Substrate Enzyme Interactions of Fungal Holocellulose Breakdown Systems


ARTICLES

Damásio AR, Pessela BC, Mateo C, Segato F, Prade RA, Guisan JM, Maria de Lourdes Teixeira de Moraes Polizeli MLTM (2012) Immobilization of a recombinant endo-1,5-arabinanase secreted by Aspergillus nidulans strain A773 Journal of Molecular Catalysis B: Enzymatic 77: 39– 45

Damásio AR, Ribeiro LF, Ribeiro LF, Furtado GP, Segato F, Almeida FB, Crivellari AC, Buckeridge MS, Souza TA, Murakami MT, Ward RJ, Prade RA, Polizeli MLTM. (2012) Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus. Biochimica et Biophysica Acta 1824(3):461-467

Ray A, Saykhedkar S, Ayoubi-Canaan P, Hartson SD, Prade R, Mort AJ. (2012) Phanerochaete chrysosporium produces a diverse array of extracellular enzymes when grown on sorghum. Applied Microbiology Biotechnology. 93:2075-2089

Segato F e Prade RA 2011 A biologia molecular dos sistemas de expressão desenvolvidos para a produção de enzimas de interesse industrial. In “A Biologia Molecular e a Produção de Enzimas de Interesse Comercial”  (Ed, Said S) Cap. 3 (In Press) - Invited Publication (Peer-reviewed)

Damásio ARL, Tony M. Silva TM, Almeida FBR, Squina FM, Ribeiro DA, Leme AFP, Segato F, Prade RA, Jorge JA, Terenzi HF, Polizeli MLTM (2011) Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application. Process Biochemistry 46:1236–1242

Squina FM, Mort AJ, Decker SR, Prade RA (2009) Xylan decomposition by Aspergillus clavatus endo-xylanase. Protein Expression & Purification 68(1):65-71

Mort A, Gao S, Wu X and R Prade 2005. The need for pure enzymes that degradecell wall polysaccharides and how to get them. Plant Biosystems 139: 84-87

Fu J, Prade R and Mort A 2001 Expression and action pattern of the rhamnoghalacturonase of Botryotinia fuckeliana in Pichia pastoris Carbohydrate Res 15: 73-81

Prade R Zhan D, Ayoubi P and Mort A 1999 Pectins, Pectinases and Plant-Microbe Interactions Biotechnology and Genetic Engineering Reviews 16: 361-391  - Invited Publication (Peer-reviewed)

Prade R 1996 Xylanases: From Biology to BioTechnology Biotechnology and Genetic Engineering Reviews 13:101-131  - Invited Publication (Peer-reviewed)

Milagres A and Prade R 1994 Production of xylanases from Penicillium janthinellum and its use in the recovery of cellulosic textile fibers Enzyme and Microbial Technology 16:627-632

Milagres A, Lacis L and Prade R 1993 Characterization of xylanase production by a local isolate of Penicillium janthinellum Enzyme and Microbial Technology 15:248-253

Plants produce elaborate cell walls that confer them protection and mechanical resistance.  For example, woody plants produce load-bearing and robust stems capable of carrying a tree and its cup up to 100 feet.  In addition, plant cell walls confer protection against environmental factors such as wind water sun and the attack of pathogens.  Thus the cell wall is constructed with dense and recalcitrant materials; cellulose, hemicellulose, pectin and lignin.  Three out of these four major components of plant cell walls are constituted by polysaccharides, which incorporate sugars as their basic repeating unit representing a massive pool of chemical energy locked up in these structural molecules.  Plant cell wall polysaccharides are the major constituent of biomass (up to 60%) and the main sugar sink in nature constituted of holocellulose (65-85%) cross-linked with the phenolic polymer lignin (15-35%) (Gray et al., 2006).

Plants utilize large multimeric cellulose synthase complexes to construct recalcitrant linear glucose containing cellulose molecules (Endler & Persson, 2011) and Golgi glycosyltransferases to synthesize heterogeneous branched sugars, xylose, arabinose, galactose, mannose, rhamnose, galacturonic- and glucuronic-acid containing hemicellulose and pectin (Scheller & Ulvskov, 2010, Mellerowicz & Sundberg, 2008); polysaccharides that come in a variety of structural combinations each of which confer a specific property to the assembled cell wall.  Thus, this carbohydrate heterogeneity and structural complexity is the barrier to the rational use of plant biomass for biofuel production, which requires simple sugars suitable for biotransformation.  Technological solutions are needed to reverse the synthesis complexity and render monomeric sugars useful in the production of biofuels (Otero et al., 2007).

However, plants along with robust and refractory cell walls die and are completely recycled in nature, polysaccharides are deconstructed and sugars used by microorganisms that live in soil and forest floors (Wei et al., 2009).  Decomposition of woody materials is a recalcitrant process, which takes place in soil, perhaps employs many enzymes produced by several microorganisms (Chassard et al., 2010).  Bacteria are major components of soil environments and are equipped with many of the enzymes needed to degrade plant cell walls (Rattanachomsri et al., 2011, Hess et al., 2011).  Fungi are also present in soil, rich in polysaccharide degrading enzymes and active in recycling plant cell walls (Liu et al., 2011a, Liggenstoffer et al., 2010).  Some of the organisms active in plant cell wall degradation work best under anaerobic conditions and consortium studies have identified fungi and bacteria and their enzymes involved in cell wall recycling (Fontes & Gilbert, 2010, Liggenstoffer et al., 2010, Tamaru et al., 2010, Morrison et al., 2009)

Anaerobic fungi are known to inhabit cell wall recycling environments and they show a cellulosome type architecture that is similar to the ones found in anaerobic bacteria (Bomble et al., 2011, Hess et al., 2011, Noach et al., 2010).  Even though, we know that filamentous fungi are active in recycling plant cell walls, they secrete free enzymes into the medium and so far have not been investigated in aerobic microbial communities.  Crystalline cellulose is a significant component to plant cell walls, specially of woody plants and so far only aerobic fungi have been found that produce enzymes capable of degrading the crystalline portion of cellulose (Liu et al., 2011b, Himmel & Bayer, 2009, Beckham et al., 2010).

Here we survey eight Aspergillii with fully sequenced genomes, for cellulose and hemicellulose degrading enzymes.  Multiplicity of genes (gene duplication and functional redundancy) encoding cellulases and hemicellulases is widely encountered in fungi, illustrating the effort in acquiring protein functions capable of degrading the recalcitrant plant cell wall.  As a result, combination of genetic multiplicity and functional redundancy creates a complex and overlapping repertoire of enzymes for which we provide a detailed comparative analysis.

Filamentous fungi are known for their ability in secreting large amounts of proteins, as well as encoding the majority of carbohydrate-degrading enzymes which function synergistically, achieving complete decomposition of plant cell wall polysaccharides.

Industrial biotechnology of biomass conversion and bioenergy has significantly been pushed forwarded by scientific achievements in functional genomics, particularly in the areas of genome analysis, transcriptome, proteomics, and metabolomics (Otero et al., 2007).  A promising model is an integrated biorefinery core that serves both the energy and chemical commodities market, which relies on innovative pathways to produce along with bioenergy, chemical compounds for high- value products industries, such as the feed, food, cosmetic or pharmaceutical purposes and biological precursors, such as new polymers and their uses (Kamm & Kamm, 2004, Vertes et al., 2006).

Significant effort in the fields of enzyme catalysis of cellulose and hemicellulose will be required to extrapolate the full energetic value of lignocellulose.  A major current challenge of current biotechnology, especially in the lignocellulose-to-ethanol process is to identify novel biocatalysis and enzymes for enzymatic hydrolysis from the genomes.  Therefore, the demand for identification of novel biomass degrading enzymes as well as for heterologous protein production at higher efficiency and reduced costs has catalyzed an interest in elucidating the genomic sequence of several filamentous fungi (Ferrer et al., 2005).

The Carbohydrate-Active enZymes (CAZy) database has compiled and assigned into families the diversity of glycosyl hydrolase (GH) genes, according with a classification system developed by Henrissat and coworkers (Henrissat & Bairoch, 1993), based on amino acid sequence similarity, secondary and tertiary fold conservation and stereo chemical architecture of catalytic mechanisms; i.e., inversion or retention of the anomeric configuration (Henrissat & Bairoch, 1993).  According to Jovanovic and coworkers (Jovanovic et al., 2009) from 114 GH families only 22 are critical for biomass decomposition and 20 are populated with genes from filamentous fungi, including endo and exo acting cellulases, hemicellulases, backbone degrading, debranching enzymes and exo glucosidases (Jovanovic et al., 2009).

References

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