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High-yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum
Mattheos Koffas
Rensselaer Polytechnic Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1405-0565
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
L-serine, exporter, C. glutamicum, transcriptional regulator, metabolic engineering
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Received 11 May, 2020
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Received 10 May, 2020
Reviewer #2 agreed
On 05 May, 2020
Editor assigned
On 04 May, 2020
Reviewers invited
Invitations sent on 04 May, 2020
Reviewer #1 agreed
On 04 May, 2020
Submission checks complete
On 03 May, 2020
Editor invited
On 03 May, 2020
No comments provided
Review #1 received
Received 05 Apr, 2020
Editorial decision: Minor Revision
On 05 Apr, 2020
Review #2 received
Received 03 Apr, 2020
Review #3 received
Received 03 Apr, 2020
Reviewer #3 agreed
On 26 Mar, 2020
Reviewer #2 agreed
On 24 Mar, 2020
Reviewers invited
Invitations sent on 09 Mar, 2020
Reviewer #1 agreed
On 09 Mar, 2020
First submitted
On 09 Mar, 2020
Editor assigned
On 09 Mar, 2020
Submission checks complete
On 08 Mar, 2020
Editor invited
On 08 Mar, 2020
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Subject Areas
Applied & Industrial Microbiology
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This preprint is under consideration at Microbial Cell Factories. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
High-yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum
Mattheos Koffas
Rensselaer Polytechnic Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1405-0565
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 2
Posted 18 May, 2020
No community comments so far
Review #2 received
Received 11 May, 2020
Review #1 received
Received 10 May, 2020
Reviewer #2 agreed
On 05 May, 2020
Editor assigned
On 04 May, 2020
Reviewers invited
Invitations sent on 04 May, 2020
Reviewer #1 agreed
On 04 May, 2020
Submission checks complete
On 03 May, 2020
Editor invited
On 03 May, 2020
No comments provided
Review #1 received
Received 05 Apr, 2020
Editorial decision: Minor Revision
On 05 Apr, 2020
Review #2 received
Received 03 Apr, 2020
Review #3 received
Received 03 Apr, 2020
Reviewer #3 agreed
On 26 Mar, 2020
Reviewer #2 agreed
On 24 Mar, 2020
Reviewers invited
Invitations sent on 09 Mar, 2020
Reviewer #1 agreed
On 09 Mar, 2020
First submitted
On 09 Mar, 2020
Editor assigned
On 09 Mar, 2020
Submission checks complete
On 08 Mar, 2020
Editor invited
On 08 Mar, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Applied & Industrial Microbiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6