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Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The L-serine exporters ThrE and SerE play important roles in L-serine production by Corynebacterium glutamicum. Deletion of both thrE and serE decreased L-serine titer by 60%, suggesting the existence of other L-serine exporters. A comparative transcriptomics identified NCgl0254 and NCgl0255 as novel L-serine exporters. Further analysis of the contributions of ThrE, SerE, NCgl0254, and NCgl0255 found that SerE was the major L-serine exporter in C. glutamicum and these four L-serine exporters were responsible for 79.7% of L-serine export. Deletion of one L-serine exporter upregulated the transcription levels of the other three, which might be coursed by increased intracellular concentrations of L-serine. Overexpression of NCgl0254 and NCgl0255 increased L-serine titer by 20.8% in C. glutamicum A36, while overexpression of the four L-serine exporters increased L-serine production by 31.9% (41.1 g·L -1 ) in C. glutamicum A36. The identification of novel L-serine exporters in C. glutamicum will help to improve industrial production of L-serine., (© 2023 Wiley-VCH GmbH.)

Promoters and ribosome binding sites (RBSs) are routinely applied in gene expression regulation, but their orthogonality and combinatorial effects have not yet been systematically studied in Corynebacterium glutamicum. Here, 17 core promoters and 29 RBSs in C. glutamicum were characterized, which exhibited 470-fold and 430-fold in transcriptional and translational activity, respectively. By comparing the expression of two reporter genes regulated by multiple RBSs, the RBS efficacy showed significant dependence on the gene context, besides the RBSs' strength, reflecting the poor orthogonality of RBSs. Bicistron-modified RBS (referred as bc-RBS) was adapted to C. glutamicum , which improved RBS reliability. By coupling a series of promoters with RBSs/bc-RBSs, a much broader regulation range that spanned 4 orders of magnitude was observed compared with that of a sole element, and the contribution to gene expression of RBS was more than that of promoter. Finally, promoters and RBSs were applied as built-in elements to fine-tune the gene cluster in the arginine synthesis pathway in C. glutamicum . Compared with the original strain, more arginine (1.61-fold) or citrulline (2.35-fold) was accumulated in a 7 L bioreactor by strains with the gene expression regulation system rationally engineered. We demonstrated that, via combination of well-characterized gene elements, and overall consideration for both transcription and translation, the biosynthesis pathway can be effectively balanced, and the yield of a target metabolite can be further improved.

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.

l-Serine is widely used in the pharmaceutical, food, and cosmetic industries. The direct fermentative production of l-serine from glucose in Corynebacterium glutamicum (C. glutamicum) has been achieved but the yield is generally low. In this study, the central metabolic pathway is rewired to direct higher accumulation of glycerate-3-phosphate (3-PG), the precursor for l-serine biosynthesis, thereby freeing up more carbon to enhance l-serine production and yield. To this end, the native phosphotransferase system (PTS) is inactivated by deleting the gene ptsH. Meanwhile, the PTS-independent glucose uptake pathway is activated via overexpression of iolT1, ppgk, and deletion of the transcriptional regulator gene iolR. Furthermore, metabolic changes between PTS and non-PTS strains are elucidated using gas chromatography-tandem mass spectrometry. Based on the metabonomic and enzyme activities analysis, the glycolysis pathway upstream of 3-PG is further enhanced by co-overexpressing pgi, pfkA, and gapA. The resulting strain with all of the modifications mentioned above leads to the l-serine yield of 0.30 g g -1 glucose (42.8% higher than that of the original strain), which is the highest yield in C. glutamicum by using glucose as the sole carbon source. These strategies can be expanded for the production of other value-added chemicals derived from the intermediates of glycolysis pathway in C. glutamicum., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 × 10 5 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.

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.

To increase the carbon flux of the L-serine synthesis pathway of Corynebacterium glutamicum A36, the phosphoglycerate kinase (pgk) was overexpressed to increase the accumulation of 3- phosphoglycerate, the precursor of L-serine. It had no significant effect on the L-serine titer. Furthermore, the acetohydroxyacid synthase gene ilvN of the byproduct L-valine synthesis pathway was knocked out. Only a little L-valine was accumulated after knocking out ilvN, however, the recombinant strain was not an auxotrophic strain. The titer of L-serine decreased, and the addition of L-valine was benefit to the production of L-serine. Then, L-valine was added to the medium. When L-valine was 750 mg/L, the titer of L-serine was 34. 19 g /L, with the yield of 0. 34 g / g, and the productivity was 0. 28 g /( L h), increased by 11. 8%, 13. 3% and 12. 0%, respectively, compared to the original strain A36. [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 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]