Mechanism Analysis Of The Citrate Overproducer Aspergillus Niger And Metabolic Regulation

Citrate?2-hydroxy-propane-1, 2, 3-tricarboxylic acid? is used extensively in the food, pharmaceutical industries, detergents, cosmetics, and a variety of other industrial applications. Currently, 80% of the world's citrate output is obtained through submerged fermentation using Aspergillus niger. As the production titer and yield have already achieved high amount, further improving citrate production hits a bottleneck. To overcome the problem, it is important to investigate the mechanism of citrate production by A. niger. In addition, the industrial citrate producing strain is obtained through several rounds of mutations, as a result the mycelium exhibits a short and bulbous appearance and has strongly thickened cell walls, making transformation difficult. A suitable transformation method and metabolic regulation element are needed to be studied.In this study, the transformation method was established for the industrial citrate producing A. niger strain H915-1 based on PEG-mediated transformation of protoplast. A. niger H915-1 and the mutation strain derived from H915-1 through ARTP were analyzed at genome and transcriptome level. Furthermore, a promoter, which was induced at low pH and suitable for dynamic control of A. niger for organic acid production, was characterized. Finally, the citrate titer was improved through regulation of a sugar transporter. Major results achieved in this study are highlighted as follows:?1? Protoplasting of the industrial citrate producing A. niger strain H915-1 was optimized and the transformation method was established. The optimal cell wall digest enzyme mixture was: 5 mg×m L^-1 lysing enzyme, 0.2 U×m L^-1 chitinase and 460 U×m L^-1 glucuronidase. The optimal digest condition was: using 0.7 M KCl as osmotic stabilizer to digest 15 mg mycelium with diameter of 50 ?m at 37°C. Integrated expression of heterologous genes was succeeded through co-transformation of two expression cassettes with co-integration rate of 58%. The oah gene was knocked-out in H915-1 with Ku-70 existing, which took charge in non-homologous end joining?NHEJ?, and the rate of homologous integration was 65%.?2? Two mutants, namely A1 and L2, were obtained through ARTP and high-throughput screening from A. niger H915-1. The citrate titer of H915-1, A1 and L2 were 157 g×L^-1, 117 g×L^-1 and 76 g×L^-1, respectively. The genomes of the three strains were sequenced, assembled and annotated. 35.98 Mb, 34.64 Mb and 36.45 Mb genomes were finally obtained. There existed a total of 59 differences in gene families, 1210 sites of gene deletion, and single-nucleotide polymorphisms?SNPs? and 52 sites of structural variation?SV?. Among these changes, aconitase from central metabolism pathway and succinate-semialdehyde dehydrogenase from ?-aminobutyric acid pathway were mutated.?3? The transcriptome data of H915-1 at 4 time-points during citrate-producing-phase were compared with the data of cell-growth-phase. A total of 479 genes were found with different expression level. The major genes of central metabolism pathway were identified. The triosephosphate isomerase was up-regulated, the pyruvate kinase and most enzymes of TCA cycle were down-regulation. In addition, the ?-aminobutyric acid?GABA? pahway was up-regulatd. Furthermore, 2 ATP-citrate lyases were up-regulated, which constituted a futile cycle with r TCA and TCA cycle. Finally, the expression of 35 transporters was up-regulated, which contained 3 organic anion transporters and a monocarboxylate transporter.?4? A low pH induced gene, gas, was screened through transcriptome analysis and the promoter of gas was predicted. The reporter gene of synthetic green fluorescent protein?s GFP? was used for varifing of Pgas expression pattern. The expression level of s GFP under the control of Pgas was extremely low at pH 5.0 but high at pH 2.0, which was the same amount with that under the control of Pgpd A. The expression of s CAD gene promoted by Pgas provided the H915-1 transformants the ability of itaconate production, and the titer achieved 4.92 g×L^-1, which increased to 5 times compared to that of Pgpd A-CAD transformants, the s CAD expression level at 24 h and 108 h increased for 2.37 and 3.23 times than that at 8 h. The inducible pattern of Pgas was analyzed further on m RNA level through q PCR. Pgas was only influenced by pH, but not influenced by acid type and concentration. Finally, two transcription factors, XP₀01388781.2 and XP₀01396281, were identified as Pgas regulators through DNA pull-down.?5? The sugar transporters were analyzed based on transcriptome data, phylogenetic tree analysis and sequences alignment. The evm.model.unitig₀.1770 gene, whose relationship was the closest with Kl HGT1, was predicted containing 11 trans-membrane domains with N-terminus in membrane and C-terminus in cytosol, and named as HGT1. The growth test on glucose limited medium showed the diameter of HGT1 transformants were 50% to 150% larger than the diameter of H915-1. After addition of 30 g×L^-1glucose into fermentation medium, the glucose consuming time of HGT1 transformants reduced for 12 h than that of H915-1. Finally, the citrate titer of HGT1 tranformant was increased for 14.7% compared to H915-1, the fermentation time reduced for 6 h and the maximum specific citrate production rate increased for 29.5%.