Week 1 HW: Final Project Ideas

1. What do you want to make? →

Target: Levothyroxine (synthetic T4)

Levothyroxine (synthetic T4) is thyroid replacement hormone medication taken by people with hypothyroidism due to thyroidectomy, underactive thyroid or any other endocrine hormonal issues. However, the manufacturing of Levothyroxine has been essentially the same since the 1950s (Kahaly). Levothyroxine is produced by chemical synthesis that is a multi-step organic synthesis requiring iodation chemistry, purification and tight quality control.

Why does it interest me? :

Hypothyroidism is a common condition worldwide, which requires meticulous care and continuous usage with changes of dosage until optimal dose. Personally, I take daily dosage of the medication after my thyroid cancer removal surgery, which led me to wonder about improving or reworking current methods of treatments. Thus, Levothyroxine can be produced more consistently, cheaper and more sustainable with synthetic biology.

2. What living system could make it? →

Chassis: modified E.coli (S.cerevisae) which have been used prior for microbial tyrosine derivative production (Guo et al.).

1. E.coli modified to overproduce L-tyrosine by upregulating shikimate pathway
2. Express heterologous iodination enzyme or modified peroxidase analogous to thyroid peroxidase (TPO)
3. Iodinate tyrosine to mono/di-iodotyrosine
4. Oxidative coupling step to form diphenyl ether backbone of T4

Input: glucose(energy source) , inorganic iodide in media, ammonia or nitrogen source, fermentation nutrients

Output: secreted levothyroxine

Assuming:

• I am assuming that TPO-like coupling reaction can function outside the specialized thyroglobulin scaffold in body
• I am assuming that iodide uptake and toxicity are manageable for E.coli

3. How could automation help you make it? →

Opentron: for strain screening

Cloudlab: robotic fermentation platform for scaled production

Automation:

Opentron : I believe Opentron can be used for media prep, iodine dosing, sampling for mass spectrometry titer and High-Performance Liquid Chromatography I would automate it to screen strain and optimization loop

• What would it take to make it at a meaningful scale?→

CloudLab: automated fermentation, inoculation, harvesting, feeding schedules, in-line monitoring (pH, dissolved O2, product titer via automated sampling), downstream triggering

Due to demand, a consistent monitored fermentation would similarly resemble pharma level production and quality control

5. How much would you need to make, and how long would it take?

• How much? Patient number: 100 (initial goal?) → small amount of patients in hospital Dosage (Adult): 125 μg/day ( mid-range for adults take 100-150μg) (“About Levothyroxine”) Annual dosage per patient: 125 μg/day × 365 days = 45,625 μg = 45.625 mg/patient/year Total mass: 45.625mg × 100 patients = 4,562.5 mg ≈ 4.56 g/year

Fermentation batch: Assumption: Titer: 50mg T4 per liter of culture Reactor volume: 10L per batch Thus: Output per batch = 50 mg/L × 10 L = 500 mg/batch

Number of batches needed: 4,562.5 mg ÷ 500 mg/batch = 9.125≈ 10 batches

• How long? Assuming it is 1 week per fermentation and downstream purification cycle (per batch) Total: Sequential (1 reactor): 10 batches × 1 week = 10 weeks Parallel (4 reactors): 10 ÷ 4 = ~3 batches per reactor × 1 week = ~3 weeks

5. What don’t you know yet?

Assuming:

• I am assuming that TPO-like coupling reaction can function outside the specialized thyroglobulin scaffold in body
• I am assuming that iodide uptake and toxicity are manageable for E.coli
• I don’t know real achievable titer amount
• I am not sure whether purification is to the quality control or be considered safe and effective for consumption
• I am not sure whether calculations are accurate since I made a lot of general assumptions

Works Cited “About Levothyroxine.” British Thyroid Foundation, 22 Apr. 2024, https://www.btf-thyroid.org/levothyroxine. Accessed 22 Sept. 2026. Guo, Xufan, et al. “Yeast: A Platform for the Production of L‐Tyrosine Derivatives.” Yeast, vol. 40, no. 5–6, 20 Apr. 2023, pp. 214–230, 10.1002/yea.3850. Kahaly, George J. “Therapeutic Use of Levothyroxine: A Historical Perspective.” PubMed, Springer, 2021, https://www.ncbi.nlm.nih.gov/books/NBK585642/. Accessed 21 Sept. 2026.

Final Project