Monday, April 19, 2010
3-30

Improving protoplast production and purity for modification of Aspergillus niger for enzyme production

Gaihong Wu, Biorefinery Processes and Systems Group, Institute of Microbiology, Beijing, China and Shulin Chen, Department of Biological Systems Engineering, Washington State University, LJSmith 258, Pullman, WA 99164-6120.

Protoplast fusion, genome shuffling and protoplast transformation as methods to produce mutant strains can be used to modify fungus for high level of cellulytic enzyme production. Protoplast formation from filamentous fungi is often affected by many factors such as enzyme components, osmotic stable, temperature, mycelium culture etc. The existence of hypha segment in the protoplast suspension often led to the poor quality of protoplast thus influenced the results of protoplast fusion, genome shuffling and protoplast transformation. Therefore, it is important to avoid the formation of hypha segment thus improve the purity of protoplast. Considerable amount of literatures have placed emphasis on improving quantity of protoplast by determining the conditions of protoplast production since the earliest studies on the formation and development of fungal protoplasts reported more than 40 years ago. Very limited research, however, was reported on how to improve the purity of protoplast. In this study, we first optimized enzyme components and concentration, osmotic stable solution, and the amount of L-cysteine through orthogonal experiment design to increase protoplast yield. Base on the above results, we studied the effect of different modes and time of mycelium culture on the quality of protoplast from Aspergillus niger. Under the optimized conditions, protoplast production reached 107 protoplasts/ml, and the lytic time was shortened from 4h to 2h. In the meantime, the purity of protoplasts formed was also improved after later protoplast purification. We concluded that protoplast quality was affected strictly by the time of mycelium culture. This report would provide critical insight in protoplast release of other filamentous fungi, thus help improving the breeding efficiency.

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