Auxiliary active site mutations enhance the glycosynthase activity of a GH18 chitinase for polymerization of chitooligosaccharides.

Depolymerization of chitin results in chitooligosaccharides (COS) that induce immunostimulatory effects and disease protective responses and have many potential applications in agriculture and medicine. Isolation of bioactive COS with degree of polymerization (DP) larger than six from chitin hydrolyzates is hampered by their water insolubility. Enzymatic synthesis by exploiting the transglycosylation activity of GH18 chitinases offers a potential strategy to access oligomers in the range of bioactive DPs. We engineered SpChiD chitinase as a glycosynthase by mutation of the assisting residue of the catalytic triad in the substrate-assisted mechanism for polymerization of an oxazoline substrate (DP5ox). The insoluble polymer containing DP10 was partially hydrolyzed due to the significant residual hydrolase activity of the mutant enzyme. Combined mutations that strongly reduce the hydrolytic activity, in which the original catalytic triad only retains the essential acid/base residue, together with neighboring mutations in the -1/+1 subsites region, render glycosynthase-like chitinases able to produce chitin oligomers with DP10 as major product in good yields.

Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity.

Chitin deacetylases (CDAs) act on chitin polymers and low molecular weight oligomers producing chitosans and chitosan oligosaccharides. Structurally-defined, partially deacetylated chitooligosaccharides produced by enzymatic methods are of current interest as bioactive molecules for a variety of applications. Among Pochonia chlamydosporia (Pc) annotated CDAs, gene pc_2566 was predicted to encode for an extracellular CE4 deacetylase with two CBM18 chitin binding modules. Chitosan formation during nematode egg infection by this nematophagous fungus suggests a role for their CDAs in pathogenicity. The P. chlamydosporia CDA catalytic domain (PcCDA) was expressed in E. coli BL21, recovered from inclusion bodies, and purified by affinity chromatography. It displays deacetylase activity on chitooligosaccharides with a degree of polymerization (DP) larger than 3, generating mono- and di-deacetylated products with a pattern different from those of closely related fungal CDAs. This is the first report of a CDA from a nematophagous fungus. On a DP5 substrate, PcCDA gave a single mono-deacetylated product in the penultimate position from the non-reducing end (ADAAA) which was then transformed into a di-deacetylated product (ADDAA). This novel deacetylation pattern expands our toolbox of specific CDAs for biotechnological applications, and will provide further insights into the determinants of substrate specificity in this family of enzymes.

Ethanol production from chitosan by the nematophagous fungus Pochonia chlamydosporia and the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana.

Chitin is the second most abundant biopolymer after cellulose and virtually unexplored as raw material for bioethanol production. In this paper, we investigate chitosan, the deacetylated form of chitin which is the main component of shellfish waste, as substrate for bioethanol production by fungi. Fungal parasites of invertebrates such as the nematophagous Pochonia chlamydosporia (Pc) or the entomopathogens Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) are biocontrol agents of plant parasitic nematodes (eg. Meloidogyne spp.) or insect pests such as the red palm weevil (Rhynchophorus ferrugineus). These fungi degrade chitin-rich barriers for host penetration. We have therefore tested the chitin/chitosanolytic capabilities of Pc, Bb and Ma for generating reducing sugars using chitosan as only nutrient. Among the microorganisms used in this study, Pc is the best chitosan degrader, even under anaerobic conditions. These fungi have alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) encoding genes in their genomes. We have therefore analyzed their ethanol production under anaerobic conditions using chitosan as raw material. P. chlamydosporia is the largest ethanol producer from chitosan. Our studies are a starting point to develop chitin-chitosan based biofuels.

Arabidopsis thaliana root colonization by the nematophagous fungus Pochonia chlamydosporia is modulated by jasmonate signaling and leads to accelerated flowering and improved yield.

Pochonia chlamydosporia has been intensively studied in nematode control of different crops. We have investigated the interaction between P. chlamydosporia and the model system Arabidopsis thaliana under laboratory conditions in the absence of nematodes. This study demonstrates that P. chlamydosporia colonizes A. thaliana. Root colonization monitored with green fluorescent protein-tagged P. chlamydosporia and quantitative PCR (qPCR) quantitation methods revealed root cell invasion. Fungal inoculation reduced flowering time and stimulated plant growth, as determined by total FW increase, faster development of inflorescences and siliques, and a higher yield in terms of seed production per plant. Precocious flowering was associated with significant expression changes in key flowering-time genes. In addition, we also provided molecular and genetic evidence that point towards jasmonate signaling as an important factor to modulate progression of plant colonization by the fungus. Our results indicate that P. chlamydosporia provides benefits to the plant in addition to its nematophagous activity. This report highlights the potential of P. chlamydosporia to improve yield in economically important crops.

CAZyme content of Pochonia chlamydosporia reflects that chitin and chitosan modification are involved in nematode parasitism.

Pochonia chlamydosporia is a soil fungus with a multitrophic lifestyle combining endophytic and saprophytic behaviors, in addition to a nematophagous activity directed against eggs of root-knot and other plant parasitic nematodes. The carbohydrate-active enzymes encoded by the genome of P. chlamydosporia suggest that the endophytic and saprophytic lifestyles make use of a plant cell wall polysaccharide degradation machinery that can target cellulose, xylan and, to a lesser extent, pectin. This enzymatic machinery is completed by a chitin breakdown system that involves not only chitinases, but also chitin deacetylases and a large number of chitosanases. P. chlamydosporia can degrade and grow on chitin and is particularly efficient on chitosan. The relevance of chitosan breakdown during nematode egg infection is supported by the immunolocalization of chitosan in Meloidogyne javanica eggs infected by P. chlamydosporia and by the fact that the fungus expresses chitosanase and chitin deacetylase genes during egg infection. This suggests that these enzymes are important for the nematophagous activity of the fungus and they are targets for improving the capabilities of P. chlamydosporia as a biocontrol agent in agriculture.

Cell wall composition plays a key role on sensitivity of filamentous fungi to chitosan.

Chitosan antifungal activity has been reported for both filamentous fungi and yeast. Previous studies have shown fungal plasma membrane as main chitosan target. However, the role of the fungal cell wall (CW) in their response to chitosan is unknown. We show that cell wall regeneration in Neurospora crassa (chitosan sensitive) protoplasts protects them from chitosan damage. Caspofungin, a ß-1,3-glucan synthase inhibitor, showed a synergistic antifungal effect with chitosan for N. crassa but not for Pochonia chlamydosporia, a biocontrol fungus resistant to chitosan. Chitosan significantly repressed N. crassa genes involved in ß-1,3-glucan synthesis (fks) and elongation (gel-1) but the chitin synthase gene (chs-1) did not present changes in its expression. N. crassa cell wall deletion strains related to ß-1,3-glucan elongation (Δgel-1 and Δgel-2) were more sensitive to chitosan than wild type (wt). On the contrary, chitin synthase deletion strain (Δchs-1) showed the same sensitivity to chitosan than wt. The mycelium of P. chlamydosporia showed a higher (ca. twofold) ß-1,3-glucan/chitin ratio than that of N. crassa. Taken together, our results indicate that cell wall composition plays an important role on -sensitivity of filamentous fungi to chitosan.

Tolerance to chitosan by Trichoderma species is associated with low membrane fluidity.

The effect of chitosan on growth of Trichoderma spp., a cosmopolitan genus widely exploited for their biocontrol properties was evaluated. Based on genotypic (ITS of 18S rDNA) characters, four isolates of Trichoderma were identified as T. pseudokoningii FLM16, T. citrinoviride FLM17, T. harzianum EZG47, and T. koningiopsis VSL185. Chitosan reduces radial growth of Trichoderma isolates in concentration-wise manner. T. koningiopsis VSL185 was the most chitosan tolerant isolate in all culture media amended with chitosan (0.5-2.0 mg ml(-1) ). Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) were determined showing that T. koningiopsis VSL185 displays higher chitosan tolerance with MIC value >2000 µg ml(-1) while for other Trichoderma isolates MIC values were around 10 µg ml(-1) . Finally, free fatty acid composition reveals that T. koningiopsis VSL185, chitosan tolerant isolate, displays lower linolenic acid (C18:3) content than chitosan sensitive Trichoderma isolates. Our findings suggest that low membrane fluidity is associated with chitosan tolerance in Trichoderma spp.