Engineering dispersed mycelium morphology in Aspergillus niger for enhanced mycoprotein production via CRISPR/Cas9-mediated genome editing

Area of Science:

• Biotechnology
• Industrial Microbiology
• Synthetic Biology

Background:

• Filamentous fungi are crucial for industrial fermentation, but their morphology impacts yield.
• Aspergillus niger forms pellets, limiting mass transfer and industrial use.
• Optimizing morphology and fermentation is key for enhanced microbial protein production.

Purpose of the Study:

• To engineer Aspergillus niger for improved microbial protein production.
• To overcome limitations of pellet formation in submerged cultures.
• To enhance biomass and protein content through genetic and process optimization.

Main Methods:

• CRISPR/Cas9 genome editing to disrupt α-1,3-glucan synthase (agsA, agsB) and galactosaminogalactan synthase (sph3, uge3) genes.
• Morphological engineering to achieve complete dispersion of fungal pellets.
• Fermentation optimization using Response Surface Methodology (RSM) with Box-Behnken design.

Main Results:

• Engineered A. niger (AnΔABSU) showed dispersed morphology, a 77.52% increase in biomass, and 39.98% higher protein content.
• Transcriptomics revealed upregulated nutrient transporters, altered cell wall integrity, and enhanced amino acid biosynthesis.
• Optimized fermentation yielded 16.67 g/L biomass and 45.91% protein content, significant improvements over the wild-type.

Conclusions:

• Genetic modification and fermentation optimization successfully created a high-efficiency microbial protein cell factory.
• Morphological engineering is a viable strategy to enhance fungal fermentation processes.
• This integrated approach offers a novel paradigm for industrial biotechnology applications.