Abstract
Modification of C4-dicarboxylate transport processes is an important strategy for the development of efficient malic acid producing cell factory in Aspergillus niger. However, there is a lack of identification and functional research of malic acid transport proteins, which seriously hinders the construction of high-yield malic acid metabolic engineering strains. A C4-dicarboxylate transport protein (DCT) DCT1 is identified as major malic acid transport protein and exhibits significant elevation in malic acid production when overexpressed. DCT1 is found by homology searches and domain analyses with SpMAE1 from Schizosaccharomyces pombe as the template. Phylogenetic and domain analyses show that DCTs belong to voltage-dependent slow-anion channel transporter (SLAC1) family and are members of Tellurite-resistance/Dicarboxylate Transporter (TDT) Family. DCT1 disruption dramatically decreases malic acid titer by about 85.6% and 96.2% at 3 days and 5 days compared with the parent strain, respectively. Meanwhile, the citric acid titers increase by 36.4% and 13.7% at 3 days and 5 days upon DCT1 deficiency. These results suggest that DCT1 is the major malic acid transporter in A. niger. Overexpression of dct1 with its native promoter significantly improves malic acid production yielding up to 13.86 g/L and 30.79 g/L at 3 days and 5 days, respectively, which is 36.8% and 22.8% higher than those in the parent strain. However, the citric acid has no significant change during the 5-day fermentation. These results demonstrate the importance of C4-dicarboxylate transporters for the efficient production of malic acid. Furthermore, enhancement of malic acid transport process is a feasible approach of efficient malic acid production in this citric acid producing A. niger strain.
Key points
• A dicarboxylate transporter DCT1 is identified as a major malic acid transporter.
• DCT1 deficiency results in significant decrease of malic acid.
• DCT1 overexpression leads to increased titers of malic acid.
• Enhancement of malic acid transport is vital for malic acid production in A. niger.
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Funding
Funding for this research was provided by the Tianjin Science and Technology Committee (18JCQNJC78700 and 18YFZCSY01360), the National Natural Science Foundation of China (21706192), the Foundation of Tianjin University of Science and Technology (2016LG12), and the program of Distinguished Professor of Tianjin 2015 and Industrial Microbial Strain Selection and Fermentation Technology Public Service Platform Project (17PTGCCX00190).
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WC designed all the experiments, analyzed the data, and wrote the manuscript. LY, ML, and XL generated all the genetic constructs and conducted experiments. YX and ZX contributed with scientific discussions and commented on the manuscript. HL supervised the work and revised the manuscript. All authors read and approved the final manuscript.
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Cao, W., Yan, L., Li, M. et al. Identification and engineering a C4-dicarboxylate transporter for improvement of malic acid production in Aspergillus niger. Appl Microbiol Biotechnol 104, 9773–9783 (2020). https://doi.org/10.1007/s00253-020-10932-1
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DOI: https://doi.org/10.1007/s00253-020-10932-1