Part:BBa_K5481003
LjIFS-CrCPR-GmHID
We synthesize three key enzyme genes, cytochrome P450 enzyme reductase from Catharanthus roseus, CrCPR), 2-hydroxyisoflavone synthase (2-HIS) from Lotus japonicus (bird foot bean), AKA Isoflavone synthase (LjIFS), 2-hydroxyisoflavone synthase HID (GmHID) from Glycine max (soybean), used to produce genistein Flavonoids
Description
Genistein was first isolated from Genista tinctoria and is commonly found in legumes such as soybeans, kudzu, and Sophora. It exhibits various pharmacological activities, including estrogenic activity, insulin secretion stimulation, anti-tumor effects, and antioxidant properties. Genistein’s structure is similar to estradiol, allowing it to selectively bind to estrogen receptors, stimulate estrogen production, and increase bone density and uterine weight in animal studies. Additionally, it improves glucose metabolism by enhancing insulin receptor sensitivity and promoting insulin secretion. Kaempferol also inhibits tumor cell proliferation by downregulating cyclooxygenase-2 (COX-2) expression, preventing cancer cells from entering mitosis. Its antioxidant properties are comparable to those of vitamin E, and it has the potential to prevent fat accumulation. These characteristics suggest that kaempferol has promising applications in estrogen replacement therapy, diabetes management, cancer prevention, and antioxidation. Currently, the synthesis of genistein relies primarily on plant extraction or chemical synthesis. However, plant extraction is limited by seasonal and geographical factors, resulting in low yields and the need for organic solvents. Chemical synthesis involves complex steps, harsh conditions, and expensive metal catalysts, which restrict the production of flavonoids. In recent years, the development of synthetic biology has promoted the microbial synthesis of genistein. Researchers have attempted de novo synthesis by introducing isoflavone synthase (IFS) and cytochrome P450 reductase (CPR) into yeast or E. coli. However, the production yield remains low, especially in E. coli, due to limited CPR expression and inefficient electron transfer, which reduces overall catalytic efficiency. Furthermore, plant-derived P450 enzymes are typically membrane-bound proteins, and the lack of similar membrane structures in E. coli further hampers their functional expression in bacterial systems. This issue can be mitigated by truncating the N-terminal of IFS and adding hydrophilic tags like 2B1 or KKK. The KKK-tLjIFS variant showed the best conversion of naringenin to genistein, achieving a yield of 45.34 mg/L in Saccharomyces cerevisiae.
This composite part is a gene assembly that produces genistein. It contains the last three genes involved in the biosynthetic pathway for genistein. This device contains CrCPR (BBa_K5481000), LjIFS (BBa_K5481001), and GmHID (BBa_K5481002). Each of these genes are codon-optimized and domesticated for EcoRI，XbaI，SpeI and PstI restriction sites. Isoflavone synthase(IFS), requires the involvement of NADPH and O2 for the reaction to occur. IFS first oxidizes C-3 to form free radicals and secondly the benzene ring is rearranged (migrated) from C-2 to C-3. Cytochorome P450 reductase (CPR), Carrying molecular oxygen for further oxidation and finally adding a hydroxy group at C-2. resulting in the formation of 2,4',5,7-tetrahydroxyisoflavanone. 2-hydroxyisoflavanone dehydratase （HID), A dehydration reaction occurs on the basis of 2,4',5,7 tetrahydroxyisoflavones. A molecule of water on C-2 is removed to form the final product, genisteins.
Figure 1. Chemical synthesis of genistein.
Usage and Biology
We used the pSB1A3 plasmid backbone, with the J23100 promoter and B0034 ribosome binding site. To enhance mRNA expression efficiency, we employed a dual-promoter strategy. The construct includes the B0015 double terminator and the recombinant plasmid pSB-IFS-CPR-HID (BBa_K5481003) was transformed into Escherichia coli BL21 for expression.
Figure 2. The gene circuit of pSB-IFS-CPR-HID.
Characterization
Content
Test of Genistein-producing Strain
Figure 3. Gel electrophoresis of CrCPR, LjIFS, GmHID gene.
We chose BL21 as the host strain.We incubated the engineered bacteria overnight at 37℃ in LB medium containing ampicillin antibiotics. It was washed using PBS. Subsequently transferred to 50 mL M9 medium. The OD600 value was adjusted to 1. The culture was incubated for 24 h at 30 ℃. Since genistein has an absorption peak at 250 nm, we planned to detect the content of genistein by measuring the absorbance at 250 nm of the sample. Prior to this, we purchased a genistein standard and configured different concentrations of 0 μM, 50 μM, 100 μM, 150 μM, 200 μM. The absorbance values at 250 nm were detected for each concentration and the standard curve of genistein was plotted. Figure 4 presents the standard curve for genistein, illustrating the linear relationship between absorbance and genistein concentration. The regression equation obtained is Y = 0.02631X + 0.3105 , with a correlation coefficient (R) of 0.9831, indicating a strong linear correlation. This standard curve can be used to accurately determine the concentration of genistein in unknown samples by measuring their absorbance.
Figure 4. Genistein standaed curve.
Figure 5 shows the genistein concentrations in different bacterial strains. BL21 and BL21/pSB1A3 were used as blank and negative controls, respectively, while the experimental group consisted of the engineered genistein-producing strain BL21/LjIFS-CrCPR-GmHID. The results indicate that there is no significant difference in genistein concentration between the two control strains, BL21 and BL21/pSB1A3. However, the genistein concentration in the experimental group was significantly higher, reaching 1.97 ± 0.38 μM. This confirms that the engineered strain is capable of producing genistein. The detection of genistein in the control groups is likely due to experimental error or the absorbance of other compounds at 250 nm, which may have influenced the measurements.
Figure 5. Genistein production in genistein-producing strain.
Truncated LjIFS Enhances Genistein Production
We optimized the codons of the three key enzyme genes (LjIFS, CrCPR, GmHID) and outsourced their synthesis to a biotech company. The N-terminal 21 amino acids of LjIFS were truncated and replaced with alanine, resulting in a truncated version of LjIFS(BBa_K5481004). In this modified system, segments of LjIFS were replaced while retaining the GmHID and CrCPR genes to enhance genistein biosynthesis. The constructed plasmid pSB-LjtIFS-CrCPR-GmHID was successfully transformed into E. coli BL21.
Figure 6. Gel electrophoresis of truncated LjIFS(LjtIFS).
After cultivating the engineered strain at 30°C for 24 hours, we measured the concentration of genistein by detecting the absorbance at 250 nm.The bacterial culture was collected and treated with ethyl acetate to dissolve the genistein. After centrifugation, the organic phase supernatant was collected, concentrated, and resuspended. The absorbance at 250 nm was then measured using a microplate reader. Finally, we converted the results into the two sets of data shown in the figure below using a standard curve. Experimental results showed that the genistein concentration of truncated LjIFS strain (LjtIFS) was significantly higher than the wild-type LjIFS strain, reaching 17.33 ± 2.37 μM. This indicates that truncating the N-terminal sequence of LjIFS and replacing it with alanine can significantly increase genistein production.
Figure 7. Genistein production in the truncated LjIFS strain.Figure 7 shows the genistein concentrations for both the wild-type LjIFS and the truncated LjIFS strains. After 24 hours of incubation at 30℃, the genistein concentration of truncated LjIFS strain was significantly higher than the wild-type LjIFS strain, reaching 17.33 ± 2.37 μM.
Effect of Different Tags on LjIFS Activity
To examine the impact of different N-terminal tags on LjIFS activity, we modified LjIFS by adding hydrophilic tags, such as KKK and HHHH, to improve the solubility and functionality of the protein. Four variants were tested: a truncated LjIFS without a tag, and three modified versions with the 17A(BBa_K5481006), KKK(BBa_K5481007), and HHHH(BBa_K5481008) hydrophilic tags replacing the N-terminal 21 amino acids.
During the validation process, we initially used regular Taq polymerase for PCR. However, as shown in Figure 8A, the gel electrophoresis results displayed significant smearing. After consulting with our advisor, we learned that several factors could contribute to this issue, such as excessive enzyme amounts, low-quality polymerase, high dNTP or Mg²⁺ concentrations, low annealing temperatures, or excessive cycle numbers. Based on this feedback, we decided to switch to a more suitable polymerase. Our advisor recommended using Long Taq polymerase, which is optimized for amplifying longer DNA fragments. As shown in Figure 8B, this adjustment allowed us to successfully amplify the target fragment without smearing！
Figure 8. (A)Gel electrophoresis of different tags on LjIFS. (B)Gel electrophoresis-2 of different tags on LjIFS.
These engineered strains were cultured at 30°C for 24 hours, and the genistein concentration produced by each strain was measured. The results demonstrate varying effects on genistein production among the different LjIFS variants. The truncated LjIFS with the 17A tag showed a decrease in genistein concentration, producing 11.35 ± 1.28 μM of genistein, compared to the truncated LjIFS(LjtIFS) baseline of 17.44 ± 5.62 μM. In contrast, the variants with KKK and HHHH tags exhibited significant increases in genistein production. Notably, the LjIFS strain with the KKK tag produced the highest concentration, reaching 130.66 ± 14.68 μM, representing a substantial improvement.These findings suggest that adding hydrophilic tags such as KKK and HHHH to the N-terminus of LjIFS enhances its solubility and activity. Among these, the KKK tag proved to be the most effective.
Figure 9. Genistein production in engineered strains with different LjIFS tags.
OmpAL Tag Enhances CrCPR Functionality
Initially, we designed a project to use CrCPR in E. coli to convert naringenin into genistein. However, we discovered that this gene is difficult to stabilize and maintain activity in E. coli, potentially due to its hydrophobic N-terminal tail, which functions as a membrane anchor in plant cells. In E. coli, the absence of an endoplasmic reticulum may reduce protein solubility and activity. The specific method we decided to use is to employ OmpAL (bacterial outer membrane protein A) as the signal peptide to enhance the activity and stability of CrCPR, so that CrCPR can play its role more completely.
Figure 10. Gel electrophoresis of OmpAL-CPR.
Using the same method, we tested two versions of CPR—one with the OmpAL sequence and one without—under identical cultivation conditions. The final results demonstrated that the CPR with the OmpAL sequence significantly increased genistein production, reaching 176.97 ± 15.30 μM, compared to the lower yield from the CPR without the OmpAL sequence .
Figure 11. Genistein production in CrCPR strains with and without OmpAL Tag.
Potential application directions
Our developed LjtIFS-CrCPR-GmHID system successfully achieves efficient conversion of naringenin to genistein by overcoming the challenges of expressing IFS and CPR in _Escherichia coli_. By performing N-terminal truncation of IFS, adding hydrophilic tags like KKK, and introducing the OmpAL tag in CPR, we significantly increased the yield of genistein. This multi-tag strategy not only enhances genistein production but also provides an effective method for expressing plant-derived or anchor proteins in E. coli.
Future iGEM teams can utilize our multi-tag strategy to achieve efficient expression and functionalization of complex or hard-to-express proteins in bacterial systems. By experimenting with different types of tags and linkers, teams can optimize protein expression levels, stability, and activity, thereby achieving better results in metabolic engineering projects. This system has potential in industrial applications, especially in the cosmetics, pharmaceutical, and skincare industries, providing a sustainable method for producing natural phytoestrogens like genistein.
References
[1] Liu X, Li L, Zhao G R. Systems metabolic engineering of Escherichia coli coculture for de novo production of genistein[J]. ACS Synthetic Biology, 2022, 11(5): 1746-1757.
[2] Scott E E, Spatzenegger M, Halpert J R. A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function[J]. Archives of Biochemistry and Biophysics, 2001, 395(1): 57-68.
[3] Hwang Y, Noh M H, Jung G Y. Recent advancements in flavonoid production through engineering microbial systems[J]. Biotechnology and Bioprocess Engineering, 2024: 1-14.
[4] Tang H, Wang S, Li X, et al. Prospects of and limitations to the clinical applications of genistein[J]. Discovery Medicine, 2019, 27(149): 177-188.
[5] Spagnuolo C, Russo G L, Orhan I E, et al. Genistein and cancer: current status, challenges, and future directions[J]. Advances in nutrition, 2015, 6(4): 408-419.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 3592
Illegal NheI site found at 3935
Illegal NheI site found at 3958
Illegal NotI site found at 3200 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3315
Illegal AgeI site found at 2671
Illegal AgeI site found at 4767 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1523
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