Topoisomerase II-Like variants enhance acid tolerance and low-pH l-malic acid biosynthesis in metabolic engineered Aspergillus nidulans
Year: 2025
Type: article
Abstract: 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., CaCO 3 ) that increase costs and environmental burdens greatly. In this study, we firstly made a engineered Aspergillus nidulans strain as a production chassis through metabolic engineering by overexpression of fructose-6-phosphate kinase gene ( pfkA ) combined with deletion of pyruvate decarboxylase gene ( pdcA ) so that the titer of L-malic acid was increased by 20.3 %. Secondly, overexpression of glucose transporter gene ( mstE) further boosted production to the highest reported shake-flask titer (44.3 g/L) for A. nidulans . 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 CaCO 3 ) while maintaining original titers. Whole-genome sequencing comparison identified mutations in gene ( ANIA_04699) 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 ( ANIA_04699) as a key acid-tolerance determinant, contributes a major role enabling CaCO 3 reduction without titer loss. Meanwhile, phylogenetic studies showed there are about 71 - 75 % homology across selected Aspergillus 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 A. nidulans as a platform for low-pH organic acid production and identifies topoisomerase II variants as new targeted candidates for acid tolerance engineering.
Authors: Lingling Pei, Yan Dong, Ziqing Chen, Chi Zhang, Ling Lu
Institution: Nanjing Normal University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,500
Awards: G2156817582, G3161785076, G8868351120
OpenAlex ID: W4413428217