To achieve the production of key metabolites through synthetic biology, we conducted a series of experimental works focusing on HMB synthesis, serotonin synthesis, and safety system design. First, we successfully constructed engineered strains capable of de novo HMB synthesis, and verified their yield and impact on host growth using HPLC. Further, we optimized the HMB production level by employing thioesterase. Second, we constructed a serotonin synthesis system by co-expressing TDC and TPH in Escherichia coli, and confirmed serotonin production and secretion through ELISA, while screening TDCs from different sources to enhance the yield. Finally, we designed and validated an arabinose-inducible suicide system, providing a biosafety safeguard for the application of the engineered strains. Overall, the experimental results demonstrate that we not only successfully achieved the synthesis of the target metabolites, but also conducted systematic explorations in yield optimization and biological safety, laying a solid foundation for future applications.

Strain Construction

Figure 1 Construction of HMB-Producing Strains

(A) Plasmid design for HMB synthesis (B) Plasmid map of HMB synthesis construct

To obtain strains capable of efficient de novo HMB synthesis, we designed a gene circuit containing the key genes for HMB production (Figure 1A). E. coli BL21 was chosen as the host chassis to construct the engineered strain, and the following genes were cloned into BL21: acetoacetyl-CoA thiolase (AtoB, BBa_25VQVUES) from E. coli, 3-hydroxymethylglutaryl-CoA synthase (MvaS, BBa_25PLCDZA) from Staphylococcus aureus, glutaconyl-CoA decarboxylase (AibAB, BBa_2554PQS6 and BBa_25EFNVLR) from Corynebacterium glutamicum, and **3-methylglutaconyl-CoA hydratase (LiuC, BBa_25P2LT86)*. The resulting engineered strain was named BL21-HMB.

First, sequences of AtoB, MvaS, AibAB, and LiuC were retrieved from NCBI, and codon optimization was performed according to E. coli codon preference, while removing EcoRI, XbaI, SpeI, and PstI restriction sites (sequence verified using SnapGene®) and synthesized by Generalbial (China). The target fragments were PCR-amplified, and their correct size was confirmed by gel electrophoresis (Figure 2A). The fragments were then cloned into the pSB1A3 vector (BioBricks™ standard RFC#10) using one-step seamless cloning (Seamless Cloning Kit, D7010, Beyotime). The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, plated on LB agar containing 100 μg/mL ampicillin (Amp⁺, pH 7.0), and incubated at 37°C for 16 h to obtain single colonies (Figure 2B). Single colonies were inoculated into 5 mL LB/Amp⁺ liquid medium, cultured at 37°C, 220 rpm until OD₆₀₀ = 0.6–0.8, and verified by colony PCR (2× Taq Master Mix, Vazyme) to confirm positive clones.

Figure 2 Verification of HMB-Related Fragments and Single Colony Screening

(A) Agarose gel electrophoresis of PCR-amplified HMB pathway genes (B) Single colonies of engineered strains

The results show that we successfully PCR-amplified the fragments of AtoB, MvaS, AibAB, and LiuC, and cloned them into the pSB1A3 vector. Finally, single colonies were obtained on LB agar plates containing ampicillin (Amp⁺).

HMB Production Assay

To verify the ability of the engineered strains to produce HMB, we established an HPLC detection method based on literature reports. The HPLC procedure is as follows:

First, to determine the maximum absorption wavelength (λmax) of the target compound, a full-wavelength scan was performed using an Agilent 1200 series high-performance liquid chromatography (HPLC) system equipped with a diode array detector (DAD) under the above chromatographic conditions. The detector scan range was set from 200–400 nm, with a bandwidth of 4 nm and a sampling frequency of 1 Hz, continuously collecting spectral data throughout the retention time. The UV spectrum at the peak apex of the target compound was extracted to obtain its characteristic absorption curve, allowing the determination of the maximum absorption wavelength (λmax). Based on our experiment, the detection wavelength for HMB was determined to be 208 nm.

Figure 3 Determination of HMB Detection Wavelength

Next, HMB quantification was performed using an Agilent 1200 series HPLC system equipped with a C18 reverse-phase column (250 mm × 4.6 mm, 5 μm). The column temperature was maintained at 30°C. The mobile phase consisted of eluent A (0.02 mol/L potassium dihydrogen phosphate, pH 3.0) and eluent B (acetonitrile). The elution gradient was set as follows: initial 95% A; linear transition to 40% A from 8–8.5 min; hold at 40% A from 8.5–13.0 min; linear return to 95% A from 13.0–13.5 min; hold at 95% A from 13.5–22.0 min. The flow rate was 1.0 mL/min, the injection volume was 10 μL, and the detection wavelength was set at 208 nm (Table 1).

A series of HMB standard solutions ranging from 0.05–2.00 mg/L were measured at 208 nm to construct a standard curve, which was then used to calculate the actual HMB content in the samples (Figure 4) [1].

Table 1. HPLC Program for HMB Detection

Time (min)	Eluent A	Eluent B	Notes
0–8.0	95%	5%	Initial conditions
8.0–8.5	95%→40%	5%→60%	Linear gradient
8.5–13.0	40%	60%	Hold at constant composition
13.0–13.5	40%→95%	60%→5%	Linear return to initial conditions
13.5–22.0	95%	5%	Hold at initial conditions until the end of analysis

Figure 4 HMB Standard Absorption Peak at 208 nm

As an intestinal probiotic, HMB production needs to be stable and sustained. Therefore, we chose to test fermentation at 12 h as the detection time point to ensure continuous HMB production.

To evaluate the HMB yield of the engineered strains in M9 medium after 12 h, the strains were activated in LB/Amp⁺ medium and then inoculated into M9 medium. Cell growth was monitored in real time via OD₆₀₀ measurements. Samples were taken at different time points, centrifuged, and the supernatant was collected for HMB quantification. Each experiment was performed in triplicate, and Student’s t-test was used for comparison between groups, with p < 0.05 considered statistically significant.

The results showed that the engineered E. coli produced 65.13 mg/L HMB after 12 h cultivation at 37°C, whereas the wild-type BL21 did not produce detectable HMB (Figure 5).

图5 HMB生产测试

Next, by comparing the growth curves of the wild-type and engineered strains, we evaluated the potential impact of HMB synthesis on the engineered bacteria. First, the growth of the engineered strain was not significantly inhibited, indicating that the intracellularly synthesized HMB did not exert direct growth toxicity. Second, these results suggest that the metabolic burden imposed by the introduction of the exogenous metabolic pathway was within the tolerable range of the engineered strain and did not significantly impair its normal proliferation (Figure 6).

Figure 6 Growth Curves of HMB-Producing Strains

Finally, to quantify the carbon source utilization efficiency and the effectiveness of the metabolic pathway in the engineered strains, we dynamically monitored the glucose consumption and HMB production every 6 hours over a 24-hour period, and performed correlation analysis. The results showed a significant positive correlation between the two variables, indicating that as time progressed, HMB was continuously produced while glucose was steadily consumed (Figure 7).

This observation directly demonstrates that glucose, as the carbon source, was efficiently channeled into the de novo HMB synthesis pathway, rather than being redundantly consumed by side metabolic pathways or non-productive biomass growth, thereby validating the functional integrity of HMB production and the rationality of the engineered metabolic pathway. More importantly, these results provide core experimental data for the subsequent calculation of carbon flux efficiency and carbon source utilization rate — the efficient allocation of carbon is not only key to increasing HMB yield, but also aligns with the requirements for low-consumption, high-efficiency biosynthesis systems in resource-limited environments, such as space stations or closed ecological systems.

Figure 7 Carbon Utilization Analysis During HMB Production

In this study, we successfully constructed engineered strains capable of HMB synthesis. The results showed that the engineered strain produced approximately 65 mg/L HMB after 12 h of cultivation in M9 medium, and HMB production did not have a significant impact on host growth. Further analysis revealed a positive correlation between HMB yield and glucose consumption, demonstrating that the engineered strain can efficiently convert carbon sources into the target metabolite.

Thioesterase-Mediated Enhancement of HMB Production

Based on literature research and communication, thioesterases are known to enhance the conversion of HMB-CoA to HMB [2]. Therefore, we tested TesB (BBa_25YQAEQ1), YciA (BBa_25BAALSN), and MenI (BBa_25A7QNVZ) from E. coli (Figure 8A, B, and C).

The results showed that upon heterologous expression of tesB, the HMB yield reached 148.32 mg/L after 12 h. Expression of YciA had the most pronounced effect, producing 185.86 mg/L HMB after 12 h. In contrast, MenI showed the lowest enhancement among the three, yielding 101.29 mg/L HMB after 12 h; however, this was still higher than the strain without thioesterase expression (Figure 8D).

Figure 8 Thioesterase-Mediated Enhancement of HMB Production

(A) Agarose gel electrophoresis verification of tesB sequence (B) Agarose gel electrophoresis verification of YciA sequence (C) Agarose gel electrophoresis verification of MenI sequence (D) HMB production under different thioesterases

Strain Construction

First, we constructed a serotonin synthesis system. The serotonin-related genes TDC (BBa_25P80OSX) and TPH (BBa_25G48CDM) were obtained from NCBI and synthesized by Generalbial (China) after codon optimization for E. coli. Restriction sites EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with RFC#10 standard and ensure compatibility with pET28a(m) cloning. The genes were then cloned into the pET28a(m) vector via the NdeI and XhoI restriction sites.

The recombinant plasmid was subsequently transformed into E. coli DH5α and BL21. Positive clones were selected on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin (Kana) and sequencing verification was performed (Beijing, Qingke) to obtain the recombinant engineered strains.

Figure 9 Construction of Serotonin-Producing Strains

(A) Construction of the serotonin synthesis pathway (B) Plasmid map of the serotonin synthesis construct (C) Agarose gel electrophoresis verification of serotonin-related gene amplification

Serotonin Production Assay

To evaluate serotonin (5-HT) production, the engineered strains were inoculated at a 1:100 ratio into 20 mL of modified M9 medium, which contained 1× M9 salts (A510881, Sangon Biotech), 0.2% (w/v) glucose, 0.1% (w/v) casamino acids (C304284, Aladdin), 1 mM MgSO₄, 50 pM FeCl₃, and 50 mg/L L-tryptophan (A64769, Innochem). When the culture reached OD₆₀₀ = 0.6, 0.5 mM IPTG was added to induce gene expression, and incubation continued for 20 h.

Samples (1 mL) were collected every 5 h, filtered through a 0.22 μm hydrophilic PES membrane (BS-PES-45, Biosharp), and stored at -20°C. Serotonin concentration in the culture medium was measured using a 5-HT ELISA kit (JL53170, Jonln), and the standard curve was used to calculate the serotonin titer.

The results demonstrated that the engineered E. coli produced 1.85 mg/L serotonin, whereas the wild-type strain and the strain expressing TDC only did not produce detectable serotonin.

Figure 10 Serotonin Production Assay

(A) Serotonin titer measured every 6 hours over 24 hours (B) Functional verification of serotonin-related enzymes

Screening of TDC from Different Sources to Enhance Serotonin Production

Through interviews with experts, we learned that TDC from different sources may affect serotonin yield. Therefore, we tested TDC genes derived from rice, Catharanthus, and pig. The results showed that Catharanthus-derived TDC had the best performance, increasing the serotonin titer to 2.62 mg/L.

Figure 11 Screening of TDC from Different Sources

In this study, we successfully constructed a serotonin synthesis system by cloning and expressing TDC and TPH genes in E. coli. The results showed that the recombinant strains were able to synthesize and secrete serotonin in the culture medium, with a maximum titer of 1.85 mg/L, whereas the wild-type strain and the strain expressing TDC alone did not produce detectable serotonin. Furthermore, TDC from different sources had a significant impact on serotonin yield, with Catharanthus-derived TDC performing best, increasing the titer to 2.62 mg/L.

Strain Construction

To prevent potential side effects of the engineered strains and their products in vivo, or to allow users to terminate bacterial activity on demand, we designed a safety system. An arabinose-inducible promoter (BBa_K808000) was used to drive the expression of E. coli endogenous mRNA-cleaving toxin MazF, enabling engineered cell self-lysis in the presence of arabinose.

Figure 12 Construction of the Safety System

(A) Genetic circuit diagram of the safety system (B) Plasmid map of the safety system construct (C) Agarose gel electrophoresis verification of safety system gene expression

Arabinose-Inducible Promoter Assay

First, we tested the inducible function of the arabinose promoter pBAD using mRFP as a reporter. The results showed that as the arabinose concentration increased, the Fluorescence/OD₆₀₀ value continuously rose, indicating that the arabinose promoter can effectively induce protein expression in the presence of arabinose.

Figure 13 Verification of Arabinose-Inducible Promoter Function

Safety System Assay

Next, we synthesized the E. coli toxin gene MazF and performed codon optimization to meet the BioBrick RFC#10 standard. Using overlap PCR, the optimized mazF was placed downstream of the arabinose-inducible promoter PBAD, forming the PBAD-mazF expression cassette. This cassette was cloned into the standard plasmid vector pSB1A3 via XbaI and SpeI restriction sites and transformed into E. coli DH5α competent cells.

To evaluate the system’s effect on host growth under induction, we combined arabinose induction with growth curve monitoring. Frozen DH5α-PBAD-MazF engineered strains and wild-type DH5α controls were inoculated into 5 mL LB medium containing 100 μg/mL Ampicillin and grown overnight at 37°C, 150 rpm for activation. The next day, overnight cultures were 1:100 inoculated into 20 mL fresh LB medium with an initial OD₆₀₀ = 0.1. When cells reached OD₆₀₀ ≈ 0.4, the experimental group was induced by adding 2% (w/v) L-arabinose (A83229, Innochem) to activate PBAD-driven mazF expression.

Cell growth was continuously monitored using a FlexStation 3 microplate reader (Molecular Devices, USA) to generate growth curves. The results showed that under L-arabinose induction, the growth of DH5α-PBAD-MazF was significantly inhibited, whereas the non-induced group and wild-type controls showed no significant growth changes.

These results demonstrate that the PBAD-MazF system can effectively activate toxin expression under arabinose induction, suppressing host growth, and validate its feasibility as a controllable “suicide switch” for biosafety control in synthetic biology.

Figure 14 Verification of the Suicide System

In summary, to prevent potential side effects of the engineered strains and their products in vivo, or to allow users to terminate bacterial activity on demand, we designed a controllable safety system. This system uses the arabinose-inducible promoter PBAD (BBa_I13453) to drive the expression of the E. coli toxin MazF, enabling engineered cell self-lysis in the presence of arabinose.

We successfully constructed the BL21-HMB engineered strain capable of de novo HMB synthesis. The results showed that this strain produced approximately 65 mg/L HMB after 12 h cultivation in M9 medium, with HMB production positively correlated with glucose consumption and no significant effect on host growth. Furthermore, by introducing the thioesterase YciA, HMB titer was enhanced to 185.86 mg/L, significantly improving synthetic efficiency.

Next, we established a serotonin synthesis system by successfully cloning TDC and TPH genes into E. coli. The recombinant strains were able to synthesize and secrete serotonin in the culture medium, reaching a maximum titer of 1.85 mg/L. TDC from different sources had a significant impact on serotonin yield, with Catharanthus-derived TDC increasing the titer to 2.62 mg/L.

Finally, to ensure the biosafety of the engineered strains, we designed an arabinose-inducible MazF suicide system. Under arabinose induction, this system could be activated to trigger self-lysis of engineered cells, enabling controllable regulation of growth and function.

In conclusion, we not only achieved efficient synthesis of HMB and serotonin, but also established a controllable safety mechanism, laying a solid foundation for the application of synthetic biology in health intervention and functional probiotic development.

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[2] Huang, S. J., Lai, M. J., Chen, A. Y., & Lan, E. I. (2024). De novo biosynthesis of 3-hydroxy-3-methylbutyrate as anti-catabolic supplement by metabolically engineered Escherichia coli. Metabolic engineering, 84, 48–58.