A Novel L-Serine Exporter and Its Positive Regulator in Corynebacterium glutamicum

Abstract

19Exporters play an essential role in the fermentative production of amino acids. In 20 Corynebacterium glutamicum, ThrE, which can export L-threonine and L-serine, is the 21 only identified L-serine exporter so far. In this study, a novel L-serine exporter NCgl0580 22 was identified and characterized in C. glutamicum ΔSSAAI (SSAAI), and named as SerE 23 (encoded by serE). Deletion of serE in SSAAI led to a 56.5% decrease in L-serine titer, 24 whereas overexpression of serE compensated for the lack of serE with respect to L-serine 25 titer. A fusion protein with SerE and enhanced green fluorescent protein (EGFP) was 26 constructed to confirm that SerE localized at the plasma membrane. The function of SerE 27 was studied by peptide feeding approaches, and the results showed that SerE is a novel 28 exporter for L-serine and L-threonine in C. glutamicum. Subsequently, the interaction of a 29 known L-serine exporter ThrE and SerE was studied, and the results suggested that SerE 30 is more important than ThrE in L-serine export in SSAAI. Probe plasmid and 31 electrophoretic mobility shift assays (EMSA) revealed NCgl0581 as the transcriptional 32 regulator of SerE. Comparative transcriptomics between SSAAI and the NCgl0581 33 deletion strain showed that NCgl0581 regulated the transcription of 115 genes in C. 34 glutamicum, among which the transcriptional level of NCgl0580 decreased 280-fold in a 35 3 NCgl0581 deletion strain, indicating that NCgl0581 is a positive regulator of SerE. Thus, 36 this study provides a novel target for L-serine and L-threonine export engineering as well 37 as a novel global transcriptional regulator NCgl0581 in C. glutamicum. 38 Importance 39 Exporters are gaining increasing attention for improving industrial production of amino 40 acids. This study identified a novel exporter NCgl0580 for L-serine and L-threonine in C. 41 glutamicum, and its positive regulator (NCgl0581), which was shown to be a novel global 42 transcriptional regulator in C. glutamicum. This study provides a new target for 43 engineering efflux of L-serine and L-threonine, expands the exporter and transcriptional 44 regulator family, and enriches our understanding of amino acid transport system in C. 45 glutamicum. 46 KEYWORDS L-serine, exporter, C. glutamicum, transcriptional regulator, metabolic 47 engineering 48 INTRODUCTION 49 L-serine has been identified as one of the top 30 most interesting building blocks for a 50 range of chemicals and materials, and is used in cosmetic, pharmaceutical, and food 51 industries (1, 2). Metabolic engineering of Corynebacterium glutamicum (C.glutamicum) 52for L-serine production has been focused on its terminal synthesis pathways and 53 4 degradation pathways, and proven to be very useful for improving L-serine production in 54 this organism (3-6); however, the L-serine productivity is still low for large-scale L-serine 55 production. 56Efflux is often overlooked as a bottleneck in metabolic pathways, and amino acid 57 efflux transporters are increasingly gainin...

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