In this project, we followed the Design-Build-Test-Learn cycle to produce kaempferol and GABA in engineered E. coli . First, we designed a polycistronic plasmid to express the enzymes F3H and FLS for kaempferol production from naringenin. Fusion enzyme technology with different linkers was introduced to enhance efficiency. After building and transforming the plasmids into E. coli BL21, we tested various conditions, such as temperature and substrate concentration. The optimized strain with the TPTP2 linker achieved a kaempferol yield of 93.35 ± 4.03 mg/L, while GABA production reached 2.48 ± 0.23 g/L at pH 4.6. These results provide insights into improving enzyme expression and production efficiency.

Kaempferol has been shown to significantly increase antioxidant enzyme activity and reduce oxidative stress markers, thereby protecting neurons from oxidative damage and alleviating depression symptoms. However, the de novo synthesis of kaempferol is complex and challenging. Fortunately, current technology enables efficient production of naringenin via synthetic biology, with yields reaching up to 391 mg/L. Therefore, we have decided to use metabolic engineering techniques to produce kaempferol using naringenin as a substrate.The conversion of naringenin to kaempferol involves two key enzymes: flavanone 3-hydroxylase (F3H) and flavonol synthase (FLS).

Based on this, we employed a polycistronic strategy, linking the coding sequences of F3H and FLS with a promoter and ribosome binding site (RBS B0034), and constructed the recombinant plasmid p23b-F3H-B0034-FLS.. This plasmid was then transformed into E. coli BL21 for expression, to test whether it could successfully produce kaempferol using naringenin as a substrate.

This design optimizes gene expression and metabolic pathways, aiming to enhance the bio-production of kaempferol and provide a potential solution for its industrial application.

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.

This result proves that the practice of the basic gene circuit design of this project is correct, and successfully opens up an innovative path for the low-cost production of kaempferol by industrial strains. Currently, the kaempferol yield is only about 40 mg/L, which falls short of our expected target. Therefore, we plan to implement fusion enzyme technology to optimize the engineered strain and enhance kaempferol production efficiency.

Through research, we discovered that fusion enzyme technology offers several advantages. It can significantly improve reaction efficiency by reducing substrate diffusion loss and can also reduce the metabolic burden on host cells, thus enhancing resource utilization. Additionally, fusion enzyme technology optimizes the spatial arrangement of enzymes, making the reaction chain more compact, which further boosts product yield. 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 4A). 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 7 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.

To investigate the effects of temperature and bacterial density on kaempferol production, we cultured the engineered strain BL21/p23b-F3H-GGGS-FLS in LB medium with 50 μg/mL ampicillin at 37℃, and added naringenin, followed by incubation at 180 rpm for 24 hours. We analyzed kaempferol yield under different induction temperatures (16℃, 20℃, 30℃, 37℃, 42℃) and initial bacterial densities (OD600= 0.2, 0.6, 1, 1.5, 2.0). Results (Figure 8) showed that kaempferol production peaked at 30℃ and bacterial density OD600 = 1.0, with lower temperatures slowing cell growth and shifting resources toward kaempferol synthesis. At higher bacterial densities, nutrient depletion and increased competition reduced kaempferol yield. Therefore, 30℃ and OD600 = 1.0 were optimal conditions for maximizing kaempferol production.

To optimize kaempferol yield and conversion rate, we investigated the effect of different naringenin concentrations on production. The engineered strain BL21/p23b-F3H-GGGS-FLS was cultured in LB medium with 50 μg/mL ampicillin at 37°C. Naringenin at concentrations of 125, 250, 500, 1000, and 2000 mg/L was added, and the cultures were incubated at 180 rpm for 24 hours. Kaempferol yield was measured at 368 nm, and the conversion rate was calculated as kaempferol concentration/naringenin concentration. Results (Figure 9) showed that kaempferol production increased with naringenin concentration, peaking at 72.39 ± 6.63 mg/L at 1000 mg/L before decreasing at higher concentrations. The highest conversion rate (0.12 ± 0.01) occurred at 500 mg/L. Thus, 1000 mg/L is optimal for yield, while 500 mg/L is best for conversion efficiency.

In this cycle, we utilized fusion enzyme technology, linking F3H and FLS with GGGS to construct the engineered strain and measured its kaempferol yield. We separately investigated the effects of temperature, cell density, and substrate concentration, identifying 30°C, an initial OD600 of 1.0, and a naringenin concentration of 1000 mg/L as the optimal conditions for maximizing production. Under these conditions, kaempferol yield reached up to 72.39 ± 6.63 mg/L. In the next cycle, we plan to further explore the impact of different linker types and copy numbers on kaempferol production.

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℃.

To evaluate the impact of different linkers on kaempferol production, recombinant strains carrying various linkers (F3H-GGGS-FLS, F3H-GGGS2-FLS, F3H-TPTP-FLS, and F3H-TPTP2-FLS) were inoculated into fresh LB medium containing 50 μg/mL ampicillin. The cultures were grown at 37°C until the bacterial density reached OD600 = 1.0. Afterward, 500 mg/L of naringenin was added to each strain, followed by incubation at 30°C for 24 hours. Kaempferol content was quantified using the method described previously. Results (Figure 12) demonstrated that the recombinant strain with the TPTP (rigid linker) produced 76.96 ± 4.19 mg/L of kaempferol, significantly higher than the strain with the GGGS (flexible linker), which produced 58.53 ± 6.33 mg/L. Furthermore, the strain with the TPTP2 (double copy) linker achieved the highest kaempferol yield at 93.35 ± 4.03 mg/L, surpassing the single-copy TPTP strain. These results suggest that the TPTP2 (double copy) linker provides the most efficient expression, offering a promising approach for optimizing kaempferol production.

In this cycle, we tested the effect of different linkers on kaempferol production, with results showing that the engineered strain using the TPTP2 (double copy) linker achieved the highest yield at 93.35 ± 4.03 mg/L. This indicates that adjusting the type and copy number of linkers can significantly enhance kaempferol production efficiency.

Based on these findings, our next step is to investigate the impact of multi-copy expression of the FLS and F3H genes on kaempferol yield. By increasing the copy number of these two key enzymes, we aim to further improve the efficiency of kaempferol biosynthesis.

To further increase kaempferol production, we plan to design a multi-copy expression system for the key enzymes FLS and F3H. The design involves constructing dual and multi-copy gene expression systems, utilizing strong promoters and efficient ribosome binding sites (RBS B0034) to drive gene expression, with terminators (B0015) separating the gene modules to prevent promoter interference. The plasmids will contain multi-copy FLS and F3H genes, such as the p23b-F3H-FLS+F3H and p23b-F3H-FLS+FLS plasmids, ensuring efficient expression of dual or multi-copy genes. Next, we will express these constructs in engineered strains and measure product yields to evaluate whether multi-copy expression significantly enhances kaempferol production. This design aims to further improve kaempferol biosynthesis efficiency by increasing the expression of key enzymes.

In this experiment, we used the TPTP2 linker-based plasmid as the core to construct different copy numbers of F3H and FLS expression systems. First, the F3H and FLS genes were synthesized, codon-optimized, and cloned into the pET23b plasmid through EcoRI and XhoI restriction sites, creating the p23b-F3H-TPTP2-FLS fusion enzyme expression plasmid. To investigate the effect of multi-copy genes on kaempferol production, we further constructed two multi-copy expression plasmids.

The design includes two plasmids: p23b-F3H-TPTP2-FLS+FLS (BBa_K5482027) and p23b-F3H-TPTP2-FLS+F3H (BBa_K5482028). In the p23b-F3H-TPTP2-FLS+FLS plasmid, an additional FLS gene module was added, while in the p23b-F3H-TPTP2-FLS+F3H plasmid, an additional F3H gene module was introduced. Each gene module is driven by a strong promoter and separated by the terminator B0015 to avoid promoter interference.

After construction, the plasmids were verified by sequencing and transformed into E. coli BL21 for kaempferol production testing.

To evaluate the impact of additional gene copies on kaempferol production, recombinant strains carrying different gene combinations (p23b-F3H-TPTP2-FLS+FLS, BBa_K5482007 and p23b-F3H-TPTP2-FLS+F3H, BBa_K5482008) 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 measured by detecting the absorbance of the samples at 368 nm using a microplate reader. Results (Figure 14) showed that adding an extra copy of FLS to the original F3H-TPTP2-FLS construct caused kaempferol production to decrease from 87.70 ± 5.37 mg/L to 34.87 ± 4.86 mg/L. In contrast, adding an extra copy of F3H significantly increased kaempferol yield from 87.70 ± 5.37 mg/L to 113.36 ± 9.47 mg/L. These findings suggest that introducing an additional copy of F3H can effectively boost kaempferol production, whereas adding an extra FLS copy has an inhibitory effect. This provides valuable guidance for optimizing gene copy number to maximize kaempferol production efficiency in future experiments.

To explore why adding F3H to the original plasmid increased kaempferol production, while adding FLS reduced it, we conducted experiments to investigate the process and results in more detail. 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 dissolved in 0.1% DEPC water. RNA purity was verified with a Nanodrop 2000, yielding an OD260/OD280 ratio of around 1.8, indicating good quality. RNA was then reverse transcribed into cDNA using the HiScript 1st Strand cDNA Synthesis Kit. The cDNA concentration was adjusted to 100 ng/μL, mixed with TB Green® dye, and real-time qRT-PCR was performed using QuantStudio 5 to measure mRNA levels. The 16S rRNA gene served as an internal reference, and the 2−ΔΔCt method was used to calculate relative mRNA expression. Results (Figure 15) showed that the mRNA expression level of F3H-TPTP2-FLS was significantly higher than that of F3H-TPTP2-FLS+FLS and F3H-TPTP2-FLS+F3H, with mRNA levels decreasing from 9 to 5. This suggests that using multicopy genes reduced mRNA expression compared to single-copy constructs. From this, we inferred two possible reasons for the observed effects: F3H is likely a rate-limiting enzyme in kaempferol synthesis, and increasing its expression enhances the conversion of flavanones to kaempferol. Conversely, plasmids carrying multicopy genes impose a heavy metabolic burden, reducing energy availability, lowering mRNA levels, and diminishing kaempferol production when FLS is overexpressed.

In this experiment, by comparing recombinant strains with different gene copy numbers, we discovered that kaempferol production is limited by the expression level of the key enzyme F3H. As a rate-limiting enzyme in the kaempferol synthesis pathway, increasing the expression of F3H effectively boosted kaempferol yield. On the other hand, adding multiple copies of FLS resulted in a decrease in yield. Further experiments revealed that this was due to the significant metabolic burden imposed by multicopy plasmids on the engineered bacteria, leading to reduced mRNA levels and lower gene expression efficiency.

From this, we learned two important lessons: first, F3H is the rate-limiting enzyme in kaempferol synthesis, and increasing its expression can significantly enhance yield. Second, although multicopy genes may enhance the activity of certain enzymes, in systems with high plasmid burden, the excessive energy consumption can actually inhibit overall production. Therefore, future experimental designs will need to carefully balance gene copy numbers and the metabolic load on host cells to achieve more efficient kaempferol production.

To achieve efficient production of GABA (γ-aminobutyric acid), we designed a metabolic engineering-based GABA production system. The core of this experiment is utilizing glutamate decarboxylase (GAD) to convert glutamate into GABA. We synthesized and optimized the GadB gene to be better suited for expression in E. coli , and cloned it into the pET23b plasmid to construct the recombinant plasmid p23b-GadB. This plasmid was transformed into E. coli BL21, and through culturing in glutamate-containing medium, the GAD enzyme converted glutamate into GABA. We then extracted crude enzyme from the bacterial cultures and measured GABA content to assess conversion efficiency. To optimize GABA production, we focused on optimizing the pH conditions, identifying the optimal pH for maximum GABA yield.

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 17).

To catalyze GABA synthesis, we used crude enzyme extracts from the engineered E. coli BL21/p23b-GadB strain. First, we harvested 2 mL of bacterial culture and centrifuged it at 8000 rpm for 10 minutes to collect the bacterial pellet. The pellet was resuspended in 2 mL acetic acid-sodium acetate buffer (pH 4.6) and 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. For the GABA synthesis reaction, 2% glutamic acid was added to 1 mL of crude enzyme solution, and the mixture was incubated at 37°C for 3 hours. After the reaction, GABA content was measured using a GABA detection kit, with absorbance at 640 nm recorded using a microplate reader. A standard curve was generated (Y = 0.5130*X - 0.05257, R² = 0.98) to calculate GABA concentration based on absorbance.BL21 and BL21/pET23b were used as control groups, while BL21/p23b-GadB served as the experimental group. By constructing the recombinant strain p23b-GadB, we achieved successful GABA production, with the recombinant strain yielding 2.32 ± 0.21 g/L of GABA (Figure 18).

To further enhance GadB enzyme activity, we tested the GABA concentration under different pH conditions. The BL21/p23b-GadB pellet was resuspended in buffers of various pH values, including acetate-sodium acetate buffer (pH 3.6 and pH 4.6), PBS (pH 7.4), and Tris-HCl buffer (pH 9.2). The cells were sonicated to obtain the crude enzyme solution, and 2% glutamic acid was added to 1 mL of enzyme solution, followed by incubation at 37°C for 3 hours. GABA production was then measured using a GABA detection kit .The results showed that GadB enzyme activity varied significantly with pH. GABA production was highest at pH 4.6 (2.48 ± 0.23 g/L) and pH 3.6 (2.17 ± 0.12 g/L), while production decreased sharply at pH 7.4 (0.61 ± 0.35 g/L) and pH 9.2 (0.03 ± 0.02 g/L). This indicates that GadB functions best under acidic conditions, with optimal activity at pH 4.6(Figure 19) .

Through this experiment, we learned that GadB enzyme activity is highly dependent on the pH of the environment. The enzyme functions optimally in acidic conditions, particularly at pH 4.6, where GABA production reached its peak. Additionally, we successfully demonstrated that the recombinant BL21/p23b-GadB strain could efficiently produce GABA, with yields significantly higher in acidic buffers compared to neutral and alkaline conditions.

Looking forward, while we have successfully tested GABA production in E. coli BL21, we aim to apply this system in probiotic strains like E. coli Nissle 1917. This strain is a safe, well-established probiotic, and future experiments will focus on producing GABA using Nissle 1917. Ultimately, our goal is to integrate the GABA production pathway into probiotic products, such as adding it to beverages like fruit tea, to create functional foods with health benefits.

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