Human Practice

2023_bit-china

Human Practice

The HP work aims to validate the significance and feasibility of the project.In the context of the HP work, "Wuli" corresponds to the feasibility of gene circuits and light-controlling elements in the laboratory. "Shili" corresponds to the advantages and significance of implementing the project on a large-scale production. "Renli" corresponds to downstream perfume companies and perfume consumers.

Wuli

During the brainstorming phase, we conducted a literature review, including relevant literature on the cell synthesis pathway of terpenoids and light-regulated gene elements. From this, we determined that we could synthesize sclareol and santalol through the metabolic pathway of yeast and designed a gene circuit.

During the process, we came across literature on red light regulation using photosensitive pigments by Dr. Haiyang Jia, assistant researcher of University of Bristol, and consulted him. Dr Jia believed that red light regulation could be combined with terpenoids synthesis, but the use of photosensitive pigments to guide protein assembly in this literature required many gene elements to achieve signal transduction, which could make the gene circuit too large. Therefore, he advised against using it.

Shili

In this part, we tried to contact fragrance companies and got in touch with Mr. Fengqing Wang, a technical consultant from BioCatSyn (Shanghai) Biotechnology Co., Ltd. We believe that "the development of the fragrance industry is technology-driven, while the development of the perfume industry is driven by cultural concepts," which was also affirmed by Mr. Wang Fengqing. Therefore, our light-controlled biologically synthesized technology and products should be targeted towards the fragrance industry.

Mr. Wang Fengqing believes that the fragrance industry faces three main challenges: unit yield, purification difficulty, environmental issues, and patent protection.

1) The inability to increase unit yield is the main reason currently limiting the wider use of biologically synthesized production. However, by introducing light-controlled promoters in large-scale production, the unit yield can be enhanced.

2) Santalol and sclareol are lipophilic substances, so their purification can avoid the difficulties associated with extracting water-soluble substances. The choice of these two products can partially mitigate purification costs.

3) When factories are established, they need to undergo environmental investigations. The pollutants generated by our project mainly include waste liquid and yeast residues, while chemical synthesis produces waste gas, waste liquid, and waste residues. Considering that biologically synthesized production does not emit waste gas, it is easier to meet the environmental requirements of factories.

4) The routes used in chemical synthesis are relatively easy to protect after patent application. However, in biologically synthesized production, even if a patent has been applied for, it is difficult to provide evidence and protect rights when the strain is misappropriated. This reminds us of our team's project in 2014, where BIT-China designed a gene password lock that can only be activated when the inducer is added in the correct order. By collaborating with fragrance companies, we may be able to help them better protect their interests.

And we emailed researcher Weiqiang Zhou from China National Cereals, Oils and Foodstuffs Corporation (COFCO) to confirm if scaled-up production equipment with light-regulated modules could be realized. Zhou Weiqiang believed that this could be easily achieved based on modern fermentation equipment.

Additionally, we also communicated with Professor Fengping Yi from Shanghai Institute of Technology, who has rich experience in the synthesis of fragrances. She affirmed our idea of synthesizing terpenoid compounds through biological methods, which can replace the pollution caused by chemical synthesis and has innovative potential in the fragrance industry. Furthermore, if we can replace some expensive natural ingredients, considerable economic benefits can be gained.

Renli

According to a preliminary investigation of Chinese perfume enthusiasts' opinions and the scent of the fragrance we want to synthesize, it is ensured that the biologically synthesized fragrance components are not objectionable and have commercial potential. Through the communication with Le Li, the founder of a perfume brand, it is realized that the target users of the fragrance components are fragrance and detergent companies that use fragrance bases to blend scents.

Regarding the audience for our perfume, we conducted a survey on a Chinese perfume enthusiast website called "Nose Time." We received some responses and summarized the following points: Perfume enthusiasts care more about the scent, appearance, cost-effectiveness, and qualities of perfumes. Whether the raw materials are biologically synthesized or naturally extracted is not their main concern. Additionally, they do not object to gene editing but require clear safety information. Moreover, perfume enthusiasts who are familiar with the fragrance industry pointed out that cost control and the scrutiny of allergenicity and toxicity by European and American institutions have led many perfume raw materials to come from chemically or biologically synthesized pure products rather than natural plant extracts. (We would like to thank "Nose Time" for their help in understanding fragrance and aroma!)

To confirm a mixture of santalol and sclareol can produce a pleasant aroma, we consulted Jinli Jia, the Cultural and Artistic Director at Shenzhen Yuetmu Holdings Limited. She reflected that sclareol wss often used as a substitute for ambergris in perfumes, presenting a cool feeling at low concentrations, which is important in high-end perfumes. However, at high concentrations, it can have an unpleasant animal odor. For santalol, it mainly presents an elegant and warm woody scent at various concentrations. The combination of santalol and perillalcohol can create a classic oriental fragrance with sweetness, strong penetration, and long-lasting aroma.

Initially, we thought our technology should be provided to perfume brands, so we contacted Mr. Le Li, the general manager of perfume brand UTTORI. Mr. Li Le expressed that in recent years, there has been a growing number of consumers who are paying attention to the significance of fragrance in their lives. As the perfume and fragrance market in China gradually expands, it is evident that there is ample room for future development. However, the fragrance industry in China is still in its early stages, with significant gaps in related synthetic technologies. Emerging fragrance companies possess abundant creativity, and the fragrance industry in China urgently needs this creativity. Moreover, he suggested that our synthesized product is too complex to be directly added to solvents to make perfume and that two products can be synthesized in different proportions to serve as a fragrance base.

Integrated Human Practices

In the Integrated Human Practice works, we are striving to further develop the three parts of the WSR system approach, namely, to explore the relationships between these three parts, and ultimately enhance our work.

The development of these three elements will be incorporated into our project's Integrated Human Practice, and our work will be divided into three stages:

Wuli: how to improve gene pathways at the laboratory level;

Shili: how to scale up production while considering technical selection and efficiency;

Renli: Focusing on the relationship between our project and people, including how to make our users understand and accept our products.

Wuli

In this section, we consulted with experts in the field, researchers from COFCO and the iGEM team Heidelberg, through various channels to seek their advice and explore the theoretical feasibility of our gene circuit and experiments. With their help, we successfully resolved some issues encountered during the experiments. Additionally, they also mentioned some details that we had not considered.

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(1) During the project's topic selection group meeting, Professor Shaowei Zhang from the National University of Defense Technology provided a method to improve the repair of photosensitive proteins. Although he suggested using a single-frequency LED or a filter to achieve an environment of single-frequency light exposure, he also pointed out that the light-responsive elements in the gene circuit can be activated within the range of 400-450 nm. He also mentioned that the required pulse light can be programmed to address.

Professor Zhenmin Ling from Lanzhou University provided valuable suggestions regarding experimental design, urging us to consider the specific impact of product accumulation on the antibacterial properties of the engineered bacteria. The positive affirmation and suggestions from the two professors during the topic selection meeting greatly boosted our confidence in the project and provided significant help for the subsequent design of the light control system.

(2) During offline discussions with researchers from COFCO, they did not have many objections to our designed gene circuit. However, they raised questions and provided some suggestions regarding modeling and experiments.

They believed that although we have calculated in the model that the knockout genes will not affect cell growth, it is necessary to verify in the experimental stage that the knockout genes will not affect product yield and cell viability. Additionally, they mentioned the antibacterial properties of santalol and sclareol, which can have a toxic effect on cells. Therefore, before establishing the synthetic product in Saccharomyces cerevisiae, we need to experimentally verify that the yeast cells are not harmed within a certain range of santalol and sclareol concentrations. Otherwise, the yeast cells may die due to the accumulation of the final product.

These details were overlooked in our experimental design, and in response to these suggestions, we made improvements in our subsequent experiments and modeling processes.

(3)We also had an online meeting with the iGEM team Heidelberg. We introduced each other's projects for this year, and then discussed our gene circuits. They suggested replacing the promoter in our gene circuit with a stronger one to enhance product yield. After careful consideration, we replaced the original Pgal10 promoter in the gene circuit with Pgal1-s, a stronger promoter that enhances its response to transcription factors. During this meeting, we and Heidelberg decided to collaborate on the EDU project, which mainly focuses on creating a four-panel comic.

(4) When we were conducting experiments to knock out the functional gene gal4, we encountered several failures. Upon the recommendation of our PI, we consulted Dr. Ying Wang, member of the Molecular Microbiology and Bioengineering Professional Committee of the Chinese Society for Microbiology, who specializes in yeast research. Dr. Wang examined the primers we designed, the extension and annealing temperatures set during PCR, and the PCR operation itself. She suggested using the genome as a template for fragment PCR. Finally, following Dr. Wang's advice, we successfully knocked out the functional gene gal4.

Shili

The aim of our project is to achieve scale production of adjustable ratio fragrance base and provide this technology to fragrance companies for technology transfer. To expand production, it is necessary to ensure the feasibility of equipment and purification schemes. To this end, we contacted Beijing InnoChem Science & Technology Co., Ltd., COFCO, BioCatSyn (Shanghai) Biotechnology Co., Ltd., and Dr. Zhiping Zhao and Dr. Xin Li from the School of Chemistry and Chemical Engineering at Beijing Institute of Technology. With their help, we found the most suitable equipment design plan for our project to develop into a large-scale production, and also designed a method to simultaneously purify two types of products from Saccharomyces cerevisiae.

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Beijing InnoChem Science & Technology Co., Ltd. is a major reagent commercial company in northern China with extensive commercial experience and unique business insight. We were fortunate to contact Mr. Boying Huang, Sales Manager of Beijing InnoCare Technology Co., Ltd., who provided us with advice on cost and equipment issues. After understanding our project, he appreciated the novelty of our controllable ratio synthesis of two fragrances in Saccharomyces cerevisiae, but also emphasized the need to consider the warranty costs during equipment operation while considering the cost of production equipment. Therefore, in the subsequent equipment design, we also paid attention to the difficulty of warranty.

After offline communication with Beijing InnoChem Science & Technology Co., Ltd., they showed great interest in our project. They believe that our project is innovative and meaningful, and they are looking forward to seeing us excel in it. As a result, they have provided us with financial support, for which we are extremely grateful.

To expand the production of the feasible projects in the experiments, we first made a preliminary design of the fermentation tank through literature research. After offline communication with Dr. Weiqiang Zhou from COFCO, we realized that the reason why photo-controlled synthesis is not widely used in the chemical production field is that the penetration of light in the broth must be considered. For a large fermentation tank, it is difficult to simultaneously irradiate all the broth. In addition, single-frequency light equipment is often expensive, so we can adjust the effective frequency range of photo-control to a certain range.

In order to make the equipment design more rigorous, after communication with Dr. Zhiping Zhao, Chairman of the Beijing Membrane Society, we obtained a preliminary design that is more feasible.

In terms of purification, we first consulted a researcher from COFCO who is responsible for product purification. He roughly observed the structural characteristics of the two products' chemical formulas and judged that the common intracellular extraction method could be used. However, since the two products have strong volatility, there may be a loss of yield during the purification process due to volatility.

We then had a discussion with Mr. Fengqing Wang, a technical consultant from BioCatSyn (Shanghai) Biotechnology Co., Ltd., who also noticed the volatility issue of the two synthesized products. He suggested that we should use the biphasic fermentation method to avoid product volatility and use the ratio of the final distillation or rectification product as the ratio of the final product.

In response to the suggestions made by COFCO and BioCatSyn regarding product purification, we have also designed an ideal purification scheme. This scheme aims to minimize the loss of volatiles while simultaneously extracting both products.

Renli

In the "Humanity" section, we need to focus on the links between the project and various groups. We mainly divide this section into three parts:

1) the promotion of mixed fragrances in fragrance and daily chemical enterprises;

2) consumer attitudes towards products containing biologically synthesized ingredients;

3) the impact of biologically synthesized ingredients on natural plant extracts.

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1) Our project is positioned for large-scale production, and the product will be supplied to enterprises that have a demand for fragrance bases, such as fragrance and daily chemical enterprises that use fragrance bases as odor base materials. Professor Fengping Yi from Shanghai Institute of Technology also analyzed the possibility of promoting mixed fragrances in the market for us. We also contacted Ms. Qili Huang, Chairman of Beijing InnoCare Technology Co., Ltd., who provided advice from the perspective of project commercialization and sales.

During our discussion with Professor Yi, she believed that biosynthesis and controllable ratio synthesis are both very innovative in the flavor and fragrance industry. Existing single-component products may be difficult to replace with new single-component products, but controllable ratio mixed fragrances may be favored in the market in a new way. In addition, Professor Yi suggested that we must pay attention to the smell of the mixture of sclareol and santalol at different mixing ratios, and we must also pay attention to how our project can reduce costs compared to existing production. These two aspects have a decisive impact on the potential expansion of our project in the market, and the project's output is also particularly important.

Ms. Huang provided us with many suggestions for commercializing our project in a simple and easy-to-understand way. She believed that finding the right product positioning and finding suitable publicity strategies are very important. We can appropriately emphasize our technology by packaging it in our publicity. For example, in our promotion, we can appropriately emphasize the advanced, superior, low-carbon, environmentally friendly, and innovative features of our biosynthesis technology. In addition, enterprises often have a good impression and trust in projects and teams with a university background, so we can emphasize the university background of our project in our promotion. Ms. Huang also reminded us that we cannot simply use low prices to open up sales channels in the initial stage of product promotion. Because the subsequent costs are likely to increase, lowering costs at the beginning will make it difficult to raise prices later without losing customers to maintain profits.

2) We considered how our project's products could be promoted in fragrance and daily chemical enterprises, and the product design of fragrance and daily chemical enterprises almost entirely depends on consumer interests and needs. Therefore, when considering the relationship between our project and people, we conducted a survey of the general public to understand their views on biologically synthesized fragrance ingredients.

We conducted surveys among the general public through questionnaires and street interviews. From the responses of the interviewees and the results of the questionnaires, it is evident that people have a generally low level of awareness about biologically synthesized products. Their fear and resistance towards genetically edited microorganisms and their products mostly stem from a lack of credible scientific information sources. Additionally, people have a low level of interest in the fragrance ingredients used in perfumes, cosmetics, and detergents, and they often do not care about the production methods of these ingredients.

Therefore, we can conclude that individual consumers in China do not pay much attention to the synthetic methods of the ingredients in the perfumes, cosmetics, and detergents they purchase. Hence, we do not need to worry about strong resistance from them towards biologically synthesized ingredients. However, from this survey, we also found that the term "natural extract" is readily accepted by the public, while "biologically synthesized" raises doubts. This indicates that there may still be some misunderstandings about biologically synthesized products among the public, and we need more effective scientific communication to dispel these misunderstandings.This is exactly what we are trying to achieve within the EDU.

From another perspective, the low level of awareness among Chinese consumers about biologically synthesized products may also indicate a huge potential for biologically synthesized products in the consumer market. With sufficient scientific communication and safety verification, biologically synthesized products are likely to successfully penetrate the consumer market and become popular among consumers.

3) In addition, we consulted with BioCatSyn (Shanghai) Biotechnology Co., Ltd. to understand their views on the relationship between biocatalysis, chemical synthesis, and natural plant extraction, as well as how our project can impact the fragrance industry.

We know that natural fragrances are extracted from animals and plants, and the initial production of natural fragrances was an industry chain related to natural raw materials. When fragrances achieve large-scale biosynthesis production, it may have an impact on raw material providers, such as farmers who collect materials from the wild or grow raw material plants on a large scale. Therefore, we also asked BioCatSyn about how they consider this issue.

Mr. Wang Fengqing said that if biosynthesis can be successfully industrialized, it will definitely have an impact, but this issue needs to be viewed from multiple perspectives. If the cost of biosynthesis production of these fragrances is significantly reduced and efficiency is significantly improved, it may have an impact on enterprises, growers, and collectors who are in the industry's position of supplying plant raw materials. However, for the santalol that we want to synthesize in our project, the planted and collected sanders can also be used in the furniture and ornaments market, and the powder of sanders is used in traditional incense, so biosynthesis and chemical synthesis will not occupy their living space. For workers in factories that extract plant ingredients, their work experience and skills in the factory should also be sufficient to support them in working in similar factories.