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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. [ABSTRACT FROM AUTHOR]

L-Serine is widely used in pharmaceutical, food and cosmetic industries, and the direct fermentation to produce L-serine from cheap carbon sources such as glycerol is greatly desired. The production of L-serine by engineered Escherichia coli from glycerol has not been achieved so far. In this study, E. coli was engineered to efficiently produce L-serine from glycerol. To this end, three L-serine deaminase genes were deleted in turn, and all of the deletions caused the maximal accumulation of L-serine at 0.06 g/L. Furthermore, removal of feedback inhibition by L-serine resulted in a titer of 1.1 g/L. Additionally, adaptive laboratory evolution was employed to improve glycerol utilization in combination with the overexpression of the cysteine/acetyl serine transporter gene eamA, leading to 2.36 g/L L-serine (23.6% of the theoretical yield). In 5-L bioreactor, L-serine titer could reach up to 7.53 g/L from glycerol, demonstrating the potential of the established strain and bioprocess. [ABSTRACT FROM AUTHOR]

L-Serine is widely used in the pharmaceutical, food, and cosmetics industries. Although direct fermentative production of L-serine from sugar in Corynebacterium glutamicum has been achieved, the L-serine yield remains relatively low. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the L-serine yield based on engineered C. glutamicum ΔSSAAI strain. Subsequently, we developed a novel high-throughput screening method using a biosensor constructed based on NCgl0581, a transcriptional factor specifically responsive to L-serine, so that L-serine concentration within single cell of C. glutamicum can be monitored via fluorescence-activated cell sorting (FACS). Novel L-serine-producing mutants were isolated from a large library of mutagenized cells. The mutant strain A36-pDser was screened from 1.2 × 105 cells, and the magnesium ion concentration in the medium was optimized specifically for this mutant. C. glutamicum A36-pDser accumulated 34.78 g/L L-serine with a yield of 0.35 g/g sucrose, which were 35.9 and 66.7% higher than those of the parent C. glutamicum ΔSSAAI-pDser strain, respectively. The L-serine yield achieved in this mutant was the highest of all reported L-serine-producing strains of C. glutamicum. Moreover, the whole-genome sequencing identified 11 non-synonymous mutations of genes associated with metabolic and transport pathways, which might be responsible for the higher L-serine production and better cell growth in C. glutamicum A36-pDser. This study explored an effective mutagenesis strategy and reported a novel high-throughput screening method for the development of L-serine-producing strains. [ABSTRACT FROM AUTHOR]

l -Serine is a non-essential amino acid that has wide and expanding applications in industry with a fast-growing market demand. Currently, extraction and enzymatic catalysis are the main processes for l -serine production. However, such approaches limit the industrial-scale applications of this important amino acid. Therefore, shifting to the direct fermentative production of l -serine from renewable feedstocks has attracted increasing attention. This review details the current status of microbial production of l -serine from renewable feedstocks. We also summarize the current trends in metabolic engineering strategies and techniques for the typical industrial organisms Corynebacterium glutamicum and Escherichia coli that have been developed to address and overcome major challenges in the l -serine production process. [ABSTRACT FROM AUTHOR]

Pyruvate kinase (PYK) is an important enzyme in the intermediary metabolism and has attracted much attention as a target for metabolic engineering of Corynebacterium glutamicum . Genome sequencing revealed that the 308 residue of PYK was mutated from methionine in model strain C. glutamicum ATCC14067 to isoleucine in L-serine-producing strain C. glutamicum SYPS-062. Consequently, a significantly lower PYK activity (77%) was noted in C. glutamicum SYPS-062, when compared with that in C. glutamicum ATCC14067. To confirm the role of this point mutation, pyk in both C. glutamicum SYPS-062 and C. glutamicum SYPS-062-33aΔSSAA was reversely mutated to restore the PYK enzyme activity, which led to a 33.1% and 28.8% decrease in L-serine titer, respectively. This is the first report to show that the (Met-308→Ile) mutation site of pyk is closely associated with its activity and apparently affected L-serine production. Furthermore, pyk was deleted in strain C. glutamicum SYPS-062-33aΔSSAA, and the resulting strain did not show alteration in growth rate and presented a 12% increase in L-serine production. [ABSTRACT FROM AUTHOR]

Microbial fermentative production of l -serine has attracted increasing attention, and in our previous study, Corynebacterium glutamicum- SYPS - 062ΔSSA has been successfully screened and engineered to produce l -serine from sucrose. It was noticed that there was significant fructose accumulation during l -serine fermentation process using sucrose as substrates, which is the preferred carbon resource for this strain. Lack of fructokinase may be responsible for poor fructose utilization. In the present study, a shortcut pathway for fructose utilization has been constructed via heterologous expression of scrK (fructokinase). Furthermore, pfkA (phosphofructokinase) was over-expressed to avoid over accumulation of fructose-6-phosphate, the resulting strain showed significantly improved cell growth and l -serine accumulation. In a 5-L bioreactor, the engineered C. glutamicum strain produced 30.6 g/L l -serine (0.99 mol/mol sucrose yield, with a theoretical maximal yield of 4 mol/mol sucrose), whereas the reference strain produced 20.54 g/L l -serine (with a yield of 0.67 mol/mol sucrose) at 120 h. After two feedings of sucrose, the l -serine production increased up to 37.0 g/L at 96 h. [ABSTRACT FROM AUTHOR]

The direct fermentative production of l-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low l-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing l-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both l-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products l-alanine and l-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards l-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as l-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the l-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of l-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of l-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve l-serine productivity. [ABSTRACT FROM AUTHOR]

A comparative genomic analysis was performed to study the genetic variations between the l-serine-producing strain Corynebacterium glutamicum SYPS-062 and the mutant strain SYPS-062-33a, which was derived from SYPS-062 by random mutagenesis with enhanced l-serine production. Some variant genes between the two strains were reversely mutated or deleted in the genome of SYPS-062-33a to verify the influences of the gene mutations introduced by random mutagenesis. It was found that a His-594 → Tyr mutation in aceE was responsible for the more accumulation of by-products, such as l-alanine and l-valine, in SYPS-062-33a. Furthermore, the influence of this point mutation on the l-serine production was investigated, and the results suggested that this point mutation led to a better growth profile and a higher l-serine production in the high-yield strain 33a∆SSAAI, which was derived from SYPS-062-33a by metabolic engineering with the highest l-serine production to date. [ABSTRACT FROM AUTHOR]

The direct fermentative production of l -serine using Corynebacterium glutamicum from renewable biomass attracts increasing attention. This study was designed to enhance l -serine production in a wild-type C. glutamicum SYPS-062 by using random mutagenesis, optimization of media, and metabolic engineering. The resulting mutant, C. glutamicum SYPS 062-33a, accumulated 65.4% more l -serine (11.0 ± 0.25 g/L) and exhibited higher growth and production of other amino acids (such as l -alanine, l -valine) compared with the parent strain. Interestingly, this mutant harbors a better ability to consume sucrose, which is the main composition of economical molasses. To further enhance l -serine production, single sdaA deletion and serA modification was performed, which led to 14.76 ± 0.82 g/L and 19.42 ± 1.20 g/L l -serine, respectively. While double sdaA deletion and serA modification led to 21.27 ± 1.87 g/L l -serine. In addition, when the modified serA gene was overexpressed in C. glutamicum SYPS-062-33aΔSS, the resultant strain accumulated 24.43 ± 0.95 g/L and 21.6 ± 1.2 g/L l -serine in flask and 5-L bioreactor, respectively. Unlike the previous finding, this study demonstrated that the key to further enhance l -serine production may be the removal of the feedback inhibition of PGDH by l -serine in a high l -serine-producing strain C. glutamicum SYPS-062-33a. [ABSTRACT FROM AUTHOR]