Figure 1 Experimental Workflow

Based on the codon preference of Escherichia coli, the HMB synthesis-related genes AtoB, Mvas, LiuC, and AibA were codon-optimized, while EcoRI, XbaI, SpeI, and PstI restriction sites were removed (sequence verification was performed using SnapGene®). The optimized genes were synthesized by Generalbial (China) and cloned into the pSB1A3 vector (BioBricks™ standard RFC#10). A polycistronic expression cassette was constructed using the constitutive promoter J23100 (BBa_J23100) and ribosome binding site B0034 (BBa_B0034), and then assembled into the vector via one-step seamless cloning (Seamless Cloning Kit, D7010, Beyotime). The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells and plated on LB agar plates containing 100 μg/mL ampicillin (Amp⁺) (pH 7.0), followed by incubation at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL LB/Amp⁺ liquid medium, incubated at 37°C, 220 rpm until OD₆₀₀ = 0.6–0.8. Positive clones were verified by colony PCR (2×Taq Master Mix, Vazyme). Sequencing verification was performed by Qingke Biotechnology (Beijing, China). The sequencing-correct BL21-HMB strain was inoculated into 5 mL LB/Amp⁺ medium and cultured at 37°C, 200 rpm. The growth curve was monitored to mid-log phase (OD₆₀₀ ≈ 0.6) using FlexStation 3 (Molecular Devices, USA). Finally, 1 mL of bacterial culture was mixed with 1 mL of 50% glycerol and stored at -20°C for future use.

100 μL of -20°C glycerol stock of the HMB-producing strain was inoculated into 5 mL LB medium containing 100 μg/mL ampicillin (LB/Amp⁺) and cultured at 37°C, 150 rpm overnight to reactivate the strain. The next day, the overnight culture was centrifuged at 8,000×g, 4°C for 10 min to collect the cell pellet. The pellet was washed three times with phosphate-buffered saline (PBS, pH 7.4) and resuspended in 50 mL M9 minimal medium (A510881, Sangon Biotech) supplemented with 20 g/L glucose, 0.1 g/L thiamine hydrochloride (A8084, Innochem), 1 mM magnesium sulfate (MgSO₄), 50 μM calcium chloride (CaCl₂), and 100 μg/mL Amp.

The bacterial growth density was monitored in real time using a FlexStation 3 multifunctional microplate reader (Molecular Devices, USA) at 600 nm (OD₆₀₀). At designated time points, 1 mL of culture was centrifuged at 18,000×g, 4°C for 2 min to collect the supernatant for HMB quantification.

HMB content was analyzed by high-performance liquid chromatography (HPLC), following a previously reported method with slight modifications. The analysis was performed using an Agilent 1200 HPLC system equipped with a C18 reverse-phase column (250 mm × 4.6 mm, 5 μm) at 30°C. The mobile phase consisted of eluent A (0.02 mol/L potassium dihydrogen phosphate, pH 3.0) and eluent B (acetonitrile), with the following gradient: initial 95% A; 8–8.5 min linear transition to 40% A; 8.5–13.0 min hold at 40% A; 13.0–13.5 min linear return to 95% A; 13.5–22.0 min hold at 95% A. The flow rate was 1.0 mL/min, injection volume was 10 μL, and detection wavelength was 208 nm.

A standard curve was generated using HMB standard solutions (0.05–2.00 mg/mL), which was then used to calculate the actual HMB content in the samples.

Table 1. HPLC Program for HMB Detection

1 mL of fermentation broth was centrifuged at 10,000×g for 5 min to collect the supernatant, which was stored at -20°C. The glucose content of the samples was determined using a Glucose Assay Kit (60408ES60, Yeasen, China). The total glucose consumption was then calculated.

Detection samples were prepared according to Table 2.

Table 2. Sample Loading in 96-Well Plate (Colorimetric Measurement Using Microplate Reader)

Gently shake the microplate and incubate at 37°C for 10 min. Measure the absorbance of each well at 505 nm using a microplate reader.

Based on the codon preference of Escherichia coli, the thioesterase genes tesB, YciA, and MenI were codon-optimized, while restriction sites EcoRI, XbaI, SpeI, and PstI were removed (sequence verification via SnapGene®). The optimized genes were synthesized by Generalbial (China) and cloned into a plasmid. A polycistronic expression cassette was constructed using the constitutive promoter J23100 (BBa_J23100) and the ribosome binding site B0034 (BBa_B0034). Plasmid transformation, engineered strain selection, and HMB production testing were performed as described above.

TDC and TPH genes were synthesized by Generalbial (China) and codon-optimized for E.coli, with removal of EcoRI, XbaI, SpeI, PstI, NdeI, XhoI restriction sites to comply with RFC#10 standard and pET28a(m) cloning compatibility. Genes were cloned into the pET28a(m) vector via NdeI and XhoI restriction sites. E.coli BL21 (DE3) (D1009S) and DH5α (D0351) supercompetent cells were purchased from Beyotime (China). Recombinant plasmids were transformed into DH5α and BL21. Positive clones were selected on LB agar plates containing 100 μg/mL kanamycin (Kana, 1.5% agar) and sequencing-verified (Qingke Biotechnology, Beijing, China). The engineered strains were stored at -80°C with 25% (v/v) glycerol. Cultivation conditions: 37°C, 150 rpm in LB broth containing 100 μg/mL Kana (G3102, Servicebio, China).

The engineered strain was inoculated at a 1:100 ratio into 20 mL modified M9 medium, containing 1× M9 salts (A510881, Sangon Biotech), 0.2% (w/v) glucose, 0.1% (w/v) casein amino acids (C304284, Aladdin), 1 mM MgSO₄, 50 pM FeCl₃, and 50 mg/L L-tryptophan (A64769, Innochem). Kanamycin final concentration: 100 μg/mL. Cultured at 37°C, 180 rpm for 3–5 h until OD₆₀₀ = 0.6, then 0.5 mM IPTG was added and cultivation continued for 20 h.

1 mL of fermentation broth was filtered through a 0.22 μm hydrophilic PES membrane (BS-PES-45, Biosharp), and samples were collected every 5 h, stored at -20°C. Serotonin content was analyzed using a 5-HT ELISA Kit (JL53170, Jonln), and concentration was calculated from a standard curve.

Experimental Procedure:

1. Remove the required microplate strips from room temperature-equilibrated foil bags; store remaining strips at 4°C.
2. Sample addition: Add 50 μL of each sample or standard solution per well; blank wells receive 50 μL universal diluent. Immediately add 50 μL Biotin-antibody working solution per well. Cover the plate and incubate at 37°C for 60 min. (Recommendation: dilute samples at least 1:1 with universal diluent to reduce matrix effects; multiply by dilution factor when calculating concentrations; replicate wells are recommended.)
3. Washing: Discard liquid; add 300 μL 1× wash buffer per well; stand 1 min; remove buffer; blot on absorbent paper; repeat 3 times (or use a plate washer).
4. Add enzyme conjugate working solution:100 μL per well, cover, incubate 37°C for 30 min.
5. Wash: Repeat step 3, 5 times.
6. Add substrate:90 μL TMB per well, cover, incubate 37°C in dark for 15 min.
7. Add stop solution:50 μL per well, measure OD at 450 nm immediately.

PBAD promoter (K808000), RBS B0034, and red fluorescent protein mRFP were synthesized. mRFP was placed downstream of PBAD-B0034, codon-optimized for E. coli, and EcoRI, XbaI, SpeI, PstI restriction sites removed to meet RFC#10 standard (Genewiz, USA). The PBAD-mRFP cassette was cloned into pSB1A3 vector via XbaI and SpeI sites. Recombinant plasmids were transformed into E. coli DH5α. Positive clones were sequencing-verified (Qingke Biotechnology, Beijing, China). Cultivation: 37°C, LB medium. Bacterial growth was monitored by OD₆₀₀ using a spectrophotometer (GenStar, China).

Inoculate the arabinose-inducible reporter strain at 1:100 into 5 mL LB + 50 μg/mL Amp, add different L-arabinose concentrations, incubate at 37°C, 180 rpm. Collect 200 μL samples at different time points. Measure fluorescence (Ex 584 nm / Em 607 nm) and OD₆₀₀ using Multiskan GO microplate reader. Calculate normalized fluorescence (Fluorescence/OD₆₀₀).

E. coli toxin protein MazF was synthesized and codon-optimized to meet RFC#10 standard. mazF was placed downstream of PBAD promoter via overlap PCR, then cloned into pSB1A3 vector via XbaI and SpeI. Recombinant plasmids were transformed into E. coli DH5α, screened on LB agar + 100 μg/mL Amp (1.5% agar), and sequencing-verified. DH5α-PBAD-mazF was stored at -20°C with 25% (v/v) glycerol, cultured at 37°C, 180 rpm in LB broth + 100 μg/mL Amp.

Frozen DH5α-PBAD-mazF and wild-type DH5α were activated overnight in 5 mL LB + 100 μg/mL Amp at 37°C, 150 rpm, then inoculated 1:100 into 20 mL LB + Amp (OD₆₀₀ = 0.1). At OD₆₀₀ ≈ 0.4, add 0.2% L-arabinose to induce MazF expression. Monitor growth curves with FlexStation 3 (Molecular Devices, USA).

Data were analyzed using GraphPad Prism and presented as mean ± SD. Multiple group comparisons: one-way ANOVA with Tukey’s post hoc test. Two-group comparisons: Student’s t-test. p < 0.05 considered statistically significant.