Aspergillus nidulans has been more extensively characterized than other Aspergillus species considering its morphology, physiology, metabolic pathways, and genetic regulation. As it has a rapid growth rate accompanied by simple nutritional requirements and a high tolerance to extreme cultural conditions, A. nidulans is a promising microbial cell factory to biosynthesize various products in industry. However, it remains unclear for whether it is also a suitable host for synthesizing abundant L-malic acid. In this study, we developed a convenient and efficient double-gene-editing system in A. nidulans strain TN02A7 based on the CRISPR-Cas9 and Cre-loxP systems. Using this gene-editing system, we made a L-malic acid-producing strain, ZQ07, derived from TN02A7, by deleting or overexpressing five genes (encoding Pyc, pyruvate carboxylase; OahA, oxaloacetate acetylhydrolase; MdhC, malate dehydrogenase; DctA, C4-dicarboxylic acid transporter; and CexA, citric acid transporter). The L-malic acid yield in ZQ07 increased to approximately 9.6 times higher (up to 30.7 g/L titer) than that of the original unedited strain TN02A7, in which the production of L-malic acid was originally very low. The findings in this study not only demonstrate that A. nidulans could be used as a potential host for biosynthesizing organic acids, but also provide a highly efficient gene-editing strategy in filamentous fungi.
Keywords: Aspergillus nidulans; CRISPR−Cas9; Cre-loxP; L-malic acid; metabolic engineering; rTCA.