News and literature review

In February, we followed a krill fishery story online which made it clear that the global Antarctic krill fishery is set to expand again in 2020, with the industry expanding rapidly, driven by major countries such as China, Norway and Chile, with Chinese krill catches reaching a ‘more than expected’ 118,000 tons, more than double the 50,000 tones caught in 2019, compared with the 50,000 tons caught in China's krill catch reached 118,000 tones ‘beyond expectations’, more than doubling from 50,000 tons in 2019. Antarctic krill is also valuable as a marine ecologically important species for use in food and as a substitute for fishmeal. However, climate change and the transition of fishing fleets to krill populations pose a threat, affecting the Antarctic ecosystem and species that rely on krill for food, such as whales and penguins. Efforts are needed to balance commercial interests with ecological protection [1].

Inspired by this, we read extensively in the literature to explore the specific impacts of krill fishing on marine ecology, and what we found was this: Commission for the Conservation of Antarctic Marine Living Resources(CCAMLR) divides the Antarctic ocean into three large areas and sets a cap on krill fishing based on 2010 estimates of krill stock size (totaling about 60.3 million tons) in one of these areas - Area 48 on the Atlantic side - and the size of the krill population in the Antarctic. -the size of the krill stock (totaling approximately 60.3 million tons) was estimated in 2010 to set a catch limit. On this basis, the Commission set an absolute catch limit - known as the ‘precautionary catch limit’ - of 5.61 million tons per year [2]. Although the actual annual catch falls far short of this cap, Philip Trathan, head of conservation biology at the British Antarctic Survey in Cambridge, UK, points out that fisheries management strategies that allow what appears to be a relatively small amount of fishing over a large area of sea may still ultimately prove unsustainable. This limit is set on the assumption that vessels will catch krill evenly throughout the area. However, this may not be the case in the real world. They will certainly choose the areas where they are most certain to make a profit. And those areas might happen to be where penguins, seals and whales go. In other words, the amount of catch that the krill fishery is allowed to take, and that is within prudent limits, may just happen to be taken from the mouths of the very marine life that feeds on krill. This is important because changes in the size of krill populations from year to year - including fluctuations due to the impact of fishing - are thought to affect predators that feed on krill, particularly penguins [3]. A 2020 study of penguin populations on South Shetland Island, about 120 kilometers north of the Antarctic Peninsula, found a correlation between the level of krill fishing in the waters around the island and declining penguin health [4].

We also looked at the effects of climate change on Antarctic krill populations. Our review of the literature showed that climate change is having a significant impact on Antarctic krill populations. Sea ice, which is vital for krill larvae as it provides shelter and food in the form of ice algae, is decreasing due to rising temperatures. In addition, rising ocean temperatures have increased krill metabolism, resulting in smaller adult krill and potential challenges to reproduction. These pressures are compounded by changes in phytoplankton, the main food source for krill. Together, these factors are threatening krill populations , which are critical to the Antarctic food web, affecting predators such as penguins and whales [5] [6] [7].

We hope to find a solution that meets the growing public demand for krill in the context of the impact of climate change on Antarctic krill stocks, while at the same time reducing the number of krill harvested by countries and protecting the marine ecosystem.

Liao Yu Group

In April, we contacted several Chinese companies specializing in Antarctic krill research and development to gain a better understanding of the whole process of research and development, production, and sales in the Antarctic krill industry. Finally, we enlisted the help of China's Liao Yu Group for a site visit. We learned from the Liao Yu Group that krill is mainly used to produce krill oil, a type of oil extracted from Antarctic krill (Euphausia superba), which is rich in omega-3 fatty acids, phospholipids, and antioxidants (such as astaxanthin).

The production of krill oil involves four processes: harvesting, separation, extraction, and encapsulation, which begins with the harvesting of Antarctic krill in Antarctic waters and requires rapid processing to prevent degradation of the krill's nutrients. After capture, krill is usually steamed to separate the oils and proteins, and then the oils are extracted using mechanical pressing, solvent extraction, or supercritical CO2 extraction. The extracted krill oil is then refined to remove impurities and prevent oxidation.

To protect the astaxanthin and omega-3 fatty acids in the oil, krill oil is usually concentrated. Strict quality control during production ensures the purity and safety of the krill oil, which is then encapsulated in a soft gel capsule to prevent oxidation and facilitate consumption by consumers. The entire production process must be efficient and fast to maintain the nutritional value of the krill oil .

Liao Yu Group staff pointed out that due to the lack of long-term in-depth research on krill, China is facing a lot of bottlenecks in technology, backward processing technology, so the high value-added krill products cannot be reflected. China still lacks independent intellectual property rights of krill meal processing equipment, the transformation of their own krill ship with a slightly modified fish meal production line, low production efficiency, shrimp meal from the rate of only 60% of the international advanced level, and backward processing technology, shrimp meal quality is low, and some even fail to meet the quality requirements for the extraction of shrimp oil, affecting the sale of products, which in turn affects the economic benefits of the fishing industry. In addition to technical problems, the production of krill oil is also facing the problem of sustainability, each bottle of krill oil production to consume about 1,000 krill, fishing krill fishing boats need to set out to the Antarctic, fishing boats need to stay in the Antarctic 1-2 years, which means that the staff also need to stay in the Antarctic for 1-2 years, time-consuming and labor-intensive, and Liao Fishery Group staff stressed that they are now in short supply of krill production.

Based on this information, we had an intra-team discussion to find a solution to the unsustainable and inefficient production of krill oil by bio producing it.

Mr. Xiaohan zhang

At the end of April, we consulted with Mr. Xiaohan zhang, the manager of Suzhou Yuekang Technology, to find out about other bio-production krill oil solutions on the market and to incorporate various suggestions for our bio-production. He confirmed our idea and told us that it was groundbreaking, that there was no precedent for bio-production of krill oil on the market and that there were no solutions for chemically synthesized krill oil, which is extracted naturally from krill oil

.

Ms.Gabby

In May we contacted Gabby, a nutritionist in the USA, to get her advice on bio-produced krill oil. She was very supportive of our use of bio-produced krill oil and said that she would be happy for her therapeutic diet patients to take krill oil, but that people would not be able to afford it in the long term due to the high price of krill oil. Inspired by this, we are planning market research to determine the price range of krill oil and a questionnaire to determine public acceptance of krill oil.

Dr.Yang

In addition to consulting a dietitian, we went to the Fourth Affiliated Hospital of Suzhou University to consult a cardiovascular expert in the hope of incorporating the expert's opinion on krill oil, Dr Yang said that he believed that krill oil, fish oil and other similar nutritional supplements nutritional supplements cannot be used as a means of treatment, but can only be used as a means of protection, and need to be eaten over a long period of time and in a regular diet in order to be able to play a role in the young people's groups. Dr Yang believes that supplements are ineffective when we do not eat regularly, and he believes that supplements may not be easily absorbed by the elderly. Combining Dr Yang's as well as Ms. Gabby's opinions, we initially constructed our idea. Through extensive review of the literature, we found that the Omega-3 fatty acids in krill oil existed in the form of phospholipids rather than triglycerides, and that this unique molecular structure allowed for a higher utilization rate, more effective absorption by the body, production of components with higher absorption rates, and a reduced metabolic burden on the elderly [8].

Thus, we finally decided to bio produce PC-DHA in large quantities and reduce the price of PC-DHA.

market research

To ensure that PC-DHA, the key ingredient in our bioengineered krill oil, can effectively meet market demand, we are laying the groundwork for future commercialization. First, we conducted market research to find out the current size of the krill oil market and the growth rate of the krill oil market. Then we learned about the price range of krill oil products by visiting retail stores and pharmacies, analyzed the average unit price of krill oil, and better understood the price sensitivity of consumers, which helped us understand the specific views of current consumers on krill oil through questionnaires and street interviews.

According to our research, we come to the following conclusions

• Krill Oil Market Size (2024): $183.2 million
• Estimated Market Value (2033): $357.7 million
• Global Market Growth Rate (CAGR 2024-2033): 7.7%

Reasons for growth

• The COVID-19 pandemic has created opportunities for manufacturers of behavioral health products, particularly omega-3 supplements. As a result, consumers have recently become more inclined to take daily supplements to prevent severe respiratory syndromes such as coronavirus. This has led to a significant increase in the demand for omega-3 supplements, thereby driving the growth of the omega-3 products market [9].
• The rising number of deaths from cardiovascular disease worldwide is driving the growth of the omega-3 market. Rates of cardiovascular disease have increased in western sub-Saharan Africa and South Asia. Omega-3 fatty acids, found in supplements and certain whole foods such as fish, nuts and seeds, have long been known to have health benefits, particularly for heart health. However, the rise in chronic diseases such as heart disease, arthritis, stroke and cancer are expected to accelerate global demand for omega-3 products. According to the World Health Organization (WHO), chronic diseases kill more than 14 million people between the ages of 30 and 70 every year [10].
• The increasing use of omega-3 fatty acids in pet food and pharmaceuticals has provided a significant boost to the market. Increasing consumer awareness of the health benefits of omega-3 fatty acids is further driving the demand for innovation among market players [11].
• Consumers are becoming increasingly aware of the health benefits of omega-3 fatty acids in krill oil, such as promoting cardiovascular health, reducing inflammation, and improving cognitive function. The growing demand for supplements from natural and sustainable sources, coupled with the increasing prevalence of chronic diseases and lifestyle-related health issues, is further driving the market expansion. Technological advancements in krill oil extraction and production, such as improved cold extraction technology and packaging methods, provide superior product quality, stability, and bioavailability, driving market growth. In addition, the increasing use of krill oil in functional foods and food and beverages, as well as the growing popularity of personalized nutrition solutions, are creating new opportunities for market players to reach a wider consumer base [12].

Based on our presentation of the summarized price range of krill oil products, random street interviews were conducted with about 15 people from different ages, genders, and educational backgrounds. They all provided valuable insights and suggestions for improvement. During our interviews, we displayed the krill oil product price range table that we made to provide some background information, but most of the people said that they had not heard of krill oil, instead they were more aware of fish oil, and slightly younger interviewees said that they usually supplemented their pets with fish oil in their lives and did not ingest it as a health supplement, and that they thought that the price of the krill oil that we displayed was too high and they would not choose to buy it, while slightly older interviewees said they would consider the source of the DHA as a primary consideration, with price as a secondary factor, and that they had concerns about the safety of our biologically produced DHA. We were lucky enough to talk to a pregnant woman during our street interviews. We explained to her the critical role of DHA in the development of infants, and she affirmed the importance of DHA products for mothers-to-be and newborns, and at the same time, she emphasized that she would carefully screen for the source of DHA when choosing a product, rather than considering price as a factor.

• 45.68% + 33.33% of the population consume fish oil and krill oil supplements, and 51.56% + 39.84% of the population believe that fish oil and krill oil are useful for health.

This affirms the significance of our project.

• 63.28% of people will consider price as the main factor when making a purchase

This reveals that we need to address the issue of cost through our bioproduction.

• 60.16% of people consider the source of the product first when purchasing

This suggests that we need to increase public acceptance of biomanufactured products.

Based on the above information, we decided to choose Schizochytrium as the chassis for our production, because Schizochytrium limacinum is currently internationally recognized as the best DHA production strain.

Optimization and outlook

After our conversations with female entrepreneurs, Professor Jie Zhang, and Dr Ziwen Xie from the Faculty of Science, they made suggestions to further improve the design of EcoMeGa beyond the scope of this year's iGEM project, and their suggestions to us could be that EcoMeGa is safer and more effective. The three suggestions we investigated included:

• We need to set up a program to transfer the pathway for PC-DHA production in Saccharomyces cerevisiae to Schizochytrium limacinum in the future.
• We need to test the safety of our product PC-DHA.
• We need to test the functionality of PC-DHA against the blood-brain barrier.

According to these three suggestions, we design the future research plan. After validating the enzyme activity, we're going to perform gene editing on schizochytrium limacinum. Firstly, we will knock out the original gene encoding the LACS enzyme in schizochytrium limacinum, and then integrate our modified enzyme into the schizochytrium limacinum genome using homologous recombination. We have proven production increase in lab environment and will conduct further measurement on production to verify whether there will be a consistent increment in the actual amount of product produced per unit time. Secondly, with our established model and simulated gene modification, we will further perform knockout to the gene of acetyl coenzyme A carboxylase (ACC) to reduce the influence of this pathway on the PC-DHA pathway produced by schizochytrium limacinum, and test if the actual production of PC-DHA increases accordingly to our prediction.

After we have extracted the target protein PC-DHA, we need to conduct experiments for safety testing of PC-DHA, and we plan to conduct in vitro safety testing of PC-DHA as well as in vivo safety testing to initially assess the potential risks of PC-DHA to human health and animals.Through multi-level safety tests combined with in vitro and in vivo experiments, the safety of PC-DHA can be comprehensively evaluated to ensure its application safety in the prevention and treatment of cardiovascular and cerebrovascular diseases. If the test results show that PC-DHA has good safety, clinical trials can be considered in the future to further evaluate its effectiveness and safety in human intake.

To determine the efficacy of PC-DHA on the blood-brain barrier, male Sprague-Dawley rats (8 weeks old, 200-250 g) available from Harlan Laboratories (Indianapolis, IN) were selected. Animals were housed (2 per cage) in temperature-controlled rooms (22±2°C) with a 12-hour light/dark cycle and fed ad lib standard laboratory chow (Teklad #7012, 5.8% fat, contains no DHA, but contains 0.3% 18:3, n-3). After one week of acclimatisation, they were randomly divided into 5 groups (10 animals each) and fed the different DHA compounds dispersed in corn oil by gavage daily for 30 days, as described below. The studies were carried out in two batches of 25 animals each (5 rats/treatment). The amount of DHA administered was 10 mg (30.4 μmol)/day in the form of TAG-DHA, EE-DHA and PC-DHA. The supplements were administered to the rats daily by gavage in corn oil for 30 days while they were fed ad lib normal rodent chow.

To prepare samples for gavage, the DHA compounds in chloroform (equivalent to 684 μmol DHA) were transferred to a glass vial and the solvent evaporated under nitrogen. The lipids were dissolved in 200 μl of ethanol and added dropwise to 5.6 ml of corn oil with stirring for 15 min at room temperature. The ethanol was then evaporated under N2 and the sample stored at 4 °C under nitrogen. Samples were warmed to room temperature and thoroughly mixed before being administered to rats by gavage (250 μl/day). Fresh dispersions were prepared every 4 days.

We used the Morris Water Maze to assess spatial learning and memory in rodents, specifically to assess hippocampal function. The MWM test consisted of two phases. (a) Acquisition phase. Rats were trained over 4 days (120 s trial duration, 4 trials per day with a 60 s inter-trial interval) to locate the position of the hidden platform (remaining on the hidden platform for >2 s). Once located, the rat was allowed to remain on the platform for 15 s before being removed from the pool. If the rat did not locate the platform within 120 s, it was gently guided to the platform and allowed to remain for 15 s. For each day of the acquisition phase, the order of quadrant entry varied, but the location of the platform remained constant. Latency to locate the platform (sec) was measured. (b) Probe trial. 24 h after the last acquisition trial, a single 60 s probe trial was performed with the platform removed. Latency to the target area (i.e., where the platform was located during the acquisition phase) and time spent in the target quadrant were calculated.

Rats were fasted overnight, anaesthetised with 2% isoflurane and blood collected by cardiac puncture into heparinised tubes. The animals were then perfused transcardially with ice-cold phosphate buffered saline, and the various tissues were collected and stored at -80°C until analysis. Total lipids were extracted from the tissues using the modified Bligh and Dyer procedure, after the addition of the internal standards di-17:0 PC and tri-15:0 TAG . Total fatty acids were analysed by GC/MS after conversion to methyl esters using methanolic HCl. The analysis was performed on a Shimadzu QP2010SE GC/MS equipped with a Supelco Omegawax column (30 m × 0.25 mm × 0.25μ). Temperature programming was as follows 165 °C for 1 min, increased to 210 °C at a rate of 3.5 °C/min and maintained at 240 °C for 10 min. The total ion current, in the range of 50-400 m/z, was used to quantify the methyl esters and the values are expressed as a percentage of the total fatty acids. The isomer composition of LPC-DHA in plasma was measured by LC/MS/MS using an ABSciex QTRAP 6500, as described by Sugasini and Subbaiah[13].

Industrialization

In order to achieve the industrial production of PC-DHA, we need to achieve the large-scale cultivation of Schizosaccharomyces pombe, firstly, we need to purify Schizosaccharomyces pombe, secondly, we need to understand the cultivation method of Schizosaccharomyces pombe, to design a suitable photobioreactor, and to explore the various factors affecting the growth of Schizosaccharomyces pombe.

Due to the complexity of large-scale cultivation of microalgae production, contact with a large number of people, so in the process of cultivation of infected bacteria in the chances of a substantial increase in the corresponding, impure algae will often lead to a number of negative impacts, such as to the dilution of the algae, the purpose of the product content is reduced to seriously affect the quality and quantity of the product as well as impure algal impurities affecting the product of the processing of the production process is more complex and cumbersome.

Therefore, the purification of Schizosaccharomyces pombe is an indispensable step for the large-scale cultivation of Schizosaccharomyces pombe. We chose to use dilution culture and plate scribing method, after a single colony grows on the plate scribing can be separated to get a single pure Schizosaccharomyces cerevisiae species. After obtaining a single alga using traditional plate streaking and coating methods, we used RT-qPCR, electrophoresis and Western blotting to differentiate the transgenic alga from the wild-type alga[14].

We would like to explore the cultivation of Split Potato Algae for high DHA production. Through extensive reading of the literature, we explored that for DHA production by fermentation of Split Potato Algae, glucose, glycerol, peptone, yeast powder, and monosodium glutamate are the more suitable media, while sucrose, (NH4)2SO4, NH4NO3, NaNO3, urea, and beef paste are relatively ineffective. For the batch fermentation, the optimal fermentation time was 4 d, and the optimal fermentation temperature was 23 °C; the optimal medium carbon and nitrogen source compositions were (g/L): glucose 65, glycerol 80, peptone 6, yeast powder 4, and monosodium glutamate 8; and the achievable cytosolic lipids DHA content according to this optimised medium formulation was 33.68%. After confirming the fermentation conditions, we planned to design a suitable bioreactor to mesophilically scale up the culture of Split Pot Algae[15].

Intellectual property

The previous section described the optimisation of the design, EcoMeGa for future research. As soon as we have initial results showing that PC-DHA can cross the blood-brain barrier, we will apply for a patent to prove the full concept. Discussions with stakeholders such as Dr Zhang Jie have made us aware of the importance of protecting the underlying technology. She explained that real-world implementation of the production method requires protection because patents make it illegal for others to develop, use, sell, rent or supply patented cell therapies for at least two decades. In addition, legal protection of the technology is essential to cover the high costs of development. In addition, Dr Zhang emphasised that patenting a system or technology requires it to be novel, non-obvious and industrially applicable. Because EcoMeGa is new, unpublished and innovative, we optimise the enzyme activity, significantly increase the production of PC-DHA, and finally, our EcoMeGa is practical, reduces production costs, protects the environment, and therefore we can apply for patent protection.

In order to allow our bioproduced PC-DHA products to enter the market legally, we have consulted Mr. Zhang Xiaohan, who has rich working experience in the biofood industry. He suggested that we refer to the "Requirements for Application Materials for Safety Evaluation of Genetically Modified Microorganisms for Food Processing (Trial)" issued by China Food Safety Assessment Center on September 23, 2024. According to his suggestions, After carefully reading the laws and regulations and consulting the relevant lawyers, we finally came to the conclusion that if we want PC-DHA to enter the market as a new food raw material, we first need to approve the new food raw material according to the Food Safety Law. We need to submit an application to the National Health Commission of China, and provide the relevant ingredients, safety evaluation, nutrition research and other relevant materials of the product. Because we use modified microorganisms to improve the production efficiency of PC-DHA, we need to conduct safety evaluation of genetic microorganisms in accordance with relevant laws and regulations such as the Food Safety Law and the Regulations on the Safety Administration of Agricultural Genetically Modified Organisms. If we want PC-DHA to be used as a food additive (rather than as a food raw material), we also need to apply for the divine approval of new food additives according to the "Management Measures for New Food Additives", after obtaining the permission of new food raw materials or food additives, we also need to comply with the "Food labeling Management Measures", and follow food safety regulations in the labeling and advertising of products. Make sure the information is true, comprehensive and not misleading.

Market authorization

In order to allow our bioproduced PC-DHA products to enter the market legally, we have consulted Mr. Zhang Xiaohan, who has rich working experience in the biofood industry. He suggested that we refer to the "Requirements for Application Materials for Safety Evaluation of Genetically Modified Microorganisms for Food Processing (Trial)" issued by China Food Safety Assessment Center on September 23, 2024. According to his suggestions, After carefully reading the laws and regulations and consulting the relevant lawyers, we finally came to the conclusion that if we want PC-DHA to enter the market as a new food raw material, we first need to approve the new food raw material according to the Food Safety Law. We need to submit an application to the National Health Commission of China, and provide the relevant ingredients, safety evaluation, nutrition research and other relevant materials of the product. Because we use modified microorganisms to improve the production efficiency of PC-DHA, we need to conduct safety evaluation of genetic microorganisms in accordance with relevant laws and regulations such as the Food Safety Law and the Regulations on the Safety Administration of Agricultural Genetically Modified Organisms. If we want PC-DHA to be used as a food additive (rather than as a food raw material), we also need to apply for the divine approval of new food additives according to the "Management Measures for New Food Additives", after obtaining the permission of new food raw materials or food additives, we also need to comply with the "Food labeling Management Measures", and follow food safety regulations in the labeling and advertising of products. Make sure the information is true, comprehensive and not misleading.

To the market

The next step after successful market authorization is the market entrance. For a successful market entrance strategy, you need a solid and viable business plan. For building a viable business plan to successfully enter the market, we co-created our business plan together with many professors. In the pdf below, you can read our detailed business plan.

For better enter the market, we have carried out SWOT analysis and competitive analysis on our future products

End Users

• Infants: DHA is crucial for brain development, especially during early childhood.
• The Elderly: It supports cognitive function and can slow age-related brain decline.
• Pregnant Women: DHA is essential for fetal brain and eye development.
• Pharmaceutical Researchers: DHA's role in cognitive health and disease prevention makes it a focus of research.
• Brain Workers: Enhances cognitive performance and memory.
• Athletes: May reduce inflammation, promote recovery, and support brain function under physical stress.

Safety

It is of great significance for JLU-NBBMS to hold the Synthetic Biology Biosafety Conference with six teams, focusing on how to ensure biosafety while promoting the rapid development of synthetic biology. The conference aims to discuss how to manage potential biosecurity threats, especially those posed by technologies such as gene editing and synthetic biology. At the end of the meeting, six teams wrote a white paper on synthetic biology.