Diabetes, a prominent chronic disease, significantly diminishes human life quality. With a population nearing 560 million, China reports over 140 million diabetic patients. Globally, the number of people affected by diabetes is estimated at approximately 500 million, affecting over 8% of the world's population^[1].

Pharmacological therapy is central to managing diabetes by maintaining stable blood glucose levels. The Blue Book of Oral Insulin Industry projects that China's diabetes market will reach 75.1 billion CNY by 2023, a 20% increase from the 62.4 billion CNY in 2022, and is expected to expand to 132.3 billion CNY by 2030^[2].

Currently, a plethora of new hypoglycemic drugs are being used in clinical practice. A recent systematic review and network meta-analysis published in the BMJ (SCI Q1, April 6, 2023, impact factor 93.6) on the benefits and harms of drug treatment for type 2 diabetes (T2D) reveals insights into the efficacy and safety profiles of commercially available drugs. This analysis highlights two main issues: efficacy and side effects.^[3]

Poor efficacy:The results of the literature above show that only SGLT-2 inhibitors and GLP-1 receptor agonists can reduce the core measure of all-cause mortality. Nonsteroidal mineral corticosteroid receptor antagonists and metformin may reduce mortality, while other drugs may have little or no effect on death outcomes or are uncertain^[4]. The efficacy of hypoglycemic drugs on the market is disappointing.

Side effects:In the first half of 2023, GLP-1 drugs, which accounted for about 70% of the market share in the Chinese market, achieved sales of 3.4 billion CNY^[5]. However, it was pointed out in the aforementioned literature that GLP-1 receptor agonists (1.97, 1.39 to 2.80) probably increase the risk of severe gastrointestinal adverse events^[6] (GLP-1 agonists (1.97,1.39 to 2.80) may increase the risk of serious gastrointestinal adverse events.). These persistent adverse reactions may affect the overall health and well-being of patients, reducing their treatment compliance.

So we turned our attention to hypoglycemic peptide P37, a new nature product extracted for soybean, which is now undergoing in preclinical trials for reducing blood glucose in T2D patients. From the scientific publications and preclinical trials, we concluded that P37 has a huge potential to improve blood glucose and therapeutic effect comparable to metformin in T2D treatment^[7-10]. In a preclinical trial of 170 patients, P37 formula significantly reduced fasting and postprandial blood glucose levels in patients with T2D, with a response rate of up to 83%. It has the advantage of medical/food homology, eliminating its side effects from the source, and is an ideal hypoglycemic drug in the future.

When our team consulted Dr. Chenguang Yao, who was in charge of the research and development of this drug, we found that the current preparation method of hypoglycemic peptide P37 is still extracted from soybeans with traditionally physical and chemical methods. Dr Yao mentioned that there were several pain points such as low extraction efficiency and using important food crops as raw materials, which seriously limited the production capacity of this drug. Therefore, we proposed to Dr. Yao's team that we hope to improve the P37 yield with synthetic biology. We plan to synthesize hypoglycemic peptide P37 in Pichia pastoris to solve the problem of low production efficiency and using crops as raw materials. Dr. Yao greatly appreciated and provided the support for our team's experimental conditions and served as our first instructor. Therefore, we chose to synthesize the hypoglycemic peptide P37 through synthetic biology methods in Pichia pastoris.

In order to understand the current domestic hypoglycemic drug market and diabetic patients' expectations for hypoglycemic drugs, we came to the retired staff activity center of Wuhan community to conduct voluntary blood glucose screening for retired staff (Figure 1). In total, we measured blood glucose in 100 randomly selected middle-aged and older adults, including 40 males and 60 females, 24% aged 40 to 60, 21% aged 60 to 70, 38% aged 70 to 80, and 17% aged 80 to 100 (Figure 2). According to the official blood glucose concentration, we divided the blood glucose status into five grades: excellent, good, average, poor, and extremely poor. Dates shows that 43% of the elderly had excellent blood glucose, 26% had good blood glucose, 14% had average blood glucose, 13% had poor blood glucose, and 4% had extremely poor blood glucose.

Figure 1. Voluntary blood glucose screening for retired employees at the Retired Employee Activity Center

The elderly with a history of diabetes accounted for 20%, but only 60% of them chose to use hypoglycemic drugs, and the remaining 40% stopped or refused to take hypoglycemic drugs. According to the MMAS-8 medication compliance scale, most hypoglycemic drugs on the market have the problem of low medication compliance.

Figure 2. The age distribution of respondents

When we further investigated the key factors resulting for the low medication compliance, we found that only 33.3% of the elderly people recovered their blood sugar to above normal after taking hypoglycemic drugs, and many hypoglycemic drugs had no obvious hypoglycemic effect. Among the elderly people who refused to take or stopped taking hypoglycemic drugs, 40% of them said that they had cardiovascular, liver and kidney diseases, and there were no hypoglycemic drugs suitable for them on the market or they were afraid of the side effects brought by the hypoglycemic drugs on the market. 30% of the elderly said that the price of hypoglycemic drugs was too expensive, and they could not afford to take them as a treatment for chronic disease. The remaining 30% of the elderly did not realize the harm of long-term high blood sugar and chose to ignore it (Figure 3).

Figure 3. The key factors for poor medication compliance

Therefore, we concluded that the reasons for the low medication compliance of most hypoglycemic drugs on the market now are of poor efficacy, expensive, and side effects, making the patients unsuitable for use in the elderly with basic diseases.

Poor medication compliance leads to poor outcomes. In the elderly with a history of diabetes, the curative effect is not obvious enough. Among the elderly who refuse to take or discontinue taking hypoglycemic drugs, 20% of their blood sugar is poor, and 40% of their blood sugar is extremely poor.

In our communication with Professor Hanluo Li, an expert major in biomedicine, he told us that the market urgently needs a drug that is oral, polypeptide or protein, low toxicity, low cost, and pharmaceutical/food homology.

Figure 4. Professor Hanluo Li

At present, the hypoglycemic peptide P37 developed just meets all the above requirements, and our team uses synthetic biology to produce the hypoglycemic peptide P37, which can further increase the yield and reduce the cost. At the same time, our team realized that it is far from enough to rely on our small team to carry out offline publicity if we want to improve the public's attention to diabetes.

To further promote our project, we next visited BGI and had in-depth exchanges and explored the possibility of cooperation. We know that BGI's gene therapy program for chronic diseases is to treat diabetes by adjusting the nutritional structure of food, such as adding vitamins B. We also presented our recent work about Vg biological synthesis in BGI, and one of our strengths- the homology of medicine/food, which coincided with BGI's program and it was highly praised by Chen Liu, an expert from BGI.

Figure 5. Academic exchange at BGI

After gathering all the background of the project, we began the preliminary preparation work. Hypoglycemic peptide P37 is a polypeptide, an expression product, and our team used the central dogma principle to deduce the possible base sequence of all genes that could encode this polypeptide. Using the algorithm, we designed and synthesized the target gene (Vglycin, hereafter referred to as Vg) based on the fundamental principles of achieving the highest gene stability, minimizing the risk of mutation during experimental and production processes, avoiding enzyme cleavage sites, and avoiding primer binding sites within the gene. For detailed work, please refer to the model section.

Through communication with our supervisor Dr. Chenguang Yao and analysis of objective experimental conditions in the laboratory, we determined the collection method of hypoglycolic peptide P37 as nickel column adsorption and imidazole competitive ligand elution, so we added histidine His-tag at the 3 'end. DDDDK, an enterokinase recognition site, was added at the site where hypoglycemic peptide P37 was separated from His-tag. So our initial genetic design was the recombinant plasmid pPIC9K-3His-DDDDK-Vg.

In terms of the selection of engineered bacteria, our team made reference to the clever utilization of Pichia pastoris by SCUT-China 2022 and produced two natural terpenoids, showing the great potential of Pichia pastoris to give us inspiration. Later, our team held a meeting to discuss and found that Pichia pastoris is very suitable as the engineering bacterium for our drug hypoglycemic peptide P37, a small molecular weight polypeptide.

Our team summarized three advantages of Pichia pastoris:

⚫ Firstly, because it enables the protein to fold correctly, the protein expressed has high biological activity.

⚫ Secondly, the timing of protein expression can be regulated.

⚫ Thirdly, it can sustainably express the target protein.

⚫ Finally, the target protein can be secreted out of the cell after translation.

Therefore, our team finally decided to use Pichia pastoris to syntheses natural peptides, which is also to inherit and further explore more possibilities in the iGEM team over the years.

Our team then established the engineered bacteria and gene design and started the initial wet experiment to verify, and calculate the yield through the strips and data developed by Western blotting. The following is our technical route diagram, the specific wet experiment steps are detailed in the Figure 6.

Figure 6. The technical route diagram of the expression and purification of Vg in Pichia pastoris.

After conducting wet experiments for a period, our team found that our hypoglycemic peptide P37, as a polypeptide, was unstable in eukaryotic expression system due to its small molecular weight of only 6.3kd. So we linked three Vg and referred to the dipeptide Pro-Asp promoters IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway. The Asp-Pro acid sensitive site was used instead of DDDDK enterokinase because the acid-sensitive site is better controlled and only breaks at pH 4 to 5. Therefore, our team upgraded our genes and added pH regulation to the technical route for acid hydrolysis. The new gene has taken place to pPIC9K-His-3Vg.

On July 14, 2024, our team participated in the 11th Conference of China iGEMer Community (CCiC). At the meeting, professor Ziwen Xie put forward a valuable suggestion to our team: our team needs an independent innovation iteration process. Therefore, our team thought that GLP-1 drugs are popular because of their good efficacy in the treatment and prevention of diabetes-related diseases, especially in the treatment of stroke and cardiovascular diseases. We cannot help but wonder, do our drugs also have the function of treating or preventing related diseases?

Figure 7. Exchanging on the 11th Conference of China iGEMer Community (CCiC)

Our team found that in our previous background studies, the vast majority of diabetes patients are accompanied by relatively severe disease. People with diabetes have a 2 to 4 times higher risk of cardiovascular disease than people without diabetes. This is because diabetes is often accompanied by problems such as high blood pressure, abnormal cholesterol levels, obesity and lack of physical activity, all of which are risk factors for cardiovascular disease. Moreover, diabetes is an important risk factor for stroke. In people with diabetes, atherosclerosis progresses more quickly, often leading to early cardiovascular events, including stroke. Stroke, cardiovascular disease and diabetes are common interdependent and concomitant relationships, and we have concluded through reading literature that nattokinase has a role in the treatment of stroke and cardiovascular disease^[11-13]. If our hypoglycemic peptide P37 can treat and prevent dependent diseases while lowering blood sugar, it will undoubtedly be more competitive in the current clinical drug market. In addition, nattokinase and hypoglycemic peptide P37 have the same advantages of medical/food homology. Compared with mainstream hypoglycemic drugs on the market, efficacy and clinical effects are gradually approaching without any side effects. Our genes eventually changed to pPIC9K- NK-3Vg-His.

Based on the existing research results of our team, we can develop the bioactive peptide P37 synthesized by Pichia pastoris into a new natural oral polypeptide drug for the treatment of diabetes and its complications. Through a series of experiments conducted by our laboratory, bioactive peptide P37 has shown promising research outcomes in the treatment of T2D. We believe that P37 has potential medicinal value in treating diabetes and its complications, and the development of P37 as a food and drug will revolutionize the treatment and prevention of T2D.

Therefore, our team selected backbone Jiafan Zhang, Jiayi Zhao, Xinyu Xiao, Siyuan Huang, Qiwen Huang to form an entrepreneurial group. Jiafan Zhang and Jiayi Zhao are in charge of biological experiment and technical support, Xinyu Xiao is in charge of staff management and staff training, Siyuan Huang is in charge of marketing publicity and software model building, and Qiwen Huang is in charge of market research and publicity in the team.

In building a corporate culture, we are guided by the spirit of iGEM to make real and inexpensive hypoglycemic drugs for more than 140 million diabetic patients in our country. The company is currently pricing Aglashin powdered granules at 188 CNY/box (about $26), which is dosage for one month, an average of about 6 CNY per day (about $0.83), combined with Chinese per capita monthly income of 6072.5 CNY in 2023, we fully believe that the vast majority of diabetes patients can afford it. See our Business plan for more details.

As a team of undergraduates, our innovation and entrepreneurship ability is insufficient, and relevant knowledge is still being learned, so our team needs training. Our company needs help in the initial stage of development, technology, personnel, experimental sites and funding. So, our instructor Dr. Chenguang Yao introduced us to Ms. Hongrong Liu, the boss of Wuhan Lanzhi Biotechnology Co., Ltd. Our team immediately rushed to Wuhan Lanzhi Biotechnology Co., Ltd.

Ms. Hongrong Liu from Wuhan Lanzhi Biotechnology Co., Ltd warmly received our team. Si Cheng, the leader of our team, first introduced the technical route and product advantages of our project in general, emphasizing that we have determined the unique certainty of drug ingredients, the homology of medicine and food, and the advantages of increasing production capacity by using synthetic biology. After hearing this, Ms. Hongrong Liu said that our team is committed to independent innovation and the development of domestic new drugs, which is greatly appreciated, and believes that the results of our team have the possibility of industrial mass production, and agreed to provide experiments and funds for our team.

Figure 8. Communication with Wuhan Lanzhi Biotechnology Co., Ltd

After the completion of the project design and some experimental progress, in order to further determine the feasibility of the project and obtain expert advice and feedback, we interviewed Kanghong Hu, director and second-class professor of the Sino-German Biomedical Center of Hubei University of Technology. We prepared some questions and received some useful advice from Professor Hu. These answers and suggestions have greatly promoted the perfection and improvement of our project. His feedback was very positive.

Q1: Hypoglycemic peptide P37 was found in natural soybeans, but we used Pichia pastoris genetically engineered to produce P37. What are the advantages of using Pichia pastoris compared with natural plant extraction?

A1: The extraction of hypoglycemic peptide P37 from natural plants has obvious disadvantages, that is, it cannot maintain the uniformity of substance extraction. Soybeans in different regions may have different gene sequences, and the quality of drugs cannot be ensured in the process of drug production, which is not in line with the production standards of drugs. The use of genetic engineering to produce Pichia pastoris can maintain the stability of the expressed protein, but also has the advantages of high expression efficiency, relatively simple operation, and suitable for large-scale industrial production. You were right to choose Pichia pastoris.

Q2: What is your opinion on the competitiveness of P37 in the market after the application of synthetic biology to hypoglycemic peptide P37?

A2: Hypoglycemic peptide P37 is homologous to medicine and food, can be taken orally and non-toxic, plant extraction yield is low and food consumption is high. The production of P37 by Pichia pastoris can greatly improve production efficiency, save food, and conform to the concept of green environmental protection. Therefore, the application of synthetic biology to hypoglycemic peptide P37 will greatly enhance the competitiveness of products.

Q3: In the process of plasmid construction, we found that the expression of a single Vg gene plasmid was not obvious after construction. Subsequently, we decided to increase the expression level by using the method of tandem expression of three Vg. Do you think this method is reasonable?

A3: I think your idea is reasonable and practical. In the process of antibiotic production, vaccine research and development, and protein engineering, multiple gene expression technology may be used. I support your idea, but the biological activity of the produced protein needs further testing in the future, and I hope you can make further progress.

Q4: What do you think are the most promising research directions in the field of synthetic biology in the future? What skills or knowledge can we learn at this stage so that we can have a better development?

A4: Synthetic biology has shown great potential in the fields of green energy and environment, medical applications, biomaterials and innovation, bioinformatics, etc. Your Pichia pastoris production of hypoglycemic peptide P37 project can be classified as biofuturing. As a new engine of bioeconomy, synthetic biology is driving the next generation of biofuturing. Microbial cell factories built through synthetic biology technology can achieve efficient production of cheap raw materials to high-value chemicals, and the production process is clean and environmentally friendly. These are all areas of great potential. I hope you will find your areas of interest and continue to explore them, and ultimately contribute to humanity.

Q5: Our iGEM team is committed to the research and promotion of synthetic biology. Do you have any suggestions or expectations for our iGEM team?

A5: 1. We hope that you can put forward innovative ideas and solutions in the field of synthetic biology, not only in technology, but also in application scenarios and business models. Team members give full play to their professional advantages and conduct interdisciplinary cooperation. Biology, computer science, economics and art can shine in the igem team. I look forward to your project turning out a real product.
2.You can actively participate in academic exchanges, discipline competitions, in the collision of ideas to inspiration.
3.I hope you can become the disseminators of synthetic biology knowledge, and let more people understand and participate in the research of synthetic biology through the platform of iGEM competition. I also hope that after the iGEM competition, you can continue to deepen your work in the field of synthetic biology, transform the team's research results into more long-term scientific research projects or entrepreneurial opportunities, and make contributions to human society. Finally, I wish you excellent results in the iGEM competition and contribute to the development of synthetic biology!

Figure 9. Interview with Professor Kanghong Hu

Secondary Education

In order to popularize students' understanding of biology and synthetic biology, our team visited Wuhan Yucai Senior High School to introduce the concept of synthetic biology and its role in the pharmaceutical industry and in maintaining human health.

Introduction to synthetic biology

Our team began with the knowledge points of fermentation culture, PCR, and agarose gel electrophoresis from high school biology textbooks and explained how these are applied in our iGEM project. This not only helps high school students better understand their high school curriculum but also provides a general direction for students interested in pursuing synthetic biology in the future.

Figure 10. Preach to students in Wuhan Yucai Senior High School

Feedback on high school education

In this activity, in order to make it easier for high school students to understand, we made popular and metaphorical explanations of all the professional words in the project, and explained the basic biological knowledge currently mastered by high school students. We tried to make the explanation more humorous and arouse high school students' interest in synthetic biology.

Although we got the support of high school students and teachers, we still found problems in our publicity method. Our team project mainly focused on the research and development of diabetes drugs. In our publicity, we used too much space to publicize the iGEM competition schedule and award requirements, failing to take into account that high school students have no concept of competitions during their university years. Our team's explanation in this section did not meet expectations, but they were particularly interested in our experimental part and had a strong desire for university biology experiments. Unfortunately, we were unable to prepare enough video materials. After learning from this experience, our team will pay more attention to the interests of the speakers in the next promotion.

Despite this flaw, our team still won the first prize of the Hubei University of Technology's Social Practice School in Wuhan Yucai Senior High School.