# Expression of chitosanase from Aspergillus fumigatus chitosanase in Saccharomyces cerevisiae by CRISPR-Cas9 tools

## TL;DR

Researchers successfully expressed a chitosanase enzyme in a safe yeast strain, achieving high activity and stable performance for potential industrial use in producing chitooligosaccharides.

## Contribution

This study demonstrates the first successful expression of Aspergillus fumigatus chitosanase in Saccharomyces cerevisiae using CRISPR-Cas9, avoiding methanol induction.

## Key findings

- Shake flask enzyme activity reached 2 U/ml using the GTR-CRISPR editing system.
- Optimal chitosanase activity occurred at 55°C and pH 5, with stability between 30 and 50°C.
- Enzymatic hydrolysis produced chitobiose to chitotetraose, predominantly chitotriose.

## Abstract

Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed Escherichia coli and Pichia pastoris strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U/ml. The optimal chitosanase activity was achieved at 55℃ and pH 5, with favorable stability between 30 and 50 °C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential.