CRISPR-Cas9/Safe Harbor-Targeted Overexpression of Glucan Synthase Gene <i>CmGls</i> in Edible Mushroom <i>Cordyceps militaris</i>

The membrane-integrated β-1,3-glucan synthase is the key enzyme involved in the biosynthesis of the core component β-1,3-glucan of the fungal cell wall. To date, the precise and targeted insertion of the β-1,3-glucan synthase gene into the genomes of edible fungi for safe and predictable overexpression has been extremely difficult due to the large DNA sequences (>5.0 kb) encoding the multitransmembrane domains and large molecular weights. In the present study, a large 5.9 kb DNA sequence of the membrane-bound β-1,3-glucan synthase gene <i>CmGls</i> was successfully and precisely inserted at a genomic safe harbor site <i>CmSh1</i> of the <i>C. militaris</i> genome for the first time. By comparing mycelial and fermentation performance, overexpression of the β-1,3-glucan synthase gene <i>CmGls</i> resulted in rapid radial growth with a more pronounced yellowish color and increased resistance to cell wall stresses. Overexpression of <i>CmGls</i> significantly improved exopolysaccharide production with higher molecular weights, accompanied by an increase in the transcription levels of genes associated with polysaccharide/glucan synthesis, such as <i>CmPgm, CmPgi</i>, and <i>CmUgp.</i> Our findings provide convincing proof for the elucidation of glucan biosynthetic pathways and a basis for developing safe strains with highly efficient production of polysaccharides/glucans by edible fungi.