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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 −1 ), A1 (117 g L −1 ), and L2 (76 g L −1 )-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 γ-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...

The dynamic control of gene expression is important for adjusting fluxes in order to obtain desired products and achieve appropriate cell growth, particularly when the synthesis of a desired product drains metabolites required for cell growth. For dynamic gene expression, a promoter responsive to a particular environmental stressor is vital. Here, we report a low-pH-inducible promoter, Pgas, which promotes minimal gene expression at pH values above 5.0 but functions efficiently at low pHs, such as pH 2.0. First, we performed a transcriptional analysis of Aspergillus niger, an excellent platform for the production of organic acids, and we found that the promoter Pgas may act efficiently at low pH. Then, a gene for synthetic green fluorescent protein (sGFP) was successfully expressed by Pgas at pH 2.0, verifying the results of the transcriptional analysis. Next, Pgas was used to express the cis-aconitate decarboxylase (cad) gene of Aspergillus terreus in A. niger, allowing the production of itaconic acid at a titer of 4.92 g/liter. Finally, we found that Pgas strength was independent of acid type and acid ion concentration, showing dependence on pH only. IMPORTANCE The promoter Pgas can be used for the dynamic control of gene expression in A. niger for metabolic engineering to produce organic acids. This promoter may also be a candidate tool for genetic engineering.KEYWORDS Aspergillus niger, low-pH-inducible promoter, itaconic acid, dynamic gene expression, metabolic engineering A spergillus niger is an excellent cell factory for the production of organic acids (1). The rational engineering of A. niger has attracted increasing attention, and great achievements have been made in this area (2, 3). Nevertheless, because heterologous pathways are controlled by constitutive promoters, e.g., the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA), most of this research has focused on static metabolic engineering; that is, the gene expression level is set without sensing changes in the pathway output or cellular environment (4). One disadvantage of static control is related to the trade-off between growth and the production of a desired compound; suboptimal productivity is obtained because these pathways can drain metabolites required for biomass synthesis (5). Metabolic engineering approaches are being developed to tackle this problem via a dynamic control system to compensate for changing conditions (6). In this system, the cell modulates its metabolic pathways dynamically to adjust fluxes such that the required metabolic intermediates are delivered at the appropriate levels and times to optimize growth (7).

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Mesoporous titania nanoribbons are synthesized via an optimized soft hydrothermal process and the derived titania ion-sieves with lithium selective adsorption property are accordingly prepared via a simple solid-phase reaction between Li2CO3 and TiO2 nanomaterials followed by the acid treatment process to extract lithium from the Li2TiO3 ternary oxide precursors. The physical chemistry structure are characterized by XRD, TEM/HRTEM, SAED and N2 adsorption-desorption analysis; and the lithium selective adsorption properties are tested by the adsorption isotherm measurement and demonstrated with the distribution coefficient of a series of alkaline and alkaline-earth metal ions. Though the high temperature calcinations and lithium insertion-extraction process resulted in the agglomeration of nanostructure to large bulky particles, it implied that that the low-dimensional titania nanoribbons might be functionalized to lithium ion-sieves with remarkable adsorption capacity and selectivity, promising in the utilization of lithium extraction from aqueous resources including brine or seawater.