611-150044, July 3, 2013.

 

Physiological Role of Acyl-Coa Synthetase Homologs in Lipid Metabolism in Neurospora Crassa, C. M. Roche, H. W. Blanch, D. S. Clark, N. L. Glass, Eukaryotic Cell, 12(9), pp. 1244-1257, doi: 10.1128/EC.00079-13, July 23, 2013.

Published in 2012

Conserved and Essential Transcription Factors for Cellulase Gene Expression in Ascomycete Fungi, S. T. Coradetti, J. P. Craig, Y. Xiong, T. Shock, C. Tian, N. L. Glass, Proceedings of the National Academy of Sciences 109(19):7397-402. doi: 10.1073/pnas.1200785109. PMID: 22532664, May 8, 2012.

 

Induction of Lignocellulose-Degrading Enzymes in Neurospora crassa by Cellodextrins, E. A. Znameroski, S. T. Coradetti, C. M. Roche, J. C. Tsai, A. T. Iavarone, J. H. Cate, N. L. Glass, Proceedings of the National Academy of Sciences 109(16):6012-7. doi: 10.1073/pnas.1118440109, PMID: 22474347, April 17, 2012.

 

Unravelling the Molecular Basis for Light Modulated Cellulase Gene Expression -- the Role of Photoreceptors in Neurospora crassa. M. Schmoll, C. Tian, J. Sun, D. Tisch, N. L. Glass, BMC Genomics 13:127. doi: 10.1186/1471-2164-13-127. PMID: 22462823, March 31, 2012.

 

Deciphering Transcriptional Regulatory Mechanisms Associated with Hemicellulose Degradation in Neurospora crassa. J. Sun, C. Tian, S. Diamond, N. L. Glass, Eukaryot Cell (4):482-93. doi: 10.1128/EC.05327-11. PMID: 22345350, April 11, 2012.

Published in 2011

Identification of the CRE-1 Cellulolytic Regulon in Neurospora crassa, Jianping Sun, N. Louise Glass, PLoS ONE 6(9): e25654.doi:10.1371/journal/pone.0025654, September 29, 2011.

Published in 2010

Expression and Characterization of the Neurospora crassa endoglucanase GH5-1 , Jainping Sun, Christopher Phillips, Charles Anderson, William Beeson, Michael Marletta, Louise Glass, Protein Expression and Purification, 75(2), pp. 147-154, September 6, 2010.

 


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