Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve l-serine yield in Corynebacterium glutamicum

et al. 2019

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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...

Section: Discussionmentioning

confidence: 72%

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

et al. 2019

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“…Similarly, NCgl0581-serE might also be a protein-gene pair sharing the same regulation mechanism. A serine-biosensor based NCgl0581 was reported by Binder et al [34], and based on this study, we constructed a biosensor for L-serine and found that NCgl0581 activated NCgl0580 (SerE) expression in the presence of L-serine, the expression of SerE enhancing with the L-serine titer increasing [35]. However, NCgl0581 did not activate the expression of SerE in the presence of L-alanine and L-valine.…”

Section: Discussionmentioning

confidence: 55%

High yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum

et al. 2020

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Background L-Serine has wide and expanding applications in industry with a fast-growing market demand. Strategies for achieving and improving L-serine production in C. glutamicum have focused on inhibiting its degradation and enhancing its biosynthetic pathway, however, L-serine yield remains relatively low. Exporters play an essential role in the fermentative production of amino acids. Improvements to L-serine yield should consider the improvement of L-serine export from the cell. In C. glutamicum , ThrE, which can export L-threonine and L-serine, is the only identified L-serine exporter so far.Results In this study, a novel L-serine exporter NCgl0580 was identified and characterized in C. glutamicum ΔSSAAI (SSAAI), and named as SerE (encoded by serE ). Deletion of serE in SSAAI led to a 56.5% decrease in L-serine titer, whereas overexpression of serE compensated for the lack of serE with respect to L-serine titer. A fusion protein with SerE and enhanced green fluorescent protein (EGFP) was constructed to confirm that SerE localized at the plasma membrane. The function of SerE was studied by peptide feeding approaches, and the results showed that SerE is a novel exporter for L-serine and L-threonine in C. glutamicum. Subsequently, the interaction of a known L-serine exporter ThrE and SerE was studied, and the results suggested that SerE is more important than ThrE in L-serine export in SSAAI. In addition, probe plasmid and electrophoretic mobility shift assays (EMSA) revealed NCgl0581 as the transcriptional regulator of SerE. Comparative transcriptomics between SSAAI and the NCgl0581 deletion strain showed that NCgl0581 is a positive regulator of NCgl0580. Finally, by overexpressing the novel exporter SerE combined with L-serine synthetic pathway key enzyme serA Δ197, serC and serB , the resulting strain lead to the L-serine titer of 43.9 g/L with yield of 0.44 g/g sucrose, which was the highest yield reported so far for any organism.Conclusions This study provides a novel target for L-serine and L-threonine export engineering as well as a novel global transcriptional regulator NCgl0581 in C. glutamicum .

“…Similarly, NCgl0581-serE might also be a protein-gene pair sharing the same regulation mechanism. A serine biosensor based NCgl0581 was reported by Binder et al [34], and accordingly, we constructed a biosensor for L-serine and found that NCgl0581 activated NCgl0580 (SerE) expression in the presence of Lserine, with expression of SerE enhancing with increasing L-serine titer [23]. However, NCgl0581 did not activate the expression of SerE in the presence of L-alanine and L-valine.…”

Section: Discussionmentioning

confidence: 75%

“…Subsequently, L-serine exporter serE, serAΔ197, serC and serB were overexpressed in strain A36 to obtainA36-serE-serAΔ197-serC-serB. A36 was stemmed from SSAAI by using ARTP mutation, which produced 34.78 g/L L-serine [23], As shown in Fig. 6, the tandem expression strain A36-serE-serAΔ197-serC-serB shared similar typical growth curves as the parent strain A36 in the overall process.…”

Section: High Yield Production Of L-serine Through Sere Combined Withmentioning

confidence: 88%

High-yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum

et al. 2020

Preprint

Self Cite

Background: L-Serine has wide and increasing applications in industries with fast-growing market demand. Although strategies for achieving and improving L-serine production in Corynebacterium glutamicum (C. glutamicum) have focused on inhibiting its degradation and enhancing its biosynthetic pathway, L-serine yield has remained relatively low. Exporters play an essential role in the fermentative production of amino acids. To achieve higher L-serine yield, L-serine export from the cell should be improved. In C. glutamicum, ThrE, which can export L-threonine and L-serine, is the only identified L-serine exporter so far.Results: In this study, a novel L-serine exporter NCgl0580 was identified and characterized in C. glutamicum ΔSSAAI (SSAAI), and named as SerE (encoded by serE). Deletion of serE in SSAAI led to a 56.5% decrease in L-serine titer, whereas overexpression of serE compensated for the lack of serE with respect to L-serine titer. A fusion protein with SerE and enhanced green fluorescent protein (EGFP) was constructed to confirm that SerE localized at the plasma membrane. The function of SerE was studied by peptide feeding approaches, and the results showed that SerE is a novel exporter for L-serine and L-threonine in C. glutamicum. Subsequently, the interaction of a known L-serine exporter ThrE and SerE was studied, and the results suggested that SerE is more important than ThrE in L-serine export in SSAAI. In addition, probe plasmid and electrophoretic mobility shift assays (EMSA) revealed NCgl0581 as the transcriptional regulator of SerE. Comparative transcriptomics between SSAAI and the NCgl0581 deletion strain showed that NCgl0581 is a positive regulator of NCgl0580. Finally, by overexpressing the novel exporter SerE, combined with L-serine synthetic pathway key enzyme serAΔ197, serC, and serB, the resulting strain presented an L-serine titer of 43.9 g/L with a yield of 0.44 g/g sucrose, which is the highest L-serine titer and yield reported so far in C. glutamicum. Conclusions: This study provides a novel target for L-serine and L-threonine export engineering as well as a new global transcriptional regulator NCgl0581 in C. glutamicum.