Despite a long and successful history of citrate production in Aspergillus niger, the molecular mechanism of citrate accumulation is only partially understood. In this study, we used comparative genomics and transcriptome analysis of citrate-producing strains-namely, A. niger H915-1 (citrate titer: 157 g L â1 ), A1 (117 g L â1 ), and L2 (76 g L â1 )-to gain a genome-wide view of the mechanism of citrate accumulation. Compared with A. niger A1 and L2, A. niger H915-1 contained 92 mutated genes, including a succinatesemialdehyde dehydrogenase in the Îł-aminobutyric acid shunt pathway and an aconitase family protein involved in citrate synthesis. Furthermore, transcriptome analysis of A. niger H915-1 revealed that the transcription levels of 479 genes changed between the cell growth stage (6 h) and the citrate synthesis stage (12 h, 24 h, 36 h, and 48 h). In the glycolysis pathway, triosephosphate isomerase was up-regulated, whereas pyruvate kinase was down-regulated. Two cytosol ATP-citrate lyases, which take part in the cycle of citrate synthesis, were up-regulated, and may coordinate with the alternative oxidases in the alternative respiratory pathway for energy balance. Finally, deletion of the oxaloacetate acetylhydrolase gene in H915-1 eliminated oxalate formation but neither influence on pH decrease nor difference in citrate production were observed.Aspergillus niger, a black aspergillus with "generally regarded as safe" status, is widely used in the biotechnological production of organic acids and industrial enzymes 1 . As a workhorse of organic acids, A. niger is a proficient producer of citrate, which is used extensively in the food and pharmaceutical industries owing to its safety, pleasant acidic taste, high water solubility, and chelating properties 2 .If A. niger is to be used as a cell factory platform, its genetic background must be thoroughly understood. The available genome sequence of A. niger offers a new horizon for both scientific studies and biotechnological applications 3 . A. niger strains NRRL 3 and ATCC 1015, which can synthesize citrate, have undergone genome-wide analysis 1,3 , and a complete genome-scale metabolic model with genomic annotation has been constructed based on the genome sequence of A. niger ATCC 1015 4 . Nevertheless, the molecular mechanism of citrate accumulation remains only partially understood 5,6 . For example, 5 citrate synthases have been identified in ATCC 9029 7 ; however, the primary gene and the timing of the activity of each enzyme during fermentation remain a mystery. Furthermore, the citrate transporter remains unknown even though the transport process is critical in the production of citrate. In addition, the role of alternative oxidases for energy balance during citrate production must be clarified 8 .In this study, the genomes of three A. niger strains with different citrate production efficiencies were sequenced, among which the genome of industrial strain A. niger H915-1 was sequenced with third-generation sequencing, which provides much longer reads an...