Construction of CRISPR/CAS9 system for industrial Saccharomyces cerevisiae strain and genetic manipulation effect on 2-phenylethanol pathway

The market demand for natural 2-phenylethanol (2-PE) continues to increase. Saccharomyces cerevisiae can synthesize 2-PE through the Ehrlich pathway. There are few studies on the improvement of the diploid industrial strains of S. cerevisiae by gene editing technology. There is no report on the comparison of genetic manipulation effect among S.cerevisiae strains with different 2-PE yield background, and the study on knockout of 2-PE downstream product synthesis gene and its effect on the yield of 2-PE have not been found.

There are precursors and energy shunts caused by two branch pathways during the synthesis of 2-PE. One is phenylacetaldehyde, the precursor of 2-PE, can be oxidized to phenylacetic acid under the action of acetaldehyde dehydrogenase [1]; The second is that the product 2-PE can be further converted into 2phenylacetoethyl ester under the action of acetyltransferase [12,27] (Fig. 1). In the oxidation process of phenylacetaldehyde, ALD3 and ALD2 genes play a key role [1,28], and acetyltransferase is encoded by ATF1. Bosu Kim et al. Knocked out ALD3 and ALD2 of S.cerevisiae W303, and when 83 mg/L L-Phe was used as substrate, the nal 2-PE yield increased by about 35% compared with that of wild type [1].
Overexpression of AAP9, ARO9 and ARO10 and knockout ALD2 and ALD3 in C.glycerinogenes resulted in an increase of 2-PE production by 38.9% to 5g/L [29]. However, the blocking of the consumption pathway of 2-PE catalyzed by ATF1 has not been reported yet.
CRISPR/CAS9 gene editing technology has been widely used in genetic engineering of microorganisms including S.cerevisiae for advantages of high e ciency and the ability to edit multiple genes at the same time [30,31]. Compared with haploid laboratory yeast strains, diploid industrial S.cerevisiae strains are more robust and characterised by high levels of genomic diversity, which makes genetic engineering di cult [30]. Although CRISPR/CAS9 gene editing technology has been applied to laboratory yeast [32,33] and industrial yeast [30], the editing e ciency in industrial yeast is still at a low level. Recent studies on 2-PE production by S. cerevisiae, YS58 strain [22], S288c strain [13]and W303 strain [1] are mostly haploid experimental strains, and there are few studies on diploid strains for industrial production. Therefore, it is necessary to explore to improve the gene editing e ciency of industrial yeast in order to carry out genetic engineering of strains.
2-PE can increase the uidity of cell membranes, reduce the uptake of glucose and amino acids, and inhibit the growth of S. cerevisiae through induction of insu cient respiration [34], and its toxicity to cells becomes the main bottleneck to further increase its production. Generally, diploid strains of S.cerevisiae used for industrial production are more stress-resistant than laboratory strains and have excellent fermentation performance [35,36]. Therefore, on the basis of industrial production strains with a certain 2-PE tolerance, the use of gene editing technology for precise modi cation of metabolic pathways is an effective strategy to further increase yield. In addition, the level of NADH in the cell re ects the redox level in the cell, which plays an important role in the growth and metabolism of the cell [37,38], so the regeneration of the reducing power in the cell has also become an important method for regulating cell metabolism.
In the previous research of our group, we obtained 2-PE-producing diploid S. cerevisiae CWY-132 and realized industrial production. Through the optimization of medium composition, culture conditions and fermentation conversion process, the 2-PE level reached 3.98 g/L in shake ask [39] and 4.1 g/L in the small-scale fermentation tank [40]. This study established and optimized the CRISPR/CAS9 gene editing system of CWY-132 with 100% e ciency when manipulate Ehrlich pathway genes. This system was used for comparative study of the performances of genes manipulations related in 2-PE pathway in different production strains, and the effect of ATF1 gene knockout was reported for the rst time. In addition, the effects of cell tolerance to 2-PE and the supply of NADH on 2-PE production were also discussed.

Results And Discussion
Construction of CRISPR/CAS9 system in industrial diploid S.cerevisiae The CRISPR/CAS9 gene editing system of S.cerevisiae has been successfully established and applied, but the editing e ciency in industrial diploid strains is still not high. Stovicek et al. constructed the CRISPR/CAS9 system in industrial yeast, but the editing e ciency was only 65%~78% [30]. Therefore, it is necessary to greatly improve the gene editing e ciency of industrial S.cerevisiae strains.
The CRISPR/CAS9 system mainly includes three parts: sgRNA, Cas9 protein, and Donor DNA. The PAM site on the target gene is used as a guide mark. The sgRNA sequence can bind to the rst 20bp of the PAM site, and then guide the Cas9 protein to cut this site. Then homologous recombination repair is performed through Donor DNA to achieve targeted editing of the target gene. In this paper, a highe ciency CRISPR/CAS9 system was constructed in the industrial strain CWY132 of 2-PE, and it was found that the construction method of gRNA plasmid and the length of the Donor DNA homology arm affect the editing e ciency. Two strategies were used to construct gRNA plasmids (Fig. 2a). One was to amplify the gRNA plasmid backbone into three fragments containing homologous arms to make it homologous recombined into a new plasmid(program 1). Second, the complete gRNA plasmid was constructed in vitro by DPN I-mediated reverse PCR(program 2). The results show that the gRNA plasmid construction method shown in program 2 can greatly increase the transformation e ciency. The number of transformants is increased from zero to about hundreds in each plate. When constructing Donor DNA, ATF1 and ALD3 were used as the target genes to be knocked out, and homology arms of 60 bp, 100 bp, 500bp, and 1000 bp were set respectively. The results show that when the length of the homology arm was 60 bp, the target gene editing e ciency is 0, and when the length reached 500bp, the editing e ciency can reach 100% (Fig. 2b). In addition, the effect of different PAM sites on the system e ciency was also studied. When the homology arm length of Donor DNA is 500 bp, its knockout rate of ATF1 reached 100% at all the selected PAM sites.
In this research, we successfully constructed the CRISPR/CAS9 system in industrial diploid S.cerevisiae. The transformation e ciency was improved by optimized plasmid construction method, and the gene editing e ciency was signi cantly improved to and reached to 100% by increasing the length of the Donor DNA homology arm, and we also found that the PAM site does not affect the editing e ciency. This system with high gene editing e ciency could provide technical support for genetic manipulation in the industrial S.cerevisiae strain.
The effect of knocking out ALD2, ALD3, ATF1 on the 2-PE and ethanol production of different strains 2-PE synthesis was shunted to branch pathways in S.cerevisiae (Fig. 1). The 2-PE-producing industrial diploid S.cerevisiae strain CWY-132 and the laboratory haploid strain PK2-C were used to analyze the function of the branch pathway acetaldehyde dehydrogenase gene ALD2 ALD3 and acetyltransferase gene ATF1 of the 2-PE metabolic pathway. By using CRISPR/CAS9 technology, the above genes were single knocked out, double knocked out and overexpressed to see their changes in 2-PE and ethanol production. The knockout e ciency of these genes has reached 100%, further verifying the high e ciency of this gene editing system we established.
The 2-PE and ethanol yields of wild-type and gene-edited mutant strains were tested to study the functions and interaction effects of different genes. 5g/L L-Phe was used as a substrate for fermentation culture, and the supernatant was taken after 36 h of continuous fermentation to detect its 2-PE and ethanol content. The results showed that when inhibiting the branching pathway of acetaldehyde dehydrogenase, in the CWY-132 strain, the 2-PE yields of ALD2∆ and ALD3∆ strains were 3.02 g/L and 2.93 g/L, respectively, which were compared with 3.5 g/L of the wild type, it dropped by 14%. The 2-PE yield of the ALD2∆ALD3∆ strain was 1.65 g/L, which was about 52.8% lower than that of the wild type (Fig. 3a); In the haploid laboratory strain PK2-C, the 2-PE yields of ALD2∆ and ALD3∆ strains were 1.20g/L and 0.34g/L, respectively, which increased by about 471% and 62% respectively compared with the wild type (Fig. 3b). When inhibiting the downstream acetyltransferase gene ATF1 of 2-PE synthesis, the 2-PE yields of ATF1∆ and ATF1∆ALD3∆ of CWY-132 strain were 0.83g/L and 0.85g/L, respectively, compared with the wild type, it is reduced by about 76% (Fig. 3a). The 2-PE yield of the ATF1∆ strain of PK2-C was 0.45 g/L, which was 114% higher than the wild-type yield of 0.21 g/L (Fig. 3b).
2-PE is toxic to S.cerevisiae cells, and the combined action of ethanol can enhance the inhibition effect on the growth [34].The ethanol production of each strain was also tested and found that in CWY-132, the ALD2∆ and ALD3∆ strains were close to the wild type, and the ALD2∆ALD3∆ strain increased by about 45% compared with the wild type. In ATF1∆ and ATF1∆ALD3∆ strains, the ethanol production increased by 128% and 146% ,respectively, compared with the wild-type strain (Fig. 3a); Among the PK2-C strains, the ethanol production in ALD2∆ and ALD3∆ strains are close to the wild type, and the ATF1∆ strain is about 30% higher than the wild type ( Fig. 3b). In the CWY-132 strains with overexpression of ALD3 or ATF1, the production of 2-PE and ethanol decreased slightly (Fig. 3c).
These results demonstrate that the blocking of branch pathways in the 2-PE high-yielding industrial strain CWY-132 failed to increase the 2-PE production, on the contrary, the yield decreased. ATF1∆ and the double mutant strains ALD2∆ALD3∆ and ATF1∆ALD3∆ decreased more signi cantly. To investigate these unexpected results, the low-yielding strain PK2-C was used for further study. The results showed that the yield of 2-PE increased when these genes was knocked out, which was consistent with the results of reported studies. For example, Bosu Kim et al. found that the 2-PE yield of haploid laboratory S.cerevisiae strain W303 that knocked out ALD3 and ALD2 was 35% higher than that of wild type [1]. These results prove that in the regulation of 2-PE biosynthesis, by blocking the branch pathway and downstream product synthase, the effect is different in strains with different yield backgrounds.
The knock-out effect on the synthase acetyltransferase of the downstream substance of 2-PE is more obvious. In the CWY-132 strain of ATF1 gene deletion, its colony on the plate is smaller and grow more slowly in liquid media when compared with the wild-type strain (Fig. 4a 4b), and the 2-PE production is greatly reduced, indicating that ATF1 involved in yeast growth and plays a crucial role in regulation of 2-PE catalytic conversion. Studies by other groups have shown that the acetyltransferase encoded by S.cerevisiae ATF1 is a key enzyme in acetate synthesis [41],and deletion or overexpression of ATF1 will signi cantly affect the production of alcohols and acetates [42,43].
The synthesis level of alcohols in S.cerevisiae is related, and the yeast performs coordinated regulation. The results of this study show that in different strains of relatively high-yield of 2-PE, the increase or decrease of 2-PE production is often accompanied by corresponding changes in ethanol, and the trend is generally opposite. For example, in the CWY-132 strain, the 2-PE dropped signi cantly after the ATF1 gene was knocked out, while the ethanol increased signi cantly. The ALD2∆ALD3∆ strain also has a similar situation. However, this phenomenon did not occur in the low-yield haploid strain PK-2C, indicating that yeast cells have global regulation of the total alcohols stress substances in the cell to reduce cytotoxicity.
The effect of initial L-Phe concentration, correlation between 2-PE and ethanol, and NADH on 2-PE production With CWY-132 as the starting strain, the initial addition amount of L-Phe was set at 0.1g/L, 1g/L, 3g/L and 5g/L. The 2-PE production of different engineered yeast strains under L-Phe concentrations was studied. The result showed that the 2-PE production of ALD2∆, ALD3∆ and ALD2∆ALD3∆ strains increased by 20%, 23% and 29% respectively under the substrate of 1g/L L-Phe (Fig. 5a). When L-Phe concentration was increased to 3g/L, the 2-PE decreased compared with the wild type (Fig. 5a). In particular, the ALD2∆ALD3∆ strain reduced the 2-PE production by nearly 50% under the 5g/L L-Phe substrate (Fig. 5a). However, the 2-PE production of ATF1∆ and ATF1∆ALD3∆ strains at the addition of 1g/L, 3g/L and 5g/L L-Phe substrate has been at a very low level (Fig. 5a).
It can be seen from the metabolic pathway ( Fig. 1) that there is a certain connection between the ethanol and the 2-PE metabolic pathway. Ethanol can be converted into ethyl acetate catalyzed by acetyltransferase, and acetaldehyde can be converted into acetic acid by acetaldehyde dehydrogenase. Therefore, when these two enzymes are knocked out, the ethanol content is greatly increased. The results of L-Phe addition study further veri ed that the production of 2-PE and ethanol are always in a negative correlation (Fig. 5b 5c). On the basis of the ALD2∆ALD3∆ strain, the pyruvate decarboxylase PDC1 in the ethanol metabolism pathway was further knocked out, and the result found that ethanol production decreased signi cantly, while 2-PE production increased by 50% (Fig. 3a). These results further indicate that 2-PE is in competition with ethanol synthesis, and the increase in ethanol production will lead to a decrease in 2-PE production. The initial 2-PE yield of strain CWY-132 reached 3.4 g/L, and the tolerance of this strain to 2-PE on the plate was also 3.4 g/L (Fig. 6a). The dual toxicity of ethanol and 2-PE to the strain further damages the viability of the strain, which in turn affects the yield of 2-PE.
Study has shown that glutathione (GSH) can enhance the tolerance of Candida glycerinogenes to 2-PE [44]. In order to verify that increasing the 2-PE tolerance of S. cerevisiae can increase the yield, the addition of 0.1 mM GSH to the culture media increased the tolerance of CWY-132 to 2-PE from 3.4 g/L to 3.6 g/L (Fig. 6a). Adding 0.1 mM GSH during the fermentation process can increase the production of 2-PE by about 10% in the WT, ALD2∆, and ALD3∆ of CWY-132 (Fig. 6b). The result suggests that the toxic effect of 2-PE is a key limiting factor affecting the further increase of 2-PE production.
The growth of CWY-132 mutants and wild type was compared. The growth curve measurement and YPD plate streaking culture results showed that the growth of the ATF1∆ strain and the ALD3∆ATF1∆ strain was more weakened compared with the wild type, and the biomass was signi cantly reduced. The growth of ALD2∆ or ALD3∆ strain showed no signi cant changes, but the biomass of ALD2∆ALD3∆ strain decreased (Fig. 4a 4b). The growth and biomass of PK2-C mutant strains did not change (Fig. 4c 4d). These results indicate that blocking the branch pathways in the 2-PE synthesis pathway, especially the terminal shunt pathway, has an inhibitory effect on the growth of industrial diploid S. cerevisiae with high production of 2-PE.
The redox level re ects some metabolic activities related to cell growth and biosynthesis, and controls cell metabolism. The levels of NAD + and NADH are key indicators of redox status [38]. When the production of ethanol increases, it can oxidize NADH in the cell and break the balance of NADH/NAD + in the cell [37,45], resulting in slow cell growth and further affecting the production of 2-PE. Measurement of the NADH content in different strains constructed, we found that the NADH content of the ALD2∆ALD3∆, ATF1∆ and ATF1∆ALD3∆ strains all decreased by 10%-15% compared with the wild type ( Fig. 6c). After 2g/L NAD + was added to the fermentation process, the 2-PE yield increased by 10% (Fig. 6b). Therefore we speculated that the metabolism of ethanol and 2-PE in S.cerevisiae strains have something in common. The blocking of the 2-PE synthesis branch pathway leads to an increase in ethanol production, which in turn leads to an imbalance between NADH/NAD + in the cell. Dysregulation causes weakened cell growth, which in turn reduces 2-PE production.
These results indicate that strains with different yield backgrounds should be optimized for the amount of substrate L-Phe. For strains that require a higher concentration of L-Phe, the 2-PE yield cannot be increased or even decreased after knocking out the shunted genes. The results also indicate that ethanol and 2-PE exert dual toxicity to S. cerevisiae, which suggests that the overcome of the low tolerance of strain to 2-PE is a key problem. Construction of tolerance improved strains could be an effective strategy in further increasing the production of 2-PE. In addition, redox state should also be considered to improve the supply of NAD + .

Conclusions
In this study, a CRISPR/CAS9 gene editing system was constructed in the diploid S.cerevisiae that produces 2-PE, and the editing e ciency reached 100% after optimization. When using this system to knock out the branch pathway of the Ehrlich pathway related genes of the 2-PE synthesis of different strains, it is found that the effect is quite different in different strains. The production of 2-PE decreased in the industrial strains, while increased in the low-yielding haploid laboratory strains due to the blocking of the branching pathway. These results suggest that the future application of gene editing strategies to improve the production of S.cerevisiae 2-PE should fully consider the strain background. In addition, the dual toxicity of 2-PE and ethanol to S.cerevisiae is an important obstacle to the continued synthesis of 2-PE. Enhancing the strain's tolerance to 2-PE is effective strategy in future for 2-PE strain improvement and production. Moreover, it is also necessary to comprehensively consider the supply of the cell NADH/NAD + in catalytic conversion for the 2-PE synthesis. This study deepened the understanding of the regulation of 2-PE synthesis by S.cerevisiae, and provided strategies and references for further development of new strategies for high-yield strain breeding.

Materials And Methods
Yeast strains and culture conditions The S. cerevisiae strains used in this study are shown in Table 1. S. cerevisiae CWY-132 was a industrial strain. Yeast strains were cultured at a constant temperature of 30°C in a complete medium YPD (1% yeast extract, 2% peptone, 2% glucose, if making a solid medium, add 2% agar powder). The overnight cultured seed culture solution was inoculated into the fermentation medium (5g/L L-phenylalanine ,30g/L glucose, 0.5g/L magnesium sulfate, 5g/L potassium dihydrogen phosphate, 1.5g/L yeast extract) at cell densities of 10 7 cells/mL, and the lling amount was 50mL/250mL, and the fermentation was continued for 36 h at 30°C and 200 rpm.

2-PE tolerance determination
The tolerance of 2-PE was analyzed by dilution gradient plate method. Dilute the exponentially growing S.cerevisiae cells to 10 7 cells/ml, and then spot 5 μL of 10-fold serial cell dilution on solid YPD plates containing 3.2 g/L, 3.4 g/L, and 3.6 g/L 2-PE. Cultivate at a constant temperature of 30°C.
Construction of CRISPR/CAS9 system All plasmids used in this study are shown in Table 1, and primers are shown in Table 2. The Cas9 plasmid was transformed into S. cerevisiae CWY-132 and PK2-C respectively, and then the transformants containing the Cas9 plasmid were re-used as the starting strain for the next step of gene manipulation. The PAM site is obtained through the website http://yeastriction.tnw.tudelft.nl . The gRNA plasmids used in this study were obtained by inverse PCR (Fig. 2a). Take the construction of gRNA plasmids when ALD3 is knocked out as an example. Using the original gRNA plasmids as templates, PCR was performed with primers ALD3 g-F and ALD3 g-R. The resulting PCR reaction solution was subjected to template elimination treatment with Dpn I enzyme, and then transformed into E. coli Dh5α after DNA puri cation, and cultured in selective medium overnight. The obtained plasmid was con rmed by sequencing and used for transformation. The other gRNA plasmids are obtained in the same way. When constructing DONOR DNA, homology arm lengths of 60 bp, 100 bp, 500 bp, and 1000 bp were set respectively. The fragments of 60 bp and 100 bp homology arm length were chemically synthesized, and the homology arm lengths of 500 bp and 1000 bp were obtained by overlap extension PCR. Take the 500bp Donor DNA homology arm of ALD3 gene as an example. First, the wild-type S.cerevisiae genome was used as a template, and the two homologous arm fragments on the left and right were ampli ed with primer pairs ALD3 L500-F, ALD3 L-R and ALD3 R-F, ALD3 R500-R, respectively. The fragment obtained is recovered and puri ed by gel and used as a template again. The primers ALD3 L500-F and ALD3 R500-R are used for another round of overlap extension PCR to connect to complete Donor DNA (Fig. 7a). Subsequently, the constructed donor DNA and gRNA plasmid were co-transformed into S.cerevisiae strains containing Cas9, positive transformants on dual antibiotic plates containing hygromycin and G418 were selected. The gene deletion or integration in transformants was determined by PCR. The general operation ow of the CRISPR/CAS9 system is shown in Fig 7b.

Yeast transformation
The plasmids and DNA fragments were transformed into yeast cells by the PEG/lithium acetate method [46]. Transformed yeasts were cultured in YPD medium supplemented with 40 μg/mL G418 sulfate and 80 μg/mL hygromycin for selection.
Analytical method for 2-PE Take out 1mL of fermentation broth, 10000 r/min, centrifuge for 10 minutes, take the supernatant liquid and analyze by gas chromatography(GC). Sample pretreatment: add 200 uL internal standard solution (methyl isobutyl methanol 1 g/L aqueous solution) to the supernatant, add 500uL ethyl acetate, shake and mix, centrifuge at 5000 rpm/min for 1 min, take the upper organic phase perform testing. Standard curve preparation: prepare 0.5, 1.0, 5.0, 10.0 g/L of 2-phenylethanol aqueous solution of standard concentration, and treat as above. The determination conditions for analyzing the concentration of 2-PE in the fermentation broth by GC are: Shimadzu GC2014 gas chromatograph; FID detector; column Rtx-1 (30m*0.25mm*0.25um, Restek); carrier gas is nitrogen; split ow; The split ratio is 49.0; the column ow rate is 1.45 mL/min; the purge ow rate is 3 mL/min; the temperature of detector is 250°C; the oven is maintained at 80°C for 1 min, and then increased to 200°C by 20°C/min; the sample volume is 1 uL.

NADH detection
The double antibody sandwich method was used to determine the level of microbial nicotinamide adenine dinucleotide (NADH) in S.cerevisiae. Collect the cells through centrifuge and discard the supernatant. The ratio of extract solution (volume) to cell number (10 4 cells) was 500:1. The cells are broken by ultrasound (ice bath, 200W, ultrasound 3s, repeat 30 times at an interval of 10s), centrifuge at 8000g for 10 min at 4°C, take the supernatant, and put it on ice for testing. Coat the microwell plate with puri ed microbial NADH antibody to make solid phase antibody, and add NADH to the micro-wells of the coated monoclonal antibody in sequence, and then combined with HRP-labeled NADH antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme and into the nal yellow under the action of acid. The color intensity is positively correlated with the microbial nicotinamide adenine dinucleotide (NADH) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of microbial nicotinamide adenine dinucleotide (NADH) in the sample was calculated from the standard curve.

Plasmid elimination
In this study, the elimination of gRNA plasmid and Cas9 plasmid was achieved by passage. The initial yeast was cultured overnight in 5 mL YPD liquid without any antibiotics, and then passaged every 12h.
After fth Generation, the plasmid elimination rate can reach more than 90%.

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Informed consent for publication was obtained from all participants.

Availability of data and materials
All data generated or analyzed during this study are included in this article.

Competing interests
No competing interests.   F   TCCAAAACATACATTCTTCAGTATC  CACATGAAAATTTTTAAAGGTATGG  GCTACCTCTTAATGTGTCACAAG  CTTACATAATGATAACTATCACATTTTCTTTTGGCTTATTTTCACG  CGTGAAAATAAGCCAAAAGAAAATGTGATAGTTATCATTATGTAAG  TTACTAATTCCTTAACCCTTAGG  CTGACACCCGGATAATTAAGAAGTG  TGGTATAATCAAGCGTATTTAGAAG  ATATCAGTCAAGCATCATGTGAGATGAGAGCTGATAAATTGATGGTATTT  AAATACCATCAATTTATCAGCTCTCATCTCACATGATGCTTGACTGATAT  GCATTTGTTTGTAGCTTTGCATTG  ATAGCATGCTCCTATTATAGATGTG  GTATGCTCTTCTGACTTTTCGTG  AACTAATAATTAGAGATTAAATCGCTTTTGATTGATTTGACTGTGTTA  TAACACAGTCAAATCAATCAAAAGCGATTTAATCTCTAATTATTAGTT     a Two methods of gRNA plasmid construction, the left is the scheme one, the right is the scheme two. b The in uence of the length of the DONOR DNA homology arm on the editing e ciency of the CRISPR/CAS9 gene editing system. The data represents the average of three independent experiments, and the deviation of the average is between 5% and 10%. The asterisk indicates a signi cant difference when p<0.05, which is based on the analysis of variance statistical test. 2-PE and ethanol production of wild-type strains and engineered strains. a The 2-PE production and ethanol production of wild-type (CWY-132) and ALD2∆, ALD3∆, ALD2∆ALD3∆, ALD2∆ALD3∆PDC1∆, ATF1∆, ATF1∆ALD3∆ strains. b The 2-PE production ethanol production of wild type (PK2-C) and ALD2∆, ALD3∆ and ATF1∆ strains. c 2-PE production and ethanol production of wild-type (CWY-132) and over-expressing ATF1 and over-expressing ALD3 strains. The data represents the average of three independent experiments, and the deviation of the average is between 5% and 10%. The asterisk indicates that when p<0.05, the 2-PE and ethanol production of wild-type and mutant strains are signi cantly different, which is based on the statistical test of variance analysis.