Whole Genome Sequencing and Comparative Genomic Analysis of a High-yielding γ-aminobutyric Acid-producing Lactobacillus brevis TCCC13007

doi:10.13560/j.cnki.biotech.bull.1985.2025-0843

Abstract

Abstract:

Objective Lactobacillus brevis TCCC13007 was isolated from northeastern Chinese pickled cabbage as a high γ-aminobutyric acid (GABA)-producing strain. Whole-genome sequencing and assembly were performed to analyze its complete genomic sequence information. Comparative genomic analysis was conducted to investigate the molecular mechanism underlying the high GABA production of strain TCCC13007. Method Sequencing was conducted by employing second-generation Illumina HiSeq technology and third-generation sequencing on the PacBio platform. Combined second- and third-generation sequencing data were used for correction, and whole genome sequencing of strain TCCC13007, as well as gene prediction, functional annotation, and comparative genomic analysis. Result The TCCC13007 genome had a total length of 2.58 Mb with a GC content of 45.72% and contained 2 581 protein-coding genes. Comparative genomic analysis revealed that TCCC13007 was most closely related to the low-GABA-producing strain YSJ3, and the two had strong synteny alongside minor genomic rearrangements such as insertions, inversions, and translocations. Compared to YSJ3, the gadA gene in TCCC13007 was noticeably relocated within the genome, and a mutation at position 104 of the GadR protein sequence resulted in a glycine substitution. Additionally, TCCC13007 harbored 308 unique genes. These unique genes appeared to synergistically enhance glutamate precursor supply, maintain the acidic intracellular environment required for glutamate decarboxylase (GAD) activation, optimize transmembrane transport systems, ensure cofactor and energy supply, and modulate regulatory networks, thereby conferring high GABA synthesis capacity on TCCC13007. Conclusion Whole-genome sequencing and comparative genomic analysis of strain TCCC13007 reveal that its high GABA-producing characteristic is not due to significant genome expansion, but may be attributed to a more refined and efficient gene combination and its regulatory network.

Key words: γ-aminobutyric acid (GABA), Lactobacillus brevis, whole-genome sequencing, gene functional annotation, comparative genomics

MIAO Hao-xiang, ZHANG Ying, GUO Shi-peng, ZHANG Jian. Whole Genome Sequencing and Comparative Genomic Analysis of a High-yielding γ-aminobutyric Acid-producing Lactobacillus brevis TCCC13007[J]. Biotechnology Bulletin, 2025, 41(11): 166-176.

Figures/Tables 8

References 32

[1]	许芮菁, 姜铖, 刘明川, 等. γ-氨基丁酸的检测方法及其功能性食品研究进展 [J]. 食品工业, 2025, 46(2): 150-155.
	Xu RJ, Jiang C, Liu MC, et al. Research progress on detection method and functional food of γ-aminobutyric acid [J]. Food Ind, 2025, 46(2): 150-155.
[2]	李瑶, 王雷, 孙鑫, 等. γ-氨基丁酸生物学功能研究进展 [J]. 农业与技术, 2024, 44(10): 12-14.
	Li Y, Wang L, Sun X, et al. Research progress on biological function of γ-aminobutyric acid [J]. Agric & Technol, 2024, 44(10): 12-14.
[3]	贾世杰, 刘江花, 李国梁. γ-氨基丁酸生物合成研究进展 [J]. 食品研究与开发, 2023, 44(23): 174-181.
	Jia SJ, Liu JH, Li GL. Recent advances in gamma-aminobutyric acid biosynthesis [J]. Food Res Dev, 2023, 44(23): 174-181.
[4]	赵静宇. 一种γ-氨基丁酸双组分生物传感器的设计与构建 [D]. 济南: 山东大学, 2023.
	Zhao JY. Design and construction of a two-component biosensor for gamma-aminobutyric acid [D]. Jinan: Shandong University, 2023.
[5]	林杨, 孙建, 顾美英, 等. 富含γ-氨基丁酸功能性乳酸菌饮品的配方及稳定性研究 [J]. 中国酿造, 2021, 40(8): 215-221.
	Lin Y, Sun J, Gu MY, et al. Formulation and stability of functional lactic acid bacteria beverage rich in γ-aminobutyric acid [J]. China Brew, 2021, 40(8): 215-221.
[6]	王世依, 赵毅雯, 贾田丽, 等. 微生物法合成γ-氨基丁酸的研究进展 [J]. 华中农业大学学报, 2024, 43(4): 94-101.
	Wang SY, Zhao YW, Jia TL, et al. Progress in microbial synthesis of gamma-aminobutyric acid [J]. J Huazhong Agric Univ, 2024, 43(4): 94-101.
[7]	金玥茹昕. 采后L-谷氨酸处理对早酥梨果实贮藏品质的影响 [D]. 锦州: 渤海大学, 2024.
	Jin Y. Effects of L-glutamate treatment after harvest on storage quality of zaosu pear fruit [D]. Jinzhou: Bohai University, 2024.
[8]	郭新月. 葡萄糖流加刺激短乳杆菌合成GABA及SPN-PCR基因组步移方法的建立 [D]. 南昌: 南昌大学, 2024.
	Guo XY. Stimulation of GABA synthesis by Lactobacillus brevis through glucose feeding and the establishment of the SPN-PCR genome walking method [D]. Nanchang: Nanchang University, 2024.
[9]	徐鹏. 以鱼蛋白胨为底物发酵生产γ-氨基丁酸及应用研究 [D]. 杭州: 浙江大学, 2023.
	Xu P. Gamma-aminobutyric acid fermentation technology using fish peptone as substrate and the applications [D]. Hangzhou: Zhejiang University, 2023.
[10]	Rehman A, Di Benedetto G, Bird JK, et al. Development of a workflow for the selection, identification and optimization of lactic acid bacteria with high γ-aminobutyric acid production [J]. Sci Rep, 2023, 13(1): 13663.
[11]	Zhang Y, Zhu MJ, Lu WJ, et al. Optimizing levilactobacillus brevis NPS-QW 145 fermentation for gamma-aminobutyric acid (GABA) production in soybean sprout yogurt-like product [J]. Foods, 2023, 12(5): 977.
[12]	Zhang JM, Liu DY, Zhang CC, et al. The impact of Levilactobacillus brevis YSJ3 and Lactiplantibacillus plantarum JLSC2-6 co-culture on gamma-aminobutyric acid yield, volatile and non-volatile metabolites, antioxidant activity, and bacterial community in fermented cauliflower byproducts [J]. Food Chem, 2024, 432: 137169.
[13]	Banerjee S, Poore M, Gerdes S, et al. Transcriptomics reveal different metabolic strategies for acid resistance and gamma-aminobutyric acid (GABA) production in select Levilactobacillus brevis strains [J]. Microb Cell Fact, 2021, 20(1): 173.
[14]	Marques TM, Patterson E, Wall R, et al. Influence of GABA and GABA-producing Lactobacillus brevis DPC 6108 on the development of diabetes in a streptozotocin rat model [J]. Benef Microbes, 2016, 7(3): 409-420.
[15]	Cataldo PG, Villena J, Elean M, et al. Immunomodulatory properties of a γ-aminobutyric acid-enriched strawberry juice produced by Levilactobacillus brevis CRL 2013 [J]. Front Microbiol, 2020, 11: 610016.
[16]	Phuengjayaem S, Pakdeeto A, Kingkaew E, et al. Genome sequences and functional analysis of Levilactobacillus brevis LSF9-1 and Pediococcus acidilactici LSF1-1 from fermented fish cake (Som-fak) with gamma-aminobutyric acid (GABA) production [J]. Funct Integr Genomics, 2023, 23(2): 158.
[17]	Jang SE, Han MJ, Kim SY, et al. Lactobacillus brevis G101 inhibits the absorption of monosodium glutamate in mice [J]. J Microbiol Biotechnol, 2014, 24(11): 1592-1596.
[18]	宫璐婵. 谷氨酸脱羧酶系统对短乳杆菌酸耐受性的影响及其调控机制与应用 [D]. 无锡: 江南大学, 2019.
	Gong LC. The effect of glutamate decarboxylase system on acid resistance and its regulatory mechanisms as well as application in Lactobacillus brevis [D]. Wuxi: Jiangnan University, 2019.
[19]	Liu DY, Zhang JM, Chen J, et al. Carrot-based fermentation juice rich in sleep-promoting components improved sleep in mice [J]. Front Nutr, 2022, 9: 1043055.
[20]	Huang J, Fang H, Gai ZC, et al. Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis [J]. Biochem Biophys Res Commun, 2018, 503(3): 1703-1709.
[21]	Kanehisa M, Goto S, Kawashima S, et al. The KEGG resource for deciphering the genome [J]. Nucleic Acids Res, 2004, 32(): D277-D280.
[22]	罗婷婷, 姚潇潇, 詹欣艺, 等. 基于代谢组学探讨氯胺酮致小鼠认知障碍的机制 [J]. 中国药房, 2025, 36(12): 1436-1441.
	Luo TT, Yao XX, Zhan XY, et al. Exploration of the mechanism of cognitive impairment induced by ketamine in mice based on metabolomics [J]. China Pharm, 2025, 36(12): 1436-1441.
[23]	徐绮思. 呋虫胺在黄酒酿造过程中残留行为及其对风味品质的影响 [D]. 保定: 河北农业大学, 2023.
	Xu QS. Residual behaviour of dinotefuran in Huangjiu brewing and its effect on flavour quality [D]. Baoding: Hebei Agricultural University, 2023.
[24]	Chen R, Xiang YH, Yu XP, et al. Comparative transcriptome analysis of global effect of ddh and lysE deletion on 4-hydroxyisoleucine production in Corynebacterium glutamicum [J]. Syst Microbiol Biomanuf, 2022, 2(3): 542-554.
[25]	陈芬. 基于香菇菇脚饲料化转化的益生菌发酵工艺及其代谢组学研究 [D]. 重庆: 重庆大学, 2022.
	Chen F. Study on probiotic fermentation technics and metabolomics of feed bioconversion using Lentinus edodes feet [D]. Chongqing: Chongqing University, 2022.
[26]	Thangaraj K, Li JJ, Mei HL, et al. Mycorrhizal colonization enhanced sorghum bicolor tolerance under soil water deficit conditions by coordination of proline and reduced glutathione (GSH) [J]. J Agric Food Chem, 2022, 70(14): 4243-4255.
[27]	陈琳. 罗伊氏乳杆菌果糖代谢通路分析及其益生特性研究 [D]. 杭州: 浙江工商大学, 2020.
	Chen L. Analysis of fructose metabolic pathway and probiotic characteristics of Lactobacillus reuteri . WHH1689 [D]. Hangzhou: Zhejiang Gongshang University, 2020.
[28]	赵天源, 王菁, 王雨露, 等. 甜菜碱神经保护作用的研究进展 [J]. 生物技术进展, 2025, 15(2): 220-225.
	Zhao TY, Wang J, Wang YL, et al. Research progress on neuroprotective effects of betaine [J]. Curr Biotechnol, 2025, 15(2): 220-225.
[29]	栗晓静. 干酪乳杆菌外源蛋白分泌系统的优化及潜在应用 [D]. 济南: 山东大学, 2024.
	Li XJ. Optimization and potential application of heterologous protein secretion system in Lacticaseibacillus casei [D]. Jinan: Shandong University, 2024.
[30]	Wagner M, Blum D, Raschka SL, et al. A new twist in ABC transporter mediated multidrug resistance-Pdr5 is a drug/proton co-transporter [J]. J Mol Biol, 2022, 434(14): 167669.
[31]	王瑞敏. 酿酒酵母生产γ-氨基丁酸的代谢工程改造及发酵优化 [D]. 济南: 山东大学, 2024.
	Wang RM. Metabolic engineering and fermentation optimization for gamma-aminobutyric acid production by Saccharomyces cerevisiae . [D]. Jinan: Shandong University, 2024.
[32]	Si MR, Chen C, Zhong JY, et al. MsrR is a thiol-based oxidation-sensing regulator of the XRE family that modulates C. glutamicum oxidative stress resistance [J]. Microb Cell Fact, 2020, 19(1): 189.

短乳杆菌菌株 L. brevis strain	基因组大小 Genome size （Mb）	GC含量 GC content （%）	蛋白编码基因 Protein-coding genes	质粒数量 Number of plasmids	登陆号 Accession number	GABA产量 GABA yield （g/L）	参考文献 References
TCCC13007	2.58	45.72	2581	5	PRJNA1290704	发酵180，转化280	本研究
CD0817	3.10	50.35	2990	4	PRJNA495163	404	［8］
NPS-QW-145	2.55	45.80	2391	0	PRJNA318996	2.302	［11］
YSJ3	2.5	46	2449	7	PRJNA804971	0.035	［12］
ATCC 14869	2.5	46	2630	0	PRJNA38325	0.05	［13］
DPC 6108	2.9	45	2743	0	PRJNA339712	16	［14］
CRL 2013	2.6	45.5	2543	0	PRJNA378171	26.9	［15］
LSF9-1	2.5	45.5	2370	0	PRJNA916067	16.935	［16］
G101	2.7	45.5	2467	0	PRJNA369571	3.542	［17］
ATCC 367	2.34	46.04	2180	2	PRJNA404	6.44	［18］

短乳杆菌菌株 L. brevis strain	基因组大小 Genome size （Mb）	GC含量 GC content （%）	蛋白编码基因 Protein-coding genes	质粒数量 Number of plasmids	登陆号 Accession number	GABA产量 GABA yield （g/L）	参考文献 References
TCCC13007	2.58	45.72	2581	5	PRJNA1290704	发酵180，转化280	本研究
CD0817	3.10	50.35	2990	4	PRJNA495163	404	［8］
NPS-QW-145	2.55	45.80	2391	0	PRJNA318996	2.302	［11］
YSJ3	2.5	46	2449	7	PRJNA804971	0.035	［12］
ATCC 14869	2.5	46	2630	0	PRJNA38325	0.05	［13］
DPC 6108	2.9	45	2743	0	PRJNA339712	16	［14］
CRL 2013	2.6	45.5	2543	0	PRJNA378171	26.9	［15］
LSF9-1	2.5	45.5	2370	0	PRJNA916067	16.935	［16］
G101	2.7	45.5	2467	0	PRJNA369571	3.542	［17］
ATCC 367	2.34	46.04	2180	2	PRJNA404	6.44	［18］