To successfully survive in plants, endophytes need strategies to avoid being detected by the plant immune system, as the cell walls of endophytes contain easily detectible chitin. It is possible that endophytes “hide” this chitin from the plant immune system by modifying it, or oligomers derived from it, using chitin deacetylases (CDA). To explore this hypothesis, we identified and expressed a CDA from Pestalotiopsis sp. (PesCDA), an endophytic fungus, in E. coli and characterized this enzyme and its chitosan oligomer products. We found that when PesCDA modifies chitin oligomers, the products are partially deacetylated chitosan oligomers with a specific acetylation pattern: GlcNAc-GlcNAc-(GlcN)n-GlcNAc (n ≥ 1). Then, in a bioactivity assay where suspension-cultured rice cells were incubated with the PesCDA products (processed chitin hexamers), we found that, unlike the substrate hexamers, chitosan oligomer products no longer elicited the plant immune system. Thus, this endophytic enzyme can prevent the endophyte from being recognized by the plant immune system; this might represent a more general hypothesis for how certain fungi are able to live in or on their hosts.

Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture.

Partially acetylated chito-oligosaccharides (paCOS) have diverse bioactivities that turn them into promising compounds especially for medical and agricultural applications. These properties likely arise from different acetylation patterns, but determining the sequences of paCOS and producing paCOS with patterns of interest have proven difficult. We present a novel method for sequencing submicrogram amounts of paCOS using quantitative mass spectrometry, allowing one to rapidly analyze the substrate specificities of chitosan hydrolases that can be used to produce paCOS. The method involves four major steps: (i) acetylation of free amino groups in paCOS using a deuterated reagent; (ii) labeling the reducing end with an O-tag; (iii) quantifying paCOS using [C, H]-labeled isotopologs as internal standards; (iv) sequencing paCOS by tandem MS. Eventually, this method will aid in developing enzymes with cleavage patterns optimized for producing paCOS with defined patterns of acetylation and specific bioactivities.

Chitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such as Ustilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a mycelium in planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity of U. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain.

Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e., their degrees of polymerization and acetylation, as well as their pattern of acetylation. However, paCOS generated via chemical N-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDAs) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDAs. However, compared to what we know about bacterial CDAs, we know little about the ability of fungal CDAs to produce defined paCOS with known patterns of acetylation. Therefore, we optimized the expression of a chitin deacetylase from the fungus Puccinia graminis f. sp. tritici in Escherichia coli. The best yield of functional enzyme was obtained as a fusion protein with the maltose-binding protein (MBP) secreted into the periplasmic space of the bacterial host. We characterized the MBP fusion protein from P. graminis (PgtCDA) and tested its activity on different chitinous substrates. Mass spectrometric sequencing of the products obtained by enzymatic deacetylation of chitin oligomers, i.e., tetramers to hexamers, revealed that PgtCDA generated paCOS with specific acetylation patterns of A-A-D-D, A-A-D-D-D, and A-A-D-D-D-D, respectively (A, GlcNAc; D, GlcN), indicating that PgtCDA cannot deacetylate the two GlcNAc units closest to the oligomer's nonreducing end. This unique property of PgtCDA significantly expands the so far very limited library of well-defined paCOS available to test their bioactivities for a wide variety of potential applications. IMPORTANCEWe successfully achieved heterologous expression of a fungal chitin deacetylase gene from the basidiomycete Puccinia graminis f. sp. tritici in the periplasm of E. coli as a fusion protein with the maltose-binding protein; this strategy allows the production of these difficult-to-express enzymes in sufficient quantities for them to be characterized and optimized through protein engineering. Here, the recombinant enzyme was used to produce partially acetylated chitosan oligosaccharides from chitin oligomers, whereby the pronounced regioselectivity of the enzyme led to the production of defined products with novel patterns of acetylation. This approach widens the scope for both the production and functional analysis of chitosan oligomers and thus will eventually allow the detailed molecular structure-function relationships of biologically active chitosans to be studied, which is essential for developing applications for these functional biopolymers for a circular bioeconomy, e.g., in agriculture, medicine, cosmetics, and food sciences.

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Chitin, a linear polymer of N-acetyl-d-glucosamine, and chitosans, fully or partially deacetylated derivatives of chitin, are known to elicit defense reactions in higher plants. We compared the ability of chitin and chitosan oligomers and polymers (chitin oligomers with degree of polymerization [DP] 3 to 8; chitosan oligomers with degree of acetylation [DA] 0 to 35% and DP 3 to 15; chitosan polymers with DA 1 to 60% and DP approximately 1,300) to elicit an oxidative burst indicative of induced defense reactions in Arabidopsis thaliana seedlings. Fully deacetylated chitosans were not able to trigger a response; elicitor activity increased with increasing DA of chitosan polymers. Partially acetylated chitosan oligomers required a minimum DP of 6 and at least four N-acetyl groups to trigger a response. Invariably, elicitation of an oxidative burst required the presence of the chitin receptor AtCERK1. Our results as well as previously published studies on chitin and chitosan perception in plants are best explained by a new general model of LysM-containing receptor complexes in which two partners form a long but off-set chitin-binding groove and are, thus, dimerized by one chitin or chitosan molecule, sharing a central GlcNAc unit with which both LysM domains interact. To verify this model and to distinguish it from earlier models, we assayed elicitor and inhibitor activities of selected partially acetylated chitosan oligomers with fully defined structures. In contrast to the initial 'continuous groove', the original 'sandwich', or the current 'sliding mode' models for the chitin/chitosan receptor, the here-proposed 'slipped sandwich' model-which builds on these earlier models and represents a consensus combination of these-is in agreement with all experimental observations.

Some of the most abundant biomolecules on earth are the polysaccharides chitin and chitosan of which especially the oligomeric fractions have been extensively studied regarding their biological activities. However, most of these studies have not been able to assess the activity of a single, defined, partially acetylated chitosan oligosaccharide (paCOS). Instead, they have typically analyzed chemically produced, rather poorly characterized mixtures, at best with a single, defined degree of polymerization (DP) and a known average degree of acetylation (DA), as no pure and well-defined paCOS are currently available. We here present data on the enzymatic production of all 14 possible partially acetylated chitosan tetramers, out of which four were purified (>95%) regarding DP, DA, and pattern of acetylation (PA). We used bacterial, fungal, and viral chitin deacetylases (CDAs), either to partially deacetylate the chitin tetramer; or to partially re-N-acetylate the glucosamine tetramer. Both reactions proceeded with surprisingly strong and enzyme-specific regio-specificity. These pure and fully defined chitosans will allow to investigate the particular influence of DP, DA, and PA on the biological activities of chitosans, improving our basic understanding of their modes of action, e.g. their molecular perception by patter recognition receptors, but also increasing their usefulness in industrial applications.