1. Abstract
Due to changes in societal dietary habits, people tend to consume high-cholesterol foods such as pizza, hamburgers, and highly processed foods. These contain cholesterol-rich ingredients like cheese, butter, and eggs. 

As a result, the phenomenon of high cholesterol-related diseases is significant. Therefore, we aim to design a probiotic powder

for use in factories to reduce cholesterol levels in food, resulting in low-cholesterol dairy products, low-cholesterol egg products (note: as an additional option for health-conscious individuals who choose to do so). This provides people with more dietary choices, allowing them to reduce cholesterol levels in their diet without excessively changing their dietary structure. This, in turn, helps address high cholesterol issues among the population and prevent more people from developing high cholesterol-related diseases.


2. Project Background
① Social Needs
• Changes in Societal Dietary Habits
In recent years, with the restructuring of the economic structure and changes in the dietary habits and lifestyles of residents in different regions, as well as varying degrees of lipid-lowering drug application, global cholesterol levels among the population have undergone significant changes.
People tend to consume highly processed foods such as pizza, hamburgers, and most highly processed foods, which contain cholesterol-rich ingredients like dairy products including cheese, butter, and eggs. As a result, the prevalence of high cholesterol-related diseases is evident.

· Serious Impact of High Cholesterol on Health
High cholesterol is the leading cause of 3.9 million deaths worldwide【5】. Half of these deaths occur in East Asia, South Asia, and Southeast Asia. In 1980, China had one of the lowest levels of non-high-density lipoprotein cholesterol (non-HDL-C) in the world, but during a 39-year study period, China had one of the highest growth rates of non-HDL-C.
 Widespread Occurrence of High Cholesterol in Society
In the figure below, the closer the colour is to dark green, the more non-HDL-C levels have decreased in that region, while the closer it is to red, the more non-HDL-C levels have increased. There's no need for further explanation; the "China red" is truly alarming. 


•  Widespread Occurrence of High Cholesterol in Society
In the figure below, the closer the colour is to dark green, the more non-HDL-C levels have decreased in that region, while the closer it is to red, the more non-HDL-C levels have increased. There's no need for further explanation; the "China red" is truly alarming. 

NCD Risk Factor Collaboration (NCD-RisC). Repositioning of the global epicentre of non-optimal cholesterol. Nature. 2020 Jun;582(7810):73-77. doi: 10.1038/s41586-020-2338-1. Epub 2020 Jun 3. PMID: 32494083; PMCID: PMC7332422.


② Types of Cholesterol:
Cholesterol is a type of lipid substance found in animals and various foods. Its role includes forming bile acids, constituting cell membranes, and synthesizing hormones. Cholesterol exists in the bloodstream within lipoproteins, mainly divided into high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C).
"Bad" cholesterol refers to the total cholesterol content in lipoproteins other than HDL-C, mainly including low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C), with LDL-C accounting for more than 70%. Excess "bad" cholesterol deposits on the blood vessel walls, affecting normal blood flow, and increasing the risk of cardiovascular diseases. On the other hand, "good" cholesterol, HDL-C, has a preventive effect against cardiovascular diseases and stroke by absorbing excess "bad" cholesterol.

③ Relationship Between Cholesterol and Cholesterol-Induced Diseases (Hypercholesterolemia):High-density lipoprotein cholesterol can help remove cholesterol from cells, while excessive low-density lipoprotein cholesterol can block arteries, thereby increasing the risk of cardiovascular diseases.【9】 Elevated levels of low-density lipoprotein cholesterol are also known as hypercholesterolemia. Hypercholesterolemia is a significant risk factor for atherosclerotic cardiovascular diseases (ASCVD), which include cerebrovascular diseases, coronary heart disease, and peripheral artery diseases. Atherosclerosis typically has no obvious symptoms until severe atherosclerosis occurs. Complications of hypercholesterolemia and atherosclerosis include myocardial infarction, ischemic cardiomyopathy, sudden cardiac death, ischemic stroke, erectile dysfunction, claudication, and acute limb ischemia. It also has adverse effects on other diseases such as diabetes, liver disease, and Alzheimer's disease.

3.Advantages of the Product:
•  Current Treatment Methods and Their Shortcomings
For individuals with high cholesterol levels, the mainstream treatment methods currently available mainly include dietary control and the use of cholesterol-lowering medications. In terms of dietary control, the primary focus is on managing cholesterol intake. Regarding medication use, cholesterol-lowering drugs can be categorized into three main types【10】:

1. Statins:

Statins are frontline drugs for treating hypercholesterolemia and are the most potent class of drugs in this category. They work by competitively inhibiting the rate-limiting enzyme HMG-CoA reductase in the endogenous cholesterol synthesis pathway, thereby blocking the synthesis of mevalonic acid, a precursor to cholesterol, and reducing cholesterol synthesis within liver cells.
However, statins can have adverse effects on various organs and tissues in the body, including the liver, nervous system, gastrointestinal tract, leading to side effects like dizziness, vomiting, etc.【6】

2. Cholesterol Absorption Inhibitors:
These drugs work by reducing the absorption of cholesterol in the intestines, thus lowering plasma cholesterol levels.
Side effects may include abdominal pain, diarrhea, bloating, headaches.

3. Bile Acid Binding Resins:
These drugs block the reabsorption of bile in the intestines, subsequently reducing cholesterol within the liver .【7】
Bile acid binding resin drugs may lead to poor fat absorption, constipation, or diarrhea, among other side effects.
In summary, these types of drugs share significant and similar side effects, such as adverse effects on the liver, gastrointestinal reactions, skin flushing, headaches, etc. Additionally, these drugs tend to be expensive.



• Benefits of Dietary Changes Over Medication
· No Apparent Side Effects:
1. Statins are primarily used to treat high cholesterol, but patients with severe conditions receiving high-dose treatment can experience elevated liver enzymes, muscle pain, myopathy, and elevated serum creatine kinase levels, among other side effects. Moreover, long-term use of such drugs has been associated with an increased risk of developing new-onset diabetes.
2. Ezetimibe is a medication with substantial cholesterol-lowering effects but is also associated with significant side effects such as constipation, which can, in severe cases, lead to intestinal obstruction, as well as indigestion, stomach pain, and diarrhea.

·Health Benefits:
Changing one's dietary structure not only reduces cholesterol levels in the body but also increases the diversity of the gut microbiota, enhances insulin sensitivity, and reduces systemic inflammation .【8】

·  Lower Cost:
Dietary therapy is generally more cost-effective compared to medication treatment, making it a more feasible option for economically challenged patients.


4. Feasibility of Our Project:
Our initial team idea was to use in vivo microbial therapy, allowing microbes to react in the human gut. However, after researching the "Food Safety Law of the People's Republic of China," "Regulations on the Administration of Health Foods," and "Provisions for the Review of Probiotic Health Foods," and consulting with experts, we discovered significant biosafety concerns associated with allowing Escherichia coli (E. coli) to react in the human gut. Furthermore, Chinese national regulations stipulate that "probiotic strains must be members of the human normal flora and can utilize their live bacteria, dead bacteria, and metabolites. Probiotic health foods must be safe and reliable for consumption, without adverse reactions. The biological, genetic, and efficacy characteristics of the production strains must be clear and stable." Chinese biosafety laws do not allow for the production of engineered probiotics that act within the human gut. Therefore, we decided to use bacterial powder designed for external use in the human body. This powder will be used in food processing factories to directly produce low-cholesterol foods.





5. Introduction to Our Research Topic:
① Genetic Circuit

Our genetic circuit includes the T7 promoter, cholesterol transport protein, oleic acid promoter, and ACS gene, IsmA gene, galU gene.

② Oleic Acid Promoter

The FadR protein has two functional domains: a DNA-binding domain (DBD) located at the N-terminus and a specific DNA sequence of approximately 17 base pairs, known as the operator binding, which inhibits transcription initiation. Therefore, we introduced oleic acid, an unsaturated eighteen-carbon fatty acid. FadR from Escherichia coli MG1655 and oleoyl-CoA, a derivative of oleic acid (OA) metabolism, exhibit specific binding affinity. This can inhibit the repression of PfadRA by FadR, thereby activating PfadRA and the expression of downstream genes【2】. Oleic acid is mainly present in the form of triglycerides in large quantities in animal and plant oils, making it convenient to obtain for further processing. 

③ Cholesterol transport protein

In order to facilitate the interaction between free cholesterol in the human body and the probiotics we are developing, and to proceed with the next transformation steps, we need a "pathway" to facilitate their binding. Currently, there are two known mainstream methods: through non-specific channels for transport on the cell membrane, and by using certain cholesterol transporter proteins. Since the first method would directly harm Escherichia coli, affecting its survival rate, we must opt for the second approach: finding a transporter protein.
Based on the work of a previous team at McGill, a characterized protein (MFS transporter protein, BBa_K4348003) has been successfully constructed. Experimental evidence shows that this protein is located in the same operon as IsmA, which we need to use. Therefore, we can use this protein as our transporter for this project. 

We have also employed two genes known for effectively degrading cholesterol, namely: 

④ IsmA Gene

Bacteria can directly metabolize cholesterol itself, producing unique metabolites such as cholestenone and coprostanol through the activity of the IsmA gene-encoded dehydrogenase. Moreover, bacteria carrying the IsmA gene can significantly reduce dietary cholesterol. The IsmA gene encodes a hydroxysteroid dehydrogenase (HSD) that participates in the first and last steps of the conversion of cholesterol into sterols in the cholesterol metabolism process【4】.


⑤ ACS Gene  The ACS gene is involved in anaerobic cholesterol degradation, specifically in the side-chain degradation process【1】. The ACS gene encodes acyl-coenzyme A synthetase (ACS), which participates in the fatty acid acylation reaction during anaerobic cholesterol degradation, binding fatty acids to CoA to form fatty acid-CoA. These reactions are critical steps in anaerobic cholesterol degradation, allowing cholesterol molecules to be further metabolized.

Warnke M, Jung T, Jacoby C, et al. Functional Characterization of Three Specific Acyl-Coenzyme A Synthetases Involved in Anaerobic Cholesterol Degradation in Sterolibacterium denitrificans Chol1S. Appl Environ Microbiol. 2018;84(7):e02721-17. Published 2018 Mar 19. doi:10.1128/AEM.02721-17
 

⑥ galU Gene
We found in the literature that extracellular polysaccharides (EPS) can increase Escherichia coli's adsorption of cholesterol. Using the GalU gene can increase the amount of EPS, this extracellular polysaccharide, in Escherichia coli (E. Coli Rosetta). The GalU gene encodes UDP-glucose pyrophosphorylase, an enzyme involved in nucleotide sugar synthesis in sugar metabolism pathways. In this reaction, UDP-glucose reacts with pyrophosphate to produce glucose-1-phosphate and inorganic phosphate. The UDP-glucose pyrophosphorylase encoded by the GalU gene converts UDP-glucose into UDP-glucose pyrophosphate, which is a crucial enzyme providing substrates for polysaccharide synthesis and plays an essential role in sugar metabolism pathways. EPS can enhance E. coli's adsorption of cholesterol, binding cholesterol around E. coli and reducing cholesterol levels in food.【11】
 

⑦ Overall working principle
In food, the T7 promoter expresses a significant amount of transporter enzymes that transport cholesterol out of the food. Subsequently, cholesterol transporter proteins can transport cholesterol into the engineered bacteria. Externally added oleic acid can activate the oleic acid promoter, initiating the expression of downstream genes. The acyl-coenzyme A synthetase (ACS) encoded by the ACS gene participates in the fatty acid acylation reaction during anaerobic cholesterol degradation, promoting the progress of anaerobic cholesterol degradation. The IsmA gene plays a role in converting cholesterol into coprostanol, which cannot be absorbed by the human body. The GalU gene increases the amount of extracellular polysaccharide (EPS) in Escherichia coli (E. Coli Rosetta). EPS enhances E. coli's adsorption of cholesterol, binding cholesterol around E. coli, and reducing cholesterol levels in food. Therefore, through these three genes, the cholesterol content in food can be effectively reduced.
 

Our product utilizes dormant engineered bacterial freeze-dried powder, which is mixed with food waiting for processing, such as dairy products, to degrade cholesterol in them and produce low-cholesterol food.


Target User Groups:
1. High Cholesterol Patients: 

This group of individuals has elevated cholesterol levels, which pose health concerns. Existing medications on the market tend to be expensive, making the demand for low-cholesterol foods urgent.
2.Health-Conscious Individuals with Some Knowledge of Preventing High Cholesterol:

 This group of people is gradually becoming more health-conscious and seeks low-cholesterol foods as a preventive measure against high cholesterol.
3. Food Processing Factories: 

As people become increasingly aware of high cholesterol issues, and with a significant population suffering from high cholesterol, there is a growing demand for low-cholesterol foods. We intend to sell our product, the dormant engineered bacterial freeze-dried powder, to food processing factories, enabling them to produce low-cholesterol foods.

After extensive discussions within our team, we have realized that the fermentation production method in the laboratory alone cannot meet our expected production volume requirements. Therefore, we have opted for a larger-scale production method and decided to seek professional contract manufacturing factories for the production and processing of our bacterial powder.To ensure the quality of our product and the smooth progress of the production process, we have contacted and inspected multiple contract manufacturing factories and have finalized a partnership with one.
The following is the process we have learned for the entire production:
Strain inoculation culture → seed tank culture → production tank culture → Discharge culture medium and add appropriate amount of carrier culture fermentation → freeze drying → grinding → screening → mixing → quality inspection → bacteria powder.

The product undergoes pasteurization through the use of the Batch method, which involves heating the mixture to 68-70°C and maintaining this temperature for 30 minutes before rapidly cooling it to 4-5°C. This ensures that the bacterial powder remains uncontaminated during production.

 Begin by passing the dairy product to be processed through a filtering membrane to remove large-molecule cholesterol.
 Mix our bacterial powder with the filtered dairy product for a certain period to ensure that as much cholesterol as possible is degraded.
 Filter the mixture again to remove any remaining solid impurities.

The genetically modified E. coli solid bacterial powder is specifically designed for the degradation of cholesterol in food and should not be used for other purposes.
During usage, maintain a clean and dry environment to prevent contamination of bacteria from other sources.
Prior to using the solid bacterial powder, carefully read the product instructions to understand storage conditions, shelf life, and other relevant information.
Precautions:
Follow hygiene requirements when using the solid bacterial powder to prevent bacterial cross-contamination, leaks, or spills.

1. Warnke M, Jung T, Jacoby C, Agne M, Feller FM, Philipp B, Seiche W, Breit B, Boll M. Functional Characterization of Three Specific Acyl-Coenzyme A Synthetases Involved in Anaerobic Cholesterol Degradation in Sterolibacterium denitrificans Chol1S. Appl Environ Microbiol. 2018 Mar 19;84(7):e02721-17. doi: 10.1128/AEM.02721-17. PMID: 29374035; PMCID: PMC5861837.
2. Ma, Yueyuan & Zheng, Xiangrui & Lin, Yi-Na & Zhang, Lizhan & Yuan, Yiping & Wang, Huan & Winterburn, James & Wu, Fuqing & Wu, Qiong & Ye, Jian-Wen & Chen, Guo-Qiang. (2022). Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas. Metabolic Engineering. 72. 10.1016/j.ymben.2022.04.003. 
3. Tok E, Aslim B. Cholesterol removal by some lactic acid bacteria that can be used as probiotic. Microbiol Immunol. 2010 May;54(5):257-64. doi: 10.1111/j.1348-0421.2010.00219.x. PMID: 20536722.
4.Kenny DJ, Plichta DR, Shungin D, Koppel N, Hall AB, Fu B, Vasan RS, Shaw SY, Vlamakis H, Balskus EP, Xavier RJ. Cholesterol Metabolism by Uncultured Human Gut Bacteria Influences Host Cholesterol Level. Cell Host Microbe. 2020 Aug 12;28(2):245-257.e6. doi: 10.1016/j.chom.2020.05.013. Epub 2020 Jun 15. PMID: 32544460; PMCID: PMC7435688.
5. NCD Risk Factor Collaboration (NCD-RisC). Repositioning of the global epicentre of non-optimal cholesterol. Nature. 2020 Jun;582(7810):73-77. doi: 10.1038/s41586-020-2338-1. Epub 2020 Jun 3. PMID: 32494083; PMCID: PMC7332422.
6.Thompson PD, Panza G, Zaleski A, Taylor B. Statin-Associated Side Effects. J Am Coll Cardiol. 2016 May 24;67(20):2395-2410. doi: 10.1016/j.jacc.2016.02.071. PMID: 27199064.
7.Rodenburg J, Vissers MN, Daniels SR, Wiegman A, Kastelein JJ. Lipid-lowering medications. Pediatr Endocrinol Rev. 2004 Nov;2 Suppl 1:171-80. PMID: 16456497.
8.Meslier V, Laiola M, Roager HM, De Filippis F, Roume H, Quinquis B, Giacco R, Mennella I, Ferracane R, Pons N, Pasolli E, Rivellese A, Dragsted LO, Vitaglione P, Ehrlich SD, Ercolini D. Mediterranean diet intervention in overweight and obese subjects lowers plasma cholesterol and causes changes in the gut microbiome and metabolome independently of energy intake. Gut. 2020 Jul;69(7):1258-1268. doi: 10.1136/gutjnl-2019-320438. Epub 2020 Feb 19. PMID: 32075887; PMCID: PMC7306983.
9.Pedro-Botet J, Pintó X. LDL-cholesterol: The lower the better. Clin Investig Arterioscler. 2019 Dec;31 Suppl 2:16-27. English, Spanish. doi: 10.1016/j.arteri.2019.10.003. Epub 2019 Dec 6. PMID: 31813618.
10.Bays HE, Dujovne CA. Drugs for treatment of patients with high cholesterol blood levels and other dyslipidemias. Prog Drug Res. 1994;43:9-41. doi: 10.1007/978-3-0348-7156-3_2. PMID: 7855253.
11.Tok E, Aslim B. Cholesterol removal by some lactic acid bacteria that can be used as probiotic. Microbiol Immunol. 2010;54(5):257-264. doi:10.1111/j.1348-0421.2010.00219.x