Co-expression of secretory and intracellular glutamate decarboxylase for plasmid-free γ-aminobutyric acid biosynthesis in Corynebacterium glutamicum

Abstract

γ-Aminobutyric acid (GABA) is a nonprotein amino acid with significant physiological functions. The production of GABA using the L-glutamate producing Corynebacterium glutamicum holds immense potential; however, the incompatibility between the optimal neutral pH required for cell growth and the acidic pH required for glutamate decarboxylase (GAD) activity severely restricts synthesis efficiency. To address this challenge, a multidimensional collaborative metabolic engineering strategy was conducted in this study. Initially, four GADs active at near-neutral pH were screened, resulting in LcGAD as the most effective candidate. Subsequently, to ensure sufficient supply of L-glutamate precursor and redirect carbon flux away from competing byproducts synthesis, the phosphoenolpyruvate carboxylase gene ppc and exogenous glutamate dehydrogenase gene gdhA were overexpressed, while acetate and lactate synthesis were blocked, significantly enhancing GABA production from 19.6 g/L to 28.9 g/L. Next, secretory LcGAD was expressed to facilitate product export; concurrently, genes encoding the aconitase repressor (acnR) and master regulator of carbon metabolism (ramB) were deleted to further enhance carbon flux toward L-glutamate biosynthesis. Furthermore, the maltooligosyl trehalose synthase gene treY and an RNA polymerase sigma factor gene sigD involved in mycolic acid synthesis were dynamically downregulated to increase cell envelope permeability. The most optimal plasmid-free strain, GAD-15, synthesized GABA both intracellularly and extracellularly and produced 41.1 g/L GABA in shake flasks and 110.1 g/L GABA in a 2.4-L bioreactor. The intracellular and extracellular synergistic synthesis strategy developed here provides a novel approach for the efficient production of GABA and other metabolites in C. glutamicum.

Keywords

γ-Aminobutyric acid / Glutamate decarboxylase / Corynebacterium glutamicum / Secretory expression / Metabolic engineering

References

[1] Beck SW, Ye DY, Hwang HG, et al. Stepwise flux optimization for enhanced GABA production from acetate in Escherichia coli. J Agric Food Chem, 2024, 72: 10420-7.

[2] Chen R, Shi F, Xiang Y, et al. Establishment of CRISPR-Cpf1-assisted gene editing tool and engineering of 4-hydroxyisoleucine biosynthesis in Corynebacterium glutamicum. World J Microbiol Biotechnol, 2023, 39: 266.

[3] Chen Z, Wang R, Song Y, et al. Expression and transformation characteristics of a novel glutamic ccid decarboxylase LcGAD10s and its application on sufu processing. Foods, 2023, 12: 3186.

[4] Choi JW, Yim SS, Lee SH, et al. Enhanced production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium glutamicum by expressing glutamate decarboxylase active in expanded pH range. Microb Cell Fact, 2015, 14: 21.

[5] Engels V, Wendisch VF. The DeoR-type regulator SugR represses expression of ptsG in Corynebacterium glutamicum. J Bacteriol, 2007, 189: 2955-66.

[6] Feng YF, Zhou YY, Duan KM. The role of extrasynaptic GABA receptors in postpartum depression. Mol Neurobiol, 2023, 61: 385-96.

[7] Ge F, Sun J, Ren Y, et al. Transcriptomic and enzymatic analysis reveals the roles of glutamate dehydrogenase in Corynebacterium glutamicum. AMB Express, 2022, 12: 4.

[8] Hu C, Shi F, Chen R, et al. Rearrangement in the regulation of sigD gene expression promotes 4-hydroxyisoleucine production in Corynebacterium glutamicum. Syst Microbiol Biomanuf, 2024, 5: 276-87.

[9] Jorge JMP, Nguyen AQD, Pérez García F, et al. Improved fermentative production of gamma-aminobutyric acid via the putrescine route: Systems metabolic engineering for production from glucose, amino sugars, and xylose. Biotechnol Bioeng, 2016, 114: 862-73.

[10] Krug A, Wendisch VF, Bott M. Identification of AcnR, a TetR-type repressor of the aconitase gene acn in Corynebacterium glutamicum. J Biol Chem, 2005, 280: 585-95.

[11] Langa S, Peirotén Á, Rodríguez S, et al. GABA- and riboflavin-enriched soy and oat beverages using selected lactic acid bacteria. Innov Food Sci Emerg Technol, 2025, 104: 104108.

[12] Li L, Dou N, Zhang H, et al. The versatile GABA in plants. Plant Signal Behav, 2021, 16: 1862525.

[13] Li H, Xu D, Tan X, et al. The role of trehalose biosynthesis on mycolate composition and L-glutamate production in Corynebacterium glutamicum. Microbiol Res, 2023, 267: 127260.

[14] Li H, Yang H, Shi F. Metabolic engineering Corynebacterium glutamicum for production of γ-aminobutyric acid by glutamate decarboxylase active at near-neutral pH and displaying at cell surface. Process Biochem, 2025, 157: 75-83.

[15] Lie Y, Farmer TJ, Macquarrie DJ. Facile and rapid decarboxylation of glutamic acid to γ-aminobutyric acid via microwave-assisted reaction: Towards valorisation of waste gluten. J Clean Prod, 2018, 205: 1102-13.

[16] Luo H, Liu Z, Xie F, et al. Microbial production of gamma-aminobutyric acid: applications, state-of-the-art achievements, and future perspectives. Crit Rev Biotechnol, 2021, 41: 491-512.

[17] Nie L, He Y, Hu L, et al. Improvement in l-ornithine production from mannitol via transcriptome-guided genetic engineering in Corynebacterium glutamicum. Biotechnol Biofuels Bioprod, 2022, 15: 94.

[18] Peerboom C, Wierenga CJ. The postnatal GABA shift: A developmental perspective. Neurosci Biobehav Rev, 2021, 124: 179-92.

[19] Schmollack M, Werner F, Huber J, et al. Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production. Biotechnol Biofuels Bioprod, 2022, 15: 138.

[20] Shi F, Li Y. Synthesis of γ-aminobutyric acid by expressing Lactobacillus brevis-derived glutamate decarboxylase in the Corynebacterium glutamicum strain ATCC 13032. Biotechnol Lett, 2011, 33: 2469-74.

[21] Shi F, Jiang J, Li Y, et al. Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis. J Ind Microbiol Biotechnol, 2013, 40: 1285-96.

[22] Shi F, Zhang M, Li Y. Overexpression of ppc or deletion of mdh for improving production of γ-aminobutyric acid in recombinant Corynebacterium glutamicum. World J Microbiol Biotechnol, 2017, 33: 122.

[23] Shi F, Luan M, Li Y. Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing γ-aminobutyrate in Corynebacterium glutamicum. AMB Express, 2018, 8: 61.

[24] Stannek L, Thiele MJ, Ischebeck T, et al. Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis. Environ Microbiol, 2015, 17: 3379-90.

[25] Sun L, Bai Y, Zhang X, et al. Characterization of three glutamate decarboxylases from Bacillus spp. for efficient γ-aminobutyric acid production. Microb Cell Fact, 2021, 20: 153.

[26] Taniguchi H, Busche T, Patschkowski T, et al. Physiological roles of sigma factor SigD in Corynebacterium glutamicum. BMC Microbiol, 2017, 17: 171.

[27] Wang Y, Zhang F, Xu J, et al Improvement of l-Leucine production in Corynebacterium glutamicum by altering the redox flux. Int J Mol Sci. 2019;20(2020). https://doi.org/10.3390/ijms20082020.

[28] Wang J, Ma W, Zhou J, et al. Microbial chassis design and engineering for production of gamma-aminobutyric acid. World J Microbiol Biotechnol, 2024, 40: 145.

[29] Wang Y, Yao C, Huang D, et al. Metabolic engineering of Corynebacterium glutamicum CGY-PG-304 for promoting gamma-aminobutyric acid production. Syst Microbiol Biomanuf, 2024, 4: 915-27.

[30] Wang J, Xu Y, Song Z, et al. Stepwise metabolic engineering of a plasmid-free Corynebacterium glutamicum for efficient production of γ-aminobutyric acid (GABA) by co-utilizing lignocellulosic feedstock-derived sugars. J Biotechnol, 2025, 406: 281-4.

[31] Wen J, Bao J. Improved fermentative γ-aminobutyric acid production by secretory expression of glutamate decarboxylase by Corynebacterium glutamicum. J Biotechnol, 2021, 331: 19-25.

[32] Wu Y, Chen Y, Wei N. Biocatalytic properties of cell surface display laccase for degradation of emerging contaminant acetaminophen in water reclamation. Biotechnol Bioeng, 2019, 117: 342-53.

[33] Xu Y, Zhang B, Bao J. Production of γ-aminobutyric acid using corncob residue as carbohydrate feedstock by engineered Corynebacterium glutamicum. Biochem Eng J, 2025, 215: 109629.

[34] Yang S, Li Y, Hong W. Construction of a co-reaction system for ethanol-promoted gamma-aminobutyric acid synthesis by Pediococcus pentosaceus. Amino Acids, 2025, 57: 36.

[35] Yao C, Shi F, Wang X. Chromosomal editing of Corynebacterium glutamicum ATCC 13032 to produce gamma-aminobutyric acid. Biotechnol Appl Biochem, 2022, 70: 7-21.

[36] Yarabbi H, Mortazavi SA, Yavarmanesh M, et al. Molecular cloning, gene overexpression and characterization of glutamate decarboxylase from Enterococcus faecium DO. LWT, 2021, 148: 111699.

[37] Zhang Y, Zhao J, Wang X, et al. Model-guided metabolic rewiring for gamma-aminobutyric acid and butyrolactam biosynthesis in Corynebacterium glutamicum ATCC13032. Biology, 2022, 11: 846.

[38] Zhu N, Xia H, Yang J, et al. Improved succinate production in Corynebacterium glutamicum by engineering glyoxylate pathway and succinate export system. Biotechnol Lett, 2013, 36: 553-60.

Rights & permissions

Jiangnan University