Dissecting key residues of a C4-dicarboxylic acid transporter to accelerate malate export in Myceliophthora

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.

Automated summary

In this study, the mechanism and preference of the C4-dicarboxylic acid transporter AoMAE in Myceliophthora thermophila were investigated. Key amino acid residues, including Arg78 and residue at position 100, were found to play crucial roles in substrate transport and protein stabilization.The mutation T100S significantly increased malate production by 68%. By introducing the variant with mutation T100S and deubiquitination, malic acid production in M. thermophila was dramatically increased from 1.22 to 54.88 g/L. These findings enhance our understanding of organic acid transporters and have implications for engineering efficient dicarboxylic acid transporters.