Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger 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.

Strategies for the Development of Industrial Fungal Producing Strains

“…PMT has become one of the most commonly used methods for the transformation of filamentous fungi due to its simplicity and high efficiency [ 100 , 101 ]. It has been successfully applied in many filamentous fungal genera such as Penicillium [ 102 , 103 , 104 , 105 , 106 , 107 ], Fusarium [ 108 ] and Aspergillus [ 101 , 109 , 110 , 111 ], including the industrial workhorses A. niger [ 112 , 113 , 114 ] and A. oryzae [ 115 , 116 ]; and Trichoderma , including T. reesei [ 117 , 118 ], among others.…”

Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review

“…The AMA plasmid can easily be recycled after several rounds of subculturing under nonselective conditions, allowing the reuse of the dominant selection marker(s) during transformation [ 94 ]. CRISPR/Cas9-based approaches involving an autonomously replicating AMA1-plasmid have been successfully established for the editing of single or multiple genes in industrial strains of Aspergillus niger [ 95 ], the edible fungus Cordyceps militaris [ 96 ], A. terreus [ 97 ], and A. oryzae [ 98 ]. For instance, an AMA1-based CRISPR/Cas9 genome-editing system was used in Paecilomyces variotii and Penicillium roquefortii for creating melanin-deficient strains by knocking out the associated PKS genes and investigating the effect of melanin on the heat and UV-C radiation resistance of conidia from these food-associated fungi [ 99 ].…”

Establishment, optimization, and application of genetic technology in Aspergillus spp.

“…At present, a considerable part of common Aspergillus gene function studies have applied the CRISPR technology, also have applied in fungal factories, such as glucoamylase-hyperproducing industrial in A . niger ( Liu D. et al, 2022 ) and kojic acid production in A . oryzae ( Chen Z. et al, 2022 ).…”