Revision as of 06:00, 30 September 2024

F3H-GGGS-FLS

Single-copy linkers (GGGS) are used to connect F3H and FLS

Description

Flavanone 3-hydroxylase (F3H，EC 1.14.11.9) is an enzyme that catalyzes the chemical reaction:a flavanone + 2-oxoglutarate + O2 ⇌ a dihydroflavonol + succinate + CO2. The 3 substrates of this enzyme are flavanone, 2-oxoglutarate, and O2, whereas its 3 products are dihydroflavonol, succinate, and CO2. This enzyme belongs to the family of oxidoreductases, specifically those acting on paired donors, with O2 as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O2 with 2-oxoglutarate as one donor, and incorporation of one atom o oxygen into each donor(Forkmann et al., 1980). Flavonol synthase (FLS, EC 1.14.11.23) is an enzyme that catalyzes the following chemical reaction:dihydroflavonol + 2-oxoglutarate + O2 ⇌ a flavonol + succinate + CO2 + H2O.The 3 substrates of this enzyme are dihydroflavonol, 2-oxoglutarate, and O2, whereas its 4 products are flavonol, succinate, CO2, and H2O. This enzyme belongs to the family of oxidoreductases, specifically those acting on paired donors, with O2 as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O2 with 2-oxoglutarate as one donor, and incorporation of one atom of oxygen into each donor.

Kaempferol is a naturally occurring flavonoid found in many plant-based foods and traditional medicinal plants. It is well-known for its potential health benefits, including antioxidant, anti-inflammatory, and antianxiety properties. Kaempferol exerts its antianxiety effects by reducing oxidative stress, inhibiting pro-inflammatory cytokines, and regulating pathways such as AKT/β-catenin signaling(Ekici et al., 2023). Despite its promising anti-anxiety effects, industrial production of kaempferol faces several challenges. Traditional extraction from plants results in low yields and time-consuming processes. To overcome these limitations, combinatorial metabolic engineering has been explored as an alternative. Recent studies have demonstrated successful reconstruction of the kaempferol biosynthesis pathway in microbial systems like Escherichia coli and Saccharomyces cerevisiae, achieving maximum yields of 58.9 mg/L (Duan et al., 2017)and 66.29 mg/L, respectively (Malla et al., 2013). Enhancing metabolite yield through metabolic engineering necessitates the reconstruction of optimal biosynthetic pathways in E. coli, as factors such as gene origin, gene dosage, enzyme expression levels, enzyme activity, and unique enzyme characteristics all influence metabolic flux and output. Consequently, selecting suitable F3H and FLS genes, alongside constructing an efficient synthesis pathway, are critical steps in boosting kaempferol production. Moreover, optimizing fermentation conditions for the engineered strains is also a crucial aspect of this process.

To improve the yield and efficiency of kaempferol biosynthesis, we designed the construct p23b-F3H-GGGS-FLS, which integrates two key enzymes—F3H and FLS—with a flexible GGGS linker between them. The addition of this linker ensures close proximity of the two enzymes, enhancing substrate channeling, minimizing by-product formation, and improving overall efficiency of the pathway.

•F3H: Catalyzes the conversion of naringenin to dihydrokaempferol. •FLS: Catalyzes the final conversion of dihydrokaempferol to kaempferol.

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.

Usage and Biology

Type: Basic Part Assembly Compatibility: RFC10 Sequence and Features: The sequence incorporates the coding regions for F3H and FLS, linked by a GGGS sequence. The part is designed to be expressed in E. coli for optimal production of kaempferol. Source: CisF3H is derived from Citrus sinensis (sweet orange), and CuFLS is sourced from Citrus unshiu (Satsuma mandarin）.

The composite part p23b-F3H-GGGS-FLS (BBa_K5482003) was designed for efficient kaempferol production in E. coli. The plasmid includes a T7 promoter for high expression, an RBS B0034 for effective translation, and the genes F3H and FLS linked by a GGGS linker, followed by the B0015 terminator to ensure proper transcription termination.After constructing and sequencing the plasmid, it was transformed into E. coli BL21.

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

Characterization

Construction of p23b-CisF3H-GGGS-CuFLS

Figure 3. Agarose gel electrophoresis of F3H-GGGS-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. Figure 4 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 4. Standard curve of kaempferol.

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 4). Notably, the absorbance of the engineered strain BL21/F3H-FLS (GGGS) exceeded the range of the kaempferol standard curve during the initial measurement, so we diluted the solution by half and measured it again. The final kaempferol concentration was calculated based on the dilution factor.The results in Figure 5 showed that the kaempferol yield of the engineered strain BL21/F3H-FLS (GGGS) using the fusion enzyme technology was significantly higher than that of the engineered strain BL21/F3H-FLS (B0034), and the yield reached 60.52 ± 5.11 mg/L. Wild-type BL2 and control strains (containing only empty plasmid pET23b), which served as the control group, were unable to synthesize kaempferol under the same manipulation and culture conditions. Therefore, employing fusion enzyme technology to link F3H and FLS can significantly enhance the yield of kaempferol produced by engineered bacteria.

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

The Effects of Temperature and Cell Density on Keampferol Production

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℃, 25℃, 30℃, 37℃, 42℃) and initial bacterial densities (OD600= 0.2, 0.6, 1, 1.5, 2.0). Results (Figure 6) 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.

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.

Optimization of Kaempferol Production -Substrate Concentration

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 7) 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. Higher naringenin concentrations may have led to enzyme instability and cell toxicity, reducing both yield and conversion rate. Thus, 1000 mg/L is optimal for yield, while 500 mg/L is best for conversion efficiency.

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.

Optimization of Kaempferol Production - Linker

Figure 8. Agarose gel electrophoresis of F3H-GGGS-FLS, F3H-GGGS2-FLS, F3H-TPTP-FLS, F3H-TPTP-FLSF3H-TPTP2-FLS.

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 9) 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.

Figure 9. Effects of different linkers on kaempferol biological production.

Effects of Multi-copy Genes on Kaempferol Production

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 10) 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.

Figure 10. 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 11) 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.

Figure 11. Relative mRNA levels of different strain.

Potential application directions

The optimized production of kaempferol using the p23b-F3H-GGGS-FLS plasmid and fusion enzyme technology holds great potential for applications in pharmaceuticals and nutraceuticals. With kaempferol’s known anti-inflammatory, antioxidant, and anti-anxiety properties, this system can be scaled for sustainable production. Future developments could focus on enhancing yields for industrial use, creating cost-effective methods for producing this valuable flavonoid in health-related products.

Reference

Ekici M, Güngör H, Mert D G. Kaempferol and Isorhamnetin alleviate Lipopolysaccharide-Induced Anxiety and Depression-Like Behavioral in Balb/C Mice: Flavonoids may reverse emotional disorders[J]. Journal of the Hellenic Veterinary Medical Society, 2023, 74(2): 5753-5764. Forkmann G, Heller W, Grisebach H. Anthocyanin biosynthesis in flowers of Matthiola incana flavanone 3-and flavonoid 3′-hydroxylases[J]. Zeitschrift für Naturforschung C, 1980, 35(9-10): 691-695. Duan L, Ding W, Liu X, et al. Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae[J]. Microbial cell factories, 2017, 16: 1-10. Malla S, Pandey R P, Kim B G, et al. Regiospecific modifications of naringenin for astragalin production in Escherichia coli[J]. Biotechnology and bioengineering, 2013, 110(9): 2525-2535.

Sequence and Features