The F7-OMT gene (BBa_259M31WV, Generalbiol, China) was synthesized and codon-optimized for E. coli. Restriction sites EcoRI, XbaI, SpeI, and PstI were removed to comply with RFC#10 standards. The promoter–RBS cassette J23100-B0034 was used as a cis-regulatory element and cloned into the pSB1A3 vector via XbaI and SpeI sites. E. coli BL21 (DE3) (D1009S) and DH5α (D0351) competent cells were purchased from Beyotime (China). Recombinant plasmids were transformed into E. coli DH5α, plated on LB agar plates (1.5% agar) supplemented with 100 μg/mL ampicillin, and positive clones were verified by sequencing (Qingke, Beijing). The resulting recombinant strain was designated DH5α-OMT. Plasmids were extracted using a commercial plasmid extraction kit (DP103, Tiangen) and subsequently transformed into E. coli BL21 (DE3), generating strain BL21-OMT. Strains were stored at −80 ℃ in 25% (v/v) glycerol. For cultivation, strains were grown at 37 ℃ and 150 rpm in LB broth (G3102, Servicebio, China) containing 100 μg/mL ampicillin.
The YdaO gene (BBa_25Z8A0VX, Generalbiol, China) was synthesized, codon-optimized for E. coli, and restriction sites avoided by RFC#10 were removed. The J23100-B0034 cassette was placed upstream, and the construct was cloned into pSB1A3 via XbaI and SpeI. PCR and overlap extension were applied to place B0034-YdaO downstream of F7-OMT, yielding the F7-OMT–YdaO co-expression sequence, which was also cloned into pSB1A3. Recombinant plasmids were transformed into E. coli DH5α and BL21, plated on LB agar containing 100 μg/mL ampicillin, and verified by sequencing. The resulting strain was designated BL21-OMT-YdaO. Strains were stored at −80 ℃ in 25% (v/v) glycerol and cultured under the same conditions as above.
To enhance methionine production in E. coli, the cysE gene encoding L-serine O-acetyltransferase (BBa_25MTY54W, Generalbiol, China) was synthesized and codon-optimized. The J23100-B0034 cassette was placed upstream of cysE, and PCR with overlap extension was used to insert J23100-B0034-cysE downstream of the OMT-YdaO module. The OMT–YdaO–CysE operon was cloned into pSB1A3 via XbaI and SpeI. Following transformation and selection, the recombinant strain was designated BL21-OMT-YdaO-CysE. Strains were stored and cultured under the same conditions described above.
Overnight cultures of engineered strains were prepared in LB medium with 100 μg/mL ampicillin at 37 ℃, 150 rpm. BL21-OMT and BL21-OMT-YdaO were inoculated into Terrific Broth (TB; BL1176A, Biosharp) supplemented with 100 μg/mL ampicillin. Cultures were incubated at 37 ℃, 180 rpm until OD600 = 0.6, followed by supplementation with 150 mg/L (2S)-naringenin (A06896, Innochem). Cultures were then shifted to 25 ℃ and incubated for 24 h. Samples (1 mL) were collected, mixed with an equal volume of methanol, centrifuged at 12,000 × g for 5 min, and filtered through a 0.22 μm nylon membrane (110414017, BKMAN). HPLC analysis was conducted using an Agilent 1200 system with a C18 column (4.6 × 250 mm, 5 μm) at 290 nm. The mobile phases consisted of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B). The gradient program was as follows: 0–10 min, 10–60% B; 10–20 min, 40–80% B; 20–25 min, 80–10% B.
To assess the effect of methyl donors on sakuranetin biosynthesis, BL21-OMT was cultured in TB with ampicillin under the same conditions as above until OD600 = 0.6. After 3 h at 37 ℃, 180 rpm, cultures were supplemented with 150 mg/L (2S)-naringenin and 1 g/L methionine (A49392, Innochem), shifted to 25 ℃, and incubated for 12 h. Samples were processed as described above and analyzed for sakuranetin content.
The TAL BBa_25USFVFA, 4CL BBa_256H06VX, CHS BBa_25NCLJJ7, and CHI BBa_25I7VX04 genes (Generalbiol, China) were synthesized, codon-optimized, and modified to remove RFC#10-restricted sites. J23100-B0034 promoters were used to independently drive TAL/4CL and CHS/CHI expression, with T0015 terminators inserted to prevent promoter interference. Constructs were cloned into pSB1A3 via XbaI and SpeI. Recombinant plasmids were transformed into E. coli BL21, generating strain BL21-Nar. Strains were stored and cultured as described above.
Frozen cultures (100 μL) were inoculated into 5 mL LB with ampicillin and incubated overnight at 37 ℃, 150 rpm. Pre-cultures were inoculated into 50 mL LB with ampicillin in 250 mL flasks (initial OD600 = 0.1) and grown to OD600 = 1 at 37 ℃, 180 rpm. Cells were harvested by centrifugation (8,000 × g, 5 min) and resuspended in M9 medium (A510881, Sangon Biotech) supplemented with 0.4% glucose, 3 mM L-tyrosine, 1 mM MgSO4, 50 μM CaCl2, and 340 mg/L thiamine. Cultures were incubated at 25 ℃, 180 rpm for 48 h. Supernatants were extracted with an equal volume of ethyl acetate, centrifuged, and the organic phase collected, dried at 37 ℃ overnight, and resuspended in 1 mL acetonitrile. Absorbance at 290 nm was measured using a plate reader (FlexStation 3, Molecular Devices, USA). Concentrations were calculated against a naringenin standard curve (0–800 mg/L). Glucose levels were determined using a glucose detection kit (60408ES60, Yeasen, China). For more information on the glucose detection kit, please refer to the following link: https://www.yeasen.com/products/detail/1904741509662253058.
The PLA2 gene (BBa_25ST8FLD, Generalbiol, China) was synthesized and codon-optimized for E. coli. Restriction sites incompatible with RFC#10 and pET28a(m) cloning were removed. The optimized sequence was cloned into the pET28a(m) vector using NdeI and XhoI restriction sites and transformed into E. coli DH5α. Positive clones were selected on LB agar plates containing 100 μg/mL kanamycin and verified by sequencing. Plasmids from verified clones were extracted and subsequently transformed into E. coli BL21 (DE3), generating the BL21-PLA2 strain. The engineered strain was stored at -80℃ with 25% (v/v) glycerol and cultured in LB broth supplemented with 100 μg/mL kanamycin.
The PLA2-overexpressing engineered strain was inoculated into 50 mL LB medium supplemented with kanamycin and cultured overnight. The next day, bacterial cultures were collected and centrifuged at 10,000 × g for 1 min. The cell pellet was resuspended in PBS and disrupted by ultrasonication (150 W, 1 s on/3 s off cycles, for a total of 20 min). The resulting lysate was transferred into a new centrifuge tube as the intracellular protein sample. Protein solutions were mixed with 5× reducing sample buffer at a ratio of 4:1 and denatured in a boiling water bath for 15 min. Proteins were separated on 15% SDS-PAGE gels at 120 V and subsequently transferred onto a PVDF membrane under cold conditions. The membrane was blocked with 5% non-fat milk in TBST at room temperature for 1 h, followed by overnight incubation at 4 ℃ with a rabbit anti-PLA2 primary antibody (1:1000; commonly sourced from Abcam or Cayman). After three 10 min washes with TBST, the membrane was incubated at room temperature for 1 h with HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody (1:1000 dilution, A0208, Beyotime). Following another three washes with TBST (10 min each), ECL reagents A and B were mixed at a 1:1 ratio. The washed PVDF membrane was placed on absorbent paper to remove excess liquid and then covered with the prepared ECL solution for 1 min to ensure full coverage. Excess liquid was gently removed, and the membrane was exposed using a chemiluminescence imaging system under preset parameters.
Overnight cultures of BL21-PLA2 were inoculated at a 1:100 ratio into M9 medium supplemented with 0.2% (w/v) glucose, 1 mM MgSO4, 50 μM CaCl2, and 100 μg/mL kanamycin. Cultures were grown at 37℃ with shaking until OD600 reached 0.6. Protein expression was induced by adding 0.5 mM IPTG and 1 mg/mL lecithin (L305002, Aladdin), and cultures were incubated for an additional 24 h.
After incubation, 1 mL of culture was centrifuged at 10,000 × g for 10 min, and the supernatant was filtered through a 0.22 μm PES membrane. The filtrate was diluted 100-fold with PBS, and lysophosphatidylcholine (LPC) levels were quantified using a commercial ELISA kit. Absorbance at 450 nm was measured with a microplate reader (FlexStation 3, Molecular Devices, USA) to determine LPC concentration.
The PBAD promoter (K808000), RBS B0034, and mRFP sequence were synthesized (Genewiz, USA), codon-optimized, and cloned into the pSB1A3 vector. The recombinant plasmid was transformed into E. coli DH5α, and colonies were screened on LB agar plates containing 50 μg/mL ampicillin. Positive clones were verified by sequencing and cultured in LB medium with 50 μg/mL ampicillin. Fluorescence (Ex 584 nm, Em 607 nm) and OD600 were measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific) after induction with varying concentrations of L-arabinose.
For the suicide system, the PBAD-B0034 regulatory module was placed upstream of the T4 holin and T4 lysozyme (T4 lysis) genes. The coding sequences were codon-optimized for E. coli and cloned into pSB1A3. Recombinant strains were grown in LB medium containing 50 μg/mL ampicillin. Cultures were induced with 0.5 mM L-arabinose, and OD600 was measured over time to evaluate cell lysis efficiency.
The cold-shock promoter PcspA (K4987003) was synthesized and cloned upstream of mRFP in the pSB1A3 vector. The mRFP sequence was codon-optimized and verified by sequencing. Recombinant strains were cultured in LB medium supplemented with 50 μg/mL ampicillin. Inductions were performed at different temperatures, and fluorescence intensity was normalized to OD600. Fluorescence was detected using a plate reader (Thermo Fisher Scientific).
For the cold-inducible suicide system, the PcspA promoter was cloned upstream of the T4 holin and T4 lysozyme genes, which were codon-optimized and inserted into the pSB1A3 plasmid. Recombinant strains were grown in LB medium containing 50 μg/mL ampicillin. Cultures were incubated at 16 ℃ under shaking conditions, and OD600 was measured at different time points to assess lysis activity.
Data were analyzed and visualized using GraphPad Prism. Results are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test for multiple comparisons and Student’s t-test for two-group comparisons. A p-value < 0.05 was considered statistically significant.
