Rare diseases often receive limited attention and resources, despite the profound impact they can have on affected individuals. Many rare diseases are congenital or genetic, collectively affecting approximately 0.65% of the population. Unfortunately, most of these conditions lack curative treatments, leaving patients reliant on medications to maintain their physical and mental well-being. However, these medications are often exorbitantly priced and come with a slew of side effects, making them financially burdensome for many families, ultimately leading some to forego treatment.

Through a stepwise investigation, we came across Phenylketonuria (PKU), one such rare disease. Our research revealed a group of individuals who, from birth, cannot consume common foods like milk, chicken, fish, meat, eggs, or even breast milk. For them, these seemingly ordinary foods are poison, leading to intellectual disabilities and even death upon ingestion. These patients must adhere to a lifelong, highly restricted diet, earning them the endearing term, "angels who don't partake in earthly pleasures."

With a foundational understanding of PKU, our team developed a strong desire to further assist these "angels." Consequently, we made the resolute decision to adopt PKU as the focal point of our iGEM project. Our goal is to engineer a novel treatment approach that offers cost-effectiveness, safety, and reliability for all PKU patients.

Phenylketonuria is a congenital autosomal recessive genetic disorder, classified as a common amino acid metabolic disorder, with an estimated prevalence in China of approximately 1 in 11,000. PKU remains incurable, necessitating lifelong treatment. With timely treatment and proper control, it typically does not affect natural lifespan.
About Phenylalanine
Phenylalanine is an essential amino acid in human metabolism, with a daily requirement for a normal child estimated at 200-500 mg. Approximately one-third is used for protein synthesis, while two-thirds are converted to tyrosine by the action of phenylalanine hydroxylase (PAH) in hepatic cells, supplying the synthesis of thyroid hormone, adrenaline, melanin, and more.
In the hydroxylation process of phenylalanine, the involvement of the coenzyme tetrahydrobiopterin (BH4) is essential.
Causes
PKU results from a deficiency of phenylalanine hydroxylase (PAH) in hepatic cells in the classical mechanism, or from a lack of tetrahydrobiopterin (BH4) in the non-classical mechanism, within the phenylalanine (PA) metabolic pathway. This deficiency leads to the inability to metabolize phenylalanine (Phe), resulting in the accumulation of Phe, a deficiency in tyrosine, and the production of phenyl-lactic acid and phenylpyruvic acid, subsequently causing a series of symptoms.

Accumulation of Phenylalanine: Excessive accumulation of phenylalanine can lead to neurotoxicity, potentially causing symptoms such as hypertension, brain damage, and seizures.
Tyrosine Deficiency: Tyrosine, originally synthesized from the metabolism of phenylalanine, plays a crucial role in the production of thyroid hormones, adrenaline, and melanin. Its deficiency can lead to intellectual disabilities, growth retardation, and light pigmentation of the skin and hair.
Excess Phenyl-Lactic Acid and Phenylpyruvic Acid: Phenylalanine is diverted into producing higher levels of phenyl-lactic acid and phenylpyruvic acid, leading to their excretion in sweat and urine, resulting in the characteristic "musty" odor.

Phenylketonuria (PKU) has three main treatment methods: a low protein diet and two medicines, KUVAN and Palynziq. PKU is caused by the inability to process phenylalanine (Phe), causing an excessive buildup of it in the patient's body. Because Phe is one of the main building blocks of protein, PKU patients need to avoid consuming proteins to decrease the accumulation hein their bodies, however, by decreasing the daily intake of protein patients risk insufficiency of other essential amino acids in proteins, raising the possibility for deficiency related conditions.

There are two FDA-approved drugs for PKU: KUVAN and Palynziq, both produced by BioMarin Pharmaceutical. KUVAN is a cofactor of PAH enzyme that assists the enzyme in breaking down Phe. Palynziq injection is a enzyme substitution therapy injecting phenylalanine ammonia-lyase (PAL), an enzyme that breaks down Phe, into the patient's body. KUVAN can be administered to children over one month and adults for certain kinds of PKY while Palynziq injections can only be administered to adults with PKU. 

Both drugs have many side effects and can cause severe, even life-threatening, allergic reactions, including side effects such as headache, nasal congestion and Inflammation of the lining of the esophagus or stomach in the case of KUVAN, and rashes, itching, and swelling of face for Palynziq In addition, these drugs must be used along with a Phe-restricted diet, which would negatively affect the patient's body due to the lack of nutrition.

Our design can operate in two different conditions: in vivo system, in which the bacteria work inside the body, and in vitro system, in which the bacteria works outside of the body. These two systems have the same transport system, but each have a slightly different biodegration system. 

Internal Transformation System
Within our in vivo transformation system, we have meticulously divided its functions into two distinct components: the transport system and the degradation system. These two systems work in perfect harmony to fulfill the objectives of our project.

Figure: Graphic Portrayal of In Vivo Transformation System. 1) Phe is phenylalanine. 2) PAL is phenylalanine ammonia-lyase. 3) TCA is trans-cinnamic acid. 

The transport system operates under the control of the T7 promoter and encompasses a critical genetic module. This module expresses the PheP transport enzyme, a specialized transporter designed to actively uptake phenylalanine (Phe) into the bacterial cells. This step is vital as it prevents Phe from being absorbed by the human body upon ingestion of our engineered bacteria.

The degradation system is poised to tackle the challenge of metabolizing Phe efficiently. This system is activated once our bacteria enter the intestinal environment, characterized by low oxygen levels. The switch is triggered by these anaerobic conditions, leading to the activation of the PepT promoter and metabolic machinery.
Within the degradation system, we've harnessed the potential of the PAL enzyme. PAL, or Phenylalanine Ammonia Lyase, plays a pivotal role by catalyzing the conversion of Phe into trans-cinnamic acid (TCA). This process marks a crucial step towards neutralizing the harmful effects of excessive Phe in the body.

1、Intestinal Absorption: TCA, the product of phenylalanine (Phe) degradation, embarks on its journey from the intestines. Here, it enters the bloodstream through the intestinal walls, carried by blood vessels.
2、Hepatic Conversion: TCA-rich blood eventually reaches the liver, where a remarkable transformation occurs. The liver enzymatically converts TCA into hippuric acid (HA), a much less harmful compound.
3、Renal Excretion: With its conversion complete, HA is transported to the bladder through the bloodstream. From there, it is excreted from the body via urine, effectively eliminating excess phenylalanine in a safe and efficient manner.

Figure: In vivo transformation system's steps to degrade phenylalanine (Phe). 1) Phe is the acronym phenylalanine. 2) PAL stands for phenylalanine ammonia-lyase. 3) TCA is the acronym trans-cinnamic acid. 4) HA stands for hippuric acid.

1. Psychological Resistance
After conducting extensive surveys involving patients and their families, it became evident that there exists significant psychological resistance to the direct consumption of Escherichia coli (E. coli) bacteria. They expressed a strong preference for avoiding direct contact between the human body and these bacteria.
Moreover, concerns were raised about the in vivo system's design, which involves degrading phenylalanine (Phe) inside the body. Many patients and their families worried whether all Phe could be fully degraded by the bacteria before the protein was absorbed by the body.

2. Lack of Objectivity
Phenylalanine (Phe) is rapidly absorbed in the body, and there's a possibility that it may be absorbed by the human body before the engineered bacteria have a chance to act. When patients self-administer treatment at home, variations in the Phe content of their food introduce uncertainty regarding the optimal dosage. If the dosage is too low, Phe may not be completely cleared, resulting in residual Phe in the body.
Additionally, legal restrictions in some countries, including China, prohibit the intake of probiotics, making it impossible to use an in vivo treatment approach.

3. Advantages
Extracorporeal transformation, conducted entirely outside the human body, offers cost-effective options even for economically disadvantaged households. Standardized production of specialized dietary products ensures a more reliable conversion of phenylalanine.

To address the specificities of inside and outside the human body environments, we made certain modifications to our gene pathways. The transport system remains unchanged.

Within the inside the human body environment, our degradation system employs an anoxia-inducible promoter adapted to the intestinal conditions.  However, in the outside the human body setting, we utilize the TCA promoter, which responds to TCA induction to activate downstream PAL gene expression.

An ensuing challenge was ensuring that our engineered bacterial strains could fully degrade phenylalanine in food.  To enhance PAL gene expression further, we designed a gene circuit incorporating a positive feedback loop and irreversible switch.

We positioned the TP901 gene downstream of the TCA promoter.  TP901 is an integrase enzyme capable of recognizing specific DNA sequences and altering their order.  We placed a reverse T7 promoter within TP901's recognition sites.

Under normal conditions when the TCA promoter is unstimulated by TCA, TP901 remains dormant, and the T7 promoter does not activate.  This design reduces the burden on bacterial cells when E. coli activation is unnecessary, such as during amplification and cultivation phases.

The TCA promoter exhibits low-level basal expression, allowing it to weakly act and transcribe phenylalanine ammonia lyase (PAL) in the absence of trans-cinnamic acid (TCA).  These trace amounts of PAL break down Phe into minimal TCA.  The TCA generated from degradation further amplifies the TCA promoter's response, leading to transcription and translation of TP901 integrase.  TP901 integrase redirects the downstream T7 promoter.

Once the robust T7 promoter is activated, this gene switch remains permanently open.  Consequently, more PAL is expressed, enabling the complete breakdown of Phe in food.

In the In Vitro process, after introducing our product, Antiphe, into the food, E. coli absorbs phenylalanine (Phe) from the food, converting it into trans-cinnamic acid (TCA) and expelling it from the bacteria. As a result, all the Phe in the treated product becomes TCA, which can be absorbed by the human body or patients. Finally, we treat and remove the residual E. coli from the food to obtain the Phe-free final product.

Target User:

Our target user group consists of patients with phenylketonuria (PKU), a genetically inherited amino acid metabolism disorder where individuals cannot metabolize phenylalanine (Phe) normally, leading to severe symptoms such as brain damage. Due to this condition, this group requires special diets of low or Phe-free food. To meet the needs of PKU patients, we plan to offer a variety of Phe-free food options, including bread, cakes, cookies, and various types of food products treated with Antiphe. We believe that these products will not only help patients manage their condition but also provide a diverse choice of food and convenience that are helpful in their daily lives.

Product Production:

The production of Antiphe involves the use of engineered E. coli bacteria that we designed. These bacteria can absorb Phe and convert it into TCA. We will cultivate these E. coli bacteria in a controlled laboratory environment and incorporate them into the food production process. After cultivating the engineered bacteria, we will transfer them to microbial fermentation factories for larger-scale cultivation before delivering them to food producers. This way, we can mass-produce foods that are low in Phe or completely Phe-free.

Product Storage and Function:

Our products undergo meticulous sterilization before leaving the factory to ensure the biological safety of the products. All E. coli are eliminated, leaving only the food components resulting from the bacteria's conversion. This means that our products can be stored at room temperature for extended periods while maintaining their quality and nutritional value. Each product will be labeled with detailed storage and consumption guidelines to assist producers and consumers in correctly storing and consuming our products.

Product Usage:

We offer a variety of flavors and types of food to meet both the needs and preferences of PKU patients. Since our products do not contain Phe, PKU patients can consume them without concerns over worsening symptoms. However, we still recommend consulting with a doctor when consuming any new food or changing dietary habits to ensure alignment with their individual health diets and needs.

Biosafety Measures:

We place a high emphasis on biosafety. Our product undergoes rigorous sterilization before leaving the factory to ensure all E. coli bacteria are killed. In addition, our production process strictly adheres to all food safety and biosafety regulations to ensure that our products are safe for consumers. Furthermore, we conduct regular product quality tests to ensure quality and safety.

In conclusion, our iGEM project represents a significant step forward in addressing the needs of PKU patients. By utilizing the In Vitro process and engineered E. coli, we've successfully created Phe-free food options that are safe, diverse, and convenient.
Our commitment to biosafety and product quality ensures that our offerings meet the highest standards. We envision a future where PKU patients can enjoy a broader range of foods without health concerns.

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