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.

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.

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.

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.

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

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.

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.

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).

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.