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✨ iGEM 2026 Synthetic Biology Project • Clean 5-HTP Biomanufacturing

Unlocking High-Yield 5-HTP Cell Factories

We engineered an optimized Escherichia coli chassis converting simple glucose feedstocks directly into 5-Hydroxytryptophan (5-HTP). By combining structural TPH enzyme mutagenesis (Y235F), BH4 cofactor regeneration, and feedback-resistant metabolic pathways, we eliminate wild seed harvesting dependencies and dangerous Peak X contamination.

5-HTP Yield +380% vs Wildtype TPH

Substrate $K_m$ 0.35 mM 3.4x Higher Affinity

Peak X Safety 0.0% Toxic Dimer Free

Meet Our Biological Agents Interactive Mascots

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Chassis Coco E. coli Host

Overexpressed YddG efflux pump & shikimate pathway shunts.

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Tippy TPH Enzyme

Engineered Y235F mutant residue removing electrostatic repulsion.

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Sparky BH4 Cofactor

Continuous DHPR salvage loop keeping BH4 cofactor active.

"Hi human! Click on any mascot above to hear our scientific breakdown!"

Interactive Biological Circuit Map

Tryptophan Supply is Insufficient ⚠️

Limited intracellular L-tryptophan pool restricts the biosynthesis of 5-HTP in wildtype chassis

Renewable Resource

🌽 Sugar Feedstocks

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Corn

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Sugarcane

Substrate Influx Glucose C₆H₁₂O₆

Assimilation: ➔ PASSIVE FLUX

🦠 Engineered E. coli Cell Factory

L-Tryptophan Biosynthesis Pathway • L-色氨酸生物合成途径 WILDTYPE BOTTLE-NECKED

Substrate Glucose

C₆

Cytoplasm Influx

Chassis Precursor Chorismate

Weak Biosynthetic Flow

Target Intermediate L-Tryptophan

Pool Level

LOW

Final Product 5-HTP

LOW 5-HTP TITER

🛑 Competing Pathways (分流消耗) DRAINING LOSS

Protein synthesis: Diverted into cellular growth
Indole degradation: Broken down via tnaA enzyme
Aromatic shunts: Lost to tyrosine/phenylalanine

⚡ Biosynthetic Pathway Bottlenecks

Without genetic optimization, wild-type E. coli shuts down tryptophan synthesis via retro-inhibition and lacks high-efficiency TPH activity.

💡 Action Required: Overexpress feedback-resistant genes & mutant TPH.

Challenges of Biosynthesis (生物合成存在的问题)

01. Low flux through tryptophan pathway: Intrinsic biosynthetic rate is slow under wildtype regulation.
02. Limited precursor availability: Essential carbon intermediates are consumed by cellular biomass.
03. Stronger competing pathways: Tryptophan is heavily degraded into indole by endogenous enzymes.
04. Result in insufficient tryptophan supply: Low substrate pool places a strict ceiling on 5-HTP yield.

Our Engineering Goal (我们的工程目标)

Strengthen tryptophan biosynthesis to relieve cellular constraints and build an industrial cell factory:

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Increase intracellular L-tryptophan pool By overexpressing feedback-resistant aroG and optimizing shikimate flux.

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Improve 5-HTP production & export By coupling mutant TPH (Y235F) with active YddG efflux pumps.

Dry Lab • Molecular Dynamics & Docking

TPH Enzyme Residue Mutagenesis Simulator 🔮

Select targeted residue mutations at position 235 of Tippy (Tryptophan Hydroxylase) to compute free binding energy ($\Delta G$) and catalytic efficiency ($k_{cat}/K_m$).

Select Mutation Variant:

Docking Simulation Output Variant Y235F

Binding Energy ($\Delta G$) -9.8 kcal Lower is More Stable

Substrate $K_m$ 0.35 mM Lower Means High Affinity

Turnover $k_{cat}$ 12.8 /s Catalytic Speed

Structural & Mechanism Analysis:

✨ Outstanding mutation! Eliminating the hydroxyl group removes local electrostatic repulsion, significantly boosting the π-π stacking interaction with tryptophan's indole ring!

Wet Lab Quality Control & Safety

Eliminating "Peak X" Toxic Contamination 🧪

Wild seed extractions of Griffonia simplicifolia harbor dangerous tryptophan dimers ("Peak X") causing Eosinophilia-Myalgia Syndrome (EMS). Biomanufacturing guarantees 100% dimer-free safety.

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Simulated HPLC Elution Chromatogram

Comparing Wildseed Extract vs iGEM Fermentation Broth

HPLC UV Absorbance (280nm) SOURCE: Wild Griffonia Seed Extract

Pure 5-HTP

⚠️ Peak X (Toxic Dimer)

0 min (Elution Start) Retention Time ($R_t$) 15 min

⚠️ Peak X Warning: Natural Griffonia seeds contain tryptophan dimers at unpredictable levels. Ingesting this extract directly without expensive chemical separation carries high risks of Neurological EMS.

Human Practices • Ethics & Community

Stakeholder Concerns Dialogue Simulator 💬

Explore how our team directly addressed socio-economic, medical purity, and biosecurity feedback from global stakeholders.

Select Stakeholder:

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Kofi Annan (Seed Harvester Representative)

Community Leader, West Africa

"If your synthetic biology factory replaces wild seed harvesting, hundreds of families in our rain forest region will lose their seasonal income. How will your project protect our community?"

Our Integrated HP Response:

"Fair-Transition Cushion: We collaborated with agro-ecological NGOs to create a transition fund, redirecting seed harvesters to high-value botanical medicinal cultivation."

iGEM 2026 Competition

Our Team & Student Researchers 🧬

Combining dry lab computational modeling, wet lab synthetic biology, and human practices.

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Dr. Sarah Chen Wet Lab Lead

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Alex Zhang Dry Lab Lead

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Elena Rostova Human Practices

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David Miller Wiki & Frontend