Dissecting key residues of a C4-dicarboxylic acid transporter to accelerate malate export in Myceliophthora.
Appl Microbiol Biotechnol
; 107(2-3): 609-622, 2023 Feb.
Article
em En
| MEDLINE
| ID: mdl-36542100
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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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transportadores de Ácidos Dicarboxílicos
/
Malatos
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Ano de publicação:
2023
Tipo de documento:
Article