Open Access

Katharina J. Ost, Mark Arentshorst, Mareike E. Dirks-Hofmeister, Bruno M. Moerschbacher, Arthur F. J. Ram

• Alpha-glucan synthases (ags) play a key role in the synthesis of alpha-1,3-glucan, a crucial component of the fungal cell wall that is (i) contributing to its structural integrity and is (ii) involved in cross-linking of different polymers, so that it influences the composition of both: the outer shell and inner membrane of fungal cells. In the filamentous fungi Aspergillus niger five ags genes are annotated, of which agsA and agsE were shown to be the most highly expressed genes during different stages of development. In addition, an agsE deletion mutant caused smaller micro-colonies as well as a shift in the secretome composition of A. niger, without affecting biomass production. Concurrently, intracellular cross-linking between chitin and beta-glucan is primarily mediated by the seven-membered cell wall-related transglycosylase gene family (crh). Although an impact on cell wall integrity has been expected when deleting the entire crh gene cluster in A. niger, significant alterations in cell wall integrity became only evident when the crh gene cluster deletion was combined with the reduction of alpha-glucan and galactomannan by deleting respective agsE or ugmA. With this background, we aimed to further explore the impact of ags gene deletions – both individually and as an entire family – on fungal cell wall integrity of A. niger. Using targeted CRISPR/Cas9 technology, we engineered various ags-deficient strains, including the deletion of the entire gene family (ΔagsA-E) in a mutant strain lacking all chitin-glucan cross-linking enzymes (Δcrh, TLF39). Subsequent morphological and biochemical characterization of these mutants pinpoint the importance of agsE for maintaining cell wall stability and suggesting its potential influence on protein production and/or secretion. These findings therefor provide not only new insights into fungal biology but also potential targets for biotechnological applications.