Chitinolytic Activity of Serratia Marcescens Chitinase in Response to Various Species of Pathological Fungi

Abstract

Fungi producing harmful mycotoxins flourish on various crops. Such fungal infections significantly reduce sustainability and food production in developing countries, where mycotoxin exposure from lack of advanced food storage are responsible for severe economic losses and 40% of diseases. Our team developed a modified enzyme chitinase capable of breaking down chitin cell walls. Specifically, Serratia Marcescens Chitinase works against multiple families of fungi. By GSTChiA Chitinase genes with a signal sequence from araC, we successfully generated an Escherichia coli line that secretes chitinase against Rhizoctoniasolani Solani, Alternaria raphani, and many other pathogenic fungi. Expression of GSTChiA was further quantified through analysis of chitin compounds. This project will provide an easily accessible method capable of combating major pathogens, saving crop yield and revenue.

Background

Fungi producing harmful mycotoxins flourish on various crops. Such fungal infections significantly reduce sustainability and food production in developing countries and in agricultural industry, in which mycotoxins deplete inadequately preserved food storages and cause 40% of diseases. Chitin is the second most common polysaccharide found in nature, next to cellulose, and is an important component of fungal cell wall. Chitinase is an enzyme that actively breaks down chitin, and different strains of chitinase are optimized to effectively degrade the cell walls of different strains of chitin. Serratia Marcescens Chitinase acts against Rhizoctonia solani, Bipolaris sp, Alternaria raphani, Alternaria brassicicola, and Fusarium oxysporum which impact a wide range of agricultural products including various herbs, cruciferous vegetables, radish species, bananas, and potatoes. The construct of this project combines a ChiA sequence from Serratia Marcescens and a Glutathione S-Transferase (GST) sequence making it is possible to easily pull down protein and produce ChiA on a small scale. Finally, Serratia Marcescen Chitinase-- when combined with the GST, FLAG-tag, and a pBAD vector-- can be mutated via Error Prone PCR, as proven in this experiment, meaning that it is possible to evolve the enzyme via this process.