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Optimization of export mechanisms for valuable extracellular products is important for the development of efficient microbial production processes. Identification of the relevant export mechanism is the prerequisite step for product export optimization. In this work, we identified transporters involved in malate export in an engineered l-malate-producing Escherichia coli strain using cheminformatics-guided genetics tests. Among all short-chain di- or tricarboxylates with known transporters in E. coli, citrate, tartrate, and succinate are most chemically similar to malate as estimated by their molecular signatures. Inactivation of three previously reported transporters for succinate, tartrate, and citrate, DcuA, TtdT, and CitT, respectively, dramatically decreased malate production and fermentative growth, suggesting that these transporters have substrate promiscuity for different short-chain organic acids and constitute the major malate export system in E. coli. Malate export deficiency led to an increase in cell sizes and accumulation of intracellular metabolites related to malate metabolism. [ABSTRACT FROM AUTHOR]