Industrializing a Bacterial Strain for <scp>l</scp>-Serine Production through Translation Initiation Optimization

Rennig, Maja; Mundhada, Hemanshu; Wordofa, Gossa Garedew; Gerngross, Daniel; Wulff, Tune; Worberg, Andreas; Nielsen, Alex Toftgaard; Nørholm, Morten H. H.

doi:10.1021/acssynbio.9b00169

Cited by 23 publications

(19 citation statements)

References 45 publications

(111 reference statements)

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“…However, Lserine titer did not significantly change when serAΔ197 and serC were respectively overexpressed in A36 (with L-serine titer of 31.1 and 32.78 g/L, respectively). In a recent study, 50 g/L L-serine was produced with glucose as the carbon source in E. coli, which is the highest L-serine production reported so far; however, the yield was 0.36 g/g glucose [36], which is lower than that obtained in the present study (0.44 g/g sucrose). Therefore, fine controlling of the three enzymes of L-serine biosynthesis pathway could possibly further enhance L-serine production.…”

Section: Discussioncontrasting

confidence: 85%

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

Zhang

Gao

Chen

et al. 2020

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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.

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Section: Discussioncontrasting

confidence: 85%

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

Zhang

Gao

Chen

et al. 2020

Preprint

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“…Interestingly, we found that L-serine titer increased significantly by overexpressing serB in A36, with L-serine titer of 37.9 g/L, which was 24% higher than that of A36, serB encoded phosphoserine phosphatase (PSP, EC 3.1.3.3), catalyzed the last step of Lserine biosynthesis, however, L-serine titer had not significantly change when serAΔ197, serC was overexpressed in A36 respectively (with L-serine titer of 31.1 g/L and 32.78 g/L) (Fig.S5), and these results were in consistent with the previous report completely, in which overexpression of the mutant allele serAΔ197 in C. glutamicum13032 either alone or in combination with overexpression of serC and serB did not result in significant L-serine accumulation [3],which might be due to the poor accumulation and intracellular conversion of L-serine in C. glutamicum13032. In a recent study, 50 g/L of L-serine was produced with glucose as carbon source in E. coli, with a yield of 0.26-0.30 g/g glucose [37], in which 50 g/L is the highest reported so far for L-serine production, however, the yield was also lower than our present study (0.44 g/g sucrose). It is possible that fine controlling the three enzymes of the L-serine biosynthesis pathway to enhance L-serine production further.…”

contrasting

confidence: 76%

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

Zhang

Gao

Chen

et al. 2020

Preprint

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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 .

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“…OD and serine were measured continuously for 24 hr. At the end of cultivation, serine titers reached 0.55 g/L (Figure 5a), slightly lower than the 0.62 g/L achieved when using lac‐T7 (Rennig et al, 2019). A decrease in growth rate and final OD could be seen for the serine‐producing strain, which may be due to carbon being utilized for serine production instead of growth.…”

Section: Resultsmentioning

confidence: 62%

“…We chose to only apply one of the cassettes, as mCherry production levels of trp‐T7 and trp*‐T7 were similar according to flow cytometry (Figure 3b). The strain was transformed with pSEVA27‐sl‐ serA mut CB TIR1 (hereafter pSEVA‐serACB) harboring an expression‐optimized serine operon under control of pT7 (Rennig et al, 2019), resulting in strain trp‐T7‐ser.…”

Section: Resultsmentioning

confidence: 99%

“…The glucose feed was started after 9 hr (at OD ~9) and growth and serine production were monitored for approximately 42 hr (Figure 5b). At the end of the fermentation, 26 g/L l ‐serine had been produced, approximately half of the 48 g/L reached when using lac‐T7 (Rennig et al, 2019). The productivity was 0.73 g/Lh (Figure 5e), approximately 70% of that reached with the lac‐T7 system.…”

Section: Resultsmentioning

confidence: 99%

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An autoinducible trp‐T7 expression system for production of proteins and biochemicals in Escherichia coli

Landberg

Mundhada

Nielsen

2020

Biotech & Bioengineering

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Inducible expression systems can be applied to control the expression of proteins or biochemical pathways in cell factories. However, several of the established systems require the addition of expensive inducers, making them unfeasible for large‐scale production. Here, we establish a genome integrated trp‐T7 expression system where tryptophan can be used to control the induction of a gene or a metabolic pathway. We show that the initiation of gene expression from low‐ and high‐copy vectors can be tuned by varying the initial concentration of tryptophan or yeast extract, and that expression is tightly regulated and homogenous when compared with the commonly used lac‐T7 system. Finally, we apply the trp‐T7 expression system for the production of l‐serine, where we reach titers of 26 g/L in fed‐batch fermentation.

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Industrializing a Bacterial Strain for l-Serine Production through Translation Initiation Optimization

Cited by 23 publications

References 45 publications

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

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

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

An autoinducible trp‐T7 expression system for production of proteins and biochemicals in Escherichia coli

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