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Article

Efficient Production of (R)-3-Aminobutyric Acid by Biotransformation of Recombinant E. coli

by
Hongtao Zhang
1,†,
Qing Xu
1,†,
Jiajia Lv
1,
Jiaxing Zhang
1,2,*,
Tongyi Dou
3,
Shengping You
1,
Rongxin Su
1,2,4,5 and
Wei Qi
1,2,4,5
1
Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
2
State Key Laboratory of Chemical Engineering and Low-Carbon Technology, Tianjin 300072, China
3
School of Life Science and Medicine, Dalian University of Technology, NO. 2 Dagong Road, New District of Liaodong Bay, Panjin 124221, China
4
Collaborative Innovation Centre of Chemical Science and Engineering (Tianjin), Tianjin 300072, China
5
Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Catalysts 2025, 15(5), 466; https://doi.org/10.3390/catal15050466
Submission received: 12 April 2025 / Revised: 6 May 2025 / Accepted: 7 May 2025 / Published: 9 May 2025
(This article belongs to the Special Issue Biocatalysis—Enzymes in Industrial Applications)
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Abstract

:
(R)-3-aminobutyric acid is an important raw material for dolutegravir production, which is a key antiretroviral medicine for AIDS treatment. Currently, the industrial production of (R)-3-aminobutyric acid relies on chiral resolution methods, which are plagued by high pollution and low yield efficiency. Here, we report an efficient pathway for (R)-3-aminobutyric acid production via engineered aspartase-driven biotransformation in recombinant E. coli. The engineered aspartase mutants, obtained through rational design based on catalytic mechanisms, were specifically employed to catalyze the production of (R)-3-aminobutyric acid from crotonic acid. The engineered T187L/N142R/N326L aspartase mutant exhibited the highest enzyme activity of 1516 U/mg. Through cell permeabilization, the system achieved (R)-3-aminobutyric acid yield of 287.6 g/L (96% productivity) within 24 h. Subsequent scale-up in a 7 L fermenter achieved a final product yield of 284 g/L (95% productivity) within 24 h. Economic balance showed that the cost of industrial production (¥116.21/kg) is about 1/4 of the laboratory production (¥479.76/kg). In summary, the engineered aspartase-mediated bioconversion pathway using recombinant E. coli offers an industrially viable approach for (R)-3-aminobutyric acid production, featuring mild reaction conditions, environmental sustainability, streamlined processing, high yield, and cost-effective substrates.

Graphical Abstract

1. Introduction

The second-generation integrase strand transfer inhibitors (INSTIs) have significantly advanced the global fight against AIDS [1,2,3], with dolutegravir being the preferred option and serving as a cornerstone therapy due to its high efficacy and tolerability [4,5]. The synthesis route of dolutegravir has high stereochemical requirements, where the six-membered rings’ chiral framework needs to be constructed using chiral intermediates such as (R)-3-aminobutyric acid [6,7,8]. Thus, developing efficient, low-cost routes to synthesize high-purity (R)-3-aminobutyric acid is a key step in reducing dolutegravir production costs and expanding its application scope. Currently, traditional chemical routes for (R)-3-aminobutyric acid rely on synthesizing 3-aminobutyric acid from raw materials like R-3-amino-3-methyl-tert-butyl propionate or methyl crotonate, followed by chiral resolution [9,10]. These enantioselective preparation methods face challenges such as difficulty in chiral control, low yields, issues in cost, and environmental sustainability, making it difficult to meet the modern pharmaceutical industry’s demand for green and efficient production. In contrast, its chiral isomer S-3-aminobutyric acid has already achieved high-purity production through a chemoenzymatic one-pot method [11,12]. The market urgently needs the development of low-cost, environmentally friendly production processes for (R)-3-aminobutyric acid.
Microbial fermentation offers a sustainable and economically viable approach for synthesizing (R)-3-aminobutyric acid. Recent advances in metabolic engineering and synthetic biology have unlocked the potential of microbial systems to produce enantiopure compounds directly from renewable feedstocks [13,14,15,16,17]. Aspartase, leveraging the dual α/β-amino acid properties of its substrate aspartic acid, serves as the key biocatalyst for β-amino acid biosynthesis such as 3-aminobutyric acid [18,19,20,21]. The key residues in aspartase active pocket responsible for substrate binding and catalysis have been elucidated [22,23,24,25]. Substrates bind to the active pocket through a high-energy enediolate-like conformation, where the β-carboxylate group forms an extensive hydrogen-bond network with Thr101, Ser140, Thr141, and Ser319 to stabilize the structure [26]. Residues Thr101, Asn142, and His188 coordinate the amino group of L-aspartate. Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate [27]. Upon substrate binding, the SS loop transitions from an open to closed conformation, enabling Ser318 to approach the Cβ of the substrate. This proximity facilitates protonation to form an enolate intermediate, followed by ammonia elimination to generate crotonic acid as the product [28]. The elucidation of the catalytic mechanism provides a foundation for aspartase engineering. However, to achieve the industrial-scale application of aspartase for (R)-3-aminobutyric acid production, further improvements in its function are still required.
Rational redesign is an effective strategy in enzyme engineering, especially improving its catalytic efficiency and product release [29,30,31,32,33,34], providing a possibility for tailoring biocatalysts toward stereospecific chemical synthesis. For aspartase, previous studies have utilized rational design methods to enhance the promiscuous activity toward non-natural substrates such as crotonic acid [35,36,37]. Andreas et al. obtained the Bacillus sp. YM55-1-derived aspartase mutant BSASP-C6 through screening of binding site residues, enabling catalysis of crotonic acid to produce enantiopure (R)-3-aminobutyric acid with a 60% conversion rate after 100 h [38], making green synthesis of (R)-3-aminobutyric acid feasible. Li et al. redesigned residues within the active pocket, yielding 14 mutants with catalytic activity toward crotonic acid [19]. Building upon BSASP-C6, Wang et al. adjusted the optimal pH of aspartase to 8.0 and improved thermal stability by modifying surface electrostatic potential from −60 to −88 via surface charge engineering [9]. The mutant achieved an (R)-3-aminobutyric acid yield of 556.1 g/L under whole-cell catalysis and fed-batch conditions, representing a 1.41-fold increase over the template strain. Despite significant progress in enzyme engineering for (R)-3-aminobutyric acid biosynthesis, current systems still suffer from critical defects, including insufficient conversion rates, high catalyst loadings, and a lack of scale-up process exploration. These limitations prevent fulfillment of large-scale biomanufacturing requirements, creating a disconnect between enzyme performance and industrial scalability.
Thus, in this work, based on the catalytic mechanism, we obtained an aspartase mutant capable of specifically catalyzing the conversion of crotonic acid to (R)-3-aminobutyric acid (Figure 1) through binding pocket redesign and site-directed mutagenesis. To develop a low-cost, high-yield production process for (R)-3-aminobutyric acid, we further optimized the catalytic processes and employed industrial raw materials. During the scale-up process, we systematically evaluated fermentation parameters, cell permeability, and bioconversion efficiency to establish a foundation for pilot-scale and industrial production.

2. Results and Discussion

2.1. Structure and Catalytic Mechanism Analysis of Aspartase

Aspartase efficiently converts crotonic acid into (R)-3-aminobutyric acid, demonstrating its biocatalytic potential for β-amino acid synthesis. To study the mechanism of aspartase catalyzing (R)-3-aminobutyric acid production, we first constructed the molecular structure of aspartase by homology modelling and then performed molecular docking on aspartase and the substrate crotonic acid. We searched for similar sequences and selected those with high identity as structural models to build the molecular structure of aspartase (Figure 2a and Figure S2). The Ramachandran plot of the structure showed reliable accordance with most residues located in favorable regions (Figure 2b).
According to the aspartase structure, the residues Thr101, Ser140, Thr141, and Ser319 interact with the carboxyl group of crotonic acid, causing a potential spatial vicinity in the binding pocket. Aromatic analysis revealed that aromatic residues near the active site exhibit limited engagement in π-π interactions, instead forming edge contacts with either the substrate or adjacent residues (Figure 2c). Interaction analysis identified strong interactions between crotonic acid and multiple residues. Lys324 and His188 formed salt bridges with the carboxyl group, Asn142/Thr187/Asn326 established conventional hydrogen bonds with the carboxyl moiety, while Leu358 and Ala99 exhibited van der Waals interactions with the amino group (Figure 2d).
Thus, structural analysis suggests that modifications on residues Asn142, Thr187, and Asn326 may contribute to improving aspartase performance.

2.2. Rational Design of Aspartase to Enhance Catalytic Activity

To further improve the catalytic activity of aspartase, we employed virtual mutation and binding energy prediction on aspartase. The virtual mutation protocol suggested a series of potential mutations enhancing the enzyme/substrate interactions, including T187L, N142R, and N326L (Figure 3a). Thus, we combined the three mutations and tested with wet-lab experiments and finally achieved a highly active T187L/N142R/N326L mutant of aspartase, named ASP T187L/N142R/N326L (Figure 3b), which retained activity (1516 U/mg) 2.7-fold of the wild type (560 U/mg). The induction of the three mutation sites increased the affinity of the aspartase mutant for the substrate crotonic acid, thereby enhancing its catalytic performance. Compared to the original strain, introducing the three mutations (ASP T187L, ASP N142R, and ASP N326L) individually enhances (R)-3-aminobutyric acid production. Among these, the triple mutant (ASP T187L/N142R/N326L) exhibits the highest yield.

2.3. Catalysis of (R)-3-Aminobutyric Acid Production by Engineered Aspartase Mutant

The recombinant plasmid 22b-PT7-Asp and competent cell BL21 (DE3) were used to obtain the E. coli engineering strain BL21-22b-PT7-Asp by CaCl2 chemical transformation, which can heterologously express aspartase. Cells rich in aspartase are obtained by fermentation and expression. Following permeabilization, the treated cells serve as whole-cell biocatalysts, facilitating the conversion of crotonic acid to (R)-3-aminobutyric acid in the reaction system.
The (R)-2-aminobutyric acid and (R)-3-aminobutyric acid standards are each configured as a 100 μg/mL standard solution, and the catalytic product supernatant obtained in Section 2.2 is configured as a 100 μg/mL sample solution. The two standard solutions and sample solutions were derivatized and then analyzed by HPLC. As shown in Figure 4, the liquid phase peak can clearly distinguish different compounds derived from the derivatization reaction—DNFB-NH4Cl (peak 1), DNFB-derived DNFB-3-aminobutyrate (peak 2), and DNFB-2-aminobutyrate (peak 3). Compared with the standard solution, in addition to the presence of DNFB-NH4Cl, the liquid peak of the reaction solution shows only one peak of (R)-3-aminobutyrate. Thus, the absence of (R)-2-aminobutyric acid in the catalytic reaction product confirms that the engineered aspartase variants (Section 2.2) selectively catalyze the conversion of crotonic acid to (R)-3-aminobutyric acid.
The MALDI-TOF-MS analysis of the sample (Figure 4b) further supports this conclusion. A prominent peak corresponding to (R)-3-aminobutyric acid (m/z 104.07) is observed, while the last three minor peaks arise from the DHB matrix. Notably, no peak corresponding to the substrate, crotonic acid (m/z 84), was detected.

2.4. Optimization of Biocatalytic Process and Industrial Raw Material Substitution

The initial conditions for biocatalytic preparation of (R)-3-aminobutyric acid are described in 2.5, where the target product yield is only 8.16 g/L by using BL21-22b-PT7-Asp T187L/N142R/N326L (Figure 3). To enhance both the conversion rate of crotonic acid and the yield of (R)-3-aminobutyric acid, we optimized key parameters, including biocatalyst system, catalyst concentration, and substrate (crotonic acid) concentration.
Initially, equivalent concentrations (2 g/L) of whole cells, cell lysates, and permeabilized cells were employed as biocatalysts for the conversion of crotonic acid to (R)-3-aminobutyric acid. The yield of three catalyst forms is presented in Figure 5a. Among these, the permeabilized form achieved the highest yield (13.92 g/L, 58%). The whole-cell catalytic efficiency is much lower than the other two catalytic modes due to the mass transfer limitation of the cell wall and membrane. The cell lysates eliminate this problem at the expense of the inability to reuse and subsequent separation difficulties. In contrast, permeabilized cells address both mass transfer limitations and enable facile separation, thereby streamlining downstream purification processes.
Subsequent optimization of permeabilized cell loading (2–20 g/L) was performed (Figure 5b). When the concentration of permeabilized cells reached 8 g/L, both the conversion rate of crotonic acid (99%) and the yield of (R)-3-aminobutyric acid (23.52 g/L, 98%) essentially reached maximum, whereas further increases in catalyst concentration did not lead to significant enhancement in the product output. Therefore, 8 g/L catalyst concentration is used for substrate concentration optimization subsequently (Figure 5c). Both the conversion rate of crotonic acid (97%) and the yield of (R)-3-aminobutyric acid (287.6 g/L, 96%) were maintained at high levels when substrate concentration reached 250 g/L. Further increases in substrate concentration resulted in a marked reduction in both conversion efficiency and product yield. In summary, in the biocatalytic system with a concentration of 250 g/L crotonic acid and 8 g/L permeabilized cells, (R)-3-aminobutyric acid with a concentration of up to 287.6 g/L and a yield of 96% can be obtained after 24 h of reaction, which is the highest yield in the current study. The time course curve of different cell concentrations is shown in Figure S3.
To effectively reduce the production cost of (R)-3-aminobutyric acid, we explored the feasibility of replacing reagents with industrial materials. In the fermentation stage, replacement of five raw materials, including peptone, yeast powder, sodium chloride, antibiotics, and IPTG, was explored. In the biocatalysis stage, replacement of the five substrates, including crotonic acid, ammonium chloride, ammonia, magnesium chloride, and HEPES, was explored. The results are shown in Figure 6. The yield of (R)-3-aminobutyric acid produced by industrial materials is basically the same as the results obtained by reagents. The feasible substitution of industrial feedstocks significantly enhanced the economic viability of the production process, thereby establishing a crucial foundation for subsequent scale-up experiments.

2.5. High-Efficiency and Low-Cost (R)-3-Aminobutyric Acid Production in the Liter-Scale Integrated Strategy

The fermentation and catalytic system were expanded from the shake flask stage to a 7 L fermentation tank under the same fermentation and biotransformation conditions. Table 1 lists the comparison of fermentation performance and enzyme catalytic efficiency between the laboratory and the scale-up process. The enzyme activity in the 7 L fermenter reached 1496 U/mg, showing no significant difference from shake-flask cultivation. However, the (R)-3-aminobutyric acid yield after 48 h of catalysis was only 215 g/L and 72%, significantly lower than those in shake flasks (287.6 g/L, 96%, 24 h). The absence of significant differences in enzyme activity between the fermenter phase and the shake-flask phase eliminates the impact of fermentation processes on enzymatic catalytic efficiency. The low catalytic efficiency of the enzyme in the scale-up process may be closely related to the permeability treatment process. Therefore, optimizing the parameters of the permeabilization process is critical, where the key influencing factors include ethanol concentration, cell concentration in the permeabilization system, and permeabilization duration. The permeabilization process was then optimized, establishing the optimal permeabilization conditions (35% ethanol concentration, 2 g/L cell concentration, permeabilization for 20 min, Figure 7).
Following these optimizations, the biotransformation process achieved a yield of 284 g/L (R)-3-aminobutyric acid with 95% conversion after 24 h, demonstrating performance comparable to the shake-flask cultivation phase. This represents the highest yield attained in full-scale reactions within the current research scope. Subsequent rotary evaporation concentration and ethanol washing yielded (R)-3-aminobutyric acid with 99% purity (in Supplementary Materials), thereby establishing an end-to-end biocatalytic production process for this chiral building block.
Table 2 shows the total cost of producing 1 kg (R)-3-aminobutyric acid with reagents and industrial materials. The cost of all industrial materials (¥116.21) in the scale-up stage is less than 1/4 of the reagent’s cost (¥479.76), which effectively shows a reduction in production costs. The scale-up process developed has reached industrial production levels, enabling efficient and cost-effective manufacturing of (R)-3-aminobutyric acid through recombinant E. coli biotransformation.

3. Materials and Methods

3.1. Strains, Media, and Reagents

E. coli DH5α (TransGen Biotech, Beijing, China) was grown at 37 °C for cloning and plasmid construction in Lysogeny broth (LB) medium (10 g tryptone, 5 g yeast extract, and 10 g sodium chloride per 1 L). E. coli BL21 (DE3) (TransGen Biotech, Beijing, China) was used as the host for overexpression of aspartase with induction of IPTG, and grew at 37 °C in LB medium, the same conditions as ref. [39]. Standard products such as (R)-3-aminobutyric acid and R-2-aminobutyric acid were purchased from Sigma Aldrich, Saint Louis, MO, USA. All other chemicals are of analytical grade and are purchased from Sigma Aldrich, Saint Louis, MO, USA.

3.2. Rational Design of Aspartase

A rational design method for the improvement in the aspartase activity was performed with Discovery Studio 2016. First, the 3D structure of aspartase was built by homology modelling module. The built-in “BLAST” module was used to search for similar sequences and those with Bit Score > 400 were retained. Then homology models were created based on the structure of the sequences, and the model with the lowest PDF total energy was selected as the aspartase structural model, and the Ramachandran plot was generated. Next, the substrate crotonic acid was constructed and prepared, and then docked to the activated site by the “CDOCKER” module. The binding pocket was set with a 20 Å radius around the active residues. Then, the interactions between aspartase and crotonic acid were analyzed, and the 2D interaction figure was illustrated. Finally, the residues within the binding pocket were selected, and a virtual mutation study was performed with the “Calculate Mutation Energy (Binding)…” module. The mutations with enhanced ΔΔG were selected for experimental catalytic activity testing.

3.3. Construction of Engineered E. coli Strain for (R)-3-Aminobutyric Acid Production

The wild-type aspartase from Bacillus sp. YM55-1 (AspB) was constructed in the pET-22b expression vector via homologous recombination, yielding the recombinant plasmid 22b-PT7-Asp. This recombinant plasmid was subsequently transformed into E. coli BL21(DE3) competent cells through the heat shock method, thereby establishing the engineered strain BL21-22b-PT7-Asp. Site-directed mutations, acquired through catalytic mechanism analysis and active site engineering, were introduced into the native template via whole-plasmid PCR amplification using primers containing the desired codon mutations. All DNA manipulations were performed following standard procedures [40]. The constructed plasmids were confirmed by DNA sequencing, which was performed by Genewiz (Tianjin, China). Primers, plasmids, and strains used in this study are listed in Table S1 and Figure S1.

3.4. Cultivation in Shaken Flasks

The engineered aspartase E. coli strains obtained above were inoculated into 5 mL LB liquid medium with 100 μg/mL ampicillin. After cultivating at 37 °C with 220 rpm of agitation speed for 12 h, the liquid bacterial germ was inoculated at a 1% inoculum volume to 100 mL LB liquid medium with 100 μg/mL ampicillin, then cultured at 37 °C with 220 rpm of agitation speed. Then, the induction was initiated by adding IPTG to a final concentration of 0.1 mM when the OD600 value reached 0.6. Induction was performed at 30 °C for 6 h. Subsequently, all cultures were centrifuged at 5000 rpm for 20 min to collect the bacterial pellet containing aspartase for cell density and aspartase activity measurement.

3.5. Preparation of Biocatalyst

Whole cells, permeabilized cells, and cell lysates were prepared as biocatalysts in this study. For the preparation of whole-cell biocatalyst, the bacterial precipitation, which was obtained by centrifuging the cultures at 5000 rpm for 20 min, was directly used. For the preparation of permeabilized cells, the cells were harvested by centrifuging the 100 mL fermentation broth at 5000 rpm for 5 min. Then, the precipitate was resuspended in 35% (v/v) ethanol. After standing at room temperature for 10 min, the permeabilized cells were obtained by centrifuging the above mixture at 6000 rpm for 10 min. The precipitation after centrifugation was prepared for the subsequent bioconversion of (R)-3-Aminobutyric acid. For the preparation of cell lysates, the bacterial precipitation was obtained by centrifuging the cultures at 5000 rpm for 20 min. Then the precipitate was resuspended in 50 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES, pH 8.0). After ultrasonic lysis, the cell lysate biocatalyst was obtained, which could be added to the subsequent catalytic system for the preparation of (R)-3-aminobutyric acid in the appropriate proportion.

3.6. Biocatalytic Production of (R)-3-Aminobutyric in Shaken Flasks

The substrate crotonic acid was dissolved in the mixture of 100 mM HEPES and 2 mM MgCl2, and then NH4Cl solution with the same concentration of substrates was added. The solution pH was adjusted to 8.0 using ammonia. The three biocatalysts obtained in 2.4 were resuspended in the substrate solution. The concentration of crotonic acid is 20 g/L, and the final cell concentration is 2 g/L. The reaction mixture was incubated at 37 °C, 200 rpm. Samples were taken at 0, 12, 24, and 48 h and heated at 80 °C for 10 min to stop the reaction. Supernatant was obtained by centrifugation for subsequent product testing.

3.7. Optimization of Catalytic Reaction Conditions

Several biocatalytic reaction parameters were optimized to ensure the most efficient crotonic acid conversion and (R)-3-aminobutyric acid yield. The influence of different cell concentrations (2, 4, 8, 12, 16, 20 g/L), biocatalyst system (whole-cells, permeabilized cells, cell lysates) and crotonic acid concentration (20, 50, 100, 150, 250, 350 and 450 g/L) on crotonic acid conversion and (R)-3-aminobutyric acid yield under the same strain and fermentation culture conditions were explored. Fermentation and bioconversion conditions were the same as those in Section 2.3, Section 2.4 and Section 2.5.

3.8. Replacement of Industrial Materials

The industrial grade materials (yeast powder, tryptone, NaCl, inducer, antibiotic, crotonic acid, HEPES, MgCl2, NH4Cl, ammonia) were used during the process of fermentation and bioconversion. The control group was conducted with lab-grade purity reagents. The effect of industrial materials on the conversion rate of crotonic acid and the yield of (R)-3-aminobutyric acid was explored. Fermentation and bioconversion conditions were the same as those in Section 2.3, Section 2.4 and Section 2.5.

3.9. Product Preparation in Small-Scale Process

The process to produce (R)-3-aminobutyric acid was amplified to a liter-scale integrated strategy, including fermentation, permeability, and biotransformation.
Fermentation: The engineered strain BL21-22b-PT7-Asp T187L/N142R/N326L was cultured for 12 h at 37 °C in 5 mL LB liquid medium and then transformed for seed culture into a 500 mL shake flask containing 100 mL LB medium with 100 μg/mL ampicillin. Bioreactor cultivations (3.5 L of LB medium with 100 μg/mL ampicillin) were performed in a 7 L fermenter (NBS Bioflo 415, New Brunswick Scientific, Edison, NJ, USA) at 0.1 MPa tank pressure, 0.75 vvm aeration rate, and 220 rpm agitation speed. The fermentation was inoculated with 100 mL of seed culture and carried out at 37 °C. When the OD600 value reached 0.8–1.2 g/L, IPTG was added to the fermentation medium, and the induction temperature was kept at 37 °C. When the OD600 reached 4–4.5, samples were taken for the determination of aspartase activity.
Permeabilization: After fermentation, the bacteria were collected by centrifuging at 2000× g for 45 min, and the cells were resuspended in 350 mL of 35% (v/v) ethanol (Permeabilized cell concentration is 20 g/L). After standing at room temperature for 10 min, permeabilized cells were obtained by centrifuging the above mixture at 2000× g for 45 min. The centrifuged precipitate is used for the subsequent biotransformation of (R)-3-aminobutyric acid. The optimized permeabilized cell concentration during scale-up is 2 g/L with a treatment time of 20 min.
Bioconversion: The bioconversion was carried out in the 7 L fermenter. Crotonic acid was dissolved in the mixture of 1 L HEPES buffer (100 mM), MgCl2 (2 mM), and NH4Cl (250 g/L). The solution pH was adjusted to 8.0 using ammonia. Then the permeabilized cell biocatalyst obtained by centrifugation was resuspended in the above solution to a final cell concentration of 8 g/L, and the reaction system was incubated at 37 °C at 200 rpm. Samples were taken at 0, 12, 24, and 48 h, and heated to 80 °C for 10 min to stop the reaction, then the supernatant was obtained by centrifuging for quantitative detection of substrate and product.

3.10. Activity Assay

The fermentation broth was taken with 1 mL for activity measurement. After centrifugation, the precipitation was resuspended in 1 mL HEPES buffer mixture containing 100 mM HEPES, 300 mM crotonic acid, 300 mM NH4Cl, and 2 mM MgCl2, at pH 8.0 adjusted by ammonia. The mixture was incubated in a 37 °C rotary shaker at 200 rpm for 30 min, then heated to 80 °C for 10 min to stop the reaction and used for quantitative detection of substrate and product. The enzyme activity was defined as follows: 1 U/mg was defined as 1 nmol of (R)-3-aminobutyric acid produced by 1 mg of cell per minute, which is modified from others’ definition.

3.11. Product Detection Method

Crotonic acid and (R)-3-aminobutyric acid concentration were detected by Agilent High Performance Liquid Chromatography (Agilent 1260 Infinity III LC System, Santa Clara, CA, USA) using an Agilent Eclipse Plus C18 column (5 μm, 4.6 × 250 mm). The sample was centrifuged at 12,000 rpm for 2 min, and then 1 mL of supernatant was used for HPLC after diluting to a certain multiple.
The crotonic acid concentration detection process was as follows: the diluted sample could be directly used for HPLC after passing through the membrane. The temperature of the C18 column was set to 30 °C. The mobile phase A was methanol, and the mobile phase B was 0.1% TFA in water. The ratio of the mobile phase was 35:65 (v/v) with a constant flow rate of 1 mL/min over a total runtime of 15 min. The detection wavelength was set to 226 nm using a UV detector, and it peaked at 5.7 min.
The (R)-3-aminobutyric acid concentration detection process was as follows: the diluted sample could be used for HPLC after derivatization. Firstly, 25 µL of sample was mixed with 10 µL of 1 M Na2CO3 and 40 µL of 1-fluoro-2,4-dinitrobenzene derivative (DNFB, 36.7 mM in acetone), then incubated at 40 °C for 1.5 h. The reaction was stopped by adding 20 µL of 1 M HCl, and the precipitate was subsequently removed by centrifugation. The sample can be applied for HPLC after mixing with 100 µL of 20% acetonitrile. The temperature of the C18 column was set to 30 °C. The mobile phase A was 0.1% TFA in water, and the mobile phase B was 0.1% TFA in acetonitrile. The ratio of the mobile phase was 75:25 (v/v) with a constant flow rate of 1 mL/min over a total runtime of 25 min. Detection wavelength was set to 360 nm using a UV detector, and the peak appeared at 12.5 min.

3.12. Data Processing and Statistical Validation

All experimental determinations were conducted with three biologically independent replicates. Central tendency was quantified using arithmetic means, with dispersion metrics represented as standard deviation (SD) error bars.

4. Conclusions

Rational design provides an effective strategy for engineering aspartase into an efficient biocatalyst for β-amino acid production [19,38]. Through rational design, the crotonic acid-converting activity of aspartase was enhanced to 1516 U/mg, enabling efficient whole-cell catalysts. This approach shows promise for integration with other protein engineering strategies (e.g., surface charge modification [9]) to further develop mutants with elevated catalytic activity. Additionally, we noticed the potential impact of divalent metal ions on aspartase activity [24,41] and supplemented the reaction system with Mg2+ concentrations. The relationship between metal ion concentration and aspartase activity warrants further investigation.
Investigations into catalyst forms revealed that permeabilization treatment significantly improved the (R)-3-aminobutyric acid production capacity of whole-cell catalysts. This treatment preserved cellular integrity and enzyme stability while enhancing substrate/product permeability, thereby addressing mass transfer limitations—a finding aligned with other membrane permeability studies [42,43]. The permeabilized cells retained the advantages of whole-cell catalysts (easy separation/reusability) while achieving catalytic efficiency comparable to lysed cells or purified enzymes. Successful industrial medium replacement and liter-scale amplification demonstrate the feasibility of (R)-3-aminobutyric acid production. High substrate conversion (≥95%) and negligible byproducts simplified purification: ethanol washing after solvent evaporation yielded high-purity (R)-3-aminobutyric acid (data not included in this article).
In summary, we engineered an industrially compatible whole-cell catalyst for (R)-3-aminobutyric acid biosynthesis. The catalyst incorporates rationally designed aspartase mutants with 1516 U/mg activity toward crotonic acid conversion. Post-permeabilization treatment enabled 284 g/L (R)-3-aminobutyric acid production from 250 g/L crotonic acid within 24 h (95% yield). Liter-scale trials achieved comparable performance, reducing production costs to ¥116.21/kg. Compared with previous work [9,37], through rational design engineering and catalytic process optimization, we achieved near-quantitative conversion efficiency (~96%) at equivalent substrate loading (~250 g/L) in AspB enzyme, while demonstrating scalable production at liter-scale with maintained productivity (>94%) even when employing cost-effective industrial feedstocks. This work establishes a scalable and cost-effective biocatalytic platform for (R)-3-aminobutyric acid production, with promising potential to streamline industrial dolutegravir synthesis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/catal15050466/s1. Figure S1. Complete plasmid map of the plasmid harboring the aspartase mutant T187L/N142R/N326L. Figure S2. Sequence similarity and Bit Scores of sequence templates. Figure S3. Time course curve of different cell concentrations on the production of (R)-3-aminobutyric acid in the biotransformation system. Figure S4. Time-course curve of the liter-scale catalytic system operating under buffer-free and ammonium chloride-free conditions. Table S1. Primers used in this study. Table S2. Plasmids used in this study. Table S3. Strains used in this study. Table S4. Solubility of reaction system components in ethanol. Table S5. Variation in product and impurity profiles during purification.

Author Contributions

Conceptualization, W.Q. and S.Y.; methodology, J.L. and H.Z.; software, T.D.; validation, H.Z. and Q.X.; investigation, S.Y.; resources, R.S.; writing—original draft preparation, H.Z. and J.L.; writing—review and editing, J.Z. and T.D.; visualization, J.Z. and J.L.; supervision, S.Y.; project administration, W.Q.; funding acquisition, W.Q. and S.Y. All authors have read and agreed to the published version of the manuscript. H.Z. and Q.X. contributed equally to this manuscript.

Funding

This research was funded by the National Key Research and Development Program of China, grant number 2022YFC2106300, the National Natural Science Foundation of China, grant numbers 22278314 and 22078239, the Beijing–Tianjin–Hebei Basic Research Cooperation Project, grant number B2021210008, and Enterprise Horizontal Entrustment, grant number 2023GKF-0146.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
LBLuria–Bertani
IPTGIsopropyl-beta-D-thiogalactopyranoside
AIDSAcquired immune deficiency syndrome

References

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Catalysts 15 00466 g001
Figure 1. Framework of the synthetic pathway of Dolutegravir, in which (R)-3-aminobutyric acid is an important precursor.
Figure 1. Framework of the synthetic pathway of Dolutegravir, in which (R)-3-aminobutyric acid is an important precursor.
Catalysts 15 00466 g001
Catalysts 15 00466 g002
Figure 2. Mechanism analyzation of aspartase catalyzing the production of (R)-3-aminobutyric acid from crotonic acid: (a) Structure of the aspartase homology model; (b) Ramachandran plot of the aspartase structure, where each point represents the φ and ψ torsion angles of a residue, and the blue and purple lines represent the favorable and unfavorable regions for residues, and the green and red points represent the favorable and unfavorable residues; (c) Binding conformation of crotonic acid molecule in the active pocket; (d) Interactions between binding pocket residues and crotonic acid.
Figure 2. Mechanism analyzation of aspartase catalyzing the production of (R)-3-aminobutyric acid from crotonic acid: (a) Structure of the aspartase homology model; (b) Ramachandran plot of the aspartase structure, where each point represents the φ and ψ torsion angles of a residue, and the blue and purple lines represent the favorable and unfavorable regions for residues, and the green and red points represent the favorable and unfavorable residues; (c) Binding conformation of crotonic acid molecule in the active pocket; (d) Interactions between binding pocket residues and crotonic acid.
Catalysts 15 00466 g002
Catalysts 15 00466 g003
Figure 3. Virtual mutation screening and enzyme activity verification: (a) Mutational energy of each mutant in virtual saturation mutagenesis of key residues in the active pocket; (b) Fermentation and catalytic performance of strains derived with different site-directed mutations.
Figure 3. Virtual mutation screening and enzyme activity verification: (a) Mutational energy of each mutant in virtual saturation mutagenesis of key residues in the active pocket; (b) Fermentation and catalytic performance of strains derived with different site-directed mutations.
Catalysts 15 00466 g003
Catalysts 15 00466 g004
Figure 4. Specific detection of the product: (a) Liquid chromatography of two standards and the samples (Peak 1: Derivative DNFB-NH4Cl, Rt 9.87 min; Peak 2: DNFB-3-aminobutyrate, Rt 12.5 min; Peak 3: DNFB-2-aminobutyrate, Rt 15.2 min). (b) MALDI-TOF-MS of the sample obtained in this study.
Figure 4. Specific detection of the product: (a) Liquid chromatography of two standards and the samples (Peak 1: Derivative DNFB-NH4Cl, Rt 9.87 min; Peak 2: DNFB-3-aminobutyrate, Rt 12.5 min; Peak 3: DNFB-2-aminobutyrate, Rt 15.2 min). (b) MALDI-TOF-MS of the sample obtained in this study.
Catalysts 15 00466 g004
Catalysts 15 00466 g005
Figure 5. Effect on the production of (R)-3-aminobutyric acid: (a) Biocatalyst system; (b) Cell concentration; (c) Substrate concentration. The yield and production of (R)-3-aminobutyric acid, and conversion rate and residual amount of crotonic acid after reaction were tested.
Figure 5. Effect on the production of (R)-3-aminobutyric acid: (a) Biocatalyst system; (b) Cell concentration; (c) Substrate concentration. The yield and production of (R)-3-aminobutyric acid, and conversion rate and residual amount of crotonic acid after reaction were tested.
Catalysts 15 00466 g005
Catalysts 15 00466 g006
Figure 6. Industrial materials substitution in the fermentation stage and the biotransformation stage. (a) Fermentation process; (b) Biotransformation process.
Figure 6. Industrial materials substitution in the fermentation stage and the biotransformation stage. (a) Fermentation process; (b) Biotransformation process.
Catalysts 15 00466 g006
Catalysts 15 00466 g007
Figure 7. Optimization of permeabilization conditions in the bioreactor scale: (a) Ethanol concentration; (b) Cell concentration; (c) Permeabilization time.
Figure 7. Optimization of permeabilization conditions in the bioreactor scale: (a) Ethanol concentration; (b) Cell concentration; (c) Permeabilization time.
Catalysts 15 00466 g007
Table 1. Performance comparison of the production of (R)-3-aminobutyric acid by the permeabilized cell biotransformation in 500 mL shaken flasks (Lab scale) and a 7 L fermenter (Bioreactor scale).
Table 1. Performance comparison of the production of (R)-3-aminobutyric acid by the permeabilized cell biotransformation in 500 mL shaken flasks (Lab scale) and a 7 L fermenter (Bioreactor scale).
FermentationBioproduction by Permeabilized Cells
TypeOD600Aspartase (U/mg)Reaction Time (h)(R)-3-Aminobutyric Acid (g/L)Yield (%)
Lab scale
(Reagents)		4.43 ± 0.371516 ± 1924287 ± 1.2595.9 ± 0.55
Lab scale
(Industrial materials)		4.35 ± 0.211503 ± 1224281 ± 0.6593.9 ± 0.25
Bioreactor scale
(Industrial materials)		4.29 ± 0.211496 ± 1648215 ± 0.9471.8 ± 0.42
Bioreactor scale after the process optimization
(Industrial materials)		4.32 ± 0.281502 ± 2324284 ± 1.0794.9 ± 0.87
Table 2. The costs of raw materials for the bioproduction of (R)-3-aminobutyric acid (1 kg) from the reagents or industrial materials.
Table 2. The costs of raw materials for the bioproduction of (R)-3-aminobutyric acid (1 kg) from the reagents or industrial materials.
ReagentsIndustrial Materials
ComponentCost (¥)ComponentCost (¥)
Tryptone (35 g)17.50Tryptone (35 g)0.35
Yeast extract (17.5 g)4.55Yeast extract (17.5 g)0.16
Sodium chloride (35 g)0.70Sodium chloride (35 g)0.02
Seed fermentation broth (100 mL)0.65Seed fermentation broth (100 mL)0.02
Resistance (0.35 mL)3.96Resistance (0.35 mL)0.14
IPTG (0.35 mL)9.8IPTG (0.35 mL)2.4
35% ethanol (3.5 L)36.7535% ethanol (3.5 L)12.01
Butenoic acid (225 g)42.3Butenoic acid (225 g)13.95
Ammonia (230 mL)2.34Ammonia (230 mL)0.525
HEPES buffer (0.9 L)1.21HEPES buffer (0.9 L)0.05
MgCl2 (2 mL)0.05MgCl2 (2 mL)0.01
NH4Cl (2 mL)2.9NH4Cl (2 mL)0.12
Total122.71Total29.72
(R)-3-aminobutyric acid (1 kg)479.76(R)-3-aminobutyric acid (1 kg)116.21
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MDPI and ACS Style

Zhang, H.; Xu, Q.; Lv, J.; Zhang, J.; Dou, T.; You, S.; Su, R.; Qi, W. Efficient Production of (R)-3-Aminobutyric Acid by Biotransformation of Recombinant E. coli. Catalysts 2025, 15, 466. https://doi.org/10.3390/catal15050466

AMA Style

Zhang H, Xu Q, Lv J, Zhang J, Dou T, You S, Su R, Qi W. Efficient Production of (R)-3-Aminobutyric Acid by Biotransformation of Recombinant E. coli. Catalysts. 2025; 15(5):466. https://doi.org/10.3390/catal15050466

Chicago/Turabian Style

Zhang, Hongtao, Qing Xu, Jiajia Lv, Jiaxing Zhang, Tongyi Dou, Shengping You, Rongxin Su, and Wei Qi. 2025. "Efficient Production of (R)-3-Aminobutyric Acid by Biotransformation of Recombinant E. coli" Catalysts 15, no. 5: 466. https://doi.org/10.3390/catal15050466

APA Style

Zhang, H., Xu, Q., Lv, J., Zhang, J., Dou, T., You, S., Su, R., & Qi, W. (2025). Efficient Production of (R)-3-Aminobutyric Acid by Biotransformation of Recombinant E. coli. Catalysts, 15(5), 466. https://doi.org/10.3390/catal15050466

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