Topoisomerase II-Like variants enhance acid tolerance and low-pH l-malic acid biosynthesis in metabolic engineered Aspergillus nidulans

l-malic acid is a versatile compound having extensive applications across food, pharmaceutical, medical, and chemical industries while its production from microbial fermentation offers sustainable advantages including renewable feedstocks and environmental friendliness. However, a major challenge faced by microbial l-malic acid production is acid-induced pH drop during the fermentation process, requiring excessive neutralizers (e.g., CaCO₃) that increase costs and environmental burdens greatly. In this study, we firstly made an engineered <i>Aspergillus nidulans</i> strain as a production chassis through metabolic engineering by overexpression of fructose-6-phosphate kinase gene (<i>pfkA</i>) combined with deletion of pyruvate decarboxylase gene (<i>pdcA</i>) so that the titer of l-malic acid was increased by 20.3 %. Secondly, overexpression of glucose transporter gene (<i>mstE)</i> further boosted production to the highest reported shake-flask titer (44.3 g/L) for <i>A. nidulans</i>. Moreover, acid-tolerant isolates capable of growth at pH 2.8 has been successfully obtained by adaptive laboratory evolution in aforementioned engineered strains. They achieved 75 % neutralizer reduction (just adding 10 g/L CaCO₃) while maintaining original titers. Whole-genome sequencing comparison identified mutations in gene (<i>ANIA_04699</i>) encoding a protein with a topoisomerase II associated PAT I domain happened in acid-tolerance isolates. Functional validation via engineered point-mutants confirmed mutations of the gene (<i>ANIA_04699)</i> as a key acid-tolerance determinant, contributes a major role enabling CaCO3 reduction without titer loss. Meanwhile, phylogenetic studies showed there are about 71-75 % homology across selected <i>Aspergillus</i> spp. suggesting high conservation and broad applicability. Function prediction analysis based on previous verified PAT I domains imply that mutations of this topoisomerase II-Like protein might be tolerant to DNA damage induced by low pH environment. This work establishes <i>A. nidulans</i> as a platform for low-pH organic acid production and identifies topoisomerase II variants as new targeted candidates for acid tolerance engineering.