Engineering Success

1. Introduction

In the beer brewing process, in addition to the main metabolite ethanol, yeast also produces higher alcohols, esters, aldehydes, phenols, acids, and other flavor metabolites. Among them, higher alcohol refers to the general term of a class of alcohols with three or more carbon chain skeletons and is one of the important chemical substances that form the flavor and taste of wine.

Appropriate higher alcohol content can make the beer taste and aroma full, and the wine body is soft and harmonious, but if the higher alcohol content is too high, it will lead to the formation of peculiar smells in beer, which not only affects the drinking taste and flavor quality of beer, but also affects drinkers. The body produces obvious side effects, which are harmful to the health of the drinker.

The higher alcohols in the finished beer are produced by the growth and reproduction of yeast in the main fermentation stage of beer. The branched-chain amino acid aminotransferase is decomposed, that is the pathway of amino acid catabolism; the other is the α-keto acid anabolic pathway. The metabolite pyruvate generated from glucose through the emp pathway can also generate α-keto acid after a series of reactions that is the pathway of higher alcohol anabolic.

For this problem, researchers have taken various measures to solve this problem. For example, the higher alcohol metabolism gene of Saccharomyces cerevisiae is modified to achieve the purpose of reducing the production of higher alcohol.

This project intends to reduce the production of higher alcohols from two types of genetic modification, we designed to edit the acetate metabolism genes. The alcohol acetyltransferases encoded by ATF1, ATF2, and Lg-ATF1 genes can catalyze the reaction. At the same time, acetate compounds can also undergo hydrolysis reactions under the catalysis of the hydrolase encoded by the IAH1 gene to generate corresponding higher alcohols. Therefore, we replaced the BAT2 gene in the S. cerevisiae genome with ATF1 through the homologous recombination way.

2. Design

The ATF1 gene is inserted into the S. cerevisiae expression vector pYES2 and transformed into the S. cerevisiae strain, which we called SFA-1 (Figure 1). In other words, this plasmid is based on the targeted modification of the aminotransferase encoding gene, and then targeted modification of the acetate metabolism gene.

Figure 1. The schematic map of the pYES2-ATF1 plasmid

3. Build

In order to build our plasmids, The ATF1 was amplified from the genome of S. cerevisiae, and the TEF1 promoter and the CYC1 terminator were amplified from the pHCas9-Nours plasmid. Next, we fused the three fragments by PCR and extracted the recombinant DNA fragment (Figure 2). Then, we digested the target fragments and the pYES2 vector with SpeI and SalI, and we used T4 DNA ligase to ligate the fragments and the vector. Then we transformed the recombinant plasmids into E. coli DH5α competent cells and coated on the LB (Amp+) solid plates.

Figure 2. Gel electrophoresis results of the target gene fragments.

We verified the colonies through colony-PCR (Figure 3), and then we inoculated single colonies (1, 2, 5, and 7) and we send the constructed recombinant plasmid to a sequencing company for sequencing.

Figure 3. verification of the recombinant plasmids by colony-PCR.

3. Functional testing

a) Construct the engineered S. cerevisiae strain

We extracted the correct pYES2-ATF1plasmid and transformed it into the S. cerevisiae competent cells through the Lithium acetate transform method. Next, we verified the colonies through colony-PCR (Figure 4).

Figure 4. verification of the engineered S. cerevisiae by colony-PCR

b) Growth Curve measurement

The plasmid was transformed into the S. cerevisiae and we named the transformants SFA-1. We inoculated the engineered S. cerevisiae colonies (SFA-1) in the 5mL YEPD medium and grew at 30℃ and 180 r/min for 12 h. The above culture 1:100 culture liquid was connected to three bottles of 50mL liquid YEPD medium and incubated under the same conditions. The absorbance value at 600 nm was measured at 2,4,8,16,24 and 32 h after culture (Table 1). This result indicates that the genetically modified S. cerevisiae SFA-1 showed no harmful effects on the growth of the organism itself. Therefore, giving support for applications in the wine fermentation industry (Table 1).

Time (h)/Type	SF-1	WT
2	0.076	0.016
4	0.106666667	0.049
7	0.18	0.109
16	1.007333333	0.639
24	2.558333333	1.105
32	2.554666667	1.233666667

Table 1. Raw data were obtained at each time period 2, 4, 7, 16, 24, and 32 hours, respectively. Each strain of WT and SFA-1 was tested with Spectrophotometer three times, to obtain an OD600 absorbance level.

4. Learn

We have already collected the figures from our experiments. Because of the importance of ATF1 in the aroma profile of the wine, the yeast with a constitutively overexpressed ATF1 gene produces more isoamyl acetate and ethyl acetate compared to a wild-type strain. In our project, we overexpressed the ATF1 in the S. cerevisiae to construct the engineered S. cerevisiae strain to control the content of higher alcohols during fermentation.

In the future, with improved engineering strains, the better quality of the wine would spread all over the world. ATF1, which we verified its activity in our project, provides a good choice for future research. We believe that the engineered S. cerevisiae which has reduced the production of higher alcohols could be used to improve the factories in the future.