Novel insights into L-serine exporters in Corynebacterium glutamicum from gene mining and functional analysis

Amino acid exporters play an important role in regulating amino acid production by Corynebacterium glutamicum, and over 90% of amino acid export is attributed to exporters in this species. ThrE was reported to be an O_SCPLOWLC_SCPLOW-serine exporter, and SerE was identified as an O_SCPLOWLC_SCPLOW-serine exporter in our previous study. However, when both ThrE and SerE were deleted, the O_SCPLOWLC_SCPLOW-serine titer was decreased by 60%, suggesting other O_SCPLOWLC_SCPLOW-serine exporters may exist. In the present study, NCgl0254 and NCgl0255 were identified as novel O_SCPLOWLC_SCPLOW-serine exporters through comparative transcriptomics and gene functional analyses. The contributions of the four exporters (ThrE, SerE, NCgl0254 and NCgl0255) in O_SCPLOWLC_SCPLOW-serine export were studied by gene deletion, gene overexpression, amino acid export assay and real-time quantitative PCR (RT-qPCR). The results showed that SerE is the major O_SCPLOWLC_SCPLOW-serine exporter in C. glutamicum. Fermentation and amino acid export assays of SSAAI, SSAAI-serE-thrE-ncgl0254-ncgl0255 and SSAAI{Delta}serE{Delta}thrE{Delta}ncgl0254{Delta}ncgl0255 indicated that the four O_SCPLOWLC_SCPLOW-serine exporters undertake most of the O_SCPLOWLC_SCPLOW-serine export, and their overexpression enhanced export of O_SCPLOWLC_SCPLOW-serine in SSAAI. When one O_SCPLOWLC_SCPLOW-serine exporter was deleted, the transcription level of the other three exporters was upregulated. However, the decrease in O_SCPLOWLC_SCPLOW-serine titer caused by deletion of one exporter was not fully compensated by upregulation of the other three exporters at the transcription level, indicating that O_SCPLOWLC_SCPLOW-serine production by C. glutamicum may be determined by cooperative efficiency of all four O_SCPLOWLC_SCPLOW-serine exporters, with each being interdependent. IMPORTANCEThis work identified the novel O_SCPLOWLC_SCPLOW-serine exporters NCgl0254 and NCgl0255, and revealed their roles in O_SCPLOWLC_SCPLOW-serine export alongside the O_SCPLOWLC_SCPLOW-serine exporters. All four O_SCPLOWLC_SCPLOW-serine exporters are interdependent and undertake most of the O_SCPLOWLC_SCPLOW-serine export, but SerE is the major O_SCPLOWLC_SCPLOW-serine exporter. The findings expand our knowledge of amino acid exporters in C. glutamicum, and the approach can be employed for exploring of bacterial exporters of unknown function.