Characterization of novel glycosyl hydrolases discovered by cell wall glycan directed monoclonal antibody screening and metagenome analysis of maize aerial root mucilage.

Tania Pozzo,Shawn M Higdon,Michael G Hahn,Alan B Bennett,Sivakumar Pattathil,Olga A Zabotina

doi:10.1371/journal.pone.0204525

Abstract

An indigenous maize landrace from the Sierra Mixe region of Oaxaca, Mexico exhibits extensive formation of aerial roots which exude large volumes of a polysaccharide-rich gel matrix or “mucilage” that harbors diazotrophic microbiota. We hypothesize that the mucilage associated microbial community carries out multiple functions, including disassembly of the mucilage polysaccharide. In situ, hydrolytic assay of the mucilage revealed endogenous arabinofuranosidase, galactosidase, fucosidase, mannosidase and xylanase activities. Screening the mucilage against plant cell wall glycan-specific monoclonal antibodies recognized the presence of carbohydrate epitopes of hemicellulosic polysaccharides like xyloglucan (both non-fucosylated and fucosylated), xylan (both substituted and unsubstituted xylan domains) and pectic-arabinogalactans, all of which are potential carbon sources for mucilage microbial residents. Mucilage metagenome annotation using MG-RAST identified the members forming the microbial community, and gene fragments with predicted functions associated with carbohydrate disassembly. Data from the in situ hydrolytic activity and monoclonal antibody screening assays were used to guide the selection of five full length genes with predicted glycosyl hydrolase function from the GenBank database that were similar to gene fragments of high relative abundance in the mucilage metagenomes. These five genes were then synthesized for recombinant production in Escherichia coli. Here we report the characterization of an α-N-arabinofuranosidase (GH51) and an oligosaccharide reducing-end xylanase (GH8) from Flavobacterium johnsoniae; an α-L-fucosidase (GH29) and a xylan β-1,4 xylosidase (GH39) from Spirosoma linguale, and a β-mannosidase (GH2) from Agrobacterium fabrum. Biochemical characterization of these enzymes revealed a β-Mannosidase that also exhibits a secondary activity towards the cleavage of galactosyl residues. We also describe two xylanases (GH8 and GH39) from underexplored glycosyl hydrolase families, one thermostable α-L-Fucosidase (GH29) and a thermostable α-N-Arabinofuranosidase (GH51).

Highlights

The large increase in shotgun metagenomic sequence data from environmental samples collected around the world provides extensive information regarding the taxonomic distribution of microbial communities
Because mucilage polysaccharides from the aerial roots of Sierra Mixe maize have the potential to be utilized as a carbon source by the resident microbial community, we sought to gather insight regarding the enzymatic machinery that is likely to be associated with polysaccharide disassembly within the aerial root mucilage environment
While the endogenous carbohydrate active enzymes (CAZymes) activities detected within the mucilage cannot be concluded to be fully attributed to microbial CAZyme activities because of the possibility that plant-derived CAZymes may populate the aerial root mucilage, this experiment was crucial for motivating further investigation because it revealed the presence of active enzymes likely to be involved in the catabolism of mucilage polysaccharide

Summary

Introduction

The large increase in shotgun metagenomic sequence data from environmental samples collected around the world provides extensive information regarding the taxonomic distribution of microbial communities. The application of functional metagenomics targeting the investigation of bacterial carbohydrate active enzymes (CAZymes) has recently emerged, where ecological changes that affect global carbon cycling in natural environments can be monitored. This approach has provided unique opportunities to rapidly scan the microbial functionality of any ecosystem for new pools of glycosyl hydrolase (GH) biodiversity that can be used to create biocatalysts for the improvement of biotechnological processes [7]. The proliferation and establishment of each species within the microbial community of a given environment will largely depend on a number of factors, including the presence of specific glyconutrients with high bioavailability [3, 8]

Methods

Results

Conclusion