Improving Organic Acid Secretion of Aspergillus niger by Overexpression C4-Dicarboxylic Acid Transporters

C4-dicarboxylic acids are essential organic compounds characterized by a four-carbon structure and two carboxyl groups. Their export from cells is mediated by specialized transporter proteins known as C4-dicarboxylic acid transporters (DCTs). The objective of this study was to investigate the specificity of six DCTs (DCT1-5 and C4t318) from Aspergillus niger or Aspergillus oryzae, focusing on their role in different production strategies for C4-dicarboxylic acids. The results indicate that compared to the WT strain, overexpressing dct2 or dct3 in A. niger CGMCC NO. 40550 specifically enhances the production of succinic acid, increasing its yield from 5.69 g/L to 6.28 g/L, and L-malic acid, increasing its yield from 11.02 g/L to 12.11 g/L. Additionally, dct5 appears to be involved in the transport of both succinic acid (6.19 g/L) and L-malic acid (16.33 g/L). The total acid yields of T-D3-7, which lacks the oxaloacetate hydrolase gene, were improved to 27.75 g/L, compared to 25.19 g/L for T-D3-26, due to blocking the branch of oxaloacetate metabolism. Furthermore, the heterologous expression of A. oryzae C4T318 in A. niger increased the production of L-malic acid by approximately 22.5%. Furthermore, the best results were observed when the strains T-D3-7 and T-D5-16 were scaled up in a 30 L bioreactor for 84 h. The succinic acid and L-malic acid yields of T-D3-7 and T-D5-16 reached 14.51 g/L and 70.79 g/L or 41.59 g/L and 81.08 g/L, respectively. Moreover, the purity of L-malic acid produced by T-D3-7 reached 71%. This work further clarifies the specificity of C4-dicarboxylic acid transporters and provides valuable insights for optimizing organic acid production.