Latest revision as of 06:24, 30 September 2024

CisF3H-CuFLS

The multi-cistronic strategy (RBSB0034) is used to connect F3H and FLS

Description

Considering the complexity and high cost associated with the chemical synthesis of naringenin in vitro, we have decided to utilize metabolic engineering techniques to construct a novel biosynthetic pathway in Escherichia coli to enhance the efficiency of converting naringenin to kaempferol. We designed and constructed the recombinant plasmid p23b-CisF3H-B0034-CuFLS, which relies on the expression of two key enzymes: flavanone 3-hydroxylase (F3H) and flavanol synthase (FLS). These enzymes sequentially catalyze the conversion of naringenin to kaempferol.

Figure 1. The plasmid map of p23b-F3H-B0034-FLS.

Usage and Biology

We used the CisF3H(BBa_K5482000) and CuFLS(BBa_K5482001) gene sequences to link these two genes via RBS(BBa_B0034) using a polycistronic strategy. To express CisF3H-B0034-CuFLS(BBa_K5482002) , we cloned them into the pET23b plasmid and introduced the constructs into E. coli DH5α and E. coli BL21, the former for preservation of the plasmid and the latter for protein expression. In the experiment, ampicillin was chosen as a screening marker and incubated at 37°C to ensure stable expression of the plasmids.

Figure 2. Gene circuit (using polycistronic strategy (B0034) to link F3H and FLS).

Characterization

To validate that our engineered strains can successfully produce kaempferol using naringenin as a substrate, we designed experiments to compare the performance of wild-type BL21, the pET23b empty vector strain, and the recombinant strain BL21/p23b-CisF3H-B0034-CuFLS. Each group was cultured at 30°C for 24 hours with the addition of 500 mg/L naringenin as the substrate. The kaempferol content in the fermentation broth was measured using a colorimetric method, and a standard curve was constructed using known concentrations of kaempferol standards.

We constructed a standard curve using kaempferol standards at concentrations of 1, 10, 20, 30, and 50 mg/L, measuring their absorbance at a wavelength of 368 nm (Figure 3A). The linear regression results indicated a strong linear relationship between absorbance and kaempferol concentration (R² = 0.9915), demonstrating the high precision and reliability of our detection method. The results showed (Figure 3B) that neither the wild-type BL21 nor the control strain BL21/pET23b synthesized kaempferol, while the recombinant strain BL21/p23b-CisF3H-B0034-CuFLS successfully produced kaempferol from naringenin, achieving a yield of approximately 40 mg/L. This indicates that our constructed polycistronic expression system effectively facilitated the synergistic action of flavanone 3-hydroxylase (F3H) and flavanol synthase (FLS), successfully enabling the conversion of naringenin to kaempferol.

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

The experimental results further confirm the effectiveness of metabolic engineering techniques in the modification of Escherichia coli. By rationally designing the gene circuitry and optimizing expression conditions, the recombinant strain exhibited a high yield of kaempferol, demonstrating its potential application value for future industrial production.

In summary, our experiments validated the production capability of the recombinant strain and laid the foundation for further enhancing the biosynthetic efficiency of kaempferol.

Potential application directions

The metabolic engineering of Escherichia coli for the production of kaempferol provides a feasible direction for developing new antidepressant products. Kaempferol exhibits significant antioxidant, anti-inflammatory, as well as anxiolytic and antidepressant properties, making it suitable for the formulation of functional beverages or dietary supplements aimed at alleviating mood fluctuations and depressive symptoms. In the future, this technology could be applied for large-scale production of natural kaempferol components to meet the growing demand for natural, side-effect-free mood regulation products, offering a novel approach to addressing the increasingly serious mental health issues. The biosynthetic technology developed in this project not only presents economic advantages but also possesses eco-friendly characteristics, promising broad applications in the food and pharmaceutical sectors and advancing the development and utilization of natural medicines.

Reference

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

Sequence and Features