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 efficiency in industrial diploid strains is still not high. Stovicek et al. constructed the CRISPR/CAS9 system in industrial yeast, but the editing efficiency was only 65%~78%. Therefore, it is necessary to greatly improve the gene editing efficiency 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 first 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 high-efficiency 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 efficiency. 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 efficiency. 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 efficiency is 0, and when the length reached 500bp, the editing efficiency can reach 100% (Fig. 2b). In addition, the effect of different PAM sites on the system efficiency 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 efficiency was improved by optimized plasmid construction method, and the gene editing efficiency was significantly 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 efficiency. This system with high gene editing efficiency 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 efficiency of these genes has reached 100%, further verifying the high efficiency 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.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 significantly. 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. 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,and deletion or overexpression of ATF1 will significantly 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 significantly after the ATF1 gene was knocked out, while the ethanol increased significantly. 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 verified 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 significantly, 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. 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 significantly reduced. The growth of ALD2∆ or ALD3∆ strain showed no significant 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 reflects 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. 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+.