iGEM 2024 — Team jiashu-southchina

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Notebook

Summary (June 1 - August 15, 2024)

System 1: Kaempferol Production Strain Construction and Optimization

System 2: GABA Production Strain Construction and Optimization

Daily Plan Summary Table (June 1 - August 15, 2024)

System 1: Kaempferol Production Strain Construction and Optimization

System 2: GABA Production Strain Construction and Optimization

# Summary (June 1 - August 15, 2024)

This experiment focuses on two major systems: the construction and optimization of strains for kaempferol production and GABA production. Both systems involve genetic engineering techniques and optimization of expression conditions to maximize product yield.

## System 1: Kaempferol Production Strain Construction and Optimization

1. Objective: To construct and optimize E. coli  strains that can produce kaempferol through the expression of the genes CisF3H and CuFLS.

2. Key Steps:

a. Synthesize the CisF3H and CuFLS genes and clone them into the pET23b plasmid.

b. Transform the plasmids into E. coli  strains DH5α for cloning and BL21 for expression.

c. Induce kaempferol production using naringenin and analyze its content using a UV spectrophotometer.

d. Optimize production conditions by testing different parameters, including temperature, initial cell density, naringenin concentration, and linker effects.

3. Expected Results: Efficient kaempferol production with optimal conditions identified for temperature, cell density, and naringenin concentration.

## System 2: GABA Production Strain Construction and Optimization

1. Objective: To construct and optimize E. coli  strains that can produce GABA through the expression of the GadB gene.

2. Key Steps:

a. Synthesize the GadB gene and clone it into the pET23b plasmid.

b. Transform the plasmids into E. coli  strains DH5α for cloning and BL21 for expression.

c. Optimize GABA production through enzyme activity tests under different pH conditions.

d. Measure GABA production levels using an assay kit and analyze enzyme activity at various pH levels.

3. Expected Results: Maximum GABA production with an optimal pH range for enzyme activity identified.

# Daily Plan Summary Table (June 1 - August 15, 2024)

## System 1: Kaempferol Production Strain Construction and Optimization

|
|  |

Date |

Experiment Title |

Experiment Objective |

Key Steps |

Expected Results |

Remarks |

|
|  |

June 1 June 5 |

Synthesis and Cloning of CisF3H and CuFLS |

Synthesize and clone genes into pET23b |

Gene synthesis, cloning into pET23b using restriction sites |

Genes successfully cloned into plasmid |

Prepare for transformation |

|
|  |

June 6 June 10 |

Transformation of Plasmid |

Transform the plasmid into E. coli  BL21 |

Transform CisF3H and CuFLS into BL21 strains |

Transformants confirmed by colony screening |

Prepare for plasmid extraction |

|
|  |

June 11 June 13 |

Plasmid Verification |

Verify plasmid sequence integrity |

Plasmid extraction, sequencing |

Plasmid sequences confirmed to be correct |

Begin kaempferol production experiments |

|
|  |

June 14 June 18 |

Induction of Kaempferol Production (F3H-B0034-FLS) |

Measure kaempferol production from F3H-B0034-FLS strain |

Add naringenin to induce production, analyze kaempferol production |

Kaempferol production measured |

Prepare for constructing the next strain |

|
|  |

June 19 June 25 |

Construction of F3H-GGGS-FLS Strain |

Link F3H and FLS with GGGS linker and clone |

Clone and transform F3H-GGGS-FLS into BL21, confirm via sequencing |

F3H-GGGS-FLS strain successfully constructed |

Prepare for kaempferol production measurement |

|
|  |

June 26 June 30 |

Induction of Kaempferol Production (F3H-GGGS-FLS) |

Measure kaempferol production from F3H-GGGS-FLS strain |

Add naringenin, incubate, and analyze kaempferol production |

Kaempferol production measured |

Compare production levels with F3H-B0034-FLS |

|
|  |

July 1 July 5 |

Temperature Optimization |

Optimize production temperature |

Test different temperatures (16°C, 25°C, 30°C, 37°C, 42°C) |

Optimal temperature for production identified |

Proceed with cell density optimization |

|
|  |

July 6 July 10 |

Cell Density Optimization |

Optimize initial cell density |

Test different cell densities (OD600 = 0.2, 0.6, 1.0, 1.5, 2.0) |

Optimal cell density identified |

Proceed with naringenin concentration optimization |

|
|  |

July 11 July 15 |

Naringenin Concentration Optimization |

Optimize naringenin concentration |

Test naringenin concentrations (125, 250, 500, 1000, 2000 mg/L) |

Optimal naringenin concentration determined |

Proceed with linker optimization |

|
|  |

July 16 July 20 |

Linker Effects on Kaempferol Production |

Study effects of linkers on kaempferol production |

Compare flexible (GGGS) and rigid (TPTP) linkers |

Rigid linker shows higher production |

Proceed with multi-gene optimization |

|
|  |

July 21 July 25 |

Multi-Gene Copy Effects |

Test effect of multiple gene copies on production |

Analyze the effect of adding multiple copies of F3H and FLS |

Multi-gene increases kaempferol yield |

Continue optimization trials |

|
|  |

July 26 July 30 |

The mRFP Expression Level Analysis |

mRFP Expression Level Analysis in F3H-FLS Recombinant Strains |

qRT-PCR |

The mRNA expression levels |

Prepare for data analysis |

|
|  |

August 1 August 5 |

Final Kaempferol Data Analysis |

Analyze final kaempferol production data |

Review experimental data |

Complete data analysis and prepare final report |

Finalize results |

## System 2: GABA Production Strain Construction and Optimization

|
|  |

Date |

Experiment Title |

Experiment Objective |

Key Steps |

Expected Results |

Remarks |

|
|  |

July 1 July 5 |

Synthesis and Cloning of GadB |

Synthesize and clone GadB gene into pET23b |

Gene synthesis, cloning into pET23b |

GadB gene successfully cloned into plasmid |

Prepare for transformation |

|
|  |

July 6 July 10 |

Transformation of Plasmid |

Transform GadB plasmid into E. coli  DH5α and BL21 |

Transformation into DH5α and BL21 strains |

Transformants confirmed by colony screening |

Prepare for plasmid extraction |

|
|  |

July 11 July 14 |

Plasmid Verification |

Verify the plasmid sequence integrity |

Plasmid extraction, sequencing |

Plasmid sequences confirmed to be correct |

Begin GABA production experiments |

|
|  |

July 16 July 20 |

GABA Production Test |

Test GABA production using glutamate as a substrate |

Incubate with glutamate, measure GABA with assay kit |

Initial GABA production observed |

Prepare for pH optimization |

|
|  |

July 21 July25 |

pH Optimization |

Optimize enzyme activity for GABA production |

Test different pH levels (3.6, 4.6, 7.4, 9.2) |

Optimal pH for enzyme activity identified |

Final GABA Data Analysis |

|
|  |

August 1 |

August 5

Final GABA Data Analysis |

Analyze final GABA production data |

Review experimental data |

Complete data analysis and prepare for final report |

Finalize results |

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Engineering

Abstract

Cycle 1-1 p23b-CisF3H-B0034-CuFLS

Design

Build

Test

Learn

Cycle 1-2 p23b-CisF3H-GGGS-CuFLS

Design

Build

Test

Learn

Cycle1-3 p23b-CisF3H-GGGS\*2/TPTP/TPTP\*2-CuFLS

Design

Build

Test

Learn

Cycle 1-4 p23b-CisF3H-TPTP\*2-CuFLS+F3H/FLS (Multicopy Genes)

Design

Build

Test

Learn

Cycle 2 GABA

Design

Build

Test

Learn

Reference：

# Abstract

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.

![](https://static.igem.wiki/teams/5482/engineering/img1.png)

# Cycle 1-1 p23b-CisF3H-B0034-CuFLS

## Design

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).

![](https://static.igem.wiki/teams/5482/engineering/img2.png)

Figure 1. Chemical synthesis of kaempferol with the introduction of the hydroxyl group at the C3 position of naringenin catalysed by flavanone 3-hydroxylase (F3H), and the introduction of a double bond at the C2-C3 position of dihydro's kaempferol by flavonol synthase (FLS) to form kaempferol

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.

![](https://static.igem.wiki/teams/5482/engineering/new-img3.png)

Figure 2. Gene circuit of p23b-F3H-B0034-FLS..

## Build

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℃.

![](https://static.igem.wiki/teams/5482/engineering/img4.png)

Figure 3. Agarose gel electrophoresis of CisF3H, CuFLS and F3H-B0034-FLS.

## Test

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.

![](https://static.igem.wiki/teams/5482/engineering/img5.png)

Figure 4. The production of kaempferol. (A) Standard curve of kaempferol. (B) Yield of kaempferol produced by different strains using naringenin as substrate.

Figure 4A shows that the concentration of kaempferol is proportional to the absorbance, the linear regression equation is Y=0.08453\*X-0.05387, the correlation coefficient R^2=0.9915, and the linear relationship is good. Figure 4B clearly shows that naringenin can successfully synthesize kaempferol through protein expression in the recombinant strain, and its yield reached 40.85 ± 5.51 mg/L. As the control group, the wild-type BL21 and the control strain (containing only the pET23b empty plasmid) were unable to synthesize kaempferol under the same operation and culture conditions.

## Learn

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.

# Cycle 1-2 p23b-CisF3H-GGGS-CuFLS

## Design

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.

![](https://static.igem.wiki/teams/5482/engineering/new-img6.png)

Figure 5. Gene circuit of p23b-F3H-GGGS-FLS.

## Build

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.

![](https://static.igem.wiki/teams/5482/engineering/img7.png)

Figure 6. Agarose gel electrophoresis of F3H-GGGS-FLS.

## Test

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.

![](https://static.igem.wiki/teams/5482/engineering/img8.png)

Figure 7. Kaempferol yield of F3H-GGGS-FLS engineered strain and control strain.

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.

![](https://static.igem.wiki/teams/5482/engineering/img9.png)

Figure 8. Effects of temperature and bacterial concentrations on kaempferol production by engineered BL21/p23b-F3H-GGGS-FLS. (A) Effect of temperature on kaempferol yield. (B) Effect of bacterial concentration on kaempferol yield.

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.

![](https://static.igem.wiki/teams/5482/engineering/img10.png)

Figure 9. Effects of naringenin concentrations on kaempferol production by engineered bacteria BL21/p23b-F3H-GGGS-FLS. (A) Effect of naringenin at different concentrations on kaempferol yield. (B) Effect of naringenin at different concentrations on kaempferol molar conversion rate.

## Learn

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.

# Cycle1-3 p23b-CisF3H-GGGS\*2/TPTP/TPTP\*2-CuFLS

## Design

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.

![](https://static.igem.wiki/teams/5482/engineering/new-img11.png)

Figure 10. Gene circuit with different linker.

## Build

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℃.

![](https://static.igem.wiki/teams/5482/engineering/img12.png)

Figure 11. Agarose gel electrophoresis of F3H-GGGS-FLS(same as Figure 6), F3H-GGGS2-FLS, F3H-TPTP-FLS, F3H-TPTP-FLSF3H-TPTP2-FLS.

## Test

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.

![](https://static.igem.wiki/teams/5482/engineering/img13.png)

Figure 12. Effects of different linkers on kaempferol biological production.

## Learn

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.

# Cycle 1-4 p23b-CisF3H-TPTP\*2-CuFLS+F3H/FLS (Multicopy Genes)

## Design

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.

![](https://static.igem.wiki/teams/5482/engineering/img14.png)

Figure 13 Plasmid Map. (A) Recombinant strain p23b-F3H-TPTP2-FLS+FLS. (B) Recombinant strain p23b-F3H-TPTP2-FLS+F3H.

## Build

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.

## Test

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.

![](https://static.igem.wiki/teams/5482/engineering/img15.png)

Figure 14. Effect of multiple copies of genes on the yield of kaempferol biosynthesis.

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.

![](https://static.igem.wiki/teams/5482/engineering/img16.png)

Figure 15. Relative mRNA levels of different strain.

## Learn

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.

# Cycle 2 GABA

## Design

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.

![](https://static.igem.wiki/teams/5482/engineering/new-img17.png)

Figure 16. Gene circuit of p23b-GadB.

## Build

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).

![](https://static.igem.wiki/teams/5482/engineering/img18.png)

Figure 17. Agarose gel electrophoresis of GadB.

## Test

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).

![](https://static.igem.wiki/teams/5482/engineering/img19.png)

Figure 18. The production of GABA. (A) The standard curve of GABA. (B) The influence of inserting GadB gene fragments on GABA yield.

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) .

![](https://static.igem.wiki/teams/5482/engineering/img20.png)

Figure 19. The effect of pH on GadB activity.

## Learn

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.

# Reference：

[1] Pei J, Chen A, Dong P, et al. Modulating heterologous pathways and optimizing fermentation conditions for biosynthesis of kaempferol and astragalin from naringenin in E. coli [J]. Journal of Industrial Microbiology and Biotechnology, 2019, 46(2): 171-186.

[2] Brazier-Hicks M, Edwards R. Metabolic engineering of the flavone-C-glycoside pathway using polyprotein technology[J]. Metabolic engineering, 2013, 16: 11-20.

[3] Rodriguez A, Strucko T, Stahlhut S G, et al. Metabolic engineering of yeast for fermentative production of flavonoids[J]. Bioresource Technology, 2017, 245: 1645-1654.

[4] Malla S, Pandey R P, Kim B G, et al. Regiospecific modifications of naringenin for astragalin production in E. coli [J]. Biotechnology and bioengineering, 2013, 110(9): 2525-2535.

[5]Duan L, Ding W, Liu X, et al. Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae[J]. Microbial cell factories, 2017, 16: 1-10.

[6] Leonard E, Lim K H, Saw P N, et al. Engineering central metabolic pathways for high-level flavonoid production in E. coli [J]. Applied and Environmental Microbiology, 2007, 73(12): 3877-3886.

[7] Stahlhut S G, Siedler S, Malla S, et al. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in E. coli [J]. Metabolic engineering, 2015, 31: 84-93.

[8] Xiong D, Lu S, Wu J, et al. Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor[J]. Metabolic engineering, 2017, 40: 115-123.

[9] Li H, Gao S, Zhang S, et al. Effects of metabolic pathway gene copy numbers on the biosynthesis of (2S)-naringenin in Saccharomyces cerevisiae[J]. Journal of Biotechnology, 2021, 325: 119-127.

[10] Daniel R M, Danson M J, Eisenthal R, et al. The effect of temperature on enzyme activity: new insights and their implications[J]. Extremophiles, 2008, 12: 51-59.

[11] McDermid A S, McKee A S, Marsh P D. Effect of environmental pH on enzyme activity and growth of Bacteroides gingivalis W50[J]. Infection and immunity, 1988, 56(5): 1096-1100.

[12] Zhang H B, Mao X Q, Wang Y J, et al. Optimization of culture conditions for high-level expression of dextransucrase in E. coli [J]. International Journal of Food, Agriculture and Environment, 2009, 7: 75-78.

[13] Leonard E, Lim K H, Saw P N, et al. Engineering central metabolic pathways for high-level flavonoid production in E. coli . Applied and Environmental Microbiology, 2007, 73(12): 3877-3886.

[14] Zhang, H., Wang, Y., & Gao, X. (2020). Metabolic engineering strategies for kaempferol biosynthesis in microorganisms. Biotechnology for Biofuels, 13(1), 35.

[15] Rodriguez, A., Strucko, T., Stahlhut, S. G., et al. Metabolic engineering of yeast for fermentative production of flavonoids. Bioresource Technology, 2017, 245: 1645-1654.

[16] Stahlhut, S. G., Siedler, S., Malla, S., et al. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in E. coli . Metabolic Engineering, 2015, 31: 84-93.

[17] Pei, J., Chen, A., Dong, P., et al. Modulating heterologous pathways and optimizing fermentation conditions for biosynthesis of kaempferol and astragalin from naringenin in E. coli . Journal of Industrial Microbiology and Biotechnology, 2019, 46(2): 171-186.

[18] Li, H., Gao, S., Zhang, S., et al. Effects of metabolic pathway gene copy numbers on the biosynthesis of (2S)-naringenin in Saccharomyces cerevisiae. Journal of Biotechnology, 2021, 325: 119-127.

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Experiments

Construction of kaempferol-producing engineered bacteria

Analysis of kaempferol content in engineered strains using microplate reader

Optimization of bioproduction conditions for kaempferol production

Effect of multicopy genes on kaempferol biosynthesis

Analysis of mRFP expression levels in engineered strain F3H-FLS

Construction of GABA-producing engineered bacteria

Production of GABA using crude enzyme extracts from engineered strains

Effect of pH on GadB enzyme activity

Statistical analysis

# Construction of kaempferol-producing engineered bacteria

The CisF3H and CuFLS gene sequences were synthesized and codon-optimized for E. coli. The two protein-coding genes were linked using fusion techniques (GGGS or TPTP linker sequences) or a polycistronic strategy (RBS B0034). Subsequently, the construct was cloned into the pET23b plasmid via EcoRI and XhoI restriction sites to obtain p23b-F3H-FLS (Genewiz, USA). After confirming the correct sequence by sequencing (Qingke, China), the recombinant plasmid was extracted using a plasmid extraction kit (TIANGEN, China). Next, the recombinant plasmid was transformed into E. coli DH5α and BL21. E. coli DH5α was used for plasmid storage, and E. coli BL21 was used for plasmid expression. The engineered strains were cultured in LB medium containing ampicillin (Amp) (50 μg/mL) at 37°C.

# Analysis of kaempferol content in engineered strains using microplate reader

Wild-type BL21, control strains (containing only the pET23b empty plasmid), and recombinant strains were inoculated into fresh LB medium. The media for the control and recombinant strains contained 50 μg/mL ampicillin (Amp). The cultures were then grown at 37°C until the cell density reached OD600 = 1.0. Subsequently, 500 mg/L naringenin was added to each group of strains, and the cultures were incubated at 30°C with shaking at 180 rpm for 24 hours. A 1 mL sample of the bacterial culture was taken, 5 mL of methanol was added, and the mixture was vortexed and then centrifuged (12,000 g, 1 min) to collect the supernatant. A standard curve was prepared using kaempferol standards (K812226, Macklin) at concentrations of 1, 10, 20, 30, and 50 mg/L. The absorbance of the samples and standards was measured at 368 nm using a microplate reader. A linear equation was established from the standard curve. The kaempferol content in the engineered strains was calculated based on the linear equation and the dilution factors.

# Optimization of bioproduction conditions for kaempferol production

The engineered strain BL21/p23b-F3H-GGGS-FLS was inoculated into fresh LB medium containing 50 μg/mL ampicillin and grown at 37°C. Naringenin was added to the recombinant strain and cultured with shaking at 180 rpm for 24 hours. The effects of induction temperature (16°C, 25°C, 30°C, 37°C, 42°C), initial bacterial density (OD600 = 0.2, 0.6, 1.0, 1.5, and 2.0), and naringenin concentration (125, 250, 500, 1000, and 2000 mg/L) on kaempferol production were analyzed.

# Effect of multicopy genes on kaempferol biosynthesis

The sequences p23100-B0034-CisF3H and p23100-B0034-CuFLS were synthesized and cloned into the pSB1A3 plasmid using XbaI and SpeI restriction sites (Genewiz, USA), resulting in pSB-p23100-B0034-CisF3H and pSB-p23100-B0034-CuFLS. Using a high-fidelity enzyme (PrimeSTAR HS, Takara) and primers F3H-FLS-F and F3H-FLS-R, F3H-FLS was cloned from p23b-F3H-TPTP-FLS. Primers PBC-F3H-F/R and PBC-FLS-F/R were used to clone p23100-B0034-CisF3H and p23100-B0034-CuFLS from pSB-p23100-B0034-CisF3H and pSB-p23100-B0034-CuFLS, respectively. F3H-FLS-p23100-B0034-CuFLS and F3H-FLS-p23100-B0034-CisF3H were obtained through overlap PCR. The recombinant DNA was then cloned into pET23b using EcoRI and XhoI restriction sites to obtain p23b-F3H-FLS+FLS or p23b-F3H-FLS+F3H. The recombinant plasmids p23b-F3H-FLS+FLS and p23b-F3H-FLS+F3H were transformed into E. coli DH5α and BL21. E. coli DH5α was used for plasmid storage, and E. coli BL21 was used for plasmid expression. The recombinant strains were inoculated into fresh LB medium containing 50 μg/mL ampicillin (Amp). Next, cultures were incubated at 37°C until the bacterial density reached OD600 = 1.0. Then, 500 mg/L naringenin was added to each group of strains, and they were incubated with shaking at 180 rpm at 30°C for 24 hours. Kaempferol was extracted with ethanol and analyzed using a microplate reader.

# Analysis of mRFP expression levels in engineered strain F3H-FLS

The recombinant strains were inoculated into fresh LB medium containing 50 μg/mL ampicillin (Amp). Next, the cultures were grown at 37 °C until the bacterial density reached OD600 = 0.6. Two milliliters of the culture were collected by centrifugation at 4000 × g for 5 minutes. Total RNA was extracted from the bacterial samples using the Bacterial RNA Kit (R6950, OMEGA) with β-mercaptoethanol. The RNA was dissolved in 0.1% diethyl pyrocarbonate (DEPC)-treated water. The OD260/OD280 ratio was measured using a NanoDrop 2000 (Thermo Fisher Scientific) to assess RNA purity. The extracted RNA was reverse-transcribed into cDNA using the HiScript 1st Strand cDNA Synthesis Kit (R111, Vazyme). The cDNA concentration was measured with the NanoDrop 2000 and adjusted to 100 ng/μL with ddH2O. The cDNA was then mixed with TB Green® Premix Ex Taq™ II (RR820A, Takara). Quantitative real-time PCR (qRT-PCR) was performed using the QuantStudio 5 (Applied Biosystems, Thermo Fisher Scientific). The 16S rRNA gene was used as an internal control, and the mRNA expression levels of each gene were calculated using the 2−ΔΔCt method.

**Table 1. Primer sequences used for qRT-PCR**

![](https://static.igem.wiki/teams/5482/experiments/img1.png)

# Construction of GABA-producing engineered bacteria

The GadB gene sequence was synthesized and codon-optimized for E. coli. It was then cloned into the pET23b plasmid using EcoRI and XhoI restriction sites to obtain p23b-GadB (Genewiz, USA). After verifying the correct sequence by sequencing (Qingke, China), the recombinant plasmid was extracted using a plasmid extraction kit (TIANGEN, China). The recombinant plasmid was transformed into E. coli DH5α and BL21. E. coli DH5α was used for plasmid storage, and E. coli BL21 was used for plasmid expression. The engineered strains were cultured in LB medium containing ampicillin (Amp) (50 μg/mL) at 37 °C.

# Production of GABA using crude enzyme extracts from engineered strains

Wild-type BL21, control strains (containing only the pET23b empty plasmid), and recombinant strains were inoculated into fresh LB medium at a ratio of 1:100d. The media for the control and recombinant strains contained 50 μg/mL ampicillin (Amp). The cultures were incubated overnight at 37 °C. Two milliliters of bacterial culture were collected and centrifuged at 8000 rpm for 10 minutes to collect the bacterial pellet. The cells were resuspended in 2 mL sodium acetate buffer (pH = 4.6). Then, the cells were lysed using an ultrasonic homogenizer (Biosafer1000, Saifei) under ice bath conditions (75 W, 1 s ultrasonication, 3 s intervals, total 20 min) to obtain the crude enzyme extract. In 1 mL of the crude enzyme solution, 2% glutamic acid was added and incubated at 37 °C for 3 hours. Subsequently, the GABA content was measured using a γ-aminobutyric acid (GABA) assay kit (mlbio, China). Specifically, in a 2 mL centrifuge tube, 100 μL of 0.4 M boric acid buffer (pH = 10), 10 μL of reaction supernatant, 100 μL of 6% phenol, and 100 μL of 6% sodium hypochlorite (NaClO) were added sequentially. The tubes were left at room temperature for 5 minutes, followed by heating in a 100 °C water bath for 10 minutes. After cooling, the samples were centrifuged at 8000 g for 10 minutes at 25 °C, and the supernatant was collected for measurement. Absorbance at 640 nm was measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific, USA). A standard curve was plotted to calculate the concentration of GABA.

# Effect of pH on GadB enzyme activity

The recombinant strains were inoculated into fresh LB medium containing 50 μg/mL ampicillin (Amp) at a ratio of 1:100d and incubated overnight at 37 °C. Two milliliters of bacterial culture were collected and centrifuged at 8000 rpm for 10 minutes to collect the bacterial pellet. The cells were resuspended in 2 mL of sodium acetate buffer (pH = 4.6), sodium acetate buffer (pH = 3.6), PBS (pH = 7.4), or Tris-HCl buffer (pH = 9.2). Then, the cells were lysed using an ultrasonic homogenizer (Biosafer1000, Saifei) under ice bath conditions (75 W, 1 s ultrasonication, 3 s intervals, total 20 min) to obtain the crude enzyme extract. In 1 mL of the crude enzyme solution, 2% glutamic acid was added and incubated at 37 °C for 3 hours. Subsequently, the GABA content was measured using a γ-aminobutyric acid (GABA) assay kit (mlbio, China).

# Statistical analysis

Data were analyzed and plotted using GraphPad Prism software. Results are presented as mean ± standard deviation (SD). For comparisons among multiple groups, one-way analysis of variance (ANOVA) and Tukey's post hoc test were used for difference analysis. A p-value less than 0.05 was considered statistically significant.

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Results

Abstract

p23b-CisF3H-B0034-CuFLS

p23b-CisF3H-GGGS-CuFLS

Optimization of Kaempferol Biosynthesis Conditions - Temperature and Cell Density

Optimization of Kaempferol Biosynthesis Conditions - substrate concentration

Optimization of Kaempferol Biosynthesis Conditions - Linker

Effects of multi-copy genes on kaempferol biosynthesis

Exploration of the reasons for the impact of gene copy number variation on kaempferol biosynthesis

Preliminary Extraction of Kaempferol Using Engineered Bacteria

GABA verification

Effect of pH level on the activity of GadB

Reference：

# Abstract

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.

# p23b-CisF3H-B0034-CuFLS

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.

![](https://static.igem.wiki/teams/5482/results/img1.png)

**Figure 1.** Chemical synthesis of kaempferol. The hydroxyl group is introduced at the C3 position of naringenin by flavanone 3-hydroxylase (F3H), and the double bond is introduced at the C2-C3 position of dihydrokaempferol by flavonol synthase (FLS) to form 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℃.

![](https://static.igem.wiki/teams/5482/results/new-img2.png)

**Figure 2.** Recombinant plasmid p23b-F3H-B0034-FLS construction and verification. (A) Recombinant plasmid p23b-F3H-B0034-FLS map. (B) F3H-B0034-FLS gene circuit. (C) Agarose gel electrophoresis of CisF3H . (D) Agarose gel electrophoresis of CuFLS . (E)Agarose gel electrophoresis of F3H-B0034-FLS.

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.

![](https://static.igem.wiki/teams/5482/results/img3.png)

**Figure 3.** The production of kaempferol. (A) Standard curve of kaempferol. (B) Yield of kaempferol produced by different strains using naringenin as substrate.

Figure 3A shows that the concentration of kaempferol is proportional to the absorbance, the linear regression equation is Y=0.08453\*X-0.05387, the correlation coefficient R^2=0.9915, and the linear relationship is good. Figure 3B clearly shows that naringenin can successfully synthesize kaempferol through protein expression in the recombinant strain, and its yield reached 40.85 ± 5.51 mg/L. As the control group, the wild-type BL21 and the control strain (containing only the pET23b empty plasmid) were unable to synthesize kaempferol under the same operation and culture conditions. 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.

# p23b-CisF3H-GGGS-CuFLS

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.

![](https://static.igem.wiki/teams/5482/results/new-img4.png)

**Figure 4.** Recombinant plasmid p23b-F3H-GGGS-FLS construction and verification. (A) Recombinant plasmid p23b-F3H-GGGS-FLS map. (B) F3H-GGGS-FLS gene circuit. (C) Agarose gel electrophoresis of F3H-GGGS-FLS.

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.

![](https://static.igem.wiki/teams/5482/results/img5.png)

**Figure 5.** Kaempferol yield of F3H-GGGS-FLS engineered strain and control strain.

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.

# Optimization of Kaempferol Biosynthesis Conditions - Temperature and Cell Density

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.

![](https://static.igem.wiki/teams/5482/results/img6.png)

**Figure 6.** Effects of temperature and bacterial concentrations on kaempferol production by engineered BL21/p23b-F3H-GGGS-FLS. (A) Effect of temperature on kaempferol yield. (B) Effect of bacterial concentration on kaempferol yield.

Figure 6A showed that the yield of kaempferol was different under different temperature conditions. The temperature gradually increased from low to high and then gradually decreased, and the highest yield was reached at 30℃. The reason for the low temperature but high yield was that lower temperature would slow down the growth rate of microorganisms and lead to more metabolic resources (such as carbon source, carbon source, nitrogen source, etc.) for kaempferol synthesis rather than cell growth. In this case, the microbe will use more energy and substrate to produce kaempferol.

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.

# Optimization of Kaempferol Biosynthesis Conditions - substrate concentration

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).

![](https://static.igem.wiki/teams/5482/results/img7.png)

**Figure 7.** Effects of naringenin concentrations on kaempferol production by engineered bacteria BL21/p23b-F3H-GGGS-FLS. (A) Effect of naringenin at different concentrations on kaempferol yield. (B) Effect of naringenin at different concentrations on kaempferol molar conversion rate.

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.

# Optimization of Kaempferol Biosynthesis Conditions - Linker

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.

![](https://static.igem.wiki/teams/5482/results/img8.png)

**Figure 8.** Gene circuit with different linker.

![](https://static.igem.wiki/teams/5482/results/new-img9.png)

**Figure 9.** Agarose gel electrophoresis of F3H-GGGS-FLS, F3H-GGGS2-FLS, F3H-TPTP-FLS, F3H-TPTP-FLSF3H-TPTP2-FLS.

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.

![](https://static.igem.wiki/teams/5482/results/img10.png)

**Figure 10.** Effects of different linkers on kaempferol biological production.

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.

# Effects of multi-copy genes on kaempferol biosynthesis

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.

![](https://static.igem.wiki/teams/5482/results/img11.png)

**Figure 11.**Plasmid Map. (A) Recombinant strain p23b-F3H-TPTP2-FLS+FLS. (B) Recombinant strain p23b-F3H-TPTP2-FLS+F3H.

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.

![](https://static.igem.wiki/teams/5482/results/img12.png)

**Figure 12.** Effect of multiple copies of genes on the yield of kaempferol biosynthesis.

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.

# Exploration of the reasons for the impact of gene copy number variation on kaempferol biosynthesis

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.

![](https://static.igem.wiki/teams/5482/results/img13.png)

**Figure 13**. Relative mRNA levels of different strain.

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.

# Preliminary Extraction of Kaempferol Using Engineered Bacteria

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.

![](https://static.igem.wiki/teams/5482/results/img14.png)

**Figure 14.** Extracted kaempferol product from engineered bacteria

# GABA verification

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).

![](https://static.igem.wiki/teams/5482/results/new-img15.png)

**Figure 15.** Recombinant plasmid p23b-GadB construction and verification. (A) Recombinant plasmid p23b-GadB map. (B) p23b-GadB gene circuit. (C) Agarose gel electrophoresis of GadB.

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

![](https://static.igem.wiki/teams/5482/results/img16.png)

**Figure 16.** GABA synthesis process

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.

![](https://static.igem.wiki/teams/5482/results/img17.png)

**Figure 17.** The production of GABA. (A) The standard curve of GABA. (B) The influence of inserting GadB gene fragments on GABA yield.

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).

# Effect of pH level on the activity of GadB

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).

![](https://static.igem.wiki/teams/5482/results/img18.png)

**Figure 18.** The effect of pH on GadB activity.

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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