The Ustilago maydis AA10 LPMO is active on fungal cell wall chitin.

Lytic polysaccharide monooxygenases (LPMOs) can perform oxidative cleavage of glycosidic bonds in carbohydrate polymers (e.g., cellulose, chitin), making them more accessible to hydrolytic enzymes. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. The AA10 LPMOs are active on chitin and/or cellulose and mostly found in bacteria and in some viruses and archaea. Interestingly, AA10-encoding genes are also encountered in some pathogenic fungi of the Ustilaginomycetes class, such as Ustilago maydis, responsible for corn smut disease. Transcriptomic studies have shown the overexpression of the AA10 gene during the infectious cycle of U. maydis. In fact, U. maydis has a unique AA10 gene that codes for a catalytic domain appended with a C-terminal disordered region. To date, there is no public report on fungal AA10 LPMOs. In this study, we successfully produced the catalytic domain of this LPMO (UmAA10_cd) in Pichia pastoris and carried out its biochemical characterization. Our results show that UmAA10_cd oxidatively cleaves α- and ß-chitin with C1 regioselectivity and boosts chitin hydrolysis by a GH18 chitinase from U. maydis (UmGH18A). Using a biologically relevant substrate, we show that UmAA10_cd exhibits enzymatic activity on U. maydis fungal cell wall chitin and promotes its hydrolysis by UmGH18A. These results represent an important step toward the understanding of the role of LPMOs in the fungal cell wall remodeling process during the fungal life cycle.IMPORTANCELytic polysaccharide monooxygenases (LPMOs) have been mainly studied in a biotechnological context for the efficient degradation of recalcitrant polysaccharides. Only recently, alternative roles and paradigms begin to emerge. In this study, we provide evidence that the AA10 LPMO from the phytopathogen Ustilago maydis is active against fungal cell wall chitin. Given that chitin-active LPMOs are commonly found in microbes, it is important to consider fungal cell wall as a potential target for this enigmatic class of enzymes.

Comprehensive Genome Analysis of Cellulose and Xylan-Active CAZymes from the Genus Paenibacillus: Special Emphasis on the Novel Xylanolytic Paenibacillus sp. LS1.

Xylan is the most abundant hemicellulose in hardwood and graminaceous plants. It is a heteropolysaccharide comprising different moieties appended to the xylose units. Complete degradation of xylan requires an arsenal of xylanolytic enzymes that can remove the substitutions and mediate internal hydrolysis of the xylan backbone. Here, we describe the xylan degradation potential and underlying enzyme machinery of the strain, Paenibacillus sp. LS1. The strain LS1 was able to utilize both beechwood and corncob xylan as the sole source of carbon, with the former being the preferred substrate. Genome analysis revealed an extensive xylan-active CAZyme repertoire capable of mediating efficient degradation of the complex polymer. In addition to this, a putative xylooligosaccharide ABC transporter and homologues of the enzymes involved in the xylose isomerase pathway were identified. Further, we have validated the expression of selected xylan-active CAZymes, transporters, and metabolic enzymes during growth of the LS1 on xylan substrates using qRT-PCR. The genome comparison and genomic index (average nucleotide identity [ANI] and digital DNA-DNA hybridization) values revealed that strain LS1 is a novel species of the genus Paenibacillus. Lastly, comparative genome analysis of 238 genomes revealed the prevalence of xylan-active CAZymes over cellulose across the Paenibacillus genus. Taken together, our results indicate that Paenibacillus sp. LS1 is an efficient degrader of xylan polymers, with potential implications in the production of biofuels and other beneficial by-products from lignocellulosic biomass. IMPORTANCE Xylan is the most abundant hemicellulose in the lignocellulosic (plant) biomass that requires cooperative deconstruction by an arsenal of different xylanolytic enzymes to produce xylose and xylooligosaccharides. Microbial (particularly, bacterial) candidates that encode such enzymes are an asset to the biorefineries to mediate efficient and eco-friendly deconstruction of xylan to generate products of value. Although xylan degradation by a few Paenibacillus spp. is reported, a complete genus-wide understanding of the said trait is unavailable till date. Through comparative genome analysis, we showed the prevalence of xylan-active CAZymes across Paenibacillus spp., therefore making them an attractive option towards efficient xylan degradation. Additionally, we deciphered the xylan degradation potential of the strain Paenibacillus sp. LS1 through genome analysis, expression profiling, and biochemical studies. The ability of Paenibacillus sp. LS1 to degrade different xylan types obtained from different plant species, emphasizes its potential implication in lignocellulosic biorefineries.

Genome analysis of Streptomyces sp. UH6 revealed the presence of potential chitinolytic machinery crucial for chitosan production.

Chitosan and its derivatives have numerous applications in wastewater treatment as bio-coagulants, flocculants and bio-adsorbents against both particulate and dissolved pollutants. Chitinolytic bacteria secrete an array of enzymes, which play crucial role in chitin to chitosan conversion. Consequently, there is a growing demand for identification and characterization of novel bacterial isolates with potential implications in chitosan production. We describe genomic features of the new isolate Streptomyces sp. UH6. Analysis of the 6.51 Mb genome revealed the GC content as 71.95% and presence of 6990 coding sequences of which 63% were functionally annotated. Further, we identified two possible chitin-utilization pathways, which employ secreted enzymes like lytic polysaccharide monooxygenases and family-18 glycoside hydrolases (GHs). More importantly, the genome has six family-4 polysaccharide deacetylases with probable role in chitin to chitosan conversion, as well as two chitosanases belonging to GH46 and GH75 families. In addition, the gene clusters, dasABC and ngcEFG coding for transporters, which mediate the uptake of N,N'-diacetylchitobiose and N-acetyl-d-glucosamine were identified. Several genes responsible for hydrolysis of other polysaccharides and fermentation of sugars were also identified. Taken together, the phylogenetic and genomic analyses suggest that the isolate Streptomyces sp. UH6 secretes potential chitin-active enzymes responsible for chitin to chitosan conversion.

Efficient conversion of crystalline chitin to N-acetylglucosamine and N,N'-diacetylchitobiose by the enzyme cocktail produced by Paenibacillus sp. LS1.

Direct conversion of crystalline chitin to N-acetylglucosamine and the related chitooligomers through enzymatic approaches is gaining importance owing to their various biological applications. Here we report the crystalline chitin degradation ability of chitinolytic cocktail produced by the isolate Paenibacillus sp. LS1. Growth studies of the isolate in presence of different chitin substrates revealed preference for ß-chitin and colloidal chitin. FE-SEM micrographs showed formation of visible perforations on the crystalline chitin particles by the isolate. Further, zymogram analysis revealed the presence of six potential chitinase isozymes. The enzyme-cocktail produced by the isolate was optimally active at 50 °C in 50 mM sodium acetate, pH-4.0. Time-course hydrolysis of crystalline α- and ß-chitin with the Paenibacillus sp. LS1 enzyme-cocktail produced N-acetylglucosamine and N,N'-diacetylchitobiose as the predominant products. Taken together, our results confirm that the Paenibacillus sp. LS1 enzyme-cocktail would have potential application in eco-friendly enzymatic approaches for efficient saccharification of crystalline chitin.