Synthesis Of Glutamic Acid Research Articles

Improved Glutamic Acid Production Capacity of Corynebacterium glutamicum by the ARTP Mutagenesis Method

Glutamic acid is an important amino acid that is used widely in the fields of food, medicine, and agriculture. One of the methods of glutamic acid production is direct microbial fermentation, so the genetic stability and glutamic-acid-producing capacity of the producing strain are the keys to improving glutamic acid concentration. Experiments were carried out using Corynebacterium glutamicum GL−6 as the parental strain, with two iterations of mutagenesis by atmospheric and room temperature plasma (ARTP) and screening with agar plates tolerant to high sugar and malonic acid, and the best strains with stable phenotypes were verified by fermentation in 20 L tanks. The results show that the optimal mutagenesis time of ARTP was 140 s, with lethality and positive mutation rates of 93.0% and 15.6%, respectively. The concentrations of the high-sugar and malonic acid agar plates were 240 g/L and 35 g/L, respectively. A mutant strain, P−45, with improved glutamic acid production capacity and genetic stability, was obtained through two rounds of iterative mutagenesis screening. The concentration of this strain in the Erlenmeyer flasks was 17.7 g/L, which was 18.8% higher than that of the parental strain, GL−6, and could be inherited stably for 10 generations. In the glutamic acid synthesis pathway, the upregulation of the gene encoding citrate synthase (cs), gene encoding isocitrate dehydrogenase (icdh), and gene encoding glutamate dehydrogenase (gdh), and the downregulation of the gene encoding oxoglutarate dehydrogenase complex (odhc) increased the carbon flows of the TCA cycle and its branch metabolic flow to glutamic acid synthesis. P−45 showed a glutamic acid concentration of 147.0 g/L under fed-batch fermentation conditions in 20 L tanks, which was 81.5% higher than the starting strain, GL−6. This study provides a new technical solution for improving microbial metabolites and genetic stability.

Mechanism of Cytotoxic Action of Gold Nanorods Photothermal Therapy for A549 Cell

Photothermal therapy has developed into an important field of tumor treatment research, and numerous studies have focused on the preparation of photothermal therapeutic agents, tumor targeting, diagnosis, and treatment integration. However, there are few studies on the mechanism of photothermal therapy acting on cancer cells. Here we investigated the metabolomics of lung cancer cell A549 during gold nanorod (GNR) photothermal treatment by high-resolution LC/MS, and several differential metabolites and corresponding metabolic pathways during photothermal therapy were found. The main differential metabolites contained 18-hydroxyoleate, beta-alanopine and cis-9,10-epoxystearic acid, and phosphorylcholine. Pathway analysis also showed metabolic changes involving cutin, suberine, and wax biosynthesis, pyruvate and glutamic acid synthesis, and choline metabolism. Analysis also showed that the photothermal process of GNRs may induce cytotoxicity by affecting pyruvate and glutamate synthesis, normal choline metabolism, and ultimately apoptosis.

Effects of Exogenous Chlorinated Amino Acetic Acid on Cadmium and Mineral Elements in Rice Seedlings.

To explore the effect of exogenous application of chlorinated amino acetic acid on cadmium (Cd) transport characteristics in rice seedlings, X24 and Z35 rice were taken as the research objects to carry out hydroponics experiments, and the changes of Cd content in rice seedlings, rice mineral elements and amino acid content in rice were analyzed. The results showed that exogenous application of 1.2 mmol·L-1 chlorinated amino acetic acid inhibited cadmium in shoots and roots of rice seedlings; Cd content in shoots and roots were reduced by up to 62.19% and 45.61%, respectively. The majority of cadmium was in the cell wall of shoots and roots; this decreased with the increase of the concentration of chlorinated acetic acid. In addition, the Mn content in shoots and Ca content in roots of rice seedlings increased significantly after the application of chlorinated amino acetic acid. The results of amino acid analysis showed that the contents of aspartic acid, glutamic acid and cystine in rice seedlings were increased. These results indicate that exogenous application of chlorinated amino acetic acid is beneficial to the synthesis of aspartic acid, glutamic acid and cysteine in rice seedlings, increases the content of Mn in shoots and Ca in roots of rice seedlings, and significantly alleviates cadmium stress in seedlings. This provides a theoretical basis for the development of an environmentally friendly Cd-lowering foliar fertilizer for rice.

Optimization of culture conditions of screened Galactomyces candidum for the production of single cell protein from biogas slurry

The use of single cell protein (SCP) has become a method for alleviating the shortage of protein feed that microorganisms propagate in a suitable culture medium. In this study, SCP was produced by yeast to use the nutrition contained in the biogas slurry of chicken manure. The results showed that Galactomyces candidum was the most efficient at producing SCP among the seven yeasts studied. The maximum cell dry weight (CDW) 6.79 g/L and protein content 39.39%, were obtained under the fermentation conditions of initial NH 4 + -N concentration of 2000 mg/L and a C/N ratio of 6:1 with acetate as the pH regulator. The total CDW increased to 9.24 g/L after secondary fermentation. Metal elements had a little effect on the growth of G. candidum . The addition of sulfur not only promoted the synthesis of sulfur-containing amino acid cysteine but also increased protein content by promoting the synthesis of glutamic acid and glutamine. Future experiments should focus more on achieving high-density cultivation and more efficient utilization of ammonia nitrogen in the biogas slurry. How to cite: Zhou P, Zhang L, Ding H, et al. Optimization of culture conditions of screened Galactomyces candidum for the production of single cell protein from biogas slurry. Electron J Biotechnol 2022;55. https://doi.org/10.1016/j.ejbt.2021.11.006

Adaptive changes in the ornithine cycle and amino acid synthesis in sheep liver with different meat productivity

The aim of the research was to study the ornithine cycle as the process of fixing ammonia and the formation of urea in the body of highly productive animals. In our experiments, we used a protein-deficient diet and urea as a nitrogen substitute for nitrogen-containing materials in the diet to reveal the mechanism of action of urea on animals, in particular on the biochemical processes of the ornithine cycle. There are some differences between Bukovinian sheep of the Askanian meat-wool breed and outbreds in terms of the ability to build muscle tissue. Our study reveals that the slaughter yield and the average daily gain consumption of Bukovinian-type meat of the Askanian meat-wool breed were higher in summer and in autumn, compare with purebred sheep. Sheep of the Bukovynian type of Askanian meat-wool breed have the intensity of enzymatic formation of urea in liver homogenates that is much higher in all experiments than in outbred sheep. A sharp drop in the activity of all stages of urea formation and glutamic acid synthesis in liver homogenates and significantly weakened urea formation was found in all experiments of the fourth series in comparison with the experiments in the third series. Increased muscle growth, high nitrogen deposition, and a much lower percentage of urinary excretion of ammonia and urea nitrogen, as well as higher activity of enzymes of the ornithine cycle and glutamic acid synthesis in the Bukovinian sheep type of Askanian meat-wool breed compared to outbreeds allow concluding that ammonia and urea in highly productive animals act less as finishing products of nitrogen metabolism than in low-productive animals.

Metabolic Engineering of Central Carbon Metabolism of Bacillus licheniformis for Enhanced Production of Poly-γ-glutamic Acid.

Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with wide-ranging applications in the areas of medicine, light chemical industry, wastewater treatment, and agriculture. However, the production cost of γ-PGA is high for the requirement of adding the expensive precursor L-glutamic acid during fermentation, which hinders its widespread application. In this study, in order to improve γ-PGA yield, central carbon metabolism was engineered to enhance the carbon flux of tricarboxylic acid (TCA) cycle and glutamic acid synthesis in a γ-PGA production strain Bacillus licheniformis WX-02. Firstly, pyruvate dehydrogenase (PdhABCD) and citrate synthase (CitA) were overexpressed to strengthen the flux of pyruvate into TCA cycle, resulting in 34.93% and 11.14% increase of γ-PGA yield in B. licheniformis WX-02, respectively. Secondly, the carbon flux to glyoxylate shunt was rewired via varying the expression of isocitrate lyase (AceA), and a 23.24% increase of γ-PGA yield was obtained in AceA down-regulated strain WXPbacAaceBA. Thirdly, deletion of pyruvate formate-lyase gene pflB led to a 30.70% increase of γ-PGA yield. Finally, combinatorial metabolic engineering was applied, and γ-PGA titer was enhanced to 12.02g/L via overexpressing pdhABCD and citA, repressing aceA, and deleting pflB, with a 69.30% improvement compared to WX-02. Collectively, metabolic engineering of central carbon metabolism is an effective strategy for enhanced γ-PGA production in B. licheniformis, and this research provided a promising strain for industrial production of γ-PGA.

Evaluation of Alpha-Ketoglutarate Supplementation on the Improvement of Intestinal Antioxidant Capacity and Immune Response in Songpu Mirror Carp (Cyprinus carpio) After Infection With Aeromonas hydrophila.

As an intermediate substance of the tricarboxylic acid cycle and a precursor substance of glutamic acid synthesis, the effect of alpha-ketoglutarate on growth and protein synthesis has been extensively studied. However, its prevention and treatment of pathogenic bacteria and its mechanism have not yet been noticed. To evaluate the effects of alpha-ketoglutarate on intestinal antioxidant capacity and immune response of Songpu mirror carp, a total of 360 fish with an average initial weight of 6.54 ± 0.08 g were fed diets containing alpha-ketoglutarate with 1% for 8 weeks. At the end of the feeding trial, the fish were challenged with Aeromonas hydrophila for 2 weeks. The results indicated that alpha-ketoglutarate supplementation significantly increased the survival rate of carp after infection with Aeromonas hydrophila (P < 0.05), and the contents of immune digestion enzymes including lysozyme, alkaline phosphatase and the concentration of complement C4 were markedly enhanced after alpha-ketoglutarate supplementation (P < 0.05). Also, appropriate alpha-ketoglutarate increased the activities of total antioxidant capacity and catalase and prevented the up-regulation in the mRNA expression levels of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 (P < 0.05). Furthermore, the mRNA expression levels of toll-like receptor 4 (TLR4), and nuclear factor kappa-B (NF-κB) were strikingly increased after infection with Aeromonas hydrophila (P < 0.05), while the TLR4 was strikingly decreased with alpha-ketoglutarate supplementation (P < 0.05). Moreover, the mRNA expression levels of tight junctions including claudin-1, claudin-3, claudin-7, claudin-11 and myosin light chain kinases (MLCK) were upregulated after alpha-ketoglutarate supplementation (P < 0.05). In summary, the appropriate alpha-ketoglutarate supplementation could increase survival rate, strengthen the intestinal enzyme immunosuppressive activities, antioxidant capacities and alleviate the intestinal inflammation, thereby promoting the intestinal immune responses and barrier functions of Songpu mirror carp via activating TLR4/MyD88/NF-κB and MLCK signaling pathways after infection with Aeromonas hydrophila.

Proteome and transcriptome analyses of wheat near isogenic lines identifies key proteins and genes of wheat bread quality

The regulation of wheat protein quality is a highly complex biological process involving multiple metabolic pathways. To reveal new insights into the regulatory pathways of wheat glutenin synthesis, we used the grain-filling period wheat grains of the near-isogenic lines NIL-723 and NIL-1010, which have large differences in quality, to perform a combined transcriptome and proteome analysis. Compared with NIL-1010, NIL-723 had 1287 transcripts and 355 proteins with significantly different abundances. Certain key significantly enriched pathway were identified, and wheat quality was associated with alanine, aspartate and glutamate metabolism, nitrogen metabolism and alpha-linolenic acid metabolism. Differentially expressed proteins (DEPs) or Differentially expressed genes (DEGs) in amino acid synthesis pathways were upregulated primarily in the glycine (Gly), methionine (Met), threonine (Thr), glutamic acid (Glu), proline (proC), cysteine (Cys), and arginine (Arg) synthesis and downregulated in the tryptophan (trpE), leucine (leuC), citrulline (argE), and ornithine (argE) synthesis. Furthermore, to elucidate changes in glutenin in the grain synthesis pathway, we plotted a regulatory pathway map and found that DEGs and DEPs in ribosomes (RPL5) and the ER (HSPA5, HYOU1, PDIA3, PDIA1, Sec24, and Sec31) may play key roles in regulating glutenin synthesis. The transcriptional validation of some of the differentially expressed proteins through real-time quantitative PCR analysis further validated the transcriptome and proteomic results.

Highly Stable Acid-Induced Emission-Enhancing Cd-MOFs: Synthesis, Characterization, and Detection of Glutamic Acid in Water and Fe Ions in Acid

Two novel 3D fluorescent metal-organic frameworks (MOFs), [Cd(L)(bbibp)]n (1) and [Cd(L)(bbibp)0.5]n (2), where H2L = 4,4'-(4,4'-bipyridine-2,6-diyl)dibenzoic acid and bbibp = 4,4'-bis(benzoimidaz-1-yl)biphenyl, were acquired through a conventional method and characterized via IR spectra, single-crystal X-ray diffraction, elemental analysis, thermogravimetric analysis, powder X-ray diffraction (PXRD), scanning electron microscopy, N2 adsorption-desorption isotherms, and X-ray photoelectron spectroscopy (XPS). The crystal framework of Cd-MOF 1 remained stable in the range of pH = 1.0-12.0. Interestingly, the emission peak of 1 showed a red shift and exhibited a fluorescence turn-on effect in an acidic environment. X-ray diffraction measurement revealed that the crystal structure of 1 remained unchanged after immersion in a pH = 1.0 solution. In addition, Cd-MOFs 1 and 2 displayed fluorescent quenching to l-glutamic acid with high sensitivity and selectivity. Meanwhile, 1 showed high selectivity in recognizing Fe3+ under acidic conditions, which made 1 capable of detecting Fe3+ in acidic industrial wastewater. Finally, the fluorescent sensing mechanism was carefully studied by PXRD, transient fluorescence lifetime, XPS, and UV spectroscopy.

Preparation of glutamic acid cholesterol ester and its gel properties

ABSTRACT In order to overcome the problems such as low yield and complex products caused by direct esterification reaction of glutamic acid and cholesterol for the preparation of glutamic cholesterol ester (GCE), the new synthesis protocol was proposed. In this process, the amino of glutamic acid was first protected with a phthalimido group to produce followed N-phthaloyl-glutamic anhydride. Then, the glutamic anhydride was combined with cholesterol to form N-phthaloyl-glutamic cholesterol ester (NP-GCE). Finally, a de-protected GCE was obtained by hydrazine hydrate. The structures of GCE and NP-GCE were characterized by Fourier transform infrared spectra (FTIR), nuclear magnetic resonance spectrum (1H and 13C NMR) and high-resolution mass spectra (HR-MS). The gelation properties of GCE and NP-GCE were evaluated in 15 solvents. The thermodynamic parameters of gels were studied by gel-sol transition temperature. The X-ray diffraction (XRD) was used to characterize the gelation mechanism and possible accumulation models of GCE. The results indicated that the new reaction route can eliminate the interference of amino group from glutamic acid, and the yield of GCE can reach 89.62 w%, which increased by 41.99 w% in comparison with the direct synthesis of glutamic acid and cholesterol. The gelling property demonstrated that GCE performs good gel properties in 5 solvents while NP-GCE only can from gel in 3 solvents. The gel-sol transition enthalpy of GCE is greater than that of NP-GCE, revealing the thermal stability of the gel from GCE is enhanced. XRD and SEM showed that GCE gel accumulated in lamellar structure due to the π-π stacking and hydrogen bond interactions.

Synthesis of Glutamic Acid Derived Organogels and their Applications in Dye Removal from Aqueous Medium

AbstractTo develop glutamic acid derived cross‐linked polymeric organogels for dye adsorption from aqueous media, dimethyl 2‐(methacrylamido)pentanedioate (Glu‐MAC) is polymerized using diethylene glycol dimethacrylate as cross‐linker via the reversible addition‐fragmentation chain transfer (RAFT) technique to synthesize cross‐linked polymeric organogels with pendant glutamate moieties. The mechanical properties of organogels are examined by rheological study. The organogels exhibit higher value of storage modulus (G′) than the loss modulus (G′′) within the linearity limits of deformation, and strong dependence of G′ values on the extent of cross‐linking in the gel matrix. Field emission scanning electron microscopy shows porous structure of the gel matrix, therefore, organogels are able to exhibit swelling behaviors in organic solvents such as dichloromethane, N,N‐dimethylformamide, tetrahydrofuran, acetone, and methanol. Dye adsorption properties of these organogels are investigated using different aqueous dye solutions, such as crystal violet (CV), malachite green (MG), rhodamine‐B, and uranine. The gels show high dye adsorption capacities (≥97%) toward cationic dyes like CV and MG.

Catalyst: Is the Amino Acid a New Frontier for Biorefineries?

Prof. Ning Yan obtained his BS and PhD degrees from Peking University in 2004 and 2009, respectively. Thereafter, he worked as a Marie-Curie research fellow at Ecole Polytechnique Federale de Lausanne, Switzerland. He joined the National University of Singapore (NUS) as an assistant professor, established the Lab of Green Catalysis in 2012, and was promoted to the position of associate professor in 2018. Yunzhu Wang received her BE degree from NUS in 2014. She joined Prof. Yan’s group in the same year and is currently pursuing her PhD. Her research interests include nanoscale metal catalysts and their applications in producing renewable nitrogen-containing chemicals.

Synthesis of Tritiated Glutamic Acid and Pyroglutamate

It was shown that introduction of tritium directly into glutamic acid does not afford a highly labeled product (at 160°C, a specimen with a specific radioactivity of 3–5 Ci/mmol was formed; at 180°C, the specific radioactivity was 26 Ci/mmol; and at 200°C, the compound strongly degraded). Heating a mixture of 5% Pd/Al2O3 with alumina-supported sodium glutamate at 220°C for 7.5 min gave mainly [3H]pyroglutamate. After isolation and purification of [3H]pyroglutamate, its solution in 1 N HCl was heated at 105°C for 20 h. This yielded [3H]glutamic acid with a specific radioactivity of 40 Ci/mmol.

Synthesis of Tritiated Glutamic Acid and Pyroglutamate

Synthesis of Tritiated Glutamic Acid and Pyroglutamate

Enhanced synthesis of poly gamma glutamic acid by increasing the intracellular reactive oxygen species in the Bacillus licheniformis Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN-deficient strain.

Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased γ-PGA yield to 13.91gL-1, 85.22% higher than that of the wild type (7.51gL-1). However, deletion of proline dehydrogenase gene ycgM had no effect on γ-PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24μmolgDCW-1) was detected in the ycgN deficient strain WXΔycgN, while the P5C levels of WXΔycgM and the double mutant strain WXΔycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXΔycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced γ-PGA synthesis capability of WXΔycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis.

Effect of glucose on poly-\u03b3-glutamic acid metabolism in Bacillus licheniformis

BackgroundPoly-gamma-glutamic acid (γ-PGA) is a promising macromolecule with potential as a replacement for chemosynthetic polymers. γ-PGA can be produced by many microorganisms, including Bacillus species. Bacillus licheniformis CGMCC2876 secretes γ-PGA when using glycerol and trisodium citrate as its optimal carbon sources and secretes polysaccharides when using glucose as the sole carbon source. To better understand the metabolic mechanism underlying the secretion of polymeric substances, SWATH was applied to investigate the effect of glucose on the production of polysaccharides and γ-PGA at the proteome level.ResultsThe addition of glucose at 5 or 10 g/L of glucose decreased the γ-PGA concentration by 31.54 or 61.62%, respectively, whereas the polysaccharide concentration increased from 5.2 to 43.47%. Several proteins playing related roles in γ-PGA and polysaccharide synthesis were identified using the SWATH acquisition LC–MS/MS method. CcpA and CcpN co-enhanced glycolysis and suppressed carbon flux into the TCA cycle, consequently slowing glutamic acid synthesis. On the other hand, CcpN cut off the carbon flux from glycerol metabolism and further reduced γ-PGA production. CcpA activated a series of operons (glm and epsA-O) to reallocate the carbon flux to polysaccharide synthesis when glucose was present. The production of γ-PGA was influenced by NrgB, which converted the major nitrogen metabolic flux between NH4+ and glutamate.ConclusionThe mechanism by which B. licheniformis regulates two macromolecules was proposed for the first time in this paper. This genetic information will facilitate the engineering of bacteria for practicable strategies for the fermentation of γ-PGA and polysaccharides for diverse applications.

Glutamate dehydrogenase (RocG) in Bacillus licheniformis WX-02: Enzymatic properties and specific functions in glutamic acid synthesis for poly-γ-glutamic acid production.

Poly-γ-glutamic acid (γ-PGA), a natural biopolymer, is widely used in cosmetics, medicine, food, water treatment, and agriculture owing to its features of moisture sequestration, cation chelation, non-toxicity and biodegradability. Intracellular glutamic acid, the substrate of γ-PGA, is a limiting factor for high yield in γ-PGA production. Bacillus subtilis and Bacillus licheniformis are both important γ-PGA producing strains, and B. subtilis synthesizes glutamic acid in vivo using the unique GOGAT/GS pathway. However, little is known about the glutamate synthesis pathway in B. licheniformis. The aim of this work was to characterize the glutamate dehydrogenase (RocG) in glutamic acid synthesis from B. licheniformis with both in vivo and in vitro experiments. By re-directing the carbon flux distribution, the rocG gene deletion mutant WX-02ΔrocG produced intracellular glutamic acid with a concentration of 90ng/log(CFU), which was only 23.7% that of the wild-type WX-02 (380ng/log(CFU)). Furthermore, the γ-PGA yield of mutant WX-02ΔrocG was 5.37g/L, a decrease of 45.3% compared to the wild type (9.82g/L). In vitro enzymatic assays of RocG showed that RocG has higher affinity for 2-oxoglutarate than glutamate, and the glutamate synthesis rate was far above degradation. This is probably the first study to reveal the glutamic acid synthesis pathway and the specific functions of RocG in B. licheniformis. The results indicate that γ-PGA production can be enhanced through improving intracellular glutamic acid synthesis.

Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position

Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of (2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial activity. However the role of (2S, 3R) mGlu is yet to be revealed. Herein, we reported the syntheses of three daptomycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) methyl glutamine (mGln), dimethyl glutamic acid and (2S, 3R) ethyl glutamic acid (eGlu), respectively, and their antibacterial activities. The detailed synthesis of dimethyl glutamic acid was also reported.

Comprehensive transcriptome and improved genome annotation of Bacillus licheniformis WX-02

The updated genome of Bacillus licheniformis WX-02 comprises a circular chromosome of 4286821 base-pairs containing 4512 protein-coding genes. We applied strand-specific RNA-sequencing to explore the transcriptome profiles of B. licheniformis WX-02 under normal and high-salt conditions (NaCl 6%). We identified 2381 co-expressed gene pairs constituting 871 operon structures. In addition, 1169 antisense transcripts and 90 small RNAs were detected. Systematic comparison of differentially expressed genes under different conditions revealed that genes involved in multiple functions were significantly repressed in long-term high salt adaptation process. Genes related to promotion of glutamic acid synthesis were activated by 6% NaCl, potentially explaining the high yield of γ-PGA under salt condition. This study will be useful for the optimization of crucial metabolic activities in this bacterium.

Intracellular synthesis of glutamic acid in Bacillus methylotrophicus SK19.001, a glutamate-independent poly(γ-glutamic acid)-producing strain.

Bacillus methylotrophicus SK19.001 is a glutamate-independent strain that produces poly(γ-glutamic acid) (γ-PGA), a polymer of D- and L-glutamic acids that possesses applications in food, the environment, agriculture, etc. This study was undertaken to explore the synthetic pathway of intracellular L- and D-glutamic acid in SK19.001 by investigating the effects of tricarboxylic acid cycle intermediates and different amino acids as metabolic precursors on the production of γ-PGA and analyzing the activities of the enzymes involved in the synthesis of L- and D-glutamate. Tricarboxylic acid cycle intermediates and amino acids could participate in the synthesis of γ-PGA via independent pathways in SK19.001. L-Aspartate aminotransferase, L-glutaminase and L-glutamate synthase were the enzymatic sources of L-glutamate. Glutamate racemase was responsible for the formation of D-glutamate for the synthesis of γ-PGA, and the synthetase had stereoselectivity for glutamate substrate. The enzymatic sources of L-glutamate were investigated for the first time in the glutamate-independent γ-PGA-producing strain, and multiple enzymatic sources of L-glutamate were verified in SK19.001, which will benefit efforts to improve production of γ-PGA with metabolic engineering strategies.