Mutation of the LRG1 Rho-GAP gene is responsible for the hyper branching C-variant phenotype in the quorn mycoprotein fungus Fusarium venenatum A3/5

Background: Quorn mycoprotein, a protein-rich meat alternative, is produced through large-scale fermentation of the fungus Fusarium venenatum. However, a major challenge during F. venenatum fermentation is the consistent appearance of mutants called colonial variants (C-variants). These C-variants have a highly branched morphology, which ultimately lead to a less desirable final product and early termination of the fermentation process. This study aimed to identify the genetic mutations responsible for C-variant morphology. Results: We first isolated both C-variant and wild-type strains from commercial fermentation samples and characterised radial growth rates on solid media. Whole genome sequencing facilitated the identification of mutations in a gene called jg4843 in 11 out of 12 C-variant isolates, which were not observed in the wild-type isolates. The jg4843 gene was identified as the ortholog of LRG1, a Rho-GTPase activating protein that regulates the Rho1 signalling pathway affecting fungal growth. Notably, the mutations in jg4843 were primarily located in the RhoGAP domain responsible for LRG1 activity. To confirm the role of these mutations, we used CRISPR/Cas9-mediated homology-directed recombination to introduce the C-variant mutations into the wild-type isolate, which successfully recapitulated the characteristic C-variant morphology. Conclusions: This study identified mutations in the LRG1 ortholog jg4843 as the genetic cause of C-variant morphology in commercial fermentation F. venenatum isolates. Understanding this genetic basis paves the way for developing strategies to prevent C-variants arising, potentially leading to more efficient and sustainable production of Quorn mycoprotein.