Abstract
Efficient transporters are necessary for high concentration and purity of desired products during industrial production. In this study, we explored the mechanism of substrate transport and preference of the C4-dicarboxylic acid transporter AoMAE in the fungus Myceliophthora thermophila, and investigated the roles of 18 critical amino acid residues within this process. Among them, the residue Arg78, forming a hydrogen bond network with Arg23, Phe25, Thr74, Leu81, His82, and Glu94 to stabilize the protein conformation, is irreplaceable for the export function of AoMAE. Furthermore, varying the residue at position 100 resulted in changes to the size and shape of the substrate binding pocket, leading to alterations in transport efficiencies of both malic acid and succinic acid. We found that the mutation T100S increased malate production by 68%. Using these insights, we successfully generated an AoMAE variant with mutation T100S and deubiquitination, exhibiting an 81% increase in the selective export activity of malic acid. Simply introducing this version of AoMAE into M. thermophila wild-type strain increased production of malic acid from 1.22 to 54.88 g/L. These findings increase our understanding of the structure–function relationships of organic acid transporters and may accelerate the process of engineering dicarboxylic acid transporters with high efficiency.
Key points
• This is the first systematical analysis of key residues of a malate transporter in fungi.
• Protein engineering of AoMAE led to 81% increase of malate export activity.
• Arg78 was essential for the normal function of AoMAE in M. thermophila.
• Substitution of Thr100 affected export efficiency and substrate selectivity of AoMAE.
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Funding
This work was supported by funding from the Key Project of the Ministry of Science and Technology of China (2018YFA0901400 and 2018YFA0900500), the National Natural Science Foundation of China (32071424, 31972878), the Chinese Academy of Sciences (XDA21060900), the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-CXRC-022 and TSBICIP-KJGG-015) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020183).
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LJ and WT conceived and designed research. WT conducted experiments and analyzed data. YW contributed software and analyzed data. WT wrote the manuscript. TC and LJ reviewed and edited the manuscript. All authors read and approved the manuscript.
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Wu, T., Wang, Y., Li, J. et al. Dissecting key residues of a C4-dicarboxylic acid transporter to accelerate malate export in Myceliophthora. Appl Microbiol Biotechnol 107, 609–622 (2023). https://doi.org/10.1007/s00253-022-12336-9
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DOI: https://doi.org/10.1007/s00253-022-12336-9