Entrepreneurship

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ONCE_CHINA is a team focused on synthetic biology beauty and food upstream, committed to becoming a leader in synthetic biology application technology. The company's core technology is synthetic biology application product manufacturing technology. As a research-oriented, customer-centric enterprise, the company provides customized raw material solutions for downstream enterprises such as beauty and food. The company pioneered the use of Vibrio natriegens as a chassis organism and derived several pipelines on this basis.

ONCE has 15 iGEM Alumni from top domestic universities as the core of the synthetic biology application research and development technology team, is committed to becoming a leader in synthetic biology application technology, using a unique chassis biology and highly customized product platform to lay out the synthetic biology industry chain.

Unique chassis bio platform:

The pioneering use of Vibrio natriegens as a chassis organism typically yields less than 4 g/l/h compared to typical microbial chassis (Escherichia coli, Corynebacterium glutamate, Saccharomyces cerevisiae, etc.). Vibrio natriegens microorganisms are the fastest growing microorganisms known and have very high glucose uptake rates in the base medium (aerobic: ~3.90 ± 0.08 g/g/h, anaerobic ~7.81 ± 0.08 g/g/h). In addition, it enables rapid utilization of many other industry-related carbon sources, such as different sugars, alcohols, and organic acids.

Highly customized product platform:

ONCE(BEAUTY )& ONCE(AROMA)

ONCE has a rich product pipeline, is market-oriented in terms of product selection, and focuses on beauty and food additives two major consumer business groups, with a highly customized product platform and ultra-fast delivery capabilities, ONCE_CHINA competitive advantage in the trillion-dollar beauty and food additives market is self-evident.

ONCE has a clear capital market plan; The company plans to raise 2 million to 5 million yuan in this round, mainly for the construction of the base platform and sales channel development of Vibrio natriegens; Historical collaborators include Bluepha, the Chinese synthetic biology unicorn.

1.Timeline：

1.Bluepha interview: chloride ion attack on stainless steel fermenters

the early stages of the project conception and design, we wanted the project to meet real-world needs, have industrial potential, and be a scientific research project. Eventually, successfully developed lab-scale processes must be transferred to an industrial scale.

For this reason, we talked with the Senior Director from our strategic partner Bluepha. Mr. Fan Bing has been working in biofermentation for 13 years. He has been a project leader in listed companies such as Guangji, Kingsway, and Andisu and incubated projects including L-lactic acid, polyglutamic acid, industrial enzyme preparation, and protein production from gas fermentation. He has rich experience in synthetic biology industrialization.

In the discussion, Mr. Fan Bing presented to us three points about the adaptability of Vibrio natriegens for industrial production. To satisfy the demand of Vibrio natriegens for sodium ions, most cultivation media are supplemented with at least 15 g NaCl L−1. This salinity is the laboratory's most commonly used culture condition for Vibrio natrium. But it will lead to the traditional fermentation tank is not suitable for the mass fermentation production of Vibrio natriegens since chloride promotes corrosion processes on metal surfaces of a bioreactor.

In addition, during the wastewater treatment stage. Wastewater containing chlorine ions kills bacteria and seriously affects the project's reproducibility. Most importantly, the fermentation effluent's high chloride and COD content will make the production effluent treatment very costly.

2.Yancheng inspection: another way out for sodium chloride

Yancheng City is the only prefecture-level city in China named after "salt." Yancheng has a history of more than 2,000 years of sea salt development and is a city of sea salt that has been developed by sea salt culture in the true sense of the word. Yancheng is an important production base for sea salt, and its vast mudflats provide excellent conditions for "cooking sea salt." The culture conditions of V. natriegens are closely related to sodium chloride, and adding sodium chloride contradicts the large-scale industrial fermentation of V. natriegens. For this reason, we went on a field trip to Yancheng to find a way to coexist between sodium chloride addition and large-scale industrial biofermentation production.

3.A visit to the China Sea Salt Museum: A storage solution for highly concentrated sodium chloride

The history of humanity's interest in the sea can be traced back to an even longer time before the invention of "boiling the sea for salt." In the face of the surging sea and the vast expanse of the mud flats, the ancestors converged on the flat, open mud flats and used the mud flats and grass swings to create a chapter in the history of sea salt extraction. With the socio-economic development, manual salt production has receded from the historical stage, the modern salt production process and procedures have become much more efficient, and salt production has increased greatly. We imagine that if V. natriegens steel tank fermentation faces a high concentration of chloride ions corrosion, the whole process of industrial salt production, storage, and transportation will face more serious corrosion. A systematic understanding of salt's industrial production may help us solve this industry problem once and for all.

We visited the China Sea Salt Museum to see the historical changes in how salt is produced and to ask the museum staff our questions. The museum staff showed us the current FRP brine storage tanks used in industrial production. He highly recommended that we consider using Fiber reinforced plastics (FRP) for the fermentation production of V. natriegens. FRP is widely used in brine storage tanks and brine pipeline transportation in the sea salt industry. Fiber-reinforced plastics can withstand up to 30% salinity. It has excellent oxidation, acid, and alkali resistance and is resistant to aging. It is also cost-effective, environmentally friendly, and potential for industrial mass production applications.

4.The wisdom of the countryside: the inspiration of the biogas digester

Young scientists often work in academic institutions and laboratories, as does ONCE. Since ancient times, China has been known as a largely agricultural country, with 94.7% of the country's land area in rural areas, but ONCE members live and work in large cities, so while we design and complete projects based on real-world needs, we also hope to be inspired by the countryside, where we are rarely found.

During a field trip to Dongzeng Village in Binhai County, Yancheng City, we were surprised to find that Fiber reinforced plastics are already widely used for microbial fermentation in the Chinese countryside. Fiber-reinforced plastics are already being used on a large scale in constructing methane-generating pits. We were fortunate enough to witness the installation of an FRP digester in the village of Dongzeng. Through the interview with the installation engineer, we learned that Fiber reinforced plastics are widely used in the fermentation culture of microorganisms of biogas fermentation, and the whole process of biogas generation is as follows. 1. Hydrolysis stage: The fermentative bacteria use the extracellular enzymes secreted to decompose the organic matter in vitro enzymatically. 2. Acid production stage: The fermentative bacteria decompose the small molecule compounds produced in the hydrolysis stage into acetic acid, propionic acid, and butyric acid. Then hydrogen-producing acetic acid-producing bacteria convert the propionic acid and butyric acid produced by fermentative bacteria into acetic acid, hydrogen, and carbon dioxide.3, methanogenic stage: methanogenic bacteria convert hydrogen and carbon dioxide into methane, and the latter decarboxylates acetic acid to produce methane.

At the same time, we witnessed the installation of FRP microorganisms for biogas fermentation. We saw for ourselves the good overall performance of FRP fermenters, the ease of transportation and installation, the adaptability of installation, the ease of management, and the quickness of operation and construction. We were convinced to use FRP as the material for V. natriegens. industrial fermenter material. Of course, during the follow-up communication with the industry, we also learned that FRP fermenters might have defects such as the inability to install high-powered stirring devices, weaker processing performance than stainless steel, easy-to-shed impurities, etc. In the follow-up small trial and pilot scale stage, we decided to further study and implement the use of Fiber-reinforced plastics for V. natriegens according to its biological characteristics. The idea of using Fiber reinforced plastics material for V. natriegens production fermentation was further investigated in the subsequent small-scale and pilot scale-up stage.

5.Non-sterilized fermentation of V. natriegens with seawater

Carbon neutrality is one of humanity's most important common issues. ONCE is also thinking about applying its power to help the cause of carbon neutrality for all humankind.

During the Nature Matters conference, we had an in-depth discussion with the government of Yancheng City about the development of synthetic biology in the era of carbon neutrality. The Yancheng city government officials discussed two environmental dilemmas they are facing.

They revealed to us a counterintuitive conclusion: In the current biosynthetic manufacturing scenario, due to factors such as production efficiency and maturity of the manufacturing process, biosynthetic products do not perform as well as their chemical counterparts in terms of carbon neutrality throughout their life cycle, leaving a larger carbon footprint in the entire value chain. The traditional petrol-based industry has contributed most of the chemicals in our daily lives. Microbial fermentation producing chemicals from a renewable resource is environmentally friendly and sustainable to replace the petrol-based industry. However, typical microbial fermentation often faces the problem of easy contamination and thus requires complicated sterilization and high energy input.

6.Bluepha Factory Tour - Interview with Bluepha's Chief Engineer

Bluepha has developed the industrial production technology of biodegradable material PHA - Bluepha™️, which solved the problems of high production cost, low yield, and unstable performance of PHA for many years. Bluepha has systematically reduced the production cost of PHA.

In January 2022, Bluepha started the construction of a 25,000 tons per year Bluepha™ mega plant in Binhai, Yancheng City, Jiangsu Province, and has already completed the main structure's roofing. The production cost of Lansu™️ will continue to be reduced to the same level as PLA at the 10,000-ton scale. It is estimated that each ton of Bluepha™ product can bring about 2 tons of bio-carbon sequestration, helping Bluepha build a synthetic biology "zero carbon industry chain" model.

As a young force in synthetic biology in China, we were honored to be invited to visit Bluepha's super factory to experience the industrialization process of China's leading synthetic biology company. During the field trip, we interviewed Mr. Kang Zhixin, Vice President of Engineering of Bluepha, and Mr. Wang Mingjiang, General Manager of the plant. The two experienced engineers greatly appreciated our project and reminded us of two aspects. They suggested we replace the one-time feeding fermentation with batch replenishment fermentation in the laboratory to obtain the highest alpha-Bisabolol content. In addition, they reminded us to pay special attention to the physicochemical parameters, such as the water hardness of the medium, because, in their experience, certain cosmetic ingredients can undergo emulsification in inappropriate water, which can affect the extraction and processing of the ingredients.

Thanks to their caution, we achieved a record yield of 116 mg/L of alpha-Bisabolol using V. natriegens, which is about ten times higher than the highest yield of alpha-Bisabolol biosynthesis reported in the previous history.

7.STEE HILL strategic cooperation

Founded in May 2019, STEE HILL is a skincare brand based on high-end synthetic biotechnology with humanized collagen as its core ingredient and is the only up-and-coming company currently positioning synthetic biology technology in skin care in China. In 2021, STEE HILL opened its first Tmall flagship store, debuting its full line of recombinant collagen skincare products. In just five months, STEE HILL has become one of the fastest growing brands in this segment, with the main product "C3 Baby Collagen" anti-aging series, which currently has six categories of serum, water, milk, cream, and cleanser, with a total of 7 SKU's. Its founder, Laura lee, was a beauty company's CEO with rich experience in research, production, marketing, and channel management. The founder, Laura lee, was the CEO of a beauty company and had extensive management experience in research, production, marketing, and channels. As an emerging force in the synthetic biology beauty industry, we had an in-depth conversation with laura lee. We and STEE HILL were fascinated by each other's stories and decided to enter into a strategic partnership to jointly explore Clean Beauty driven by synthetic biology techniques in the bio-economy era.

In 2017, after laura lee saw the entire list of upstream factory raw material suppliers in the cosmetic industry, she found that 80% of raw materials for domestic brands came from imports, and only 20% of basic raw materials came from local Chinese sources. She believes there is no Chinese way to make big high-end cosmetic brands because big international brands and suppliers master international raw materials. Because of the real supply chain of local cosmetic ingredients in China, big international brands have a better chance than local Chinese brands to get these new ingredients first and thus gain a competitive advantage in the market.

In China, only 20% of the active ingredients in cosmetic ingredients can be supplied by local Chinese suppliers, and foreign suppliers monopolize important functional and basic ingredients. However, products need to rely more on active and functional ingredients for good efficacy performance. This dilemma was sharply exacerbated in 2019 after the outbreak of COVID-19 when local Chinese cosmetic companies were generally faced with a cut-off in raw material supply due to the embargo, and international political tensions brought about by COVID-19. The synthetic project will bring new life to China's cosmetics industry in the face of a global pandemic of COVID-19 and a dangerous geopolitical confrontation.

In our strategic partnership with STEE HILL, we have jointly recognized the importance of synthetic biology technology to the Chinese beauty industry. First, synthetic biology technology is the only possibility for Chinese beauty functional ingredients and packaging materials to overtake the competition; second, synthetic biology technology is the only technology that can truly realize the four principles of Clean Beauty; third, synthetic biology is the only discipline that can perfectly combine Clean Beauty and efficacy; fourth, synthetic biology is the only choice to realize the sustainable development of beauty industry. The efficacy of alpha-Bisabolol is proven in the market, and we will supply the alpha-Bisabolol we produce to STEE HILL as a priority and add it to STEE HILL cosmetic products as a compound ingredient after the efficacy is proven.

8. Distributors Interview: High Quality, Mass production, and On-Time Delivery rate are the primary factors when they cooperate.

Distributors tell us that traditional bisabolol production requires a lot of tree felling, and the chemical synthesis method has a large purity problem. Therefore, if it is necessary to sell in the country, quality, scale-up production and timely delivery will become the main issues.

9.Harvard Entrepreneurship Competition

At the Harvard Entrepreneurship Competition，“H-InnoPitch” Start-up Pitch Competition, global venture capital institutions highly recognize the development model of synthetic biology and believe that there should be more commercialization elements for traditional selection.

Final List

Fast-moving consumer goods are products that sell quickly at relatively low cost. These goods are also called consumer packaged goods.

FMCGs have a short shelf life because of high consumer demand (e.g., soft drinks and confections) or because they are perishable (e.g., meat, dairy products, and baked goods). These goods are purchased frequently, are consumed rapidly, are priced low, and are sold in large quantities. They also have a high turnover when they're on the shelf at the store.

For Once, we need to bring out our own MCGs productss and the SKU design could be bold.

10. MiraclePlus (Fall 2022)- Dr. LU QI helped us to figure the brand positioning. Dr. LU QI Speaking

Dr.LUQI helps us to reconsider the first product ONCEBeauty Skincare for Men.

With the improvement of China's male skin care awareness, male skin care product consumption demand continues to increase, while men pay more attention to product quality, men's high-end skin care consumption will increase rapidly, men's skin care market size will maintain rapid growth, it is expected that the market size of China's male skin care industry in 2022 is 11.3 billion yuan, with the end of the epidemic, the market demand for male skin care products will pick up, it is expected that by 2027, the market size of China's male skin care industry is expected to reach 24 billion yuan, The average compound growth rate from 2022 to 2027 is 16.26%.

According to the data of the brand network, the TOP5 men's skin care brands in China have long been covered by foreign brands. L'Oréal Group also has L'Oréal Men's, Beyond-Spring Men's, Keyan's Men and other brands. Manszoraydon in the United States is also continuing to develop, and these big names are constantly conducting market education and brand promotion. The domestic brand is only Gaofu under Shanghai Jahwa United, ranking fifth.

Among all male cosmetics consumers, young consumers aged 18-25 account for 59.50%, while the number of male consumers aged 26-30 accounts for 21.30%.

11. Brainstorming on Product Design with Alila employees during tracking - ONCE LABORATORY.

Products

Spa Alila believes in all things natural, whether it be the spa ingredients that we source locally, or international brands that complement our treatment range. Spa Alila products are made from creative blends of ingredients, such as indigenous plants, fruits, herbs and spices, that are fragrant and so fresh they are almost edible - cucumber and aloe vera to soothe, lemongrass and ginger to refresh, coffee and coconut to tone and condition.

Based on this, we decided to promote our first SKU as ocean base. considering that we can also use seawater to produce our main ingredients. Combinaing with our synsetic biology concept, we named it ONCE LABORATORY.

12. Promoting on social media and Diving activites

We started by contacting some of our diving buddies in Bali to ask them about their views on the ocean, skincare and recycling. And with them, experience underwater diving near Penida Island to cultivate recyclable and technological skincare concepts.

13. A ONCE company was founded- A LEADING SYNTHETIC BIOLOGY APPLICATIONS PLATFORM

Based on all the trails before, we decided to start a company. ONCE_CHINA is a company focused on synthetic biology beauty and food upstream, committed to becoming a leader in synthetic biology application technology. The company's core technology is synthetic biology application product manufacturing technology. As a research-oriented, customer-centric enterprise, the company provides customized raw material solutions for downstream enterprises such as beauty and food. The company pioneered the use of Vibrio sodiums as a chassis organism and derived several pipelines on this basis.

We had communicated with Sequoia China、Heli and many other Ventures shows Great interest in ONCE, and we had communication with local government (ZHANGJIAGANG, JIANGSU, CHINA)

14. Digital transformation on supply chain（Interview with Bill Yang from Pactera）

Bill is a senior consultant at Pactera, who foucused on supply chain Digtalization. With the help of Bill, we redesigned the supply chain with all stakeholders in mind.

In-depth understanding of supply chain needs, innovative BI design, to achieve high OTD, high inventory turnover.

15. Industry research on Sugar substitute（Interview with Layn）

With the intensification of obesity, dental caries, sugar taxes and other issues, the global demand for sugar substitutes in the beverage and food sector is increasing. The market for a higher sweet price ratio, safer natural flavor sugar substitute demand has risen, single product high sweetness natural sugar substitute market (steviol glycosides, luo han guo glycosides) scale exceeded one billion US dollars, CAGR maintained a rapid growth trend of more than 10%.

However, the single sugar substitute itself is not good in flavor, the market prefers compound sugar substitute, how to adjust the compound ratio and the customized service needs of downstream enterprises have become the focus of attention.

The customized service that requires a high technical threshold is precisely the advantage that distinguishes it from planting and extracting enterprises. We believe that a custom compounded natural sugar substitute large-scale production platform based on synthetic biology will lead the future.

In the selection of major categories, the unit price of more than 3000 yuan / kg of luo han guo glycosides has great consumption potential, and in the 2022 China top ten flavors released by Kerry China, we believe that the luo han guo glycosides with the flavor of "finishing herbs" will have great achievements. In the compound sugar substitute, coconut flower sugar, lucuma fructose, kiwifruit sweet protein, agave sugar, steviol glycosides are all good choices.

2 Executive Summary

3. Industry Analysis

4. Market Analysis

5. Marketing Plan

6. Product Design and Development Plan