Glucoamylase, a crucial enzyme in various industries, is predominantly produced by Aspergillus niger. Despite significant advancements in genetic manipulation of fungi, transforming industrial strains like A. niger N1 and O1, known for their low conidia production, has been challenging. This study focuses on developing efficient genetic tools for these strains, including CRISPR/Cas9 systems, to accelerate glucoamylase hyperproduction and facilitate targeted mutations in industrial fungi.
The filamentous fungus Aspergillus niger is widely used for enzyme production, particularly glucoamylase. Genetic techniques, such as Agrobacterium tumefaciens-mediated and protoplast-mediated transformations, have been successful in wild-type strains but remain laborious for industrial strains like A. niger N1 and O1 due to their unique characteristics. By optimizing protoplast release and transformation methods, this study achieved high efficiencies of 89.3% for N1 and 82.1% for O1 using PMT, and 98.2% for N1 and 43.8% for O1 using AMT. These advancements will not only aid in understanding glucoamylase hyperproduction but also enhance genetic manipulation in other industrial strains of A. niger.
The study identified suitable antibiotics for selection during transformation, with A. niger N1 and O1 showing sensitivity to hygromycin B and G418. Protoplast production was optimized in CPZ-old medium, yielding 9.2 × 10^6 protoplasts/mL, a significant improvement over other tested media. Additionally, lysing enzymes from various sources were utilized, and different enzymolysis times were tested to enhance protoplast yield. The results demonstrated the significance of medium composition and culture conditions in efficient protoplast isolation for genetic transformation.
Moreover, the development of a marker-free CRISPR/Cas9 system using an autonomously replicating plasmid enabled successful genome editing in A. niger N1. The system allowed for the creation of albino deletion mutants, showcasing the potential for targeted mutations in industrial fungi without the use of integrative selection markers. This breakthrough paves the way for precise genetic modifications in A. niger strains and other filamentous fungi, revolutionizing strain engineering and genetic manipulation in the industry.
In conclusion, the study exemplifies a paradigm shift in genetic tools for enhanced glucoamylase production in industrial Aspergillus niger strains. By overcoming the challenges posed by low conidia production and aconidial characteristics, the developed transformation methods and CRISPR/Cas9 system offer a robust platform for genetic engineering in industrial fungi. These advancements not only contribute to understanding glucoamylase hyperproduction mechanisms but also open new avenues for targeted mutation strategies in various industrial strains of A. niger and beyond.
Key Takeaways:
– Efficient genetic tools, including CRISPR/Cas9 systems, have been developed for industrial Aspergillus niger strains with low conidia production.
– Optimization of protoplast isolation methods in CPZ-old medium significantly improved protoplast yield for genetic transformation.
– Marker-free CRISPR/Cas9 system enabled successful genome editing and creation of albino deletion mutants in A. niger N1.
– These advancements revolutionize genetic manipulation in industrial fungi, facilitating targeted mutations and enhancing glucoamylase production capabilities.
Tags: secretion, fungi, yeast, genome editing
Read more on pmc.ncbi.nlm.nih.gov