Abstract:Objective Citric acid is the main metabolite of Aspergillus niger at pH≤5.0, while l-malic acid becomes the main metabolite at pH 6.0. In this study, we employed transcriptomics to analyze the differences in the expression of key genes in metabolic pathways, aiming to explore the biosynthesis mechanisms of the two organic acids.Methods The cells at 48 h and 72 h of the fermentation processes for citric acid and l-malic acid production were selected for transcriptomics analysis.Results The transcriptome data of 72 h and 48 h were compared. GO enrichment analysis showed that the upregulated genes related to the synthesis of citric acid were concentrated in carbohydrate metabolism, while those related to the synthesis of l-malic acid were concentrated in ion transport process. The acid protease genes ANI_1_62014 (aspergillin II) and ANI_1_654124 (aspartic protease pepA) showed extremely high transcription levels during citric acid synthesis, while the key genes ANI_1_2494074 [3-oxoacyl-(acyl carrier protein) synthase] and ANI_1_2488074 (biosynthetic fatty acid synthase subunit β) essential for fatty acid chain synthesis showed extremely high transcription levels in the l-malic acid synthesis pathway. The transcription level of zinc cluster transcription factor [Zn(II)2Cys6 transcription factor] was higher in the synthesis process of l-malic acid. HacA, AP-1, and AtfA in the bZIP family showed higher transcriptional levels in response to environmental low pH stress during citric acid synthesis. Compared with l-malic acid synthesis, citric acid synthesis was accompanied by upregulated transcription levels of ANI_1_66114 (hexokinase), ANI_1_2950014 (citrate synthase), and ANI_1_478154 (citrate transporter) and a downregulated transcription level of ANI_1_3136024 (isocitrate dehydrogenase). Efficient glycolysis, citric acid synthesis, and citric acid transport capacity and low isocitrate dehydrogenase level were the key factors for citric acid production. In the process of l-malic acid synthesis, cytoplasmic ANI_1_440184 (pyruvate carboxylase), cytoplasmic ANI_1_12134 (malate dehydrogenase), ANI_1_914104 (isocitrate lyase), and ANI_1_2040144 (malate transporter) showed upregulated transcriptional levels. The cytoplasmic rTCA pathway and glyoxylic acid carboxylation pathway were thereby determined to be the main pathways for l-malic acid synthesis.Conclusion This study inferred the key differential metabolic pathways for the production of citric acid and l-malic acid by analyzing integrated transcriptomic data, and screened significant differentially expressed core genes, transcription factors, and potential transporters. These results provide important clues and a theoretical basis for elucidating the regulatory mechanisms of citric acid and l-malic acid synthesis.
