Improvement

Overview

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. However, higher alcohol would accumulate and increase its toxicity in the long-term fermentation process. This makes it particularly important to control the content of higher alcohols during fermentation.

In 2006, group Igem2006_MIT designed a basic part BBa_K45006, ATF1, but they only provided the DNA sequence of it. Our composite part BBa_K4278717 is composited by the promoters, ATF1 gene fragment, NrsR gene fragment, and terminators. To control the content of higher alcohols during fermentation, by reading literature and consulting experts in related fields, we replaced the BAT2 gene with the ATF1 reading cassette in the host strain.

We amplified the DNA fragment and transformed it into S. cerevisiae to replace the BAT2 DNA fragment in the strain genome. By detecting the fermentation product of the transformants, it was further confirmed that the engineered S. cerevisiae has reduced the production of higher alcohols which could be used to improve the quality of the wine in factories in the future.

Introduction

Aminotransferases can catalyze the deamination of amino acids to form α-keto acids. According to the different types of amino acids catalyzed, aminotransferases can be divided into two categories, one is branched-chain aminotransferases enzymes encoded by BAT1 and BAT2 genes, and the other is aromatic amino acid aminotransferases enzymes encoded by ARO8 and ARO9 genes. The main component of higher alcohols produced by S. cerevisiae is converted from branched-chain amino acids, such as isobutanol. Therefore, knocking out BAT1 and BAT2 genes can effectively reduce the synthesis level of higher alcohols in S. cerevisiae.

1. BAT2-L-TEF1-ATF1-CYC1-TEFP-NrsR-TEFt-BAT2-R gene fragment amplification

Figure1 shows the schematic map of the BAT2-L-TEF1-ATF1-CYC1-TEFP-NrsR-TEFt-BAT2-R gene fragment that will replace the BAT2 gene fragment in the cell. Among them, BAT2-L and BAT2-R gene fragments are the upstream and downstream homology arms of the BAT2 gene in this fragment; the ATF1 gene is used to replace the BAT2 fragment and encode alcohol acetyltransferase in cells; TEFP-NrsR -TEFt fragment is a fragment extracted from the nourseothricin resistance gene expression cassette.

Figure 1. The schematic diagram map of the composite DNA fragment.

Firstly, we amplified five fragments, which were CYC1-TEFp-NrsR-TEFt, pTEF1, BATup, BATdown, and ATF1. The results (Figure 1) indicated that the five target DNA strands are successfully amplified.

Figure 1. Gel electrophoresis to identify the target DNA fragments. CYCter-Nours: CYC1-TEFp-NrsR-TEFt (1715 bp), pTEF1: TEF1 promoter (400 bp), BATup: the upstream homologous arm of BAT2 (503 bp), BATdown: the downstream homologous arm of BAT2 (501 bp), ATF1 DNA fragment (1575 bp).

Secondly, we fused the DNA fragments in figure2 by PCR. The following result of the gel electrophoresis shows the gene strands are successfully amplified (Figure 2).

2. Measure the growth curve of ATF1 transformants

The constructed DNA fragment was transformed into the S. cerevisiae and replaced the BAT2 gene in the genome through a recombinant way and we named the transformants SF-1. In order to measure if the integrated gene interferon the growth of the host strain, we measured the growth curve arranged 32h and compared it with the wildtype AQ yeast. This result indicates that the genetically modified Saccharomyces cerevisiae SF-1 that we aimed to reduce drunk phenomenon caused no harmful effects on the survival and growth of the organism itself. Therefore, giving support for applications in the wine fermentation industry.

The determination of the growth curve is to obtain the growth rate of the strain. Figure3 is a log2 function graph of the growth curve of strain SF-1. The y-axis is the triple log mean of the OD₆₀₀ absorbance, and the y-axis is the fermentation time. As a result, strain SF-1 can grow normally in the 16% YEPD medium. At the 32nd hour of simulated cultivation of the strain, the absorbance of OD₆₀₀ reached about 2.5, and the log2 value was about 5.07. Expected levels are envisaged in the experiment (Figure 3).

Figure 3. the graph of the growth curve of strain SF-1.

3. Functional test

We use gas chromatography to measure the content of higher alcohols. The following table shows the experimental data of various alcohols produced by wild-type AQ yeast and SF-1 yeast. We did two sets of experiments for each yeast. The experimental data showed that the ethanol content of SF-1 yeast was similar to that of wild-type AQ yeast, indicating that the knockout of the BAT2 gene and integration of the ATF1 gene had little effect on the production of ethanol.

As indicated in Table 1, the content of high alcohol produced by AQ yeast ①, AQ yeast ②, Fragments F①, Fragments F②, Plasmid pYES2-ATF1① , and Plasmid pYES2-ATF1② is 249.71, 242.59, 89.81, 97.29 mg/L, respectively.

Figure 4. The results measured by gas chromatographic

In Table1 and Figure 4, we can find that the percentage of higher alcohols produced by wild-type yeast was significantly higher than that produced by SF-1. These results indicated that replacing the BAT2 gene with the ATF1 gene of alcohol acetyltransferase could reduce the production of higher alcohols in yeast strains. In other words, the wine produced by SF-1 engineering bacteria has a low content of higher alcohols, which is not likely to cause symptoms such as headache and thirst after drinking. Our study provides solutions to problems such as hangovers and headaches after drinking alcohol, which have certain practical significance.