In this study, we engineered Escherichia coli strains for the biosynthesis of two key antidepressants, kaempferol and γ-Aminobutyric acid (GABA). For kaempferol production, we constructed recombinant strains by linking the enzymes flavanone 3-hydroxylase (F3H) and flavonol synthase (FLS) using various linkers, including GGGS, GGGS2, TPTP, and TPTP2. The use of TPTP2 significantly improved kaempferol yield, with the strain carrying the TPTP2 (double copy) linker reaching 93.35 ± 4.03 mg/L. Additionally, we optimized conditions such as temperature, cell density, and substrate concentration, with the highest kaempferol yield at 30°C and an OD600 of 1.0, using 1000 mg/L naringenin. Furthermore, we explored the effect of multi-copy FLS and F3H genes, concluding that adding multiple copies of F3H increased kaempferol yield, while adding multiple copies of FLS decreased it.

For GABA production, the recombinant strain BL21/p23b-GadB was constructed, achieving a yield of 2.32 ± 0.21 g/L. Further optimization of pH conditions demonstrated that GABA production was highest at pH 4.6. Our findings provide an efficient method for producing kaempferol and GABA through metabolic engineering and process optimization.

We focus on using metabolic engineering technology to produce kaempferol in E. coli using naringenin as a substrate. The process of converting naringenin to kaempferol involves the action of two key enzymes: First, flavanone 3-hydroxylase (F3H) introduces a hydroxyl group into naringenin; Then, flavonol synthase (FLS) forms a double bond at the C2-C3 position of dihydrokaempferol. This series of enzymatic reactions ultimately converts naringenin into biologically active kaempferol.

To verify whether F3H and FLS can synthesize kaempferol after successful protein expression in E. coli using recombinant plasmids. The CisF3H (BBa_K5482000, from Citrus sinensis) and CuFLS (BBa_K5482001, from Citrus unshiu) gene sequences were codon-optimized for E. coli and synthesized. The two genes were connected using a polycistronic strategy (RBSB0034) and cloned into the pET23b plasmid via EcoRI and XhoI restriction sites to create p23b-F3H-B0034-FLS (BBa_K5482002). Primers were designed, and PCR amplification was performed. The product length was verified by agarose gel electrophoresis and then sequenced. After verification, the recombinant plasmid was extracted and transformed into E. coli DH5α for preservation and E. coli BL21 for expression. The strain was cultured in LB medium with ampicillin (50 μg/mL) at 37℃.

During the experiment, we selected three strains: wild-type BL21, control strain (containing only pET23b empty plasmid) and recombinant strain (BL21/p23b-F3H-B0034-FLS). First, we inoculated the three strains into fresh LB medium and cultured the bacteria at 37℃ to OD600 = 1.0 to prevent errors in the experimental results due to different bacterial densities. Subsequently, an equal amount of 500 mg/L naringenin was added to each group of strains as a substrate, and cultured at 30℃ for 24 hours to ensure that each group of strains had sufficient time for protein expression. After 24 hours, we took 1 mL of bacterial culture medium from each of the three groups of strains, added 5 mL of methanol to each to extract kaempferol components, vortexed, and then centrifuged to collect the supernatant. Subsequently, we used kaempferol standards (K812226, McLean) with concentrations of 1, 10, 20, 30, and 50 mg/L, respectively, and used a microplate reader to detect the absorbance of each concentration of the standard at 368 nm to prepare a standard curve as a data reference for the test results. At the same time, a microplate reader was used to detect the absorbance of the samples at 368 nm to calculate the kaempferol content.

Through literature review, we identified several advantages of enzyme fusion technology, including improved metabolic efficiency and enhanced enzyme stability and activity. Therefore, we used the GGGS linker to connect the F3H and FLS enzymes, bringing them closer together in space. This reduces substrate diffusion between the enzymes and optimizes the overall metabolic process. This strategy aims to improve enzyme function and expression efficiency, ultimately increasing the yield of kaempferol.We used the same method to construct the recombinant plasmid p23b-F3H-GGGS-FLS(BBa_K5482003), and after sequencing verification, it was transformed into E. coli BL21.

The absorbance of the BL21/F3H-FLS (GGGS) strain was measured using the same method, and the kaempferol yield of the engineered strain was calculated based on the standard curve of kaempferol (Figure 3A).Notably, the absorbance of the engineered strain BL21/F3H-FLS (GGGS) exceeded the range of the kaempferol standard curve during the initial measurement, so we diluted the solution by half and measured it again. The final kaempferol concentration was calculated based on the dilution factor.

The results in Figure 5 showed that the kaempferol yield of the engineered strain BL21/F3H-FLS (GGGS) using the fusion enzyme technology was significantly higher than that of the engineered strain BL21/F3H-FLS (B0034), and the yield reached 60.52 ± 5.11 mg/L. Wild-type BL2 and control strains (containing only empty plasmid pET23b), which served as the control group, were unable to synthesize kaempferol under the same manipulation and culture conditions. Therefore, employing fusion enzyme technology to link F3H and FLS can significantly enhance the yield of kaempferol produced by engineered bacteria.

In order to investigate the relationship between temperature and bacterial density and kaempferol production, we measured the yield of kaempferol at different temperatures and bacterial densities.

We inoculated the engineered strain BL21/p23b-F3H-GGGS-FLS into a fresh LB medium containing 50 μg/mL ampicillin and grew at 37℃. naringenin was added to the recombinant strain and cultured at 180 rpm for 24h. The effects of induction temperature (16℃, 20℃, 30℃, 37℃, 42℃) and initial bacterial density (OD600= 0.2, 0.6, 1, 1.5 and 2.0) on kaempferol production were analyzed.

In addition, we also observed that bacterial density (OD600) also had a significant effect on kaempferol production by Figure 6B, which tended to increase first and then decrease with the increase of cell density. At low densities, bacteria need more time to reach sufficient quantities to turn on and maintain kaempferol production. In this case, the cells do not fully utilize the nutrients (carbon sources, nitrogen sources, etc.), resulting in reduced efficiency of naringenin conversion to kaempferol. When the bacterial density is too high, the nutrients in the medium will gradually decrease until they are exhausted. This results in insufficient nutrients and increased competitive pressure between bacteria, which affects their normal biological activity.

Finally, we can conclude that the condition of 30℃ or bacterial density OD600=1 can be selected to improve the yield of kaempferol.

In order to optimize kaempferol yield and conversion rate by exploring the influence of naringenin concentration. The concentration of the substrate, naringenin, has an important effect on the reaction rate, enzyme stability and by-product formation in the experiment to optimize the production conditions of kaempferol.

The experimental steps were roughly divided into the following steps: First, the constructed engineered strain BL21/p23b-F3H-GGGS-FLS was inoculated into fresh LB medium containing 50 ug/mL ampicillin and grew at 37℃. Secondly, naringenin at concentrations of 125, 250, 500, 1000 and 2000 mg/L was added to the recombinant strains and cultured at 180 rpm for 24h. Third, a microplate reader was used to detect the absorbance of the sample at 368 nm, so as to obtain the yield of kaempferol in different naringenin concentrations and calculate the conversion rate (kaempferol concentration/naringenin concentration).

According to Figure 7A, with the increase of naringenin concentration, kaempferol production showed a trend of first increase and then decrease, and when naringenin concentration is 1000mg/L, the yield of kaempferol production(72.39 ± 6.63 mg/L) reaches the maximum. That may because substrate inhibition (When the substrate concentration exceeds the optimal range of the enzyme, the enzyme activity decreases, resulting in reduced product production).

Moreover, Figure 7B showed that the conversion rate tended to increase first and then decrease with the increase of naringenin concentration and when naringenin concentration was 500 mg/L, the conversion rate of kaempferol reached the highest point (0.12 ± 0.01).

Finally, we can conclude that naringenin with a concentration of 1000mg/L can be selected to increase the yield of kaempferol. To improve the conversion rate of kaempferol, naringenin at the concentration of 500 mg/L can be selected.

Linkers can improve the spatial folding of fused proteins. We tested GGGS (G is glycine, S is serine; Flexible linker) and TPTP (T is threonine, P is proline; Effect of rigid linker) on the activity of fusion protein.

The CisF3H and CuFLS gene sequences were synthesized and codon optimized for E. coli. The two protein-coding genes were connected using fusion enzyme technology, with the ligating sequences being BBa_K5482004 (GGGS), BBa_K5482005 (GGGS2), BBa_K5482006 (TPTP), and BBa_K5482007 (TPTP2), respectively. These constructs were then cloned into the pET23b plasmid via the EcoRI and XhoI restriction sites. The recombinant plasmid was extracted using a plasmid extraction kit (Tiangen, China). Subsequently, the recombinant plasmid was transformed into E. coli DH5α and BL21. The engineered strains were cultured in LB medium containing ampicillin (Amp) (50 μg/mL) at 37℃.

Recombinant strains carrying different linkers (F3H-GGGS-FLS, F3H-GGGS2-FLS, F3H-TPTP-FLS, and F3H-TPTP2-FLS) were inoculated into fresh LB medium and grown at 37°C until the bacterial density reached OD600 = 1.0. Subsequently, 500 mg/L of naringenin was added to each culture, followed by incubation at 30°C for 24 hours. The kaempferol content was then determined using the previously described method.

The results showed that the kaempferol yield of the recombinant strain with the TPTP (rigid linker) (76.96 ± 4.19 mg/L) was significantly higher than that of the strain with the GGGS (flexible linker) (58.53 ± 6.33 mg/L). Additionally, the yield of the strain carrying the TPTP2 (double copy) linker (93.35 ± 4.03 mg/L) was signiticantly higher than that of the strain with the TPTP (single copy) linker.

These findings indicate that the TPTP2 (double copy) linker achieves the best expression efficiency and can be used to optimize kaempferol production.

In order to test the effect of multiple copies of FLS or F3H on the production of kaempferol by engineered bacteria, we added F3H/FLS genes to the F3H-TPTP2-FLS engineered bacteria and expressed them using J23100-B0034 (RBS). The terminator B0015 was used to separate them to avoid promoter interference.

First, the recombinant strains carrying different gene copies (p23b-F3H-TPTP2-FLS+FLS, BBa_K5482027 and p23b-F3H-TPTP2-FLS+F3H, BBa_K5482028) were inoculated into fresh LB medium and cultured at 37°C until the bacterial density reached OD600 = 1.0 . Subsequently, 500 mg/L naringenin was added to each group of strains, followed by incubation at 30°C for 24 hours. Kaempferol production was then measured by detecting the absorbance of the samples at 368 nm using a microplate reader.

The experimental results showed a significant impact on kaempferol production when additional gene copies were introduced. Specifically, after adding an extra copy of FLS to the original F3H-TPTP2-FLS construct, the kaempferol yield decreased from 87.70 ± 5.37 mg/L to 34.87 ± 4.86 mg/L, indicating a negative effect on production. In contrast, adding an additional copy of F3H resulted in a substantial increase in kaempferol production, from 87.70 ± 5.37 mg/L to 113.36 ± 9.47 mg/L (Figure 12).

These findings suggest that introducing an extra copy of F3H to the original construct can effectively boost kaempferol production. However, adding an extra copy of FLS has an inhibitory effect, reducing the overall yield. This insight provides valuable guidance for optimizing gene copy number to maximize production efficiency in future metabolic engineering efforts.

To investigate why adding F3H to the original plasmid increases kaempferol production, whereas adding FLS reduces it, we carried out experiments to further clarify the process and results.

We collected 2 mL of overnight bacterial cultures containing different plasmid constructs: F3H-TPTP2-FLS, F3H-TPTP2-FLS+FLS, and F3H-TPTP2-FLS+F3H. Total RNA was extracted using a Bacterial RNA Kit, and the RNA was dissolved in 0.1% DEPC water. The RNA purity was assessed using a Nanodrop 2000, with an OD260/OD280 ratio of around 1.8, indicating good purity. The RNA was then reverse transcribed into cDNA using the HiScript 1st Strand cDNA Synthesis Kit.

The cDNA concentration was adjusted to 100 ng/μL. We mixed the cDNA with TB Green® dye, which fluoresces upon binding to double-stranded DNA. Real-time quantitative reverse transcription PCR (qRT-PCR) was performed using QuantStudio 5 to measure the mRNA levels of different genes. The 16S rRNA gene served as an internal reference, and the 2−ΔΔCt method was used to calculate the relative mRNA expression levels.

We found that the mRNA expression level of F3H-TPTP2-FLS was significantly higher than F3H-TPTP2-FLS+FLS and F3H-TPTP2-FLS+F3H, with the mRNA level dropping from 9 to 5 (Figure 13). Therefore, we observed that when using multicopy genes, the expression level of mRNA was lower than when using single-copy genes.

To verify our hypothesis, through the above experiments, we came up with two inferences to explain why adding FLS would reduce the yield of kaempferol, while adding F3H could increase the yield of kaempferol:

F3H is likely a rate-limiting enzyme in the synthesis of kaempferol. By increasing F3H expression, we enhance the conversion of flavanones to kaempferol (its reaction rate limits the overall kaempferol synthesis rate). Increasing the expression of F3H can increase the production of flavanones, thereby promoting the synthesis of kaempferol, and may encourage the shift of the overall metabolic flow to kaempferol.

Plasmids carrying multicopy genes are heavily burdened, thus consuming a large amount of energy and increasing metabolic stress, leading to the reduction of mRNA levels and kaempferol production from FLS multicopy genes.

In this experiment, the engineered bacterial strain constructed in our laboratory was utilized for the production of kaempferol. The engineered bacteria were cultured under optimal growth conditions to induce kaempferol biosynthesis. After sufficient biomass was achieved, the bacterial culture was sent to a company for extraction of kaempferol.

γ-Aminobutyric acid (GABA) is a key inhibitory neurotransmitter in the central nervous system, known for its water solubility, thermal stability, and safety as an ingredient in food and beverages. Due to its anti-anxiety and stress-relieving properties, GABA is widely used in the food and health supplement industries. Given the need for an edible, anti-stress, and anti-anxiety compound in our project, we initiated research into the production of GABA.

We first designed and constructed a GABA production strain. The GadB gene (BBa_K376900), encoding glutamate decarboxylase (GAD), was synthesized and codon-optimized for expression in E. coli. The gene was cloned into the pET23b plasmid using the EcoRI and XhoI restriction sites, generating the recombinant plasmid p23b-GadB. The construct was verified through sequencing, and the recombinant plasmid was extracted using a plasmid extraction kit. After verification, the plasmid was transformed into E. coli strains DH5α (for plasmid storage) and BL21 (for protein expression)(Figure 15).

Glutamate decarboxylase (GAD) uses pyridoxal phosphate (the active form of vitamin B6) as a cofactor to convert glutamate into GABA (Figure 16).

To catalyze GABA synthesis, we employed crude enzyme extracts from the engineered E. coli strains. The process involved the following steps: 1. Cell Harvesting and Enzyme Extraction: We collected 2 mL of bacterial culture, centrifuged it at 8000 rpm for 10 minutes to obtain the bacterial pellet. The pellet was resuspended in 2 mL of acetic acid-sodium acetate buffer (pH 4.6). This suspension was subjected to ultrasonic treatment (75 W, 1 second pulses with 3 second intervals, for a total of 20 minutes) in an ice bath to obtain the crude enzyme solution. 2. GABA Synthesis Reaction: In 1 mL of crude enzyme solution, 2% glutamic acid was added as the substrate, and the mixture was incubated at 37°C for 3 hours. 3. Quantification of GABA: After the reaction, GABA content was measured using a GABA test kit. The principle of the GABA test kit is that phenol and sodium hypochlorite react with GABA to produce a blue-green product, which has a maximum absorbance at 640 nm. Absorbance at 640 nm was recorded using a microplate reader, and a standard curve was generated to calculate the GABA concentration in the samples.

The linear regression equation is Y = 0.5130*X - 0.05257, with an R² value of 0.98. This indicates a strong linear relationship between GABA concentration and absorbance. Therefore, we conclude that GABA concentration can be reliably calculated based on absorbance(Figure 17A).

BL21 and BL21/pET23b were used as control groups, while BL21/p23b-GadB served as the experimental group for the controlled experiments. By constructing the recombinant strain p23b-GadB, we successfully achieved GABA production, with the recombinant strain yielding 2.32 ± 0.21 g/L(Figure 17B).

To enhance GadB enzyme activity, we tested the GABA concentration in the crude enzyme solution of the engineered strain under different pH conditions. The BL21/p23b-GadB pellet was resuspended in 2 mL of either acetate-sodium acetate buffer (pH 4.6), acetate-sodium acetate buffer (pH 3.6), PBS (pH 7.4), or Tris-HCl buffer (pH 9.2). The cells were then sonicated under ice bath conditions to obtain the crude enzyme solution. For each 1 mL of crude enzyme solution, 2% glutamic acid was added and incubated at 37°C for 3 hours. The GABA concentration was then measured using the γ-aminobutyric acid (GABA) detection kit (mlbio, China).

The activity of GadB enzyme varied significantly under different pH conditions. At pH 3.6 and pH 4.6, GABA production was higher, reaching 2.17 ± 0.12 g/L and 2.48 ± 0.23 g/L, respectively. In contrast, under neutral and alkaline conditions (pH 7.4 and pH 9.2), GABA production was significantly lower, with values of 0.61 ± 0.35 g/L at pH 7.4 and 0.03 ± 0.02 g/L at pH 9.2(Figure 18).

The GadB enzyme shows optimal activity in acidic conditions, with the highest GABA production observed at pH 4.6. Enzyme activity decreases significantly in neutral and alkaline environments, indicating that GadB functions best in an acidic environment.

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