Exploring the multi-level regulation of lignocellulases in the filamentous fungus Trichoderma guizhouense NJAU4742 from an omics perspective.

BACKGROUND: Filamentous fungi are highly efficient at deconstructing plant biomass by secreting a variety of enzymes, but the complex enzymatic regulation underlying this process is not conserved and remains unclear. RESULTS: In this study, cellulases and xylanases could specifically respond to Avicel- and xylan-induction, respectively, in lignocellulose-degrading strain Trichoderma guizhouense NJAU4742, however, the differentially regulated cellulases and xylanases were both under the absolute control of the same TgXyr1-mediated pathway. Further analysis showed that Avicel could specifically induce cellulase expression, which supported the existence of an unknown specific regulator of cellulases in strain NJAU4742. The xylanase secretion is very complex, GH10 endoxylanases could only be induced by Avicel, while, other major xylanases were significantly induced by both Avicel and xylan. For GH10 xylanases, an unknown specific regulator was also deduced to exist. Meanwhile, the post-transcriptional inhibition was subsequently suggested to stop the Avicel-induced xylanases secretion, which explained the specifically high xylanase activities when induced by xylan in strain NJAU4742. Additionally, an economical strategy used by strain NJAU4742 was proposed to sense the environmental lignocellulose under the carbon starvation condition, that only slightly activating 4 lignocellulose-degrading genes before largely secreting all 33 TgXyr1-controlled lignocellulases if confirming the existence of lignocellulose components. CONCLUSIONS: This study, aiming to explore the unknown mechanisms of plant biomass-degrading enzymes regulation through the combined omics analysis, will open directions for in-depth understanding the complex carbon utilization in filamentous fungi.

Two degradation strategies for overcoming the recalcitrance of natural lignocellulosic xylan by polysaccharides-binding GH10 and GH11 xylanases of filamentous fungi.

The recalcitrance of lignocellulose forms a strong barrier for the bioconversion of lignocellulosic biomass in chemical or biofuel industries. Filamentous fungi are major plant biomass decomposer, and capable of forming all the required enzymes. Here, they characterized the GH10 and GH11 endo-xylanases and a CE1 acetyl-xylan esterase (Axe1) from a superior biomass-degrading strain, Aspergillus fumigatus Z5, and examined how they interact in xylan degradation. Cellulose-binding (CBM1) domain inhibited GH10 xylanase activities for pure xylan, but afforded them an ability to hydrolyze washed corncob particles (WCCP). CBM1-containing GH10 xylanases also showed synergism with CBM1-containing Axe1 in WCCP hydrolysis, and this synergy was strictly dependent on the presence of their CBM1 domains. In contrast, GH11 xylanases had no CBM1, but still could bind xylan and hydrolyzed WCCP; however, no synergism displayed with Axe1. GH10 xylanases and GH11 xylanases showed a pronounced synergism in WCCP hydrolysis, which was dependent on the presence of the CBM1 in GH10 xylanases and absence from GH11 xylanases. They exhibit different mechanisms to bind to cellulose and xylan, and act in synergy when these two structures are intact. These findings will be helpful for the further development of highly efficient enzyme mixtures for lignocellulosic biomass conversion.

Characterization of extracellular polymeric substances of Bacillus amyloliquefaciens SQR9 induced by root exudates of cucumber.

Bacillus amyloliquefaciens SQR9 is a plant growth-promoting rhizobacterium (PGPRs) that forms biofilm on the roots of plants and protects them from a variety of pathogens. In this study, we reported the effect of root exudates produced by cucumber (Cucumis sativus L.) at different developmental stages on the biochemical composition of the biofilm matrix of SQR9. The results showed that the amino acids present in the root exudates of cucumber were responsible for triggering biofilm formation of SQR9. In addition, when root exudates harvested at different growth phases of cucumber were used as carbon sources for biofilm formation, the resulting biofilm matrixes differed both quantitatively and qualitatively. The biofilm matrix was mostly composed of amino groups observed by confocal laser scanning microscope (CLSM) hence the proteins formed the major component of the resulting extracellular polymeric substances (EPS). The potential use of amino acid-based dietary supplements to control biofilm formation in the plants may be a viable option to improve agricultural productivity by recruiting beneficial association with PGPRs in the manufacture of bio fertilizers or bio controls.

Genome-wide transcriptomic analysis of a superior biomass-degrading strain of A. fumigatus revealed active lignocellulose-degrading genes.

BACKGROUND: Various saprotrophic microorganisms, especially filamentous fungi, can efficiently degrade lignocellulose that is one of the most abundant natural materials on earth. It consists of complex carbohydrates and aromatic polymers found in the plant cell wall and thus in plant debris. Aspergillus fumigatus Z5 was isolated from compost heaps and showed highly efficient plant biomass-degradation capability. RESULTS: The 29-million base-pair genome of Z5 was sequenced and 9540 protein-coding genes were predicted and annotated. Genome analysis revealed an impressive array of genes encoding cellulases, hemicellulases and pectinases involved in lignocellulosic biomass degradation. Transcriptional responses of A. fumigatus Z5 induced by sucrose, oat spelt xylan, Avicel PH-101 and rice straw were compared. There were 444, 1711 and 1386 significantly differently expressed genes in xylan, cellulose and rice straw, respectively, when compared to sucrose as a control condition. CONCLUSIONS: Combined analysis of the genomic and transcriptomic data provides a comprehensive understanding of the responding mechanisms to the most abundant natural polysaccharides in A. fumigatus. This study provides a basis for further analysis of genes shown to be highly induced in the presence of polysaccharide substrates and also the information which could prove useful for biomass degradation and heterologous protein expression.

Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost.

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by ß-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.

A novel biochemical route for fuels and chemicals production from cellulosic biomass.

The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing ß-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate--glucose and gluconate--can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of ß-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.