This study aimed to engineer Escherichia coli to construct and optimize production systems for two bioactive molecules: HMB (β-hydroxy-β-methylbutyrate) and serotonin (5-hydroxytryptamine, 5-HT). HMB, an important muscle-protective agent, is primarily synthesized via the leucine metabolic pathway. We designed and constructed an engineered strain containing the key genes for HMB synthesis. To enhance HMB production, thioesterases were introduced to alleviate metabolic bottlenecks.
Serotonin, a crucial neurotransmitter, is widely involved in the regulation of mood, sleep, and other physiological processes. We further designed and constructed a serotonin synthesis system, optimizing enzyme expression to increase serotonin yield. Finally, a safety system was implemented to prevent potential biosafety issues associated with the engineered strains.
1.1 Construction of the HMB Synthesis System
Design
HMB (β-hydroxy-β-methylbutyrate) is a bioactive molecule derived from leucine metabolism and has been widely applied in the prevention and mitigation of muscle atrophy. HMB exhibits significant anti-proteolytic and protein synthesis-promoting activities, making it an effective muscle-protective agent [1].
Escherichia coli (E.coli) was selected as the microbial chassis for HMB production due to its multiple advantages, including rapid growth, simple cultivation, ease of genetic manipulation, low cost, and precise genome editing capabilities, making it an ideal host for the production of proteins and other bioactive molecules [2].
The de novo HMB synthesis pathway (3-hydroxy-3-methylbutyrate) consists of five key enzymes: AtoB (acetoacetyl-CoA thiolase), MvaS (HMG-CoA synthase), AibAB (3-methylglutaconyl-CoA decarboxylase), LiuC (3-methylglutaconyl-CoA hydratase), and YciA (thioesterase). This pathway uses three molecules of acetyl-CoA as substrates to produce one molecule of HMB, demonstrating high theoretical carbon conversion efficiency and being independent of ATP or NADH.
Figure 1: De Novo Synthesis Method of HMB
Bulid
To obtain a strain capable of efficient de novo HMB synthesis, we designed a gene circuit containing the key genes for HMB biosynthesis. E. coli BL21 was chosen as the microbial chassis for constructing the engineered strain. Using genetic engineering techniques, we cloned acetoacetyl-CoA thiolase (AtoB) from E. coli, 3-hydroxy-3-methylglutaryl-CoA synthase (MvaS) from Staphylococcus aureus, glutaconyl-CoA decarboxylase (AibAB) from Bacillus subtilis, and 3-methylglutaconyl-CoA hydratase (LiuC) into the pET28A(m) plasmid, which was subsequently transformed into E. coli BL21 (Figure 2).
Figure 2 Construction of HMB-Producing Strain
We first obtained the AtoB, MvaS, AibAB, and LiuC genes from NCBI and performed codon optimization based on E. coli codon preference, while removing EcoRI, XbaI, SpeI, and PstI restriction sites (sequence verification was conducted using SnapGene®) before outsourcing synthesis. The target fragments were then PCR-amplified and verified by agarose gel electrophoresis (Figure 3A). Subsequently, the fragments were cloned into the pSB1A3 vector (BioBricks™ standard RFC#10) using one-step seamless cloning (Seamless Cloning Kit, D7010, Beyotime).
The resulting recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells and plated on LB agar containing 100 μg/mL ampicillin (Amp⁺, pH 7.0). After incubation at 37°C for 16 h, single colonies were obtained (Figure 3B). Single colonies were inoculated into 5 mL LB/Amp⁺ liquid medium and cultured at 37°C, 220 rpm until OD₆₀₀ reached 0.6–0.8. Positive clones were further verified by colony PCR (2×Taq Master Mix, Vazyme) (Figure 3B).
Figure 3 Verification of HMB-Related Fragments and Single Colony Screening
Test
To determine the maximum absorption wavelength (λmax) of the target compound, a full-wavelength scan was performed using an Agilent 1200 series HPLC system equipped with a diode array detector (DAD) under the previously described chromatographic conditions. The detector scan range was set from 200 to 400 nm, with a bandwidth of 4 nm and a sampling frequency of 1 Hz, continuously acquiring 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, which was then used to determine the λmax, selecting an appropriate wavelength for quantitative detection. The experimentally determined detection wavelength for HMB was 208 nm (Figure 4).
Figure 4 Determination of HMB Detection Wavelength
The HMB content was analyzed 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 M KH2PO4, pH 3.0) and eluent B (acetonitrile). The elution gradient was set as follows: initial 95% A; linearly decreased to 40% A from 8 to 8.5 min; maintained at 40% A from 8.5 to 13.0 min; linearly returned to 95% A from 13.0 to 13.5 min; maintained at 95% A from 13.5 to 22.0 min. The flow rate was 1.0 mL/min, and the injection volume was 10 μL. The detection wavelength was set at 208 nm (Table 1).
A series of HMB standard solutions (0.05–2.00 mg/L) were measured at 208 nm to generate a standard curve, which was subsequently used to calculate the HMB concentration in the samples (Figure 5) [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 5 HMB Standard Peaks at 208 nm
To determine the HMB production of the engineered strain in M9 medium after 12 hours of cultivation, the strains were first activated in LB/Amp⁺ medium and then inoculated into M9 medium. The cell growth was monitored in real-time by measuring OD₆₀₀. Samples were collected at different time points, centrifuged, and the supernatants were used for HMB quantification. Each experiment was performed in triplicate, and comparisons between groups were analyzed using the Student’s t-test, with p < 0.05 considered statistically significant.
The results showed that the engineered E. coli produced 65.13 mg/L HMB after 12 hours of cultivation at 37°C, whereas the wild-type BL21 did not produce detectable HMB (Figure 6).
Figure 6 HMB Production Testing
To evaluate whether HMB production affects the growth of the engineered strain, the OD₆₀₀ of both wild-type and engineered E. coli was measured every 6 hours over a 24-hour cultivation period. The results demonstrated that HMB production did not significantly affect the growth of the engineered strain (Figure 7).
Figure 7 HMB-Producing Strain Growth Curve
To quantify the carbon source utilization efficiency and metabolic pathway effectiveness of the engineered strain, the glucose consumption and HMB production were dynamically monitored every 6 hours over a 24-hour cultivation period, followed by correlation analysis. The results showed a significant positive correlation between glucose consumption and HMB production (Figure 8).
This observation directly demonstrates that glucose as a carbon source is efficiently channeled into the de novo HMB biosynthesis pathway, rather than being redundantly consumed in side metabolism or for non-productive biomass growth, thereby confirming the functional integrity and metabolic rationality of the engineered pathway.
Importantly, these results provide essential experimental data for the subsequent calculation of carbon flux efficiency and carbon source utilization rate. Efficient carbon allocation is not only crucial for increasing HMB yield but also aligns with the requirements of resource-limited scenarios, such as space stations or closed ecosystems, where low-consumption, high-efficiency biosynthetic systems are essential. Overall, the positive correlation between glucose consumption and HMB generation over 24 hours confirms the engineered strain’s capacity for de novo HMB biosynthesis (Figure 8).
Figure 8 HMB Production Time-Course Curve
In this study, we successfully constructed an engineered strain capable of HMB biosynthesis. Using the optimized HPLC method, results showed that the engineered strain produced approximately 65 mg/L HMB after 12 hours of cultivation in M9 medium, and HMB production had no significant impact on host growth. Further analysis revealed a positive correlation between HMB production and glucose consumption, demonstrating that the engineered strain can efficiently convert carbon sources into the target product.
Learn
Although we successfully established the HMB-producing strain, the initial HMB yield of 65 mg/L was insufficient to effectively counteract muscle atrophy. Therefore, through discussions with researchers and literature review, we decided to screen thioesterases from different sources to enhance HMB production.
1.2 Introduction of Thioesterases to Enhance HMB Production
Design
The core rationale for introducing thioesterases to boost HMB production is their ability to efficiently hydrolyze HMB-CoA, releasing free HMB. This alleviates metabolic flux bottlenecks, reduces feedback inhibition, and improves both accumulation and secretion of HMB, thereby increasing the overall yield [3]. Therefore, we heterologously expressed thioesterases in plasmids to evaluate their effects on HMB production.
Build
First, we obtained thioesterase genes tesB, YciA, and MenI from E. coli via NCBI. Each gene was then cloned into plasmid 2 for expression (Figure 9).
Figure 9 Thioesterase-Related Plasmid Construction
Test
First, we successfully amplified the thioesterase genes tesB, YciA, and MenI (Figure 10).
Figure 10 Thioesterase Expression Verification by Agarose Gel Electrophoresis
Subsequently, HMB production was tested using the same method. The results showed that: tesB expression led to an HMB titer of 148.32 mg/L after 12 hours. YciA expression exhibited the best performance, producing 185.86 mg/L HMB after 12 hours. MenI expression resulted in the lowest titer among the three, 101.29 mg/L, but still higher than the strain without thioesterase (Figure 11).
Figure 11 HMB Production under Different Thioesterases
Learn
We successfully screened the thioesterase YciA, which could increase HMB production to 185.86 mg/L within 12 hours. Although the constructed HMB biosynthesis system mainly aims to alleviate muscle loss through the de novo synthesis pathway, the health challenges faced by humans in space environments go far beyond muscle atrophy. Besides metabolic regulation, the balance of neurotransmitter systems is equally crucial. Therefore, we decided to design a serotonin biosynthesis system to counteract neural and emotional dysregulation caused by the space environment.
2.1 Construction of Serotonin Biosynthesis System
Design
Serotonin (5-hydroxytryptamine, 5-HT) is an important monoamine neurotransmitter that participates extensively in human signal transduction and is considered a key factor in regulating mood, sleep, appetite, cognition, and gut function. Similarly, we selected Escherichia coli as the microbial chassis to test serotonin production.
Build
Serotonin biosynthesis primarily relies on tryptophan (TRP) and is catalyzed sequentially by two key enzymes: first, tryptophan hydroxylase (TPH) converts TRP into the intermediate 5-hydroxytryptophan (5-HTP); then, tryptophan decarboxylase (TDC) decarboxylates 5-HTP to produce the active neurotransmitter 5-HT.
We first constructed the serotonin biosynthesis system by obtaining TDC and TPH genes from NCBI (synthesized by Generalbial, China) and performed codon optimization for E. coli, while removing EcoRI, XbaI, SpeI, PstI, NdeI, XhoI restriction sites to comply with the RFC#10 standard and pET28a(m) cloning compatibility. The genes were then cloned into the pET28a(m) vector using NdeI and XhoI restriction sites. The recombinant plasmids were transformed into E. coli DH5α and BL21, and positive clones were screened on LB agar plates containing 100 μg/mL kanamycin (Kana, with 1.5% agar) and verified by sequencing (Beijing, Qingke), yielding the recombinant engineered strains (Figure 12).
Figure 12 Construction of Serotonin-Producing Strains
(A) Serotonin Biosynthesis Pathway Construction (B) Serotonin Biosynthesis Plasmid Map (C) Agarose Gel Electrophoresis Verification of Serotonin-Related Enzyme Amplification
Test
To measure serotonin (5-HT) content, the engineered E. coli strain was inoculated at a 1:100 ratio into 20 mL of modified M9 medium. When the culture reached OD600 = 0.6, 0.5 mM IPTG was added to induce expression, and the culture was incubated for an additional 20 hours. 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 hours and stored at -20°C. The serotonin concentration in the culture medium was then analyzed using a 5-HT ELISA kit (JL53170, Jonln). Serotonin levels were calculated based on a standard curve. The results showed that the engineered E. coli produced 1.85 mg/L serotonin, whereas the wild-type strain and E. coli expressing only TDC did not produce detectable serotonin (Figure 13).
Figure 13 Serotonin Production Assay
(A) Serotonin production measured every 6 hours over 24 hours.
(B) Functional verification of enzymes involved in serotonin biosynthesis.
Learn
We successfully constructed a serotonin biosynthesis system, achieving a production level of 1.85 mg/L. However, for gut microbiota applications, higher product concentrations are necessary to ensure effective secretion and functional impact. Therefore, we screened TDC enzymes from different sources to further improve serotonin yield.
2.2 Screening TDC to Improve Serotonin Production
Design
By searching databases and literature, we selected TDC enzymes from rice, catharanthus, and pig to compare their serotonin production capabilities.
Build
The TDC genes from catharanthus and pig were cloned into plasmids for expression in E. coli (Figure 14).
Figure 15 TDC Amplification Verification
Test
We successfully amplified the TDC genes from catharanthus and pig (Figure 15 A, B).
Figure 15 TDC Source Screening
We then compared the serotonin production of TDCs from different sources. The results showed that: Rice-derived TDC produced 1.85 mg/L serotonin; Catharanthus-derived TDC had the best performance, producing 2.62 mg/L serotonin; Pig-derived TDC produced only 0.28 mg/L serotonin (Figure 15 C).
Learn
By screening TDCs from different sources, we successfully increased serotonin production to 2.62 mg/L. In the future, we plan to screen additional TDC sources to further enhance serotonin yield to meet our target.
3.1 Arabinose Promoter Testing
Design
To ensure that engineered genetic constructs do not escape into the environment and can be cleared from the host when needed, we used an arabinose-inducible promoter as a controllable switch to trigger bacterial cell death. The team first validated the function of the existing part BBa_K808000 in E. coli DH5α.
Build
To verify that the arabinose promoter functions properly in E. coli DH5α, we fused it with an mRFP fragment. The promoter activity was then evaluated by measuring fluorescence at different arabinose concentrations (Figure 16).
Figure 16. Construction of plasmid for testing arabinose promoter functionality
Test
We systematically tested the effect of different arabinose concentrations on mRFP expression. The results showed that the fluorescence/OD600 ratio increased significantly with higher arabinose concentrations, confirming that the arabinose promoter can dose-dependently induce target protein expression (Figure 17).
Figure 17. Dose-dependent induction of red fluorescent protein expression by different concentrations of arabinose
Learn
We successfully validated the arabinose-inducible system in E. coli DH5α, laying a solid foundation for the construction of the suicide system.
3.2 Suicide System Validation
Design
MazF is the toxin protein from the E. coli mazEF toxin-antitoxin system, which specifically cleaves bacterial mRNA at ACA sequences, blocking protein synthesis and causing rapid cell death. Therefore, we chose to use arabinose-inducible expression of MazF to quickly eliminate administered engineered bacteria in the gut, ensuring biosafety.
Build
MazF expression was placed under the control of the arabinose promoter to allow inducible activation of the suicide system.
Figure 18. Construction and Validation of the Safety (Suicide) System
(A) Gene circuit diagram of the safety system (B) Plasmid map of the safety system (C) Agarose gel electrophoresis verifying the expression of safety system genes
Test
The experimental results showed that under L-arabinose induction, the growth of the engineered strain DH5α-PBAD-MazF was significantly inhibited, whereas the uninduced group and wild-type control strain exhibited no noticeable growth changes. This indicates that the PBAD-MazF system can effectively trigger toxin expression under arabinose induction, suppress host cell growth, and validates its feasibility as a controllable “suicide switch” for biosafety control in synthetic biology.
Figure 19 Suicide System Validation
Learn
We successfully designed and constructed an L-arabinose-inducible MazF safety system and validated its feasibility in Escherichia coli DH5α.
[1] Holeček M. (2017). Beta-hydroxy-beta-methylbutyrate supplementation and skeletal muscle in healthy and muscle-wasting conditions. Journal of cachexia, sarcopenia and muscle, 8(4), 529–541.
[2] Lee S. Y. (1996). High cell-density culture of Escherichia coli. Trends in biotechnology, 14(3), 98–105.
[3] 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.
