Genome-integrated engineering of <i>Escherichia coli</i> Nissle 1917 enables sustained gut-local biosynthesis of 5-hydroxytryptophan.
The engineered probiotic strain achieved a 5-HTP titer of 863.2 mg/L and showed potential for gut-local biosynthesis, but findings are based on mouse models and may not directly translate to humans.
Where it sits
this study against the rest of the ecnoglutide corpusSummary and findings
This study developed a genome-integrated engineered probiotic system using Escherichia coli Nissle 1917 to enable gut-local biosynthesis of 5-hydroxytryptophan (5-HTP). The engineered strain achieved a 5-HTP titer of 863.2 mg/L. In vivo analysis indicated that single-dose administration led to a transient elevation in plasma 5-HTP levels.
Abstract
5-Hydroxytryptophan (5-HTP) is a functional precursor of serotonin; however, achieving sustained 5-HTP production <i>in vivo</i> by engineered microbes remains challenging. To address this, we developed a genome-integrated engineered probiotic system enabling gut-local biosynthesis of 5-HTP. <i>Escherichia coli</i> Nissle 1917 (EcN), a clinically validated probiotic with an established safety profile for intestinal application, was selected as the chassis. Constitutive expression was employed to eliminate inducer dependence, while genomic integration was used to enhance genetic stability and avoid reliance on antibiotic selection for pathway maintenance. In addition, BH4 biosynthesis and regeneration modules were incorporated to address the limited endogenous BH4 availability in the gut environment. Different administration strategies were systematically evaluated to characterize <i>in vivo</i> performance. Notably, our results showed that the hTPH2 hydroxylation module, together with the BH4 biosynthesis and regeneration system, supported 5-HTP biosynthesis in EcN. The engineered EcN strain achieved a 5-HTP titer of 863.2 mg/L, the highest level reported in EcN-based systems. Under non-selective conditions, EcN<sup>Pconst-5-HTP</sup> carrying the chromosomally integrated pathway maintained stable 5-HTP production in vitro and retained 5-HTP-producing capacity after intestinal passage, whereas plasmid loss in non-integrated constructs resulted in a pronounced decrease in production. <i>In vivo</i> analysis showed that single-dose administration induced a transient elevation in plasma 5-HTP, whereas repeated dosing generated reproducible, time-dependent fluctuations in fecal bacterial abundance and gut-local biosynthetic activity. Treatment with the engineered probiotic further improved behavioral phenotypes and modulated brain 5-HT-related metabolites in a reserpine-induced mouse model. Together, these findings provide a translational framework linking in vitro 5-HTP production by engineered probiotics to their <i>in vivo</i> functional performance.
Background
This paper addresses the challenge of achieving sustained in vivo production of 5-hydroxytryptophan (5-HTP), a precursor of serotonin, by engineered microbes. Prior research has indicated difficulties in maintaining stable production of such compounds in the gut. The significance of this study lies in its exploration of a novel probiotic system that aims to enhance the biosynthesis of 5-HTP in a clinically validated strain of E. coli.
Methods
The study utilized a genome-integrated engineered strain of E. coli Nissle 1917 with constitutive expression to eliminate inducer dependence. The primary outcome measure was the titer of 5-HTP produced, while secondary measures included plasma 5-HTP levels and fecal bacterial abundance. The administration strategies were systematically evaluated, though specific n and duration were not reported in abstract.
Results
The engineered EcN strain achieved a 5-HTP titer of 863.2 mg/L. In vivo analysis showed that single-dose administration induced a transient elevation in plasma 5-HTP. Repeated dosing led to fluctuations in fecal bacterial abundance and gut-local biosynthetic activity.
Interpretation
The findings suggest that the engineered probiotic can produce 5-HTP effectively, which is a step forward compared to previous attempts. However, the clinical significance of the transient elevation in plasma 5-HTP and the fluctuations in gut activity remains uncertain. Additionally, the study's reliance on animal models limits the applicability of the results to human populations.
Key findings
- 5-HTP titer of 863.2 mg/L, the highest level reported in EcN-based systems.
- Single-dose administration induced a transient elevation in plasma 5-HTP.
- Repeated dosing generated reproducible, time-dependent fluctuations in fecal bacterial abundance.
Limitations
- Primarily focused on in vitro and animal models.
- Not reported in abstract for specific n and duration of administration.
- Potential translational issues to human applications.