Implementation

Background Research

China's alcohol market has developed rapidly in the past ten years. Especially before the epidemic, the scale of China's alcohol circulation market has been steadily expanding. According to statistics, there are nearly 450 million drinking consumers in China at present. However, due to the impact of the epidemic, holiday parties and family gatherings have been canceled in large numbers, resulting in a decline in the consumption of alcohol products. In this regard, some experts said that the Chinese alcohol market in the post-epidemic era is about to face an inflection point like a "U" shape. Because the Chinese alcohol market has strong consumption inertia and market elasticity, China's high-end alcohol occupies the market for festive gifts, business banquets, and other markets. Therefore, if the economy enters a new round of growth cycle, social and economic activity and market demand will be activated, and Chinese alcohol may be the first beneficiary industry.

Circulation market scale (billion in CNY) of the alcohol products during 2009-2019 in China
(Blue: liquor; Orange: beer; Grey: wine; Yellow: others )

In the traditional fermentation process of brewing wine, Saccharomyces cerevisiae would produce higher alcohols, a monohydric alcohol compound that contains three or more carbon atoms. An optimum amount of higher alcohols secreted is the key factor for maintaining the aroma of the wine, which gives a characteristic for its distinct smell. However, higher alcohol would accumulate and increase its toxicity in the long-term fermentation process. Especially, the over-accumulation of Isoamyl alcohol would cause disorders in the human central nervous system, leading to headaches, nausea, vomiting, and other adverse symptoms. Therefore, it is important to reduce the production of higher alcohols during fermentation.

Product

• Experimental Design

The Principle of Product Engineering

We applied intracellular homologous recombination technology to knock out the branched-chain amino acid aminotransferase encoded by the BAT2 gene, thereby reducing the synthesis level of higher alcohols in Saccharomyces cerevisiae. At the same time, the position of the BAT2 gene was replaced by the alcohol acetyltransferase ATF1 gene. The ATF1 gene was then placed on a Saccharomyces cerevisiae expression vector and transformed into Saccharomyces cerevisiae. These operations can reduce the level of higher alcohols produced in the brewing process and further improve the quality of the wine.

• Performance Test

Figure: Fitting result of Wild-type AQ yeast and engineered yeast SF-1.

As the fermentation test results shown above, the higher alcohol production of our engineered strain SF-1 decreased by about 152.6 mg per liter compared to the wild-type strain, which is 38% of the wild-type strain. This proved that our engineered yeast achieved the experimental assumption and successfully reduced the production of higher alcohols.

• Product

Our experimental outcome and our final product (concept drawing)

• Product properties: Freeze-dried powder in solid form, vacuum-dried on the package to isolate the air.
• Method: according to the proportion, it is directly added to the brewing tank for the fermentation process.
• Storage condition: cold and dry. Recommend to be stored under -80℃ for long-term storage.

Target Users

Considering the characteristics and cost of our products, we target customers as factories producing alcoholic products. Our goal is to use our technology to empower small wineries, improve wine quality at a lower cost, and meantimes conduct technical cooperation with major wine companies for upgrading.

Per our interview with Manager Qiqian Jia from Hongqiao Winery company, factories will spend more on collecting grapes of higher quality to reduce the production of higher alcohols during fermentation. While those small factories might not bear much budget on raw materials, it will be hard for them to control the production of higher alcohols. Therefore, our product provides a quite effective and low-cost, solution for them to improve their wine quality by sorting out the root issue and reducing the production of higher alcohols.

We also look forward to cooperating with some major wine companies, by adding our gene-engineered yeast to optinize the brewing process so as to improve the wine quality.

Safety

We attach great importance to safety in the process of experimental design and execution, which fully complies with Igem's safety-related requirements. Microorganisms used in our research were Escherichia coli DH5 and Saccharomyces cerevisiae. On the experimental materials, we repeatedly confirmed the safety of the experimental materials. During the experiment, our team set up a special laboratory code of conduct to ensure the safety of the experiment and its effect on the experiment. Manipulation with microorganisms was conducted on the ultra-clean bench. The UV light was turned on after finishing the experiment to disinfect the workspace.

In addition, since our final product, the gene-edited yeast will be used for wine brewing, must be conducted many tests to ensure it meets relevant requirements and standards in the food processing industry.

Challenges

General resistance or suspicion to GMOs in society presents the biggest obstacle to our products. Based on our survey, still many people are not clearly aware of gene editing technology. The inadequate knowledge might result in some blind opposition to accepting our product, even opposing those wines through fermentation by using our engineered yeast. It will take time to change their attitudes, which can be improved by spreading scientific knowledge and promoting synthetic biology.

Another big challenge with our project is that we have to iteratively determine the stability of our product's efficacy and find the most suitable conditions. As we all know, fermentation is a process with strict requirements on the environment. This requires our products to be of high purity and free of impurities, which is also a direction we will continue to work towards in the future.

Finally, since our product will be used in product processing, it means that it must meet some government requirements for the food industry. We must obtain relevant qualification certificates before we can realize the industrial production of products, which is also a challenge we must face.

Future Plan

So far, in the laboratory, we have proven that our gene-edited yeast has presented its efficacy in reducing higher alcohols than wild yeast. Regarding the future plan for research and development, we still need to duplicate our fermentation tests to determine the stability of the efficacy of our engineered strains. In addition, we will also do more research to design various comparison group tests to explore the optimal fermentation conditions of our strains.

Besides, we also need to overcome those challenges we listed above. In order to increase public awareness of gene-editing technology, online we will take the advantage of social media platforms for posting relative knowledge and information to spread the impact of synthetic biology to a wider public. Offline we plan to raise a wine fair so as to educate the public closely and also promote our product.

To further implement our business in the real world, we also drafted our own business plan which can be found on the Supporting Entrepreneurship page. In this file, we did not only analyze the market but also settle down our own marketing strategy and financial planning.

With improved engineering strains, the better quality of the wine would spread all over the world and our product can contribute to human health.