Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome

Loredano Pollegioni,Kjell M. Vårum,Ilaria Presti,Fabrizio Beltrametti,Flavia Marinelli,Francesca Berini,Sara Sjöling,Marco Pedroli

doi:10.1186/s12934-017-0634-8

Abstract

BackgroundThrough functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase—named Chi18H8 and belonging to family 18 glycosyl hydrolases—was previously discovered. The initial extremely low yield of Chi18H8 recombinant production and purification from Escherichia coli cells (21 μg/g cell) limited its characterization, thus preventing further investigation on its biotechnological potential.ResultsWe report on how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up to a high-yielding, 30 L bioreactor process, based on a novel method of mild solubilization of E. coli inclusion bodies in lactic acid aqueous solution, coupled with a single step purification by hydrophobic interaction chromatography. Chi18H8 was characterized as a Ca2+-dependent mesophilic chitobiosidase, active on chitin substrates at acidic pHs and possessing interesting features, such as solvent tolerance, long-term stability in acidic environment and antifungal activity against the phytopathogens Fusarium graminearum and Rhizoctonia solani. Additionally, Chi18H8 was found to operate according to a non-processive endomode of action on a water-soluble chitin-like substrate.ConclusionsExpression screening of a metagenomic library may allow access to the functional diversity of uncultivable microbiota and to the discovery of novel enzymes useful for biotechnological applications. A persisting bottleneck, however, is the lack of methods for large scale production of metagenome-sourced enzymes from genes of unknown origin in the commonly used microbial hosts. To our knowledge, this is the first report on a novel metagenome-sourced enzyme produced in hundreds-of-milligram amount by recovering the protein in the biologically active form from recombinant E. coli inclusion bodies.

Highlights

Through functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase—named Chi18H8 and belonging to family 18 glycosyl hydrolases— was previously discovered
We report how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up a high-yielding process in a 30 L bioreactor, based on the recovery of the recombinant protein from E. coli inclusion bodies (IBs)
Heterologous expression of the metagenome‐sourced Chi18H8 The complete chi18H8 gene sequence (G + C ratio 64.4%) was previously identified by genetic and expression screening of a high-molecular-weight DNA metagenomic library constructed from a soil in Uppsala (Sweden), characterized as suppressive to club-root disease of cabbage [6, 7, 29]

Summary

Introduction

Through functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase—named Chi18H8 and belonging to family 18 glycosyl hydrolases— was previously discovered. We recently applied different genetic and/or activitybased screenings to metagenomes from suppressive and/ or chitin-amended agricultural soils to access novel bacterial chitinolytic enzymes (CEs) [6,7,8]. CEs are attracting an increasing interest because of their potential in biotechnological applications [12,13,14]: e.g. as biocontrol agents that antagonize chitin-containing phytopathogenic fungi, insects and nematodes in integrated pest management strategies [7, 11, 13], or as industrial biocatalysts for the production of chitin derivatives that possess interesting nutraceutical and pharmaceutical properties [14]. The mode of action of CEs is important in relation to the length, degree of acetylation and sequence of the COs they generate [17]

Results

Discussion

Conclusion