# Effect of the Deletion of Genes Related to Amino Acid Metabolism on the Production of Higher Alcohols bySaccharomyces cerevisiae

Authors: Ya-Ping Wang; Xiao-Qing Wei; Xue-Wu Guo; Dong-Guang Xiao
Journal: BioMed Research International
 (2020)

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## Abstract

The higher alcohols produced bySaccharomyces cerevisiae exert remarkable influence on the taste and flavour of Chinese Baijiu. In order to study the regulation mechanism of amino acid metabolism genes on higher alcohol production, eight recombinant strains with amino acid metabolism gene deletion were constructed. The growth, fermentation performance, higher alcohol production, and expression level of genes in recombinant and original α5 strains were determined. Results displayed that the total higher alcohol concentration in α5ΔGDH1 strain decreased by 27.31% to 348.68 mg/L compared with that of α5. The total content of higher alcohols in α5ΔCAN1 and α5ΔGAT1 strains increased by 211.44% and 28.36% to 1493.96 and 615.73 mg/L, respectively, compared with that of α5. This study is the first to report that the CAN1 and GAT1 genes have great influence on the generation of higher alcohols. The results demonstrated that amino acid metabolism plays a substantial role in the metabolism of higher alcohols by S. cerevisiae. Interestingly, we also found that gene knockout downregulated the expression levels of the knocked out gene and other genes in the recombinant strain and thus affected the formation of higher alcohols by S. cerevisiae. This study provides worthy insights for comprehending the metabolic mechanism of higher alcohols in S. cerevisiae for Baijiu fermentation.


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## Body

## 1. Introduction

Higher alcohol is one of the important flavour substances in Baijiu [1] that are involved in the formation of Baijiu taste and flavour [2]. Baijiu has 12 flavour types in China. Each flavour type of Baijiu has its unique style because of its unique content of higher alcohols and other flavour substances. For example, Maotai jiu has higher content of higher alcohols, a prominent Maotai flavour and a heavy taste, whereas Fen jiu (traditional Baijiu with light aroma) has less higher alcohol content, is fresh and clean, and tastes light and pure [3]. The appropriate content of higher alcohols brings unique aroma and mellow taste to Chinese Baijiu [4]. However, excessive higher alcohol concentration will lead to fusel oil taste and is potentially harmful to human health as it may cause hangover and cerebral paralysis [5, 6]. According to reports, higher alcohols are generated by yeast during the fermentation of alcohol [7]. Higher alcohols are produced by the decarboxylation and dehydrogenation of α-ketoacids in yeast cells [8]. As displayed in Figure 1, the metabolic pathway for Saccharomyces cerevisiae to generate higher alcohols is divided into the catabolic pathway (Ehrlich pathway) and biosynthetic pathway according to the different sources of α-ketoacids [9, 10].Figure 1
The pathway of higher alcohol formation inS. cerevisiae. Two pathways for yeast to metabolize higher alcohols are described in the figure. Amongst them, the orange dotted box represents the biosynaptic pathway, and the green dotted box represents the Ehrlich pathway. The colored dots in the figure represent n-propanol, isobutanol, isoamyl alcohol, phenylethanol, and other higher alcohols.Amino acids as a nitrogen source utilised by yeast play a critical role in the formation of higher alcohols through the Ehrlich pathway [11], and their metabolism is divided into the following steps. Firstly, amino acids are transported from the fermentation substrate to yeast cells through amino acid transporters [12–14]. For example, branched amino acids can be transported by BAP2-encoded branched amino acid-permeable enzyme [15], and aromatic amino acids are absorbed by aromatic amino acid-permeable enzymes. Then amino acids generate corresponding α-ketoacids under the action of aminotransferases, such as the branched chain amino acid (BCAA) transaminases encoded by BAT1 and BAT2 genes [16–18] and the aromatic amino acid transaminases encoded by ARO8 and ARO9 genes [19]. Then, α-ketoacids generate corresponding aldehydes under the action of decarboxylases. The metabolism of amino acids involved in this pathway can cross each other because of the wide substrate specificity of decarboxylase. Aldehydes can catalyse the formation of higher alcohols under the action of various alcohol dehydrogenases [17]. Finally, higher alcohols are discharged into the fermentation broth through simple infiltration from yeast cells. Many studies have demonstrated that amino acids are absorbed by yeast as a nitrogen source in the process of alcohol fermentation to allow fermentation to proceed as expected [20]. Amino acids in fermentation media are important for the growth of fermentation microorganisms and the formation of flavour compounds [21].Amino acids are important in the formation of flavour compounds by yeast. The deficiency of amino acids in fermentation medium will cause yeast to synthesise the amino acids it needs by using the ketoacids produced in sugar metabolism; thus, the production of ethanol, higher alcohols, esters, and other flavour substances will change when amino acids are lacking [22–24]. Aoki et al. mutated Lu’s yeast with nitrosoguanidine and obtained a strain with substantially decreased utilisation of leucine and phenylalanine [25]. The isoamyl alcohol and 2-phenyl ethanol contents of the mutated strain decreased by 65.96% and 90.70%, respectively, compared with those of the parent strain. The inactivation of BAT2 gene in haploid yeast strain can reduce the concentration of isoamyl alcohol and isobutanol [26]. Pirkov et al. confirmed that the mutants of ARO8 and ARO9 genes of the knockout haploid S. cerevisiae could not catalyse the transamination of aromatic amino acids [19]. We found that the utilisation of amino acids by yeast has an important impact on the formation of higher alcohols by yeast. Although many studies have reported on the influence of amino acid metabolism on the production of higher alcohols by S. cerevisiae, only a few have reported on the change of higher alcohol content in S. cerevisiae as a consequence of the interaction of genes involved in amino acid metabolism. Therefore, we focused on the effects and interaction of these genes on the higher alcohol production of yeast.In our study, the effect of amino acid metabolism-related genes, namely,AGP1 (encodes amino acid transporter), GAD2 (encodes glutamic acid decarboxylase), ARO80 (encodes amino acid transaminase transcriptional activator), GAT1 (encodes the transcriptional regulatory protein of nitrogen breakdown repression mechanism), CAN1 (encodes arginine permeability enzyme), BAP2 (encodes BCAA permeability enzyme), GDH1 (encodes glutamic acid dehydrogenase), and BAT2 (encodes BCAA transferase), on the concentration of higher alcohols in yeast was investigated by individually deleting each gene. The gene expression level, growth, and fermentation performance, as well as the higher alcohols produced in fermentation of the parental and recombinant strains, were determined. The regulation of amino acid metabolism in yeast on the formation of flavour compounds was analysed to provide guidance in the construction of industrial S. cerevisiae strains with desired higher alcohol concentration for Chinese Baijiu fermentation.


## 2. Materials and Methods

### 2.1. Strains, Plasmids, and Primers

All the strains and plasmid used in this study are shown in Table1.Table 1
Strains and plasmid used in this study.
Strains or plasmidsGenotypic or constructReferences or originAY15 CICC32315Commercial Baijiu yeast strainThis labα5MATα, haploid yeast strain from AY15This labTransformants△GAT1α5△GAT1::loxP-KanMX-loxPThis study△AGP1α5△AGP1::loxP-KanMX-loxPThis study△BAP2α5△BAP2::loxP-KanMX-loxPThis study△GDH1α5△GDH1::loxP-KanMX-loxPThis study△ARO80α5△ARO80::loxP-KanMX-loxPThis study△CAN1α5△CAN1::loxP-KanMX-loxPThis study△BAT2α5△BAT2::loxP-KanMX-loxPThis study△GAD1α5△gad1::loxP-KanMX-loxPThis studyPlasmidpUG6Kanr, containingloxP-KanMX-loxP disruption cassetteGüldener et al., 2002


### 2.2. Medium and Culture Conditions

Yeast strains were cultured in yeast extract-peptide-dextrose (YEPD) medium (2% peptone, 2% glucose, and 1% yeast extract) in a 30°C incubator. The YEPD medium (Promega, Madison, WI, USA) with 1 mg/mL G418 antibiotic (Promega, Madison, WI, USA) was used to screen the transformed recombinant strains using theKanMX gene. All solid culture media were added with 2% agar powder (Solarbio, Beijing, China).Sorghum hydrolysate was used for the culture of primary and secondary seed cultures of yeast cells according to the method in Li et al.’s literature [27]. Firstly, sorghum powder was weighed, added with hot water (65°C) with a material-to-liquid ratio of 1 : 4, and stirred evenly for gelatinisation. Then, high-temperature-resistant α-amylase (the addition amount was 3 U/g, and enzyme activity was 2×104 U/mL) was added. The mash was kept in an 85–90°C water bath for 1 h to liquefy, during which the mash was stirred from time to time. Then, the temperature of mash was reduced to 60°C, and saccharifying enzyme (the addition amount was 90 U/g, and enzyme activity was 10 × 104 U/mL) was added. The mash was placed in a 55–60°C water bath for 20 h and stirred evenly. Then, saccharification was carried out. After saccharification, the temperature of the hydrolysate was reduced to 40°C, acid protease (the addition amount was 4 U/g, and enzyme activity was 2×104 U/mL) was added, and the mixture was let stand for 30 min. Lastly, the hydrolysate was filtered using two layers of gauze and adjusted with water until the sorghum juice had a sugar content of 20°Bx. Each 145 mL of sorghum juice was dispensed into a 250 mL flask, then sterilised at 115°C for 20 min and cooled to room temperature. The second precultured yeast (5 mL) was inoculated to the prepared sorghum hydrolysate medium with an inoculum density of approximately 5×108 cells/mL. All fermentations were performed in triplicate.


### 2.3. Construction of Recombinant Strain with Single Gene Deletion

Gene deletion in parental strainα5 was carried out by integrating the KanMX box, which was amplified by polymerase chain reaction (PCR) with target-K-U/target-K-D as primers and pUG6 plasmid as a template. The PCR primers are listed in Supplemental Table S1 and were designed on the basis of the S. cerevisiae S288c genome sequence in the National Centre for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). The upstream and downstream homologous fragments of the target gene were amplified by PCR using target-FA-U/target-FA-D and target-FB-U/target-FB-D as primers and α5 genome as a template, respectively.The homologous recombination of the fragment and yeast genome was implemented using the LiAc/SS carrier DNA/PEG method [28]. YEPD solid medium mixed with 1 mg/mL G418 was used to preliminary screen the transformants. The exact integration of the KanMX box in a single colony was verified by diagnostic oligonucleotide PCR with verification primers (target-1-U/-1-D and target-2-U/target-2-D). Then, the right single colony was selected for purification, and second validation was carried out. Finally, the correct single colony is the right recombinant strain we constructed.


### 2.4. RNA Extraction and Quantitative Real-Time PCR (RT-qPCR)

Total RNA was extracted by a yeast RNA isolation kit and then reverse-transcribed using a PrimeScript™ RT reagent kit. Changes in gene expression level were assessed by RT-qPCR with SYBR® Premix Ex Taq™ II test kit (Tli RNaseH Plus). The kits were purchased from Takara (Takara Biotechnology, Dalian, China). The PCR primers (target-U/target-D) listed in Supplementary TableS1 were synthesised by GENEWIZ (Suzhou, China). The PCR procedure was set according to that reported by Li and Chen [1]. The result was analysed quantitatively using 2−ΔΔCt method, and the actin gene (UBC6) was used as the housekeeping gene.


### 2.5. Growth Curve Determination

Yeast strains were inoculated into 5 mL of YEPD liquid culture medium and cultured in a 30°C shaking bed incubator for 12 h. Then, 40μL of yeast cell fluid was added into 360 μL of YEPD liquid medium, and the mixture was transferred to a 96-well plate. Optical density at 600 nm was measured every 30 min by a Bioscreen automated growth curve analysis system (OY Growth Curves Ab Ltd., Helsinki, Finland).


### 2.6. Fermentation Analysis

Fermentation experiment was carried out in a 250 mL flask with 150 mL of the separated sorghum hydrolysate. The second precultured yeast seed liquid was inoculated to the sorghum hydrolysate medium. The inoculum density was approximately5×108 cells/mL. Fermentation was carried out in a 30°C incubator, and the hydrolysate was weighed every 12 h. Fermentation was concluded when the weight loss was less than 0.1 g. The hydrolysate was distilled, and the content of higher alcohols and other flavour substances in the distillate was determined by gas chromatography method according to Ma et al.’s report [29].


### 2.7. Statistical Data Analysis

All experiments were repeated three times. The growth curves and other charts were drawn using the Origin 2018 software. The heat map was drawn using Excel 2016 and Adobe Illustrator CS4 software. Data calculation and processing were performed in Excel 2016, and the results are displayed as themean±standarddeviation. The significance of the difference between experimental and control groups was analysed using a t-test (★★P<0.01, ★P<0.05).


## 2.1. Strains, Plasmids, and Primers

All the strains and plasmid used in this study are shown in Table1.Table 1
Strains and plasmid used in this study.
Strains or plasmidsGenotypic or constructReferences or originAY15 CICC32315Commercial Baijiu yeast strainThis labα5MATα, haploid yeast strain from AY15This labTransformants△GAT1α5△GAT1::loxP-KanMX-loxPThis study△AGP1α5△AGP1::loxP-KanMX-loxPThis study△BAP2α5△BAP2::loxP-KanMX-loxPThis study△GDH1α5△GDH1::loxP-KanMX-loxPThis study△ARO80α5△ARO80::loxP-KanMX-loxPThis study△CAN1α5△CAN1::loxP-KanMX-loxPThis study△BAT2α5△BAT2::loxP-KanMX-loxPThis study△GAD1α5△gad1::loxP-KanMX-loxPThis studyPlasmidpUG6Kanr, containingloxP-KanMX-loxP disruption cassetteGüldener et al., 2002


## 2.2. Medium and Culture Conditions

Yeast strains were cultured in yeast extract-peptide-dextrose (YEPD) medium (2% peptone, 2% glucose, and 1% yeast extract) in a 30°C incubator. The YEPD medium (Promega, Madison, WI, USA) with 1 mg/mL G418 antibiotic (Promega, Madison, WI, USA) was used to screen the transformed recombinant strains using theKanMX gene. All solid culture media were added with 2% agar powder (Solarbio, Beijing, China).Sorghum hydrolysate was used for the culture of primary and secondary seed cultures of yeast cells according to the method in Li et al.’s literature [27]. Firstly, sorghum powder was weighed, added with hot water (65°C) with a material-to-liquid ratio of 1 : 4, and stirred evenly for gelatinisation. Then, high-temperature-resistant α-amylase (the addition amount was 3 U/g, and enzyme activity was 2×104 U/mL) was added. The mash was kept in an 85–90°C water bath for 1 h to liquefy, during which the mash was stirred from time to time. Then, the temperature of mash was reduced to 60°C, and saccharifying enzyme (the addition amount was 90 U/g, and enzyme activity was 10 × 104 U/mL) was added. The mash was placed in a 55–60°C water bath for 20 h and stirred evenly. Then, saccharification was carried out. After saccharification, the temperature of the hydrolysate was reduced to 40°C, acid protease (the addition amount was 4 U/g, and enzyme activity was 2×104 U/mL) was added, and the mixture was let stand for 30 min. Lastly, the hydrolysate was filtered using two layers of gauze and adjusted with water until the sorghum juice had a sugar content of 20°Bx. Each 145 mL of sorghum juice was dispensed into a 250 mL flask, then sterilised at 115°C for 20 min and cooled to room temperature. The second precultured yeast (5 mL) was inoculated to the prepared sorghum hydrolysate medium with an inoculum density of approximately 5×108 cells/mL. All fermentations were performed in triplicate.


## 2.3. Construction of Recombinant Strain with Single Gene Deletion

Gene deletion in parental strainα5 was carried out by integrating the KanMX box, which was amplified by polymerase chain reaction (PCR) with target-K-U/target-K-D as primers and pUG6 plasmid as a template. The PCR primers are listed in Supplemental Table S1 and were designed on the basis of the S. cerevisiae S288c genome sequence in the National Centre for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). The upstream and downstream homologous fragments of the target gene were amplified by PCR using target-FA-U/target-FA-D and target-FB-U/target-FB-D as primers and α5 genome as a template, respectively.The homologous recombination of the fragment and yeast genome was implemented using the LiAc/SS carrier DNA/PEG method [28]. YEPD solid medium mixed with 1 mg/mL G418 was used to preliminary screen the transformants. The exact integration of the KanMX box in a single colony was verified by diagnostic oligonucleotide PCR with verification primers (target-1-U/-1-D and target-2-U/target-2-D). Then, the right single colony was selected for purification, and second validation was carried out. Finally, the correct single colony is the right recombinant strain we constructed.


## 2.4. RNA Extraction and Quantitative Real-Time PCR (RT-qPCR)

Total RNA was extracted by a yeast RNA isolation kit and then reverse-transcribed using a PrimeScript™ RT reagent kit. Changes in gene expression level were assessed by RT-qPCR with SYBR® Premix Ex Taq™ II test kit (Tli RNaseH Plus). The kits were purchased from Takara (Takara Biotechnology, Dalian, China). The PCR primers (target-U/target-D) listed in Supplementary TableS1 were synthesised by GENEWIZ (Suzhou, China). The PCR procedure was set according to that reported by Li and Chen [1]. The result was analysed quantitatively using 2−ΔΔCt method, and the actin gene (UBC6) was used as the housekeeping gene.


## 2.5. Growth Curve Determination

Yeast strains were inoculated into 5 mL of YEPD liquid culture medium and cultured in a 30°C shaking bed incubator for 12 h. Then, 40μL of yeast cell fluid was added into 360 μL of YEPD liquid medium, and the mixture was transferred to a 96-well plate. Optical density at 600 nm was measured every 30 min by a Bioscreen automated growth curve analysis system (OY Growth Curves Ab Ltd., Helsinki, Finland).


## 2.6. Fermentation Analysis

Fermentation experiment was carried out in a 250 mL flask with 150 mL of the separated sorghum hydrolysate. The second precultured yeast seed liquid was inoculated to the sorghum hydrolysate medium. The inoculum density was approximately5×108 cells/mL. Fermentation was carried out in a 30°C incubator, and the hydrolysate was weighed every 12 h. Fermentation was concluded when the weight loss was less than 0.1 g. The hydrolysate was distilled, and the content of higher alcohols and other flavour substances in the distillate was determined by gas chromatography method according to Ma et al.’s report [29].


## 2.7. Statistical Data Analysis

All experiments were repeated three times. The growth curves and other charts were drawn using the Origin 2018 software. The heat map was drawn using Excel 2016 and Adobe Illustrator CS4 software. Data calculation and processing were performed in Excel 2016, and the results are displayed as themean±standarddeviation. The significance of the difference between experimental and control groups was analysed using a t-test (★★P<0.01, ★P<0.05).


## 3. Results

### 3.1. Effect of Amino Acid Metabolism-Related Gene Deletion on the Growth and Fermentation Performance ofS. cerevisiae

The growth and fermentation performance ofS. cerevisiae were monitored during gene knockout. The growth curve, fermentation curve, and alcohol production are shown in Figures 2(a)–2(c), respectively. The growth rate of each recombinant strain showed a slight difference compared with that of the parent strain α5 (Figure 2(a)). The growth curve with larger change range compared with α5 was labelled with colour. The results in Figure 2(a) demonstrate that α5ΔCAN1 mutant entered the stable stage earlier, the final biomass of α5ΔARO80 was higher, and the final biomass of α5ΔGAT1 was lower, compared with those of α5. The growth curves of the other mutants were similar to that of α5. The results showed that the inactivation of these genes exerts no noticeable effect on the growth performance of S. cerevisiae. The loss of CO2 in the fermentation medium was weighed every 12 h during the whole fermentation process to assess the fermentation properties of the recombinant strains (Figure 2(b)). As described in Figure 2(b), the CO2 emission of the α5ΔCAN1 strain was the lowest amongst the strains, and the α5ΔGAT1 strain had lower CO2 emission compared with the parent strain α5. The CO2 emissions of the α5ΔCAN1 and α5ΔGAT1 strains decreased by 19.05% (i.e., 7.07 g) and 9.13% (i.e., 7.93 g) compared with that of the parent strain α5, respectively. The recombinant strain α5ΔGDH1 had the highest CO2 emission of 9.00 g, which was 3.09% higher than that of α5. In addition, the CO2 emission in most of the recombinant strains had no obvious difference with that of the parent strain α5. In the fermentation process, the weight loss of CO2 was reflected in the formation of alcohol. Significant differences were seen in the alcohol content amongst the recombinant strains and α5 (Figure 2(c)). As shown in Figure 2(c), the alcohol contents of α5ΔGAT1 and α5Δ CAN1 remarkably decreased by 14.34% (i.e., 7.07% (v/v)) and 5.45% (i.e., 7.80% (v/v)) compared with that of the original strain α5, respectively. The alcohol content of strain α5ΔGDH1 increased by 4.24% to 8.60% (v/v) compared with that of α5, and this result was consistent with the result of CO2 emission in Figure 2(b). The findings in Figure 2(c) indicate that GAT1, CAN1, and GDH1 deletions have different influences on the growth and fermentation performance of S. cerevisiae α5. α5ΔGAT1, α5ΔCAN1, and α5ΔGDH1 strains showed significant difference in alcohol content compared with that of α5. We consider that the reason for the difference in the alcohol consumption of the recombinant strains is the deletion of the gene related to amino acid transportation, which led to the diversity in the utilisation of amino acids by yeast and finally reflected in the discrepancy of alcohol yield.Figure 2
Growth and fermentation properties of recombinant strains andα5. (a) Growth curve of recombinant strains and α5. (b) The CO2 emission of recombinant strains and α5. (c) The alcohol content of recombinant strains and α5. The error bar represents the standard deviation of three repeated fermentation experiment data. The significant difference between the recombinant strain and the parent strain was tested and confirmed by Student’s t-test (★★P<0.01, ★P<0.05).
(a)(b)(c)


### 3.2. Effect of Amino Acid Metabolism-Related Gene Deletion on the Gene Expression Level inS. cerevisiae

The gene expression levels of the recombinant strains and the parent strainα5 were measured by RT-qPCR to explore the interaction amongst the genes (Figure 3). Diagonally, from the upper left corner to the lower right corner of Figure 3, we can see that gene knockout reduced the expression level of the knocked out gene in S. cerevisiae. Interestingly, amino acid metabolism-related genes had various expression levels in these recombinant strains. For example, the GDH1 gene was highly expressed in α5ΔGAT1 and α5ΔCAN1 strains. The expression of CAN1 and BAP2 genes was upregulated in α5△AGP1 strain compared with those in α5. Moreover, the expression of CAN1 and BAP2 was downregulated in strain α5ΔGDH1 compared with those in α5. BAT2 gene was highly expressed in the recombinant strain α5ΔCAN1 compared with that in α5. The discrepancy in the gene expression levels of different recombinant strains may have certain effects on their metabolism. Gene interaction changes the growth and fermentation properties of the recombinant strains and thus regulates the higher alcohol concentration in yeast.Figure 3
Determination of genes expression levels in recombinant strains andα5. The gene expression level in each recombinant strain was measured, compared with α5. With the change fold as the data, the heat map of the expression level of each gene in the recombinant strain was drawn by Excel 2016. Each row represents the expression level of each target gene in each recombinant strain, compared with the parent strain α5. Red indicates that the expression level of the target gene is upregulated, while green indicates that it is downregulated, respectively, compared with that of α5. The deeper the color, the larger the up/downregulation. Amongst them, the maximum upregulation multiple was 25.155, and the maximum downregulation multiple was 1.43923e−06, which was compared with α5.


### 3.3. Effect of Amino Acid Metabolism-Related Gene Deletion on the Formation of Higher Alcohols of Recombinant Strains

The concentrations of n-propanol, isobutanol, isoamyl alcohol, and 2-phenyl ethanol in recombinant strains andα5 were detected to study the influence of the knockout of amino acid metabolism-related genes on the production of higher alcohols. Remarkable differences were noticed in the higher alcohol concentrations amongst the recombinant strains and α5 (Figures 4(a)–4(d)). As shown in Figure 4(a), the concentration of n-propanol in strain α5ΔAGP1 increased by 8.02% to 13.06 mg/L compared with that of the parent strain α5. The n-propanol contents of the other recombinant strains presented decreased by varying degrees. Amongst them, the n-propanol content of strain α5ΔBAP2 decreased by 17.37% (i.e., 9.99 mg/L) compared with that of the parent strain α5. The production of n-propanol by AGP1 deletion haploid strain α5ΔAGP1 and BAP2 deletion haploid strain α5ΔBAP2 had significant differences. The isobutanol content of α5ΔCAN1 strain was the highest amongst the strains and reached 465.98 mg/L, which was higher by 596.64% compared with that of α5 (Figure 4(b)). In addition, as shown in Figure 4(b), the isobutanol contents of strains α5ΔGDH1 and α5ΔBAT2 decreased by 24.67% and 22.57% to 50.39 and 51.79 mg/L, respectively, compared with that of α5. Significant differences were found in the production of isobutanol by α5ΔAGP1 and α5ΔBAP2 compared with that of α5. The isoamyl alcohol content of BAT2 gene mutant decreased (Figure 4(c)), and this result was consistent with previously reported results [30]. The isoamyl alcohol contents of strains α5ΔGAT1 and α5ΔCAN1 increased by 19.75% (i.e., 322.91 mg/L) and 126.10% (i.e., 609.71 mg/L), respectively, compared with that of α5. There are significant differences in the production of isoamyl alcohol by α5△BAT2, α5△GAT1, and α5△CAN1, compared with that of α5. From Figures 4(b) and 4(c), we found that the amount of isobutanol and isoamyl alcohol in the single gene knockout recombinant strain had the same trend. It is noteworthy that the 2-phenyl ethanol content by the strain α5△ARO80 and α5△GDH1 decreased significantly by 50.72% (i.e., 64.59 mg/L) and 44.82% (i.e., 72.32 mg/L), respectively, compared with that of α5 (Figure 4(d)). Figure 4(d) also shows that the 2-phenyl ethanol content of recombinant strain α5ΔGAT1 increased by 46.16% to 191.56 mg/L compared with that of α5. Furthermore, similar to isobutanol and isoamyl alcohol yields, the 2-phenyl ethanol content of α5ΔCAN1 strain increased by 205.91% to 400.93 mg/L. The 2-phenyl ethanol production of α5ΔGAT1 and α5ΔCAN1 was significant different compared with that of α5.Figure 4
The formation of higher alcohol of recombinant strains andα5. (a) The n-propanol content in recombinant strains and the parental strain α5. (b) The isobutanol content of recombinant strains and α5. (c) The isoamyl alcohol content of recombinant strains and α5. (d) The 2-phenyl ethanol content of recombinant strains and α5. The error bar represents the standard deviation of three repeated fermentation experiment data. The significant difference between the recombinant strain and the parent strain was verified by Student’s t-test (★★P<0.01, ★P<0.05).
(a)(b)(c)(d)


### 3.4. Effect of Amino Acid Metabolism-Related Gene Deletion on the Formation of Total Higher Alcohols of Recombinant Strains

The impact of amino acid metabolism-related gene deletion on total higher alcohol concentration in eight recombinant strains was further analysed. Significant differences were noticed in the total higher alcohol contents amongst the recombinant strains andα5 (Figure 5). According to the content of total higher alcohols (Figure 5), isobutanol, isoamyl alcohol, and 2-phenyl ethanol are the main components of higher alcohols; therefore, the change in total higher alcohol content is consistent with the changes in the three higher alcohols. The total higher alcohol content of the α5△GDH1 strain decreased by 27.31% to 348.68 mg/L compared with that of α5. The total higher alcohol contents of α5ΔCAN1 and α5ΔGAT1 strains increased by 211.44% (i.e., 1493.96 mg/L) and 28.36% (i.e., 615.73 mg/L), respectively, compared with that of α5. Besides, the total higher alcohol contents of the other recombinant strains had little change.Figure 5
Content of total higher alcohols in the recombinant strains andα5. The significant difference between the recombinant strain and the parent strain was verified by Student’s t-test (★★P<0.01, ★P<0.05).


## 3.1. Effect of Amino Acid Metabolism-Related Gene Deletion on the Growth and Fermentation Performance ofS. cerevisiae

The growth and fermentation performance ofS. cerevisiae were monitored during gene knockout. The growth curve, fermentation curve, and alcohol production are shown in Figures 2(a)–2(c), respectively. The growth rate of each recombinant strain showed a slight difference compared with that of the parent strain α5 (Figure 2(a)). The growth curve with larger change range compared with α5 was labelled with colour. The results in Figure 2(a) demonstrate that α5ΔCAN1 mutant entered the stable stage earlier, the final biomass of α5ΔARO80 was higher, and the final biomass of α5ΔGAT1 was lower, compared with those of α5. The growth curves of the other mutants were similar to that of α5. The results showed that the inactivation of these genes exerts no noticeable effect on the growth performance of S. cerevisiae. The loss of CO2 in the fermentation medium was weighed every 12 h during the whole fermentation process to assess the fermentation properties of the recombinant strains (Figure 2(b)). As described in Figure 2(b), the CO2 emission of the α5ΔCAN1 strain was the lowest amongst the strains, and the α5ΔGAT1 strain had lower CO2 emission compared with the parent strain α5. The CO2 emissions of the α5ΔCAN1 and α5ΔGAT1 strains decreased by 19.05% (i.e., 7.07 g) and 9.13% (i.e., 7.93 g) compared with that of the parent strain α5, respectively. The recombinant strain α5ΔGDH1 had the highest CO2 emission of 9.00 g, which was 3.09% higher than that of α5. In addition, the CO2 emission in most of the recombinant strains had no obvious difference with that of the parent strain α5. In the fermentation process, the weight loss of CO2 was reflected in the formation of alcohol. Significant differences were seen in the alcohol content amongst the recombinant strains and α5 (Figure 2(c)). As shown in Figure 2(c), the alcohol contents of α5ΔGAT1 and α5Δ CAN1 remarkably decreased by 14.34% (i.e., 7.07% (v/v)) and 5.45% (i.e., 7.80% (v/v)) compared with that of the original strain α5, respectively. The alcohol content of strain α5ΔGDH1 increased by 4.24% to 8.60% (v/v) compared with that of α5, and this result was consistent with the result of CO2 emission in Figure 2(b). The findings in Figure 2(c) indicate that GAT1, CAN1, and GDH1 deletions have different influences on the growth and fermentation performance of S. cerevisiae α5. α5ΔGAT1, α5ΔCAN1, and α5ΔGDH1 strains showed significant difference in alcohol content compared with that of α5. We consider that the reason for the difference in the alcohol consumption of the recombinant strains is the deletion of the gene related to amino acid transportation, which led to the diversity in the utilisation of amino acids by yeast and finally reflected in the discrepancy of alcohol yield.Figure 2
Growth and fermentation properties of recombinant strains andα5. (a) Growth curve of recombinant strains and α5. (b) The CO2 emission of recombinant strains and α5. (c) The alcohol content of recombinant strains and α5. The error bar represents the standard deviation of three repeated fermentation experiment data. The significant difference between the recombinant strain and the parent strain was tested and confirmed by Student’s t-test (★★P<0.01, ★P<0.05).
(a)(b)(c)


## 3.2. Effect of Amino Acid Metabolism-Related Gene Deletion on the Gene Expression Level inS. cerevisiae

The gene expression levels of the recombinant strains and the parent strainα5 were measured by RT-qPCR to explore the interaction amongst the genes (Figure 3). Diagonally, from the upper left corner to the lower right corner of Figure 3, we can see that gene knockout reduced the expression level of the knocked out gene in S. cerevisiae. Interestingly, amino acid metabolism-related genes had various expression levels in these recombinant strains. For example, the GDH1 gene was highly expressed in α5ΔGAT1 and α5ΔCAN1 strains. The expression of CAN1 and BAP2 genes was upregulated in α5△AGP1 strain compared with those in α5. Moreover, the expression of CAN1 and BAP2 was downregulated in strain α5ΔGDH1 compared with those in α5. BAT2 gene was highly expressed in the recombinant strain α5ΔCAN1 compared with that in α5. The discrepancy in the gene expression levels of different recombinant strains may have certain effects on their metabolism. Gene interaction changes the growth and fermentation properties of the recombinant strains and thus regulates the higher alcohol concentration in yeast.Figure 3
Determination of genes expression levels in recombinant strains andα5. The gene expression level in each recombinant strain was measured, compared with α5. With the change fold as the data, the heat map of the expression level of each gene in the recombinant strain was drawn by Excel 2016. Each row represents the expression level of each target gene in each recombinant strain, compared with the parent strain α5. Red indicates that the expression level of the target gene is upregulated, while green indicates that it is downregulated, respectively, compared with that of α5. The deeper the color, the larger the up/downregulation. Amongst them, the maximum upregulation multiple was 25.155, and the maximum downregulation multiple was 1.43923e−06, which was compared with α5.


## 3.3. Effect of Amino Acid Metabolism-Related Gene Deletion on the Formation of Higher Alcohols of Recombinant Strains

The concentrations of n-propanol, isobutanol, isoamyl alcohol, and 2-phenyl ethanol in recombinant strains andα5 were detected to study the influence of the knockout of amino acid metabolism-related genes on the production of higher alcohols. Remarkable differences were noticed in the higher alcohol concentrations amongst the recombinant strains and α5 (Figures 4(a)–4(d)). As shown in Figure 4(a), the concentration of n-propanol in strain α5ΔAGP1 increased by 8.02% to 13.06 mg/L compared with that of the parent strain α5. The n-propanol contents of the other recombinant strains presented decreased by varying degrees. Amongst them, the n-propanol content of strain α5ΔBAP2 decreased by 17.37% (i.e., 9.99 mg/L) compared with that of the parent strain α5. The production of n-propanol by AGP1 deletion haploid strain α5ΔAGP1 and BAP2 deletion haploid strain α5ΔBAP2 had significant differences. The isobutanol content of α5ΔCAN1 strain was the highest amongst the strains and reached 465.98 mg/L, which was higher by 596.64% compared with that of α5 (Figure 4(b)). In addition, as shown in Figure 4(b), the isobutanol contents of strains α5ΔGDH1 and α5ΔBAT2 decreased by 24.67% and 22.57% to 50.39 and 51.79 mg/L, respectively, compared with that of α5. Significant differences were found in the production of isobutanol by α5ΔAGP1 and α5ΔBAP2 compared with that of α5. The isoamyl alcohol content of BAT2 gene mutant decreased (Figure 4(c)), and this result was consistent with previously reported results [30]. The isoamyl alcohol contents of strains α5ΔGAT1 and α5ΔCAN1 increased by 19.75% (i.e., 322.91 mg/L) and 126.10% (i.e., 609.71 mg/L), respectively, compared with that of α5. There are significant differences in the production of isoamyl alcohol by α5△BAT2, α5△GAT1, and α5△CAN1, compared with that of α5. From Figures 4(b) and 4(c), we found that the amount of isobutanol and isoamyl alcohol in the single gene knockout recombinant strain had the same trend. It is noteworthy that the 2-phenyl ethanol content by the strain α5△ARO80 and α5△GDH1 decreased significantly by 50.72% (i.e., 64.59 mg/L) and 44.82% (i.e., 72.32 mg/L), respectively, compared with that of α5 (Figure 4(d)). Figure 4(d) also shows that the 2-phenyl ethanol content of recombinant strain α5ΔGAT1 increased by 46.16% to 191.56 mg/L compared with that of α5. Furthermore, similar to isobutanol and isoamyl alcohol yields, the 2-phenyl ethanol content of α5ΔCAN1 strain increased by 205.91% to 400.93 mg/L. The 2-phenyl ethanol production of α5ΔGAT1 and α5ΔCAN1 was significant different compared with that of α5.Figure 4
The formation of higher alcohol of recombinant strains andα5. (a) The n-propanol content in recombinant strains and the parental strain α5. (b) The isobutanol content of recombinant strains and α5. (c) The isoamyl alcohol content of recombinant strains and α5. (d) The 2-phenyl ethanol content of recombinant strains and α5. The error bar represents the standard deviation of three repeated fermentation experiment data. The significant difference between the recombinant strain and the parent strain was verified by Student’s t-test (★★P<0.01, ★P<0.05).
(a)(b)(c)(d)


## 3.4. Effect of Amino Acid Metabolism-Related Gene Deletion on the Formation of Total Higher Alcohols of Recombinant Strains

The impact of amino acid metabolism-related gene deletion on total higher alcohol concentration in eight recombinant strains was further analysed. Significant differences were noticed in the total higher alcohol contents amongst the recombinant strains andα5 (Figure 5). According to the content of total higher alcohols (Figure 5), isobutanol, isoamyl alcohol, and 2-phenyl ethanol are the main components of higher alcohols; therefore, the change in total higher alcohol content is consistent with the changes in the three higher alcohols. The total higher alcohol content of the α5△GDH1 strain decreased by 27.31% to 348.68 mg/L compared with that of α5. The total higher alcohol contents of α5ΔCAN1 and α5ΔGAT1 strains increased by 211.44% (i.e., 1493.96 mg/L) and 28.36% (i.e., 615.73 mg/L), respectively, compared with that of α5. Besides, the total higher alcohol contents of the other recombinant strains had little change.Figure 5
Content of total higher alcohols in the recombinant strains andα5. The significant difference between the recombinant strain and the parent strain was verified by Student’s t-test (★★P<0.01, ★P<0.05).


## 4. Discussion

Higher alcohols produced by yeast have an important effect on the flavour and taste of alcoholic beverages [31, 32]. In the Ehrlich pathway, the utilisation of amino acids by yeast plays a critical role in the formation of higher alcohols [33]. In this research, we concentrated on the effect of the inactivation of genes related to amino acid metabolism on the growth, fermentation performance, and higher alcohol formation of S. cerevisiae. Eight mutants with single gene (GAT1, AGP1, BAP2, GDH1, ARO80, CAN1, BAT2, and GAD1) deletion were constructed. The gene expression level, growth properties, fermentation performance, and higher alcohol formation in each recombinant strain were determined. We reported for the first time that the inactivation of the GDH1 gene would reduce the total higher alcohol content in yeast and the knockout of CAN1 and GAT1 genes would increase the formation of total higher alcohols in S. cerevisiae. Moreover, we confirmed the influence of gene interaction on the yield of higher alcohols in S. cerevisiae.The alcohol concentration of theα5ΔGDH1 strain increased by 4.24% compared with that of α5 (Figure 2(c)). S. cerevisiae can use the nutrients in the culture medium for alcohol fermentation. The utilisation of nutrients directly affects the speed and yield of alcohol production by yeast [34, 35]. A study demonstrated that the inactivation of the GDH1 gene can eliminate the dependence of the glutamate metabolic pathway on NADPH and make yeast specifically dependent on the metabolism of NADH to change the metabolic flow of NADH in yeast cells, reduce the formation of glycerol in the fermentation process of S. cerevisiae, increase the conversion of carbon flow to ethanol, and improve the production of ethanol in the fermentation process by yeast [36]. Besides, according to the changes in the gene expression levels of amino acid metabolism-related genes in each recombinant strain, we believe that the formation of higher alcohols in S. cerevisiae is not the result of a single gene but the interaction between genes that regulate the formation of higher alcohols. For example, the knockout of the GDH1 gene will lead to the increase of alcohol content in yeast (Figure 2(c)); thus, the expression of the GDH1 gene is negatively related to alcohol production in yeast. We noticed that the GDH1 expression levels in α5ΔGAT1 and α5ΔCAN1 strains were substantially upregulated than that in α5 and the alcohol contents of α5ΔGAT1 and α5ΔCAN1 strains remarkably decreased (Figure 2(c)). Therefore, the expression of the GDH1 gene in α5ΔGAT1 and α5ΔCAN1 strains regulates alcohol production. We can explain the changes in fermentation performance caused by gene deletion by combining the gene expression levels in recombinant strains with the results of fermentation performance. The deletion of the GDH1 gene would reduce the formation of total higher alcohols by S. cerevisiae. Our result showed that the total higher alcohol content in α5ΔGDH1 strain decreased by 27.31% to 348.68 mg/L compared with that of α5 (Figure 5). GDH1 encodes glutamate dehydrogenase, and its transcription and expression are regulated by carbon and nitrogen sources. Leu3p and Gcn4p are the two key regulatory proteins that participate in the transcription regulation of BCAA metabolism in yeast. Leu3p is a pathway-specific regulator that regulates six genes involved in BCAA metabolism, including GDH1 [37]. We speculate that the knockout of GDH1 affects the metabolism of BCAA in yeast and leads to the change in higher alcohol production by yeast.Figure5 shows that the total higher alcohol content of α5ΔCAN1 increased by 211.44% to 1493.96 mg/L compared with that of α5. Notably, the knockout of CAN1 reduced the n-propanol content of the recombinant strain by 15.14% compared with that of the parent strain α5 (Figure 4(a)). By contrast, isobutanol, isoamyl alcohol, and 2-phenyl ethanol increased by 596.64%, 126.10%, and 205.91%, respectively, in α5ΔCAN1 compared with α5 (Figure 4(b)). The effect of CAN1 deletion on higher alcohol production by yeast is rarely reported. CAN1 encodes arginine transaminase, which regulates the utilisation of arginine by S. cerevisiae [38]. We suspect that the absence of CAN1 leads to the decrease in the utilisation rate of arginine in yeast, which affects the nitrogen metabolism of yeast and ultimately affects the fermentation performance and flavour quality of the final product. In addition, we noticed that the expression level of BAT2 was upregulated in the recombinant strain α5ΔCAN1 compared with α5 (Figure 3), and the isobutanol and isoamyl alcohol contents in α5ΔCAN1 strain were also higher than those of α5 (Figures 4(b)–4(c)). Therefore, according to the results of gene expression level and higher alcohol concentration in the recombinant strain, we speculate that the change in flavour substances in α5△CAN1 is caused by the knockout of CAN1 and the interaction of multiple genes, such as BAT2, which leads to the change in yeast fermentation performance. Interestingly, we also found that the n-propanol contents of strains α5ΔCAN1 and α5ΔBAP2 decreased substantially compared with that in α5 (Figure 4(a)). Hence, the downregulation of the expression of these two genes will reduce the production of n-propanol in yeast. We noticed that the n-propanol content in strain α5ΔAGP1 increased markedly compared with that in α5 (Figure 4(a)), and the expression level of CAN1 and BAP2 in α5ΔAGP1 was upregulated compared with those in α5 (Figure 3). We inferred that the rise in CAN1 and BAP2 expression led to the increase in n-propanol content of α5ΔAGP1. However, in α5ΔGDH1, the expression levels of CAN1 and BAP2 were downregulated than that of α5 (Figure 3), and the n-propanol content of α5ΔGDH1 was slightly higher than that of its parent strain α5 (Figure 4(a)), which was in contrast to the result of strain α5ΔAGP1. We consider that the expression of genes in the recombinant strain cannot explain the change in the fermentation performance of all yeast.The total higher alcohol content ofα5△GAT1 increased by 28.36% to 615.73 mg/L compared with that of α5. The effect of GAT1 deletion on the generation of higher alcohols in yeast has not been reported. GAT1 gene is the key regulator of nitrogen catabolism inhibition gene transcription in S. cerevisiae [39]. GAT1 was isolated in the cytoplasm when a good nitrogen source is added in the fermentation medium. By contrast, GAT1 is transferred to the nucleus when the poor nitrogen source is exhausted; this transfer leads to the activation of sensitive genes related to the inhibition of nitrogen catabolism [40]. This inhibition can inhibit the absorption of arginine and alanine by S. cerevisiae and stimulate the utilisation of BCAA and aromatic amino acids [40, 41]. We consider that GAT1 deletion leads to the failure of the activation of nitrogen metabolism, which affects the absorption and utilisation of nitrogen sources by yeast in the fermentation process and thus affects the generation of higher alcohols. Notably, the 2-phenyl ethanol content of α5ΔGAT1 increased by 46.16% to 191.56 mg/L compared with that of α5. We think that the consumption of aromatic amino acids by yeast leads to the increase in 2-phenyl ethanol synthesis. We also observed that the isobutanol and isoamyl alcohol contents of α5ΔBAT2 decreased by 22.57% and 22.29% compared with that of α5, respectively (Figures 4(b) and 4(c)). The BCAA aminotransferase encoded by BAT2 gene in yeast cytoplasm is responsible for catalysing the production of corresponding α-ketoacids from BCAA [12]. Li et al. took the recombinant strain α5-IAH1 as the parental strain and constructed a recombinant strain with BAT2 gene deletion and ATF1 gene overexpression simultaneously; the isobutanol and isoamyl alcohol content of this strain decreased by 59.24% and 70.89%, respectively, compared with that of the parent strain [27]. Styger et al. found that the inactivation of BAT2 makes a big difference on the formation of isobutanol and isoamyl alcohol in S. cerevisiae [42]. Their finding is consistent with our conclusion that the knockout of BAT2 gene can cut down the utilisation of BCAA in yeast and thus decrease the concentration of higher alcohols produced by yeast.The 2-phenyl ethanol content of strainα5ΔARO80 decreased substantially by 50.72% to 64.59 mg/L compared with that of α5. Aro80p, a transcription factor encoded by ARO80, responds to aromatic amino acids and activates ARO9 and ARO10 expression [43]. The absence of ARO80 will prevent the expression of ARO9 and ARO10 from being activated. Kim et al. overexpressed the transcription factor Aro80p in S. cerevisiae, which remarkably upregulated the expression levels of ARO9 and ARO10, and increased 2-phenyl ethanol production by 58% compared with those of the original strain [44]. The expression of ARO80 is positively correlated with the assimilation of aromatic amino acids. The upregulated expression level of ARO80 and the high 2-phenyl ethanol contents of α5ΔGAT1 and α5ΔCAN1 (Figures 3 and 4(d)) infer that the expression level of ARO80 in α5ΔGAT1 and α5ΔCAN1 increased and led to the increase in the 2-phenylethanol contents of α5ΔGAT1 and α5ΔCAN1. We speculate that single-gene deletion destroys the metabolism of one or several amino acids, but yeast may increase gene expression in other alternative pathways to make up for the decrease in nutrition utilisation caused by gene inactivation. The recombinant yeast strains with gene knockout and blocked expression may strengthen other metabolic pathways to compensate for the effect of gene deletion. The formation of flavour substances in S. cerevisiae is the result of the interaction of multiple genes, which is interesting and complex, and deserves further study. The interaction between genes helps us to understand the regulatory mechanism between amino acid transport and higher alcohol synthesis in S. cerevisiae and is also of great importance to guide in the engineering of bacteria with industrial application value.


## 5. Conclusion

Higher alcohol is one of the by-products produced by yeast in alcohol fermentation, and its content has an important impact on the flavour and taste of alcoholic beverages. The utilisation of amino acids by yeast plays an important role in the formation of higher alcohols. We studied the effects of amino acid metabolism-related gene deletion on yeast growth, fermentation performance, and higher alcohol formation. The interaction between amino acid metabolism-related genes was found and confirmed that gene interaction plays an important role in the regulation of higher alcohols. In particular, we found that the deletion ofCAN1 and GDH1 has an important effect on the higher alcohol content of S. cerevisiae. Our study provides valuable insights into the mechanism of higher alcohol production by S. cerevisiae in liquor fermentation. This study provides a new idea for the breeding of S. cerevisiae suitable for liquor fermentation.



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*Source: 6802512-2020-11-06.xml*