Automated genome annotation analysis of metagenomic DNA revealed sequences for novel chitin and chitosan modifying enzymes

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Jenni Stöveken, S Kolkenbrock, M Zakrzewski, A Pühler, A Schlüter and BM Moerschbacher

Westphalian Wilhelms-University Münster, Germany

Chitin and chitosan, the deacetylated form of chitin, belong to earth’s most abundant biopolymers. For chemical and pharmaceutical industries, chitosan is of increasing interest due to its unique physio-chemical properties and intriguing biological functionalities. However, the term chitosan actually refers to a family of oligo- and polymers differing in their degree of polymerisation (DP), degree of acetylation (DA), as well as their pattern of acetylation (PA), and different chitosans possess different biological activities. Chitin and chitosan modifying enzymes (CCME) provide promising tools for the industrial production of chitosans with defined DP, DA, and PA. We have, therefore, set up powerful knowledge-based and –omics-based discovery projects, i.a. a metagenomic sequencing approach, for the identification of potentially novel CCMEs. Soil with a long history of exposure to chitin and chitosan was used for the extraction of metagenomic DNA. The purified DNA was sequenced on a Genome Sequencer FLX platform from Roche applying Titanium chemistry. Management and analysis of the sequences were performed within the MetaSAMS platform and by using GenDB, an automated genome annotation system for prokaryotic genomes. Bioinformatic analysis of the obtained data revealed a rather large number of genes with homology to different CCME, including chitin synthases, chitin deacetylases, chitinases, and chitosanases. A full-length sequence putatively coding for a bacterial chitinase was selected for further investigation. Its sequence was codon-optimized for E.coli expression, synthesized, cloned into E.coli, and the corresponding recombinant protein was expressed. Chitinase activity was demonstrated by dot activity assays, establishing proof-of-principle that metagenomic sequencing followed by automated genome annotation can be used as a promising source for novel CCMEs.