Abstract
Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene function analysis is an urgent problem to be solved in A. oryzae and other filamentous fungal systems. Therefore, establishing efficient genetic transformation and multiplexed genome editing tools is significant for developing A. oryzae expression systems, and revealing its intrinsic mechanisms. In this study, the high-efficiency transformation of A. oryzae was achieved by optimizing the preparation conditions of protoplasts, and the random editing efficiency of the CRISPR/Cas9 system in A. oryzae for single and double genes reached 37.6% and 19.8%, respectively. With the aid of the selection marker, such as color or resistance, the editing efficiency of single and double genes can reach 100%. Based on the developed CRISPR/Cas9 genome editing method, the heterologous lipase gene (TLL) achieves precise integration at different genetic loci in one step. The efficient and accurate acquisition of positive transformants indicated that the morphological gene yA could be used as a helpful selection marker for genome editing in A. oryzae. In conclusion, the developed system improves the efficiency of transformation and multiplexed genome editing for A. oryzae. It provides a practical method for developing the A. oryzae high-efficiency expression system for heterologous proteins.
Funder
National Key Research and Development Program of China
the Natural Science Foundation of Jiangsu Province
Reference33 articles.
1. Improvement of the quality of soy sauce by reducing enzyme activity in Aspergillus oryzae;Ding;Food Chem.,2019
2. Safety of the fungal workhorses of industrial biotechnology: Update on the mycotoxin and secondary metabolite potential of Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei;Frisvad;Appl. Microbiol.,2018
3. Filamentous fungi for the production of enzymes, chemicals and materials;Curr. Opin. Biotechnol.,2019
4. Recent advances in the development of Aspergillus for protein production;Li;Bioresour. Technol.,2022
5. CRISPR/Cas9-based genome editing of the filamentous fungi: The state of the art;Shi;Appl. Microbiol.,2017
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