Functional Characterization of a β-1,3-Glucanosyltransferase <i>CmGel4</i> in <i>Cordyceps militaris</i> Using a Precise CRISPR-Cas9 Genome-Editing System

<i>Cordyceps militaris</i> polysaccharides, especially β-glucans, have presented significant antitumor, hypoglycemic, and immunomodulatory activities. However, the enzymes involved in the branching formation of <i>C. militaris</i> β-glucans remain to be elucidated. In the present study, a 1.69-kb β-1,3-glucanosyltransferase <i>CmGel4</i> gene putatively involved in β-glucan branching was cloned from <i>C. militaris</i> mycelia and bioinformatically analyzed. The encoded 54.12 kDa CmGel4p consisted of 515 amino acid residues and contained a typical GH72⁺ structural characteristic of a signal peptide (1-19aa), a GH72 conserved domain (20-334aa), a GPI-anchor site (485aa), and a CBM43/X8 domain (382-458aa). Using the established CRISPR-Cas9 genome-editing system, the full length of 1.69-kb <i>CmGel4</i> was precisely inserted at a genomic safe-harbor site <i>CmSh1</i>, and the GH72 conserved domain of <i>CmGel4</i> was successfully deleted in <i>C. militaris</i> genome for the first time. By comparing the mycelial growth and fermentation performance of WT, control, and <i>CmGel4</i>-overexpressed/knockout mutants, β-1,3-glucanosyltransferase gene <i>CmGel4</i> was shown to play key roles in cell growth and branching of <i>exo</i>-polysaccharides of <i>C. militaris</i>, accompanied by the transcriptional changes of genes such as <i>CmGel4, CmUgp,</i> and <i>CmPgm</i>. These findings provided the proof of β-1,3-glucanosyltransferases vital for formatting cell walls and maintaining cellular integrity, and a fine regulation strategy for precisely remodeling the β-1,3-glucan with high-branched structures in edible fungi.