Transcriptomics reveal a mechanism of niche defense: two beneficial root endophytes deploy an antimicrobial GH18-CBM5 chitinase to protect their hosts.

Effector secretion is crucial for root endophytes to establish and protect their ecological niche. We used time-resolved transcriptomics to monitor effector gene expression dynamics in two closely related Sebacinales, Serendipita indica and Serendipita vermifera, during symbiosis with three plant species, competition with the phytopathogenic fungus Bipolaris sorokiniana, and cooperation with root-associated bacteria. We observed increased effector gene expression in response to biotic interactions, particularly with plants, indicating their importance in host colonization. Some effectors responded to both plants and microbes, suggesting dual roles in intermicrobial competition and plant-microbe interactions. A subset of putative antimicrobial effectors, including a GH18-CBM5 chitinase, was induced exclusively by microbes. Functional analyses of this chitinase revealed its antimicrobial and plant-protective properties. We conclude that dynamic effector gene expression underpins the ability of Sebacinales to thrive in diverse ecological niches with a single fungal chitinase contributing substantially to niche defense.

Heterogeneously deacetylated chitosans possess an unexpected regular pattern favoring acetylation at every third position.

Chitosans are promising biopolymers for diverse applications, with material properties and bioactivities depending i.a. on their pattern of acetylation (PA). Commercial chitosans are typically produced by heterogeneous deacetylation of chitin, but whether this process yields chitosans with a random or block-wise PA has been debated for decades. Using a combination of recently developed in vitro assays and in silico modeling surprisingly revealed that both hypotheses are wrong; instead, we found a more regular PA in heterogeneously deacetylated chitosans, with acetylated units overrepresented at every third position in the polymer chain. Compared to random-PA chitosans produced by homogeneous deacetylation of chitin or chemical N-acetylation of polyglucosamine, this regular PA increases the elicitation activity in plants, and generates different product profiles and distributions after enzymatic and chemical cleavage. A regular PA may be beneficial for some applications but detrimental for others, stressing the relevance of the production process for product development.

Fast insights into chitosan-cleaving enzymes by simultaneous analysis of polymers and oligomers through size exclusion chromatography.

The thorough characterization of chitosan-cleaving enzymes is crucial to unveil structure-function relationships of this promising class of biomolecules for both, enzymatic fingerprinting analyses and to use the enzymes as biotechnological tools to produce tailor-made chitosans for diverse applications. Analyzing polymeric substrates as well as oligomeric products has been established as an effective way to understand the actions of enzymes, but it currently requires separate, rather laborious methods to obtain the full picture. Here, we present ultra high performance size exclusion chromatography coupled to refractive index and mass spectrometry detection (UHPSEC-RI-MS) as a straightforward method for the semi-quantitative analysis of chitosan oligomers of up to ten monomers in length. Additionally, the method allows to determine the average molecular weight of the remaining polymers and its distribution. By sampling live from an ongoing enzymatic reaction, UHPSEC-RI-MS offers the unique opportunity to analyze polymers and oligomers simultaneously-i.e., to monitor the molecular weight reduction of the polymeric substrate over the course of the digestion, while at the same time analyzing the emerging oligomeric products in a semi-quantitative manner. In this way, a single simple analysis yields detailed insights into an enzyme's action on a given substrate.

Customized chitooligosaccharide production-controlling their length via engineering of rhizobial chitin synthases and the choice of expression system.

Chitooligosaccharides (COS) have attracted attention from industry and academia in various fields due to their diverse bioactivities. However, their conventional chemical production is environmentally unfriendly and in addition, defined and pure molecules are both scarce and expensive. A promising alternative is the in vivo synthesis of desired COS in microbial platforms with specific chitin synthases enabling a more sustainable production. Hence, we examined the whole cell factory approach with two well-established microorganisms-Escherichia coli and Corynebacterium glutamicum-to produce defined COS with the chitin synthase NodC from Rhizobium sp. GRH2. Moreover, based on an in silico model of the synthase, two amino acids potentially relevant for COS length were identified and mutated to direct the production. Experimental validation showed the influence of the expression system, the mutations, and their combination on COS length, steering the production from originally pentamers towards tetramers or hexamers, the latter virtually pure. Possible explanations are given by molecular dynamics simulations. These findings pave the way for a better understanding of chitin synthases, thus allowing a more targeted production of defined COS. This will, in turn, at first allow better research of COS' bioactivities, and subsequently enable sustainable large-scale production of oligomers.

Reassessment of chitosanase substrate specificities and classification.

Chitosanases can be used to produce partially acetylated chitosan oligosaccharides (paCOS) for different applications, provided they are thoroughly characterized. However, recent studies indicate that the established classification system for chitosanases is too simplistic. Here, we apply a highly sensitive method for quantitatively sequencing paCOS to reassess the substrate specificities of the best-characterized class I-III chitosanases. The enzymes' abilities to cleave bonds at GlcNAc residues positioned at subsite (-1) or (+1), on which the classification system is based, vary especially when the substrates have different fractions of acetylation (F A ). Conflicts with the recent classification are observed at higher F A , which were not investigated in prior specificity determinations. Initial analyses of pectin-degrading enzymes reveal that classifications of other polysaccharide-degrading enzymes should also be critically reassessed. Based on our results, we tentatively suggest a chitosanase classification system which is based on specificities and preferences of subsites (-2) to (+2).