Preparation of Protoplast for Efficient DNA Transformation of Citric Acid Hyper-producing Aspergillus niger Industrial Strain

doi:10.13560/j.cnki.biotech.bull.1985.2015.04.025

Abstract

Abstract: Aspergillus niger is the major industrial strain for citric acid production. In spite of many successes of modern molecular biology approaches in engineering laboratory or protein-producing strains of A. niger, there is few positive report on its application for citric acid industrial strains mainly due to the hard-to-transform nature of these strains. In this study, the protoplast-PEG mediated genetic transformation system for citric acid industrial strain was extensively studied, suggesting an optimized protocol for protoplast preparation, regeneration and DNA transformation. The concentration of protoplasts reached up to 10⁶ /mL by lysing younger mycelia for 2.5 h after 48 h incubation of a proper amount of conidia spores in enrichment medium. The optimal lysing enzyme mixtures comprised of 1.5% lysing enzyme, 0.5% snail enzyme and 0.2% lysozyme. Concentration of protoplast influenced the protoplast-PEG mediated transformation efficiency, which reached the maximal when the concentration of protoplast was higher than 10⁶/mL. The genetic transformation system established in this study should pave the way to molecular biology study of the citric acid hyper-producing strains, for further understanding its acid-tolerant physiology and for rational design of the industrial strain for further improvement of the citric acid production process as well as creation of new organic acid-producing cell factories.

Key words: Aspergillus niger, citric acid, genetic manipulation system, protoplast, DNA transformation

Zhang Xiaoli, Zheng Xiaomei, Man Yun, Luo Hu, Yu Jiandong, Zheng Ping, Liu Hao, Sun Jibin. Preparation of Protoplast for Efficient DNA Transformation of Citric Acid Hyper-producing Aspergillus niger Industrial Strain[J]. Biotechnology Bulletin, 2015, 31(3): 171-177.

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