LAUSR

Simple Summary Glucoamylase is one of the most needed industrial enzymes in the food and biofuel industries. Aspergillus niger is a commonly used cell factory for the production of commercial glucoamylase. For decades, genetic manipulation has promoted significant progress in industrial fungi for strain engineering and in obtaining deep insights into their genetic features. However, genetic engineering is more laborious in the glucoamylase-producing industrial strains A. niger N1 and O1 because their fungal features of having few conidia (N1) or of being aconidial (O1) make them difficult to perform transformation on. In this study, we targeted A. niger N1 and O1 and successfully developed high-efficiency transformation tools. We also constructed a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 editing marker-free system using an autonomously replicating plasmid to express Cas9 protein and to guide RNA and the selectable marker. By using the genetic tools developed here, we generated nine albino deletion mutants. After three rounds of sub-culturing under nonselective conditions, the albino deletions lost the autonomously replicating plasmid. Together, the tools and optimization process above provided a good reference to manipulate the tough working industrial strain, not only for the further engineering these two glucoamylase-hyperproducing strains, but also for other industrial strains. Abstract The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type A. niger, the transformation of industrially used strains with few conidia (e.g., A. niger N1) or that are even aconidial (e.g., A. niger O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation of the A. niger strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The A. tumefaciens-mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 albA deletion mutants were constructed in the A. niger N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of A. niger.