Metagenome-Sourced Microbial Chitinases as Potential Insecticide Proteins

Francesca Berini,Flavia Marinelli,Gianluca Tettamanti,Marcella Reguzzoni,Morena Casartelli,Aurora Montali

doi:10.3389/fmicb.2019.01358

Francesca Berini, Flavia Marinelli + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2019.01358

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Abstract

Microbial chitinases are gaining interest as promising candidates for controlling plant pests. These enzymes can be used directly as biocontrol agents as well as in combination with chemical pesticides or other biopesticides, reducing their environmental impact and/or enhancing their efficacy. Chitinolytic enzymes can target two different structures in insects: the cuticle and the peritrophic matrix (PM). PM, formed by chitin fibrils connected to glycoproteins and proteoglycans, represents a physical barrier that plays an essential role in midgut physiology and insect digestion, and protects the absorptive midgut epithelium from food abrasion or pathogen infections. In this paper, we investigate how two recently discovered metagenome-sourced chitinases (Chi18H8 and 53D1) affect, in vitro and in vivo, the PM integrity of Bombyx mori, a model system among Lepidoptera. The two chitinases were produced in Escherichia coli or, alternatively, in the unconventional – but more environmentally acceptable – Streptomyces coelicolor. Although both the proteins dramatically altered the structure of B. mori PM in vitro, when administered orally only 53D1 caused adverse and marked effects on larval growth and development, inducing mortality and reducing pupal weight. These in vivo results demonstrate that 53D1 is a promising candidate as insecticide protein.

Highlights

Pesticides derived from chemical synthesis are massively used to control different pests that constantly threaten crop production (Atwood and Paisley-Jones, 2017)
To assay its insecticidal activity, Chi18H8 was produced in 2-L flasks and recovered from E. coli BL21 StarTM (DE3)/pET24b(+)::chi18H8 cells (Table 1) by using a newly developed process based on the mild solubilization of inclusion bodies (IBs), as recently described in Berini et al (2017b)
We tested the insecticidal activity of two recently discovered soil metagenome-sourced chitinases on the larvae of B. mori, by using a combined in vivo and in vitro approach

Summary

Introduction

Pesticides derived from chemical synthesis are massively used to control different pests that constantly threaten crop production (Atwood and Paisley-Jones, 2017). A compelling alternative is formulating cocktails of (semi)purified antibiotics and enzymes, which mimic living biocontrol agents, without presenting the limitations inherent to their use and storage Such biopesticides can be used alone or in combination with other controlling agents to enhance their efficacy (Regev et al, 1996; Karasuda et al, 2003; Liu et al, 2010). If added to chemically synthesized pesticides, biopesticides might allow the reduction of their dosage, alleviating their negative impact on the ecosystem (Karasuda et al, 2003) To this purpose, chitinases represent promising biopesticides, since they hydrolyze chitin, which is present in different plant pests, i.e., insects, fungi, and nematodes (Mavromatis et al, 2003; Neeraja et al, 2010; Hjort et al, 2014; Soares et al, 2015; Berini et al, 2016, 2017b, 2018). Based on their mode of action on chitin, they are classified as endochitinases, which split chitin randomly at internal sites, or as exochitinases that remove monomers (β-N-acetyl glucosaminidases) or dimers (chitobiosidases) of GlcNAc from the non-reducing end of chitin chains (Adrangi and Faramarzi, 2013; Berini et al, 2018)

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