Here, proteins involved in sulfur-containing amino acid uptake in Escherichia coli strains were investigated with the aim of applying the findings in fermentative amino acid production. A search of genes in an L-methionine auxotrophic strain library suggested YecSC as the putative transporter of L-cystathionine. L-Methionine production increased by 15% after amplification of yecSC in producer strains. A candidate protein responsible for L-cysteine uptake was also found by experimentation with multicopy suppressor E. coli strains that recovered from growth defects caused by L-cysteine auxotrophy. Based on the results of an uptake assay, growth using L-cysteine as a sole sulfur source, and sensitivity to L-cysteine toxicity, we proposed that YeaN is an L-cysteine transporter. L-Cysteine production increased by 50% as a result of disrupting yeaN in producer strain. The study of amino acid transporters is valuable to industrialized amino acid production and also sheds light on the role of these transporters in sulfur assimilation. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.