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Effect of induction temperatures on GAD expression and GABA production. (a) SDS-PAGE analysis of the GAD expression under different induction temperatures. M represents molecular weight markers; 1–5 represents supernatants of the cell extract from different induction temperatures (25, 28, 30, 33 and 37 °C). (b) GABA production of whole-cell bioconversion (square) and the supernatant of the cell lysate as the catalyst (diamond) within 3 h, and cell density (triangle) from different induction temperatures. The cells induced by different induction temperatures were applied as the catalysts for the bioconversion using 1 M L-Glu solution as the substrate at 45 °C. Data are presented as the mean ± SD values from three independent experiments.
Source publication
Gamma-aminobutyric acid (GABA) can be converted into 2-pyrrolidone, an intermediate in the synthesis of nylon 4 and agrochemicals, which has great potential for application in the chemical industry. The main aim of this work was to construct a simple and efficient process for industrial production of GABA from L-glutamic acid (L-Glu) by whole-cell...
Citations
... The optimal induction conditions for the whole-cell biocatalyst were explored using a single-factor experimental design. Recombinant cells were subjected to induction at various temperatures (16, 20, 25, 30, and 37°C) for varying durations (2, 4, 6, 10, and 16 h) using 1 mmol/L IPTG [40]. The experiments were replicated three times for each set. ...
Background
γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has extensive applications in the food, feed, pharmaceutical, and chemical synthesis fields. The utilization of engineered Escherichia coli in whole-cell catalysis offers a promising approach for GABA synthesis based on the rapid reaction kinetics and reduced byproduct formation. Previously, we constructed a recombinant E. coli that efficiently converts L-glutamate (L-Glu) to GABA; however, freezing and thawing of the strain and the addition of external pyridoxal 5′-phosphate (PLP) were required. The objective of this investigation was to enhance the efficiency of GABA synthesis through E. coli genetic modifications to achieve a more streamlined production process.
Results
First, the inducible expression conditions of the gad gene were optimized to 37°C for 6 h. Next, cell permeability was improved by overexpressing sulA in E. coli, which eliminated the need for the freeze-thaw treatment during GABA production. The overexpression of pdxS and pdxT from Bacillus subtilis strain 168 resulted in an ideal engineered strain without the addition of external PLP. Thus, an efficient whole-cell biocatalytic process was optimized. The ideal isopropyl β-D-thiogalactopyranoside concentration, cellular density, and reaction temperature were 0.2 mmol/L, 15 units, and 37°C, respectively, and the substrate consisted of a 4:1 ratio of L-glutamic acid (L-Glu) to L-monosodium glutamate (L-MSG). Ultimately, the optimized conditions were employed for a bioconversion procedure using whole cells in a 3 L bioreactor. The microbial strain was capable of being utilized for a minimum of two cycles with 1 mol/L substrate mixtures, thus achieving a GABA productivity of 103.1 g/L/h and a molar yield of 100.0%.
Conclusion
A whole-cell catalyst for highly efficient GABA production from a mixture of L-Glu and L-MSG was constructed by engineering E. coli, and the freeze-thaw steps and external PLP addition were not required. This research illustrates that the recently engineered strain of E. coli exhibits promise for utilization in the large-scale industrial synthesis of GABA.
... However, for the enzyme synthesis reaction, an optimum induction time of 10 hours was determined based on the analysis shown in Figure 3 (B) and (C). The levels of soluble protein expression remained constant at different induction times, while slight decreases in yield production were observed with increasing induction times [21].This indicates that induction time is a critical factor affecting recombinant E. coli growth and protein yield production [22]. ...
Ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE) is used as an intermediate in the production of angiotensin converting enzyme (ACE) inhibitors for treating various medical conditions. The study used Stereospecific carbonyl reductase (KmCR) enzyme for the asymmetric synthesis of (R)-HPBE. The optimization process involved varying IPTG concentration, induction temperatures, and induction times to determine the optimal culture conditions for producing (R)-HPBE with increased yields. The most efficient culture conditions were determined as 0.2 mM IPTG concentration, 17 °C temperature, and 10 hours of induction. Additionally, the study explored various reaction parameters, including temperature, substrate concentration, enzyme concentration, co-solvents, and their ratios, to optimize the reaction conditions for synthesizing (R)-HPBE with higher productivity. The optimal reaction conditions were found at 25 °C, substrate concentration of 10.3 g·L-1 , enzyme concentration of 50 g·L-1 , and isopropanol co-solvent with a ratio of 10%. The optimized conditions produced 62% yield of (R)-HPBE with an optical purity of ≥99.9% and a reaction time of 20 minutes. These findings provide valuable insights for potential large-scale production using the identified optimal parameter concentrations. Keywords Kluyveromyces marxianus carbonyl reductase (KmCR), (R)-2-hydroxy-4-phenylbutyrate, ethyl-2-oxo-4-phenylbutyrate, asymmetric transformation, stereospecificity Khan S et al The Pharmaceutical and Chemical Journal, 2023, 10(3):73-85 The Pharmaceutical and Chemical Journal 74 1. Introduction Ethyl (R)-2-hydroxy-4-phenylbutanoate ((R)-HPBE) is one of the important intermediate of angiotensin converting enzyme inhibitors [1]. ACE inhibitors is the key and prominent group of drugs that are used for controlling different diseases such as treating heart failure, blood pressure, preventing kidney damage, and preventing strokes, in people with diabetes or hypertension [2]. (R)-HPBE can be synthesized by resolution of racemic ethyl 2-hydroxy-4-phenylbutyrate (HPBE) with a chemical catalyst or lipase [3, 4]. The resolution conversion of the racemate can be as high as 50%. (R)-HPBE can be obtained by asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate using a microorganism as a catalyst, while the conversion of the substrate can theoretically reach 100%. The microorganism can convert a small amount of substrate because the reducing ability of cells can be suppressed by a large amount of substrate. In the current year different approaches have been reported for the preparation of (R)-HPBE, mostly in two ways: synthesis and kinetic resolution. However the resolution methods are very low because the maximum yield can be reached to 50% theoretically [4], and the chemical synthesis method normally involve in many stages and the reaction condition is stringent [5]. Ethyl 2-oxo-4-phenyl-butyrate (OPBE) is a prominent way to produce optically active (R)-HPBE, by microbial and enzymatic reduction process. meanwhile ethyl 2-oxo-4-phenyl-butyrate can be synthesized easily and quietly cheap. Numerous biocatalysts has been used for the synthesis of (R)-HPBE, such as hydrolysis and transesterification by lipase [6] and the OPBE reduction catalyzed by whole cells [5, 7] and dehydrogenase [8]. Stoichiometric volumes of NADH cofactors is require for the reduction reaction, isolated enzyme rather than whole cell were used specially to avoid cofactor addition and purification of enzyme [9]. However, during the last decade, numerous microorganisms have been recognized as an effective biocatalyst in OPBE reduction to (R)-HPBE. Chadha et al reported that, the reduction of enantioselective of OPBE to (R)-HPBE can be achieved using callus cell-free aqueous extracts of Daucus carota (wild carrot) in high yield (90%) and enantiomeric excess (ee) (99%) [10]. However, their process required a high cell/substrate ratio of 100:1, high cell numbers, and a long reaction time of 10 days. Dao et al. and Lacerda et al. reported and achieved an effective reduction of OPBE with Saccharomyces cerevisiae and Pichia angusta, respectively, producing (R)-HPBE with a reasonable enantioselectivity (81% ee) [7, 11]. Recently, Chen et al. using Candida boidinii CIOC21 [12] and described the preparation of successful (R)-HPBE by a yield (92%) and favorable ee (99%). However, comparatively low substarte concentrations is about 4.1 g·L-1 and product 3.8 g/L in their process, which limits their use in large-scale production. Furthermore, Zhang et al. obtained (R)-HPBE from 20 g/L OPBE with moderate yield (82%) and excellent ee (97.4%) [5]. In past [13] Rhodotorula mucilaginosa CCZU-G5 yeast strain was isolated from vineyard soil samples and used it for the preparation of (R)-HPBE with high enantiomeric excess and high yields. However, strong substrate inhibition was detected when the concentration of test OPBE in the monophasic aqueous system was high due to the strong hydrophobicity of the substrate and its toxicity to cells, and the concentration of substrate was high and that the bacteria transformed was only 50 mM. A two-phase water/organic solvent system is a good alternative to solve the above problems in an aqueous system. In a biphasic system, the organic solvent phase serves as a substrate reservoir and protects the cells in the aqueous phase from being harmed by a high substrate concentration.This two-stage system has attracted a lot of attention in the last decades and several successful examples have been reported [14, 15]. In a preliminary study, Gene mining was used to identify the carbonyl-reducing enzymes from Candida albicans (CaCR), Saccharomyces cerevisiae (ScCR), Kluyveromyces marxianus (KmCR), and Candida parapsilosis (CPR-C1, CPRC2). These enzymes are anticipated to solve the issue of enzymatic reactions involving large substrates, such as ketoesters and heterocyclic ketones, which serve as precursors for the synthesis of crucial pharmacological intermediates. The newly identified enzymes' biocatalytic abilities towards the tested carbonyl substrates were also assessed [16]. In this study, the biocatalytic efficiency was improved by optimize the conditions for culturing the recombinant E. coli that which temperature, time, and inducer were optimized for high production of enzyme (KmCR), to ensure a balance between expression and growth. Furthermore, the enzymatic reaction conditions were optimized for catalyzing the substrate under increased concentration and producing high yield of corresponding
... Several mutational approaches such as directed evolution and site-specific mutagenesis are considered as powerful tools for optimizing or improving enzyme properties. Several researchers have applied these approaches to improve GAD activity [84,[97][98][99][100][101] and were applied in whole-cell biocatalysts. In order to improve GAD activity over an expanded pH range, recombinant C. glutamicum cells were obtained by expressing L. brevis Lb85 GadB variants. ...
Glutamate decarboxylase (l-glutamate-1-carboxylase, GAD; EC 4.1.1.15) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the irreversible α-decarboxylation of l-glutamic acid to γ-aminobutyric acid (GABA) and CO 2. The enzyme is widely distributed in eukaryotes as well as prokaryotes, where it-together with its reaction product GABA-fulfils very different physiological functions. The occurrence of gad genes encoding GAD has been shown for many microorganisms, and GABA-producing lactic acid bacteria (LAB) have been a focus of research during recent years. A wide range of traditional foods produced by fermentation based on LAB offer the potential of providing new functional food products enriched with GABA that may offer certain health-benefits. Different GAD enzymes and genes from several strains of LAB have been isolated and characterized recently. GABA-producing LAB, the biochemical properties of their GAD enzymes, and possible applications are reviewed here.
... Several mutational approaches such as directed evolution and site-specific mutagenesis are considered as powerful tools for optimizing or improving enzyme properties. Several researchers have applied these approaches to improve GAD activity [83,[97][98][99][100] that was applied in whole-cell biocatalysts. In order to improve GAD activity over an expanded pH range, recombinant C. glutamicum cells were obtained by expressing L. brevis Lb85 GadB variants. ...
Glutamate decarboxylase (L-glutamate-1-carboxylase, GAD; EC 4.1.1.15) is a pyridoxal 5-phosphate-dependent enzyme, which catalyzes the irreversible α-decarboxylation of L-glutamic acid to γ-aminobutyric acid (GABA) and CO2. The enzyme is widely distributed in eukaryotes as well as prokaryotes, where it – together with its reaction product GABA - fulfils very different physiological functions. The occurrence of gad genes encoding GAD has been shown for many microorganisms, and GABA-producing lactic acid bacteria (LAB) have been a focus of research during recent years. A wide range of traditional foods produced by fermentation based on LAB offer the potential of providing new functional food products enriched with GABA that may offer certain health-benefits. Different GAD enzymes and genes from several strains of LAB have been isolated and characterized recently. GABA-producing LAB, biochemical properties of their GAD enzymes, and possible applications are reviewed here.
Recently, bio-based manufacturing processes of value-added platform chemicals and polymers in biorefineries using renewable resources have extensively been developed for sustainable and carbon dioxide (CO2) neutral chemical industry. Among them, bio-based diamines, aminocarboxylic acids, and diacids have been used as monomers for the synthesis of polyamides having different carbon numbers and ubiquitous and versatile industrial polymers and also as precursors for further chemical and biological processes to afford valuable chemicals. Until now, these platform bio-chemicals have successfully been produced by biorefinery processes employing enzymes and/or microbial host strains as main catalysts. In this review, we discuss recent advances in bio-based production of diamines, aminocarboxylic acids, and diacids, which has been developed and improved by systems metabolic engineering strategies of microbial host strains and optimization of microbial conversion processes including whole cell bioconversion and direct fermentative production in biorefineries.
γ-Aminobutyrate (GABA) is an important chemical by itself and can be further used for the production of monomer used for the synthesis of biodegradable polyamides. Until now, GABA production usingCorynebacterium glutamicum harboring glutamate decarboxylases (GADs) has been limited due to the discrepancy between optimal pH for GAD activity (pH 4.0) and cell growth (pH 7.0). In this study, we developed recombinant C. glutamicum strains expressing mutated GAD from Escherichia coli (EcGADmut) and GADs from Lactococcus lactis CICC20209 (LlGAD) and Lactobacillus senmaizukei (LsGAD), all of which showed enhanced pH stability and adaptability at a pH of approximately 7.0. In shake flask cultivations, the GABA productions of C. glutamicum H36EcGADmut, C. glutamicum H36LsGAD, and C. glutamicum H36LlGAD were examined at pH 5.0, 6.0, and 7.0, respectively. Finally, C. glutamicum H36EcGADmut (40.3 and 39.3 g L-1), H36LlGAD (42.5 and 41.1 g L-1), and H36LsGAD (41.6 and 40.2 g L-1) produced improved GABA titers and yields in batch fermentation at pH 6.0 and pH 7.0, respectively, from 100 g L-1 glucose. The recombinant strains developed in this study could be used for the establishment of sustainable direct fermentative GABA production from renewable resources under mild culture conditions, thus increasing the availability of various GADs.
Background
Gamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts l -glutamate ( l -Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use.
Results
Three GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards l -Glu (2.19 mM − 1 s − 1 ). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 14 times, the cells produced GABA with an average molar conversion rate of 98.6% within 14 h.
Conclusions
Three recombinant GADs from Bacillus strains have been conducted functional identification. The engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of l -Glu to GABA in a buffer-free reaction at a high l -Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA.
Fermentative production of γ-aminobutyric acid by the glutamate overproducing Corynebacterium glutamicum from cheap sugar feedstock is generally regarded as one of the most promising methods to reduce the production cost. However, the intracellularly expressed glutamate decarboxylase in C. glutamicum often showed feeble catalysis activity to convert glutamate into γ-aminobutyric acid. Here we tried to secretory express glutamate decarboxylase to achieve efficient extracellular decarboxylation of glutamate, thus improving the γ-aminobutyric acid production by C. glutamicum. We first tested glutamate decarboxylases from different sources, and the mutated glutamate decarboxylase GadBmut from E. coli with better catalytic performance was selected. Then, a signal peptide of the SEC translocation pathway directed the successful secretion of glutamate decarboxylase in C. glutamicum. The extracellular catalysis by secreted glutamate decarboxylase increased the γ-aminobutyric acid generation by three-fold, compared with intracellular catalysis. Enhancing glutamate decarboxylase expression and decreasing γ-aminobutyric acid degradation further increased γ-aminobutyric acid production by 39%. The fed-batch fermentation of the engineered C. glutamicum strain reached the record high titer (77.6 ± 0.0 g /L), overall yield (0.44 ± 0.00 g/g glucose), and productivity (1.21 ± 0.00 g/L/h). This study demonstrated a unique design of extracellular catalysis for efficient γ-aminobutyric acid production by C. glutamicum.
