Human Practices

Overview

Inspired by the news, we conducted field trips to supermarkets and markets. We understand that the omega-3 family of unsaturated fatty acids occupies a huge market. However, there are limitations to its existing extraction methods. DHA and EPA are the essential omega-3 series of polyunsaturated fatty acids , which play an irreplaceable role in human health. As one of the most widely consumed sources of nutrients in people's daily lives, eggs are rich in a variety of nutrients such as unsaturated fatty acids. We hope to build engineered strains through synthetic biology to obtain Omega-3 eggs rich in DHA and EPA. In the key experimental design steps, we make the corresponding experimental planning by consulting with the experts in the relevant fields. We also maintain close contact with other teams during the experiment. During the design process, we share many valuable information with each other. We also conducted online questionnaires and public education on synthetic biology to investigate the public knowledge of PUFAs and the awareness and acceptance of DHA eggs.

Background
1、News reports that the majority of people's Omega-3 intake is seriously insufficient

Omega-3 polyunsaturated fatty acids (PUFAs) are essential for human health, but are not produced by the body itself (Reference:Omega-3 Polyunsaturated Fatty Acids and Their Health Benefits). In addition, studies have shown that omega-3 and omega-6 fatty acids should be kept 4:1 to maintain health (Reference:A Low ω-6 to ω-3 PUFA Ratio (n-6:n-3 PUFA) Diet to Treat Fatty Liver Disease in Obese Youth). However, due to the different dietary structure in many regions, the proportion of omega-3 and omega-6 intake is often unbalanced. The Chinese Nutrition Society recommends a daily intake of at least 220 milligrams of omega-3 fatty acids, but surveys show that Chinese people consume 49 milligrams a day. Especially for children aged 5 to 12 years who are in a critical period of physical development, omega-3 intake is obviously seriously insufficient. At present, the main sources of omega-3 supplementation are fish products and health supplements, but they cannot meet the daily needs. Based on this, NNU-China members began to pay attention to the production of omega-3 PUFAs, and hope to supplement it through daily diet.

Fig1:data sheet from Chinese Nutrition Society

2、 Listening to the lecture produced the idea of exploring the microbial production of ω -3 unsaturated fatty acids

Introduction: Academician Chen Jian is an expert in fermentation and light industry biotechnology, academician of Chinese Academy of Engineering, and deputy director of environment and Light Textile Engineering Department of Chinese Academy of Engineering. He has been engaged in the research and teaching of fermentation engineering and food biotechnology for a long time.

Fig2:Academician Chen Jian

On March 8, 2022, Academician Chen Jian came to Nanjing Normal University to give a lecture on the future of food and biotechnology.

Academician Chen Jian introduced microbial metabolic engineering and related technologies of future food to us. After the lecture, the team members asked Academician Chen Jian ω- 3 Production of unsaturated fatty acids. We learned about some biological production routes and those used by oil-bearing microorganisms ω- 3. Alternative sources of unsaturated fatty acids.

At the same time, Academician Chen Jian also discussed with us the broad prospects of synthetic biology and future food. Academician Chen Jian said that "it will become one of the important trends of food industry development in the future to transform biological principles into advanced industrial elements and apply them to food processing." It is suggested that we can focus on future food and develop future food through synthetic biological technology.

Fig3:Lecture site

3、 Course study helped us to identify the carrier of ω -3 unsaturated fatty acids

Relying on professional courses, the course teacher is Mr. Yuan Kai from the Biotechnology Center of COFCO Nutrition Research Institute. He said that the fish oil capsules on the market at present have a strong fishy smell for children, and children's acceptance is not high; As one of the most extensive sources of daily nutrition intake, eggs are a supplement ω- 3 Good carrier of unsaturated fatty acids. He also said that relevant experiments and reports had verified the advantages of eggs as a carrier for DHA supplementation. DHA in egg yolk belongs to lecithin DHA, which is enriched by chicken body and has pure natural characteristics and can be absorbed efficiently.

Therefore, we decided to use metabolic engineering to construct engineering strains, so that they can co produce DHA and EPA, and then add them to the layer feed, in order to obtain Omega-3 eggs rich in DHA and EPA.

Fig4:Mr. Yuan Kai's online course

Public Engagement
1、 DHA egg market research: visit the supermarket

On July 7,2022, our NNU-China 2022 team conducted a supermarket research, and went to Hema, China Resources Suguo, China Resources Vanguard, Golden Eagle Supermarket and many other supermarkets to conduct interviews and investigations on the issues related to ω -3 eggs.

Through the visit, it was found that there are very few omega-3 eggs on sale in supermarkets at present, and the brand is relatively single. And, by comparing the prices of omega-3 eggs with other eggs, the price of omega-3 eggs is relatively high. As a result, the current sales of omega-3 eggs are not in good shape. People's enthusiasm for buying is not high. Not only that, most omega-3 eggs on the market are DHA eggs, and there are almost no EPA eggs.

Through this survey, the NNU-China 2022 team has a basic understanding of the current sales of omega-3 eggs on the market. Through interviews with customers, we have a preliminary understanding of consumer acceptance of omega-3 eggs. It lays a good foundation for the next step of the team's experimental process and the application of omega-3 eggs.

Fig5:members investigate eggs in supermarkets

2、 Current study on the intake of ω -3 series polyunsaturated fatty acids in DHA / EPA: an exclusive interview with Hu Xuechao, president of Jian Qixing Child Nutrition Research Institute

Introduction: Hu Xuechao, male, president of Jian Qixing Children's Nutrition Research Institute, President Hu has been committed to the precise supplement of DHA nutrition for Chinese babies. According to the physical characteristics, dietary methods, nutritional needs and metabolic mechanism of Chinese babies, President Hu has a systematic and in-depth study.

On July 20, 2022, representatives of some members of NNU-China2022 conducted an exclusive interview with Dean Hu Xuechao offline. Our members consulted and confirmed the specific standards for DHA/EPA in the dietary guidelines for Chinese residents. We also learned about the current intake of omega-3 polyunsaturated fatty acids such as DHA/EPA.But there is still a long way to go in the scientific exploration of omega-3 polyunsaturated fatty acids. President Hu also said that it is a very good idea for us to develop omega-3 eggs with eggs as the carrier. As a food that people eat almost every day in their daily life, eggs have greatly expanded their popularity, and it is no longer far away to supplement DHA and EPA in the human body.

Fig6:communicate with Dean Hu

Through this exchange with Dean Hu, the team has a deep understanding of the development status and prospects of functional eggs. National awareness and acceptance of DHA/EPA is generally low. However, with the popularization of national policies and extensive social publicity, omega-3 polyunsaturated fatty acid health foods will be widely recognized by the public.

Fig7:Group photo with Dean Hu

3、 Questionnaires about public acceptance of DHA eggs and awareness of synthetic biology

We sent an online questionnaire to the public about DHA egg acceptance and awareness of synthetic biology. We hope to understand the public's acceptance and expectations of DHA eggs through the questionnaire. In addition, we look forward to raising public awareness of synthetic biology today."

The number of completed questionnaires was 711. Only a few members of the public know anything about synthetic biology and ω -3 eggs. Most of the public do not know much about ω -3 eggs and their efficacy. As a result, we believe that promoting ω -3 eggs is extremely necessary. Most people, some of whom have never even been exposed to DHA eggs, are very much looking forward to daily supplementation of DHA and EPA with omega-3 eggs. Through the questionnaire, we concluded that the public's exact hope is to reduce the price of the product while ensuring quality and safety performance.

Fig8:Some results of questionnaire survey

Project Design
1、 Interview with Professor Huang

Introduction: Professor Huang He is the Vice President of Nanjing Normal University. His scientific research work takes microorganisms as the core, based on metabolic engineering and synthetic biology, mining, analyzing and transforming microbial resources, and combining fermentation engineering and other means to develop downstream products (bio-based chemicals, nutritional chemicals, pharmaceutical intermediates, etc.).

Inspired by the lectures and literature, our team does exist microbial production of ω -3 unsaturated fatty acids. For more efficient expression of ω -3 unsaturated fatty acids, we consulted Professor Huang He from Nanjing Normal University.

Professor Huang He pointed out that Yarrowia lipolytica is a model oil-producing yeast. It has a wide range of usable substrates and is rich in acetyl coenzyme a. Its lipid content reaches more than 70% of the dry weight of cells. Moreover, the whole genome has been sequenced and has a complete means of genetic operation. Therefore, Professor Huang suggested that we can choose to use Yarrowia lipolytica as a chassis cell for the production of omega-3 unsaturated fatty acids. Finally, Professor Wong also had high hopes for the success of our project and welcomed us to ask him further questions.

Fig9:Group photo with Professor Huang He

2、Ask Dr. Du Yuanhang for advice

Introduction: Dr. Du Yuanhang, Ph.D. in the School of Food and Pharmaceutical Engineering, Nanjing Normal University, his major is biological informatics.

Given that our experiments involved in the production of Yarrowia lipolytica, we need to choose the corresponding and appropriate model for simulation. The team members asked Dr. Du Yuanhang. The equation and computer simulation simulation equation perform CFD simulation, telling the computer what kind of fluid we want to and what kind of environment do we simulate the computer through corresponding instructions. Dr. Du personally took us to simulate a demonstration, which provided a lot of help for the results observation and data analysis of our subsequent experiments.

Project optimization and improvement
1. Interview with Dr. Yin Xuebin

Introduction: Dr. Yin Xuebin, chairman of Suzhou Selenium Valley Technology Co., Ltd., chairman of the National Functional Agriculture Science and Technology Innovation Alliance, and vice chairman of the International Selenium Research Society.

Fig10:Dr. Yin Xuebin

The team members briefly introduced our team project and experimental results to Dr. Yin Xuebin. Dr. Yin Xuebin expressed his appreciation for the team's experimental results. Dr. Yin Xuebin expressed his appreciation for the team's experimental results. Dr. Yin Xuebin suggested that the team could be modified through metabolic engineering on the basis of completing the efficient production of DHA. Designing the DHA/EPA production ratio on demand to accommodate different groups of people and extend the benefits.

Fig11:interview with Dr. Yin Xuebin

2. Interview with Dr. Pang Jinzhu

Dr. Pang Jinzhu, male, director of nutrition science of Inner Mongolia Mengniu Dairy Group

Fig12:Dr. Pang Jinzhu

Since the public's awareness and acceptance of DHA/EPA is generally low, we would like to understand the market prospects of DHA/EPA eggs and how to improve consumer awareness in the later stages. So we contacted Dr. Jin Zhu.

Dr. Pang said the first step is to raise awareness and provide special education for consumers. Secondly, products should be classified according to their expertise in nutrition to facilitate later industrial production. In addition, DHA is an indispensable nutrient during pregnancy and infancy. In the process of supplementation, it is also necessary to be precise and accurate, and strive to localize DHA nutrition in China.

Fig13:interview with Dr. Pang Jinzhu

Ⅰ.Discovery background

· How Our HP Inform and Shape Our Project

Omega-3 polyunsaturated fatty acids are important nutrients that can not be synthesized by the human body but are also indispensable for the human. Omega-3 unsaturated fatty acids and Omega-6 unsaturated fatty acids maintain a certain proportion in our body, maintaining our body balance. However, according to research, in recent years, the proportion of omega-3 and omega-6 in people's body is unbalanced, and the daily intake of omega-3 fatty acids per capita is far below the standard line. Especially for children aged 5-12 years who are in the critical period of physical development, the intake of omega-3 is obviously seriously insufficient. At present, the sources of omega-3 supplementation are mainly fish oil. However, in recent years, serious marine pollution has led to a decline in the quality of fish oil. Relying on fish oil is not a long-term solution. So NNU China members began to pay attention to the production of omega-3 series unsaturated fatty acids. After listening to academician of the Chinese Academy of Sciences Chen Jian's lecture, the team members had great inspiration. We believe that we can rely on synthetic biology to promote people's daily nutrition intake and promote the development of social food health.

· How We Respond to Our HP

According to the information obtained from hp and the brainstorming within the team, and in combination with the characteristic research direction of the laboratory, we roughly define the overall direction of the project as: synthesizing DHA/EPA for nutritional supplements by synthetic biology technology.

Ⅱ. Results of Our Questionnaire

· How Our HP Inform and Shape Our Project

The analysis of the questionnaire results gave us an insight into social awareness and attitudes towards polyunsaturated fatty acids and omega-3 eggs. These analytical data are consistent with our preliminary interviews with supermarkets and experts, proving the relevance and necessity of our study.

· How We Respond to Our HP

The analysis results of these questionnaire data did not directly affect our project design, so we have no way to directly provide these data like the expert interview results in our design. However, we use this valuable social survey data when building partnerships with companies. To make the most of the information in the questionnaire, we use it to communicate with biology companies. Based on the analysis of the information, we can reach a consensus and thus build a partnership.

Ⅲ. Project design

· How Our HP Inform and Shape Our Project

In order to effectively solve the problem of insufficient intake of polyunsaturated fatty acids in the general population and get specific solutions, we have met with university professors and relevant enterprises many times. In every communication, we stick to our direction and fully consider how to improve the positive impact of the project on society and how to benefit more people. Professor Yuan Kai from Biotechnology Center of COFCO Nutrition Research Institute pointed out that the best solution is to increase the content of polyunsaturated fatty acids in daily nutrition. Hu Xuechao, president of Jianqixing Children's Nutrition Research Institute, also said that the existing fish oil capsules generally have a strong fishy smell so children's acceptance is not high. As one of the most extensive sources of nutrition intake in people's daily life, eggs can be agood carrier of ω- 3unsaturated fatty acids. Omega-3 eggs may be a safe, effective and promising polyunsaturated fatty acid carrier. Therefore, we conducted further research on omega-3 eggs, which may be more acceptable to the public. After sorting out and summarizing, we found that there are two main production methods for omega-3 eggs in the market. One is to add flaxseed into the feed of laying hens to generate EPA and DHA through the internal metabolic pathway of the hens. The other is to feed laying hens directly with natural algae sources. However, both methods have the disadvantages of high cost, complex process and strong environmental dependence. Through further communication with the Biotechnology Center of COFCO Institute of Nutrition and Health, we also learned that the production of polyunsaturated fatty acids by biosynthesis is more suitable for commercial production, which has the advantages of reducing time and economic costs and improving yield and purity.

Through field research and the release of relevant questionnaires, we learned that consumers have a weak desire to buy polyunsaturated fatty acid products, and their awareness and acceptance are generally low, which greatly affects people's daily intake and supplement of DHA/EPA. It is particularly urgent to solve this problem. Through market research, we found that the prices of deep-sea fish oil and algae health products are generally high and the sales volume is low. Meanwhile, most of the special nutritional eggs on sale are DHA eggs, and there are few EPA eggs. And the price is 2-3 times that of ordinary eggs, and the DHA content is about 2%.

· How We Respond to Our HP

Synthetic biology allows us to use genes and proteins from different sources, which can further broaden our design vision, promote our projects more comprehensively and innovatively, and solve problems in an all-round way. The classic strategy of synthetic biology is to continuously produce functional substances through engineering bacteria, and finally achieve the long-term goal of high efficiency. The information obtained at this stage is the key to our project design. We listened to the suggestion of President Hu Xuechao, chose eggs as our carrier for DHA/EPA production, conducted in-depth research, and finally determined the specific direction of our project: microbial metabolic engineering to efficiently produce polyunsaturated fatty acids.

After determining the specific direction, we designed the experiment and actively carried out it. Professor Huang He pointed out that Yarrowia lipolytica could be used as the chassis cell for DHA/EPA production. On this basis, we conducted a series of investigations on Yarrowia lipolytica. And we choose the elongase–desaturase pathway of Yarrowia lipolytica as the target pathway. The team members also consulted Dr. Du Yuanhang of bioinformatics to simulate and analyze various fluid problems. Dr. Du Yuanhang personally demonstrated the CFD model, which provided great help for the smooth development of our later experiments.

IV. Project optimization

· How Our HP Inform and Shape Our Project

After determining the basic route of our project, we contacted Jianqixing Children's Nutrition Research Institute to ask about the feasibility of the project development and the market application prospect of omega-3 eggs. In order to optimize and improve the design of the subject content and provide reliable guidance for the experimental design, we also interviewed experts and professors in the field of metabolic engineering for many times. Professor Pang Jinzhu suggested that the optimization of fatty acid content should not be limited to the improvement of enzyme activity in the target metabolic pathway. It is also a good choice to inhibit the bypass pathway of fatty acids and reduce the degradation of target products. Dr. Yin Xuebinsuggests that we can adjust the proportion of DHA and EPA in eggs to meet the needs of various people, because different people have different DHA/EPA absorption capacity.

· How We Respond to Our HP

In this part, we focused on interviewing several experts in metabolic engineering and nutrition based on our experimental design. We decided to strengthen DHA/EPA synthesis pathway through push-pull strategy. According to the suggestion of Professor Pang Jinzhu, we added a design to prevent the degradation of target fatty acids on the basis of the original design. Considering the inhibition of theβ-oxidation pathway as an important part of our experiment. According to the suggestion of Dr. Yin Xuebin, we decided to regulate the copy number of the important target genes(∆-12 desaturase, ∆-9 elongase and C16/18 elongase) in DHA/EPA synthesis pathway to obtain different ratios of DHA/EPA content. It is not limited to improving DHA/EPA production through screening for different sources of elongations/disasters.

Value orientation of the project

In conclusion, we have used a number of methods to ensure that our projects have and will continue to make a good impact on the world and the specific communities we hope to help. From gathering knowledge to understand the needs and priorities of various populations, to actually adapting our projects to those needs and priorities: we put in the work to complete the cycle.

As we move forward and improve our projects, we always put safety first. The vision of our project is to engineer microorganisms to produce DHA/EPA to solve the problem of insufficient intake of polyunsaturated fatty acids in the population. Once these new methods are used on a large scale, any minor safety issue can become a serious public safety issue. Therefore, the need for security concerns is at the top of our agenda. This was reflected in the questionnaires we sent out to the public, which clearly explained the questions and surveyed the public on their acceptance.

To end it in a more cohesive way, we thought it would be interesting to discuss its prospects with actual companies that might be interested in the omega-3 eggs we were targeting. Perhaps, in a few years, regulations regarding omega-3 eggs may be revised and adjusted to allow for more synthetic biological innovation in the field. Perhaps in a few years, the producers and consumers we come into contact with will be eating polyunsaturated fatty acid products made from our oleaginous yeast.

Either way, we know that omega-3 eggs have the potential to make a real difference in the polyunsaturated fatty acid supplement industry, providing value for a supplement that has yet to be found that is more effective.

Close the loop!

When running HP campaigns, we focus heavily on translating others' suggestions into the design of our themes, thus creating a closed loop between others' expectations and our designs. These positive feedbacks are shown in the figure below.