Description

1. Background

In the traditional fermentation process of brewing beer, 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, for instance, the aroma character of liquor in Yellow Wine. 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 headache, nausea and vomiting, and other adverse symptoms. Thus, it is not advisable to take too much of it. This makes it particularly important to control the content of higher alcohols during fermentation. We noticed that when using yeast for brewing, the BAT2 gene controls the yeast to produce higher alcohols, which is the culprit of adverse reactions. In our experiment, we used the characteristics of polymerase chain reaction (PCR) and homologous recombination to achieve the synthesis of gene fragments (BAT2-L-TEF1-ATF1-CYC1-TEFP-NrsR-TEFt- BAT2-R) and conversion (conversion of BAT2 contained in the original yeast genome into ATF1, a gene that can slow down yeast metabolism and reduce the output of higher alcohol) to achieve genetic modification of yeast, and this will improve the flavor of beer, at the same time reducing the content of higher alcohol, inhibiting adverse impact on the human body after getting drunk.

2. Experiment Design

Our team aims to manufacture Saccharomyces cerevisiae to produce fewer higher alcohol in beer brewing by synthetic biology. Here, The BAT2 allelic genes were replaced by the beneficial ATF1 gene for enhancing acetate ester synthesis in the industrial brewer’s yeast strains.

Figure 1. The Principle of Product Engineering

Our experiment includes knocking out the BAT2 gene from the genome of Saccharomyces cerevisiae and replacing it with ATF1 using genetic engineering techniques.

1. BAT2 gene

The BAT2 gene is responsible for the production of higher alcohols, by replacing it, it prevents its expression and reduces the amount of higher alcohols synthesized.

2. ATF1 gene

The ATF1 gene changes other types of higher alcohols into ethyl acetate

General Experiment Procedure

The designed gene sequence contains the homologous sequence of BAT2 gene, Nourseothricin-resistance gene, and ATF1 gene sequence so that when we culture them onto the agar plate consisting of Nourseothricin, only the genetically modified genes will express resistance and be screened out while the others will fail to survive. Therefore, we extracted the ones producing less high alcohols which will lead to reduced drunk phenomenons and increases the aroma character of liquor.

Figure 2. Experiment Procedure

3. Expected Result

1. Successfully construct L-TEF1-ATF1-CYC1-TEFp-NrsR-TEFt-R
2. The L-TEF1-ATF1-CYC1-TEFp-NrsR-TEFt-R was transferred into Saccharomyces cerevisiae expression vector to reduce the level of higher alcohols in Saccharomyces cerevisiae.

4. Reference

1. Zhang J., Zhang C., Wang J., Dai L., Xiao D. (2014) Expression of the Gene Lg-ATF1 Encoding Alcohol Acetyltransferases from Brewery Lager Yeast in Chinese Rice Wine Yeast. In: Zhang TC., Ouyang P., Kaplan S., Skarnes B. (eds) Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012). Lecture Notes in Electrical Engineering, vol 249. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-37916-1_5.
2. Ma L, Huang S, Du L, Tang P, Xiao D. Reduced Production of Higher Alcohols by Saccharomyces cerevisiae in Red Wine Fermentation by Simultaneously Overexpressing BAT1 and Deleting BAT2. J Agric Food Chem. 2017 Aug 16;65(32):6936-6942. doi: 10.1021/acs.jafc.7b01974.
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4. Dai L., Zhang C., Zhang J., Qi Y., Xiao D. (2014) Effects of IAH1 Gene Deletion on the Profiles of Chinese Yellow Rice Wine. In: Zhang TC., Ouyang P., Kaplan S., Skarnes B. (eds) Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012). Lecture Notes in Electrical Engineering, vol 249. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-37916-1_42.
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