Engineering leucine dehydrogenase to enhance L-2-aminobutyric acid production in Escherichia coli

Abstract

L-2-aminobutyric acid (L-ABA) is a non-proteinogenic chiral α-amino acid and, as an important chemical feedstock and pharmaceutical intermediate, has broad application potential in the pharmaceutical and fine chemical industries. In this study, Escherichia coli TWF106 was used as the chassis strain for L-ABA production. In strain TWF106, we individually deleted rhtA, rhtC, ptsG and gabP to optimize the metabolic flux toward L-ABA production, and constructed multiple single-deletion and double-deletion strains. The reductive amination of 2-ketobutyric acid (2-KB) to L-ABA catalyzed by leucine dehydrogenase (LeuDH) is considered a potential bottleneck in this pathway; therefore, we engineered LeuDH to improve the L-ABA titer. By substituting the key residue Ser³³¹ in LeuDH with different amino acids, 19 mutants were constructed and tested for L-ABA production. Two mutants, LeuDH^S331R and LeuDH^S331C, showed the greatest improvement in L-ABA production, the L-ABA titers of TWF106-331R and TWF106-331 C reached 5.46 g/L and 5.45 g/L respectively. Molecular docking and molecular dynamics simulations revealed that mutations at Ser³³¹ altered the static binding mode and modulated the dynamic binding mechanism of the protein–ligand complex. Subsequently, by combining metabolic flux optimization with site-directed mutagenesis, TWF1604-331 C was constructed through deletion of gabP in TWF106 coupled with introduction of plasmid pB-AmDH^S331C. TWF1604-331 C could produce 6.47 g/L L-ABA. Finally, fed-batch fermentation of TWF1604-331 C was conducted in a 2-L bioreactor, achieving an L-ABA titer of 16.58 g/L.

Keywords

Escherichia coli / L-ABA / gabP / LeuDH / Molecular docking / Molecular dynamics simulation

References

[1] Balderas-Hernandez VE, Landeros Maldonado KP, Sánchez A, Smoliński A, De Leon Rodriguez A. Improvement of hydrogen production by metabolic engineering of Escherichia coli: Modification on both the PTS system and central carbon metabolism. Int J Hydrog Energy, 2020, 45(9): 5687-96

[2] Brodie MJ, Perucca E, Ryvlin P, Ben-Menachem E, Meencke HJ. Comparison of levetiracetam and controlled-release carbamazepine in newly diagnosed epilepsy. Neurology, 2007, 68(6): 402-8

[3] Chen J, Zhu R, Zhou J, Yang T, Zhang X, Xu M, et al. . Efficient single whole-cell biotransformation for L-2-aminobutyric acid production through engineering of leucine dehydrogenase combined with expression regulation. Bioresour Technol, 2021, 326: 124665

[4] Fang Y, Wang J, Ma W, Yang J, Zhang H, Zhao L, et al. . Rebalancing microbial carbon distribution for L-threonine maximization using a thermal switch system. Metab Eng, 2020, 61: 33-46

[5] Gabor E, Gohler AK, Kosfeld A, Staab A, Kremling A, Jahreis K. The phosphoenolpyruvate-dependent glucose-phosphotransferase system from Escherichia coli K-12 as the center of a network regulating carbohydrate flux in the cell. Eur J Cell Biol, 2011, 90(9): 711-20

[6] Galkin A, Kulakova L, Yoshimura T, Soda K, Esaki N. Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes. Appl Environ Microbiol, 1997, 63(12): 4651-6

[7] Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol, 2023, 11: 1118948

[8] Hu LA, King SC. Functional sensitivity of polar surfaces on transmembrane helix 8 and cytoplasmic loop 8–9 of the Escherichia coli GABA (4-aminobutyrate) transporter encoded by gabP: mutagenic analysis of a consensus amphipathic region found in transporters from bacteria to mammals. Biochem J, 1998, 330(Pt 2): 771-6

[9] Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol, 2015, 81(7): 2506-14

[10] King SC, Fleming SR, Brechtel CE. Ligand recognition properties of the Escherichia coli 4-aminobutyrate transporter encoded by gabP. Specificity of Gab permease for heterocyclic inhibitors. J Biol Chem, 1995, 270(34): 19893-7

[11] Korös A, Varga Z, Molnár-Perl I. Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography. J Chromatogr A, 2008, 1203(2): 146-52

[12] Kruse D, Krämer R, Eggeling L, Rieping M, Pfefferle W, Tchieu JH, et al. . Influence of threonine exporters on threonine production in Escherichia coli. Appl Microbiol Biotechnol, 2002, 59(2–3): 205-10

[13] Liao L, Zhang Y, Wang Y, Fu Y, Zhang A, Qiu R, et al. . Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine. Microb Cell Fact, 2021, 20(1): 3

[14] Liu W, Ma H, Luo J, Shen W, Xu X, Li S, et al. . Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli. Biochem Eng J, 2014, 91: 204-9

[15] Liu Y, Zhong X, Luo Z, Meng X, Li R, Zhong W, et al. . The identification of a robust leucine dehydrogenase from a directed soil metagenome for efficient synthesis of L-2-aminobutyric acid. Biotechnol J, 2023, 18(8): e2200590

[16] Liu J, Li R, Miao J, Sun H, Chen Q, Song H, et al. . A Cofactor Regeneration System for 2-Aminobutyric Acid Production Based on Combined Cross-Linked Enzyme Aggregates: Utilizing His-Tagged Enzymes With Low-Concentration Calcium Ions as Precipitant. Eng Life Sci, 2025, 25(3): e70013

[17] Livshits VA, Zakataeva NP, Aleshin VV, Vitushkina MV. Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli. Res Microbiol, 2003, 154(2): 123-35

[18] McCraw SL, Park DH, Jones R, Bentley MA, Rico A, Ratcliffe RG, et al. . GABA (gamma-Aminobutyric Acid) Uptake Via the GABA Permease GabP Represses Virulence Gene Expression in Pseudomonas syringae pv. tomato DC3000. Mol Plant Microbe Interact, 2016, 29(12): 938-49

[19] Niegemann E, Schulz A, Bartsch K. Molecular organization of the Escherichia coli gab cluster: nucleotide sequence of the structural genes gabD and gabP and expression of the GABA permease gene. Arch Microbiol, 1993, 160(6): 454-60

[20] Nowak C, Beer B, Pick A, Roth T, Lommes P, Sieber V. A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors. Front Microbiol, 2015, 6: 957

[21] Park JH, Lee KH, Kim TY, Lee SY. Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation. Proc Natl Acad Sci USA, 2007, 104(19): 7797-802

[22] Sheldon RA, Brady D. Green Chemistry, Biocatalysis, and the Chemical Industry of the Future. Chemsuschem, 2022, 15(9): e202102628

[23] Weber N, Hatsch A, Labagnere L, Heider H. Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae. Microb Cell Fact, 2017, 16(1): 51

[24] Xu JM, Li JQ, Zhang B, Liu ZQ, Zheng YG. Fermentative production of the unnatural amino acid L-2-aminobutyric acid based on metabolic engineering. Microb Cell Fact, 2019, 18(1): 43

[25] Xu JM, Wang M, Jin YH, Liu ZQ, Zheng YG. Development of a fermentation strategy to enhance the catalytic efficiency of recombinant Escherichia coli for l-2-aminobutyric acid production. 3 Biotech, 2021, 11(8): 387

[26] Xu J, Tao Y, Shan Q, Feng Y, Wang Y, Liu Z, et al. . Optimized Biosynthetic Pathway for Nonnatural Amino Acids: An Efficient Approach for L-2-Aminobutyric Acid Production. Biotechnol Bioeng, 2025, 122(7): 1856-72

[27] Zaprasis A, Hoffmann T, Stannek L, Gunka K, Commichau FM, Bremer E. The gamma-aminobutyrate permease GabP serves as the third proline transporter of Bacillus subtilis. J Bacteriol, 2014, 196(3): 515-26

[28] Zhang Z, Liu Y, Zhao J, Li W, Hu R, Li X, et al. . Active-site engineering of omega-transaminase from Ochrobactrum anthropi for preparation of L-2-aminobutyric acid. BMC Biotechnol, 2021, 21(1): 55

[29] Zhang L, Hong Y, Lu J, Wang Y, Luo W. Semi-rational engineering of ω-transaminase for enhanced enzymatic activity to 2-ketobutyrate. Enzym Microb Technol, 2024, 180: 110505

[30] Zhao G, Tang Y, Li Z, Liu G, Zhang D, Hu X, et al. . Engineering Corynebacterium glutamicum for efficient l-homoserine production. Bioresour Technol, 2025, 431: 132617

[31] Zhao G, Tang Y, Zhang D, Liu G, Li Z, Li R, et al. . Engineering homoserine kinase for competitive inhibition release to enhance l-threonine production in Corynebacterium glutamicum. Int J Biol Macromol, 2025, 321(Pt 4): 146536

[32] Zhao Z, Liu Y, Zhu R, Yang F, Liu Z, You J, et al. . De novo synthesis of L-2-aminobutyric acid in Escherichia coli based on multi-layered metabolic engineering strategies. Synth Syst Biotechnol, 2026, 12: 374-82

Funding

Basic Research Program of Jiangsu(No. BK20233003)

RIGHTS & PERMISSIONS

Jiangnan University