The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is found in abundance across microorganisms, plants, and animals [1]. In animals, GABA is a widely recognized inhibitory neurotransmitter within the central nervous system that has various physiological functions, including lowering blood pressure, tranquilizing the mind, and treating epilepsy [2]. Because of its distinctive properties, GABA has been widely applied in the functional food, feed, and pharmaceutical sectors [3]. Moreover, GABA serves as a starting material for the environmentally friendly production of various chemicals, including N-methylpyrrolidone and polyamide 4 [4].

With the growing market demand for GABA, many studies have focused on developing efficient biological synthesis methods using bacteria due to their straightforward reaction processes, environmental friendliness, and gentle reaction conditions [5]. Bacterial GABA biosynthesis is mediated by glutamate decarboxylase (GAD, EC 4.1.1.15), an enzyme dependent on pyridoxal 5′-phosphate (PLP). This enzyme facilitates the irreversible α-decarboxylation of L-glutamate (L-Glu) into GABA in a single step [6]. Numerous endeavors have focused on developing highly efficient bacterial strains for GABA production. These efforts encompass GABA biosynthesis through the screening of lactic acid bacteria for fermentation and implementation of metabolic engineering in Escherichia coli and Corynebacterium glutamicum. These engineering approaches involve the introduction of genes encoding GAD enzymes for de novo fermentation. Another avenue for GABA synthesis involves biotransformation within whole-cell systems. When viewed from both industrial and economic perspectives, the use of engineered E. coli for whole-cell catalysis stands out due to its advantages, including rapid reaction kinetics and minimized byproduct formation [7]. However, certain drawbacks restrict its industrial implementation. The utilization of whole-cell biocatalysts encounters significant constraints owing to the presence of the cell envelope, which serves as a formidable obstacle to efficient mass transfer, consequently hindering the movement of reactants and resulting compounds [8]. To achieve a high GABA biotransformation rate, physical or chemical treatment is required before the bioconversion reaction to increase cell permeability. This leads to the requirement for cumbersome processes or contamination due to the introduction of external chemicals [4], [9]. In addition, PLP plays a crucial role in regulating the proton translocation necessary for the GAD-catalyzed decarboxylation of L-Glu. E. coli whole-cell catalyst cannot provide sufficient PLP for this biotransformation reaction [10], [11], and supplementation with PLP has been explored to improve GABA production [11]. However, the application of exogenous PLP leads to increased expenses [12]. Furthermore, L-Glu and L-monosodium glutamate (L-MSG) can both be used as substrates for the enzymatic conversion to GABA by GAD. L-MSG is a cheaper substrate that dissolves more easily in water than L-Glu [3]. GAD activity represents a crucial mechanism underlying the resistance to acidic environments in microorganisms, which is why the majority of GADs are functional under low pH conditions [13]. Therefore, the pH level of the biotransformation system must be regulated by adding extra acidic substances when utilizing less-expensive L-MSG as a substrate [14].

In our previous study, a GAD gene (gadz11) was identified from Bacillus sp. Z11 (GenBank ID: MW703456). The optimal reaction temperature and pH value for GADZ11 were 40 °C and 5.0, respectively. The purified recombinant GADZ11 showed high catalytic activity toward L-Glu with a specific activity of 98.9 ± 6.5 U/mg. Then, we engineered an E. coli BL21(DE3) strain by overexpressing gadz11 to serve as a whole-cell biocatalyst for efficient GABA biosynthesis from L-Glu in a buffer-free reaction through the utilization of an ultra-low-temperature freeze-thaw treatment and the inclusion of PLP in the transformation process [10]. The objective of this research was to create a convenient and highly effective whole-cell biocatalyst through the sequential modification of the recombinant E. coli strain above (Fig. 1). We attempted to increase cell permeability by overexpressing sulA and constructed a PLP self-sufficient system that did not require an additional supply of PLP for the reaction. Moreover, various factors affecting GABA biosynthesis were optimized, and a bioconversion process employing whole cells was used for GABA synthesis in a 3-L bioreactor. This study provides an effective method for GABA production using an engineered whole-cell catalyst.