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Transcriptome analysis and reverse engineering verification of SNZ3Val125Ile and Pho3Asn134Asp revealed the mechanism of adaptive laboratory evolution to increase the yield of tyrosol in Saccharomyces cerevisiae strain S26-AE2

Biotechnology for Biofuels and Bioproducts volume 18, Article number: 29 (2025) Cite this article

Abstract

Background

Tyrosol is an important drug precursor, and Saccharomyces cerevisiae is one of the main microorganisms that produces tyrosol. Although excessive metabolic modification increases the production of tyrosol, it also causes a decrease in the growth rate of yeast. Therefore, this study attempted to restore the growth of S. cerevisiae through adaptive evolution and further improve tyrosol production.

Results

After the adaptive laboratory evolution of S. cerevisiae S26, three evolutionary strains were obtained. The biomass of strain S26-AE2 reached 17.82 g DCW/L in the presence of 100 g/L glucose, which was 15.33% higher than that of S26, and its tyrosol production reached 817.83 mg/L. The transcriptome analysis revealed that, upon exposure to 100 g/L glucose, the S26-AE2 strain may reduce the transcriptional regulation of glucose repression through decreased HXK2 expression. The expression of genes related to pyruvate synthesis was increased in strain S26-AE2. Meanwhile, the expression levels of most tricarboxylic acid cycle-related genes in S26-AE2 were increased when cultured with 20 g/L glucose. Furthermore, the amount of tyrosol produced by strain S26 with the SNZ3Val125Ile mutation increased by 17.01% compared with that of the control strain S26 following exposure to 100 g/L glucose.

Conclusions

In this study, a strain, S26-AE2, with good growth and tyrosol production performance was obtained by adaptive evolution. The transcriptome analysis revealed that the differences in the expression of genes involved in metabolic pathways in adaptive evolutionary strains may be related to yeast growth and tyrosol production. Further reverse engineering verified that the mutation of SNZ3 promoted tyrosol synthesis in S. cerevisiae in glucose-rich medium. This study provides a theoretical basis for the metabolic engineering of S. cerevisiae to synthesise tyrosol and its derivatives.

Graphical Abstract

Background

Tyrosol is the precursor of salidroside and hydroxytyrosol and has various pharmaceutical activities, such as anticancer, antioxidant, and anti-inflammatory activities [1, 2]. It mainly exists in olive oil, wine, and plant tissues and is a phenethyl alcohol derivative. At present, the commercial production of tyrosol mainly depends on chemical synthesis and olive leaf extraction [3,4,5]. However, the olive leaf extraction method faces the problem of a low extraction efficiency, and its production is highly dependent on the supply of olive leaf resources. More importantly, both methods are not environmentally friendly, whether extraction or chemical synthesis. In contrast, microbial cell factories exhibit many characteristics that are conducive to the low-cost, environmentally friendly, and sustainable production of tyrosol [6]. In recent years, Saccharomyces cerevisiae and E. coli have been successfully used to produce tyrosol [7,8,9,10]. These studies revealed that the tyrosol concentration was 1.3 g/L in S. cerevisiae [11] and 2.12 g/L in E. coli [12]. Although a certain amount of high yield has been achieved in S. cerevisiae, with increasing modification, the robustness of the yeast is reduced. In previous work, a strain of S. cerevisiae S26 with a high yield of tyrosol was constructed. However, excessive modification decreases the vigorous growth of yeast, which greatly limits its application in fermentation tanks and industrial production.

Adaptive laboratory evolution (ALE) is an efficient method for obtaining strains with improved traits [13]. Through natural selection, many mutants accumulate to quickly obtain strains with ideal characteristics. ALE has been applied to optimise the growth rate of various industrial-related microbial species, to improve the growth defects of engineered strains, and to study how and why the growth rate changes during evolution [14]. ALE can also be combined with a bioinformatics analysis and reverse engineering to understand the basic mechanism of molecular evolution and the adaptive changes that accumulate in the long-term selection process of microbial populations under specific growth conditions. Zongjie Dai et al. combined rational design, adaptive laboratory evolution, and reverse engineering, and observed that the specific growth rate of the best strain reached 0.218 h−1, which is close to the maximum growth rate of S. cerevisiae with purely respiratory metabolism [15]. Henri Ingelman et al. used three different and independent adaptive laboratory evolution strategies to evolve wild-type Clostridium autoethanogenum to grow faster without yeast extract and to be robust in operating continuous bioreactor cultures [16]. Kuk-Ki Hong et al. improved the growth rate of S. cerevisiae under specific conditions of galactose supplementation by adaptive evolution [17]. Ryan A. LaCroix et al. used adaptive laboratory evolution and observed that key mutations caused E. coli K-12 MG1655 to grow rapidly on glucose-based media [18]. Eugen Pfeifer et al. performed a comparative adaptive laboratory evolution experiment on Corynebacterium glutamicum ATCC 13032 and its phage-free variant MB001, thereby increasing the growth rate on the medium with the lowest glucose concentration [19].

In this study, S. cerevisiae S26 was used as the starting strain for laboratory evolution [20]. Throughout the evolutionary process, the glucose concentration progressively increased. Three strains, S26-AE1, S26-AE2, and S26-AE3, were isolated during the evolutionary phase, and strain S26-AE2, which exhibited superior fermentation characteristics and a high yield of tyrosol, was selected for further investigation. The transcriptome analysis of the evolutionary strain S26-AE2 revealed changes in the expression of genes involved in the metabolic pathway of yeast after evolution. Increases in the activity of the TCA cycle and aromatic amino acid gene expression may affect the growth recovery of S. cerevisiae and increase the tyrosol content. The single-nucleotide polymorphism (SNP) mutations PHO3Asn134Asp and SNZ3Val125Ile were screened, and reverse engineering verification was performed. The results showed that the SNZ3Val125Ile mutation was positively correlated with tyrosol production in yeast. These results provide ideas for obtaining more robust industrial S. cerevisiae strains and a theoretical basis for further modification of the strain to increase tyrosol production.

Results

Adaptive laboratory evolution of S. cerevisiae S26 for improved growth phenotypes

Adaptive laboratory evolution was performed to improve the growth phenotype of S. cerevisiae S26. The initial medium was YEPD, and during the evolutionary process, the glucose concentration in the YEPD medium progressively increased. As shown in Fig. 1, the evolutionary process underwent three stages. In stage 1, the glucose concentration was increased to 40 g/L, and the strain was named S26-AE1. At this stage, the average passage time was 16 h. In stage 2, the glucose concentration was increased from 40 to 80 g/L. A total of 20 passages were conducted in this stage, and strain S26-AE2 was obtained. The strain was passaged once every 11.75 h on average, and was shorter than stage 1. In stage 3, the glucose content increased from 80 to 125 g/L, and the strain was passaged once every 11.95 h on average. The evolutionary strain in stage 3 was named S26-AE3.

Fig. 1
figure 1

Diagram showing the adaptive laboratory evolution of S. cerevisiae strain S26. The total evolution time was 529 h, and the evolution process was divided into three stages

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The cellular morphology of the yeast was subsequently observed by scanning electron microscopy (SEM) (Fig. 2A). A small amount of cell damage was observed in S26-AE1. S26-AE2 showed obvious cell damage, which may be attributed to the premature transition of cells into the stable phase or an increase in the sugar concentration. However, with a further increase in the evolutionary sugar concentration, the S26-AE3 strain did not exhibit more cell damage. We performed spot assays of strains S26-AE1, S26-AE2, and S26-AE3 in the presence of 20 g/L and 100 g/L glucose to investigate the growth phenotypes of the strains after ALE (Fig. 2B). The growth of the evolutionary strains in media containing 20 g/L and 100 g/L glucose was better than that of the original strain, indicating that adaptive laboratory evolution was conducive to improving strain growth.

Fig. 2
figure 2

A The cellular morphology of the original strain and the evolved strains cultured in 100 g/L glucose medium for 24 h was observed by scanning electron microscopy. B Picture of the spot assay. After the original strain and the evolved strain were cultured on solid YEPD medium containing 20 g/L or 100 g/L glucose, for 24 h, images were captured

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Changes in the fermentation performance of evolutionary strains

The starting strain S26 and the evolutionary strains S26-AE1, S26-AE2, and S26-AE3 were cultured in YEPD medium supplemented with 100 g/L glucose to determine the fermentation performance of evolutionary strains. As shown in Fig. 3A, the starting strain S26 entered the stationary phase within 48 h, with an average OD600 of 36.39 and a dry weight of 15.45 g DCW/L. The biomass of S26-AE1 was 8.99% higher than that of S26 at 48 h (Fig. 3B). S26-AE2 entered a stable phase after 24 h, which was earlier than that of strain S26 (Fig. 3C). The OD600 during this period was 38.45, and the biomass of strain S26-AE2 was 17.82 g DCW/L at 48 h, which was 15.33% higher than that of strain S26. Similarly, strain S26-AE3 also entered the stationary phase after 24 h, with an OD600 of 39.14 (Fig. 3D). The reason why S26-AE2 and S26-AE3 entered the stationary phase earlier than the original strain may be an adaptation to the 100 g/L glucose environment and their rapid utilisation of glucose.

Fig. 3
figure 3

A, B, C, and D The OD600 and dry weight of strains S26, S26-AE1, S26-AE2, and S26-AE3, which were cultured in YEPD liquid medium supplemented with 100 g/L glucose. E, F, G, and H Tyrosol production by strains S26, S26-AE1, S26-AE2, and S26-AE3, which were cultured in YEPD liquid medium supplemented with 100 g/L glucose

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We further detected the tyrosol content to explore whether the adaptation of yeast to a high-glucose environment was helpful for enhancing the de novo synthesis of tyrosol. As shown in Fig. 3F, the maximal tyrosol production of S26 was 483.35 mg/L at 60 h, which was somewhat lower than that on the YEPD medium (538.41 mg/L) [20]. However, tyrosol production was not significantly different between strains S26-AE1 and S26. Surprisingly, the tyrosol production of S26-AE2 improved significantly, especially at 48 h, and its tyrosol production reached 817.83 mg/L, which was 69.20% higher than the highest yield of strain S26-AE1. However, tyrosol production in strain S26-AE3 reached 553.79 mg/L after 72 h of fermentation (Fig. 3H). The evolution of S26-AE2 was beneficial for the production of tyrosol.

We further explored the growth characteristics and physiological characteristics of the evolved strains by determining the indicators of glucose consumption and the production of ethanol, glycerol, and trehalose. The glucose consumption and ethanol production of the starting strain and the evolutionary strain were compared. The residual glucose concentration of the starting strain S26 in fermentation medium after 12 h was 89.30 g/L, and the glucose concentration of the evolutionary strain S26-AE1 was 38.05 g/L. At this time, the ethanol production was 4.20 g/L and 23.08 g/L, respectively (Fig. 4A, B). The evolved strains S26-AE2 and S26-AE3 completely consumed glucose after 12 h and produced 34.16 g/L and 36.12 g/L ethanol, respectively (Fig. 4C, D).

Fig. 4
figure 4

A, B, C, and D Glucose consumption and ethanol production by strains S26, S26-AE1, S26-AE2, and S26-AE3, which were cultured in YEPD liquid medium supplemented with 100 g/L glucose. E, F, G, and H Changes in the trehalose contents in strains S26, S26-AE1, S26-AE2, and S26-AE3, which were cultured in YEPD liquid medium supplemented with 100 g/L glucose. I, J, K, and L Changes in the glycerol contents of strains S26, S26-AE1, S26-AE2, and S26-AE3, which were cultured in YEPD liquid medium containing 100 g/L glucose

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In addition, strains S26 and S26-AE1 accumulated a certain amount of trehalose in the extracellular space during fermentation (Fig. 4E, F). Compared with that of the other strains, the trehalose production of strains S26-AE2 and S26-AE3 was significantly reduced (Fig. 4G, H). The intracellular glycerol content of strain S26 was 0.0378 mM at 24 h of fermentation, and the glycerol contents of strains S26-AE1, S26-AE2, and S26-AE3 were 0.0103 mM, 0.0243 mM, and 0.0083 mM, respectively (Fig. 4I, J, K, L). Both trehalose and glycerol play a role in preserving the osmotic pressure in cells, thereby reducing the damage caused by external stress to cells. In the process of evolution, the strains reduced the production of trehalose and glycerol, suggesting that the evolved strains gradually adapted to grow on 100 g/L glucose medium.

Transcriptome analysis of the mechanisms underlying adaptive evolution leading to increased tyrosol production in strain S26-AE2

We explored the mechanism of the increases in the S26-AE2 biomass and tyrosol production after evolution by performing a transcriptome analysis of the changes in gene expression in strains S26 and S26-AE2. The strains were cultured with 20 g/L glucose or 100 g/L glucose, and RNA was extracted for the transcriptome determination after 24 h of culture. According to the transcriptome results, the genes whose expression differed significantly are shown in volcano plots in Fig. 5. S26 had 807 upregulated genes and 583 downregulated genes in the presence of 100 g/L glucose compared with 20 g/L glucose (Fig. 5A). Compared with S26, S26-AE2 presented 36 upregulated genes and 1 downregulated gene in the presence of 20 g/L glucose (Fig. 5B). Compared with S26-AE2 cultured with 20 g/L glucose, S26-AE2 cultured with 100 g/L glucose presented 779 upregulated genes and 621 downregulated genes (Fig. 5C). Compared with the original strain S26, the evolutionary strain S26-AE2 had 9 upregulated genes and 16 downregulated genes in the presence of 100 g/L glucose (Fig. 5D). This result indicated that the metabolism of the strains differed significantly after laboratory evolution, and these differences may be due to the recovery of growth or the improvement in tyrosol production.

Fig. 5
figure 5

Volcano plots showing the gene expression levels in strains S26 and S26-AE2 cultured with 20 g/L or 100 g/L glucose. A The number of differentially expressed genes in group S26-100 compared with group S26-20. B The number of differentially expressed genes in group S26-AE2-20 compared with group S26-20. C The number of differentially expressed genes in group S26-AE2-100 compared with group S26-AE2-20. D The number of differentially expressed genes in group S26-AE2-100 compared with group S26-100. Group S26-20 represents strain S26 cultured under 20 g/L glucose conditions, and group S26-100 represents strain S26 cultured under 100 g/L glucose conditions. Group S26-AE2-20 represents strain S26-AE2 cultured under 20 g/L glucose conditions, and group S26-AE2-100 represents strain S26-AE2 cultured under 100 g/L glucose conditions

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We further studied the roles of these differentially expressed genes in metabolic pathways by mapping them to metabolic pathways. As shown in Fig. 6A, HXT5 is a low/medium-affinity glucose transporter gene, and the expression of the HXT5 gene increased significantly in strain S26-AE2 in the presence of 20 g/L glucose. Stefan Buziol et al. reported that the HXT5 gene was not expressed during the growth of yeast cells in rich media supplemented with glucose or raffinose. However, it was strongly induced during nitrogen or carbon starvation [21]. The expression levels of HXK1 and GLK1 were lower under 100 g/L glucose conditions than under 20 g/L glucose conditions in both strains S26 and S26AE-2, which is similar to the findings of previous studies (Fig. 6A) [22]. In strain S26, the expression of HXK2 under 100 g/L glucose conditions was significantly higher than that under 20 g/L glucose conditions and higher than the expression of HXK2 in strain S26AE-2. According to Rodríguez A et al. when glucose is present in the culture medium, the GLK1 gene is either not expressed or expressed at a very low level, and the transcription of the HXK1 and GLK1 genes is automatically regulated by glycolytic products and themselves [23]. HXK2 is a dual-function hexokinase that acts as a glycolytic enzyme and is involved in the transcriptional regulation of glucose-responsive genes [24]. HXK2 also plays an important regulatory role in glucose repression [25,26,27]. According to these studies, we speculated that after adaptive evolution, the decreased expression of HXK2 in strain S26-AE2 reduced the transcriptional regulation of glucose repression in cells.

Fig. 6
figure 6figure 6figure 6

Map of the metabolic pathways related to the differentially expressed genes in strains S26 and S26-AE2 under 20 g/L and 100 g/L glucose conditions. A Glycolysis pathway. B The pentose phosphate pathway. C Tricarboxylic acid (TCA) cycle. D Glyoxylic acid pathway. E The tyrosol synthesis pathway

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PGI1 (phosphoglucose isomerase 1) catalyses the interconversion of glucose 6-phosphate and fructose 6-phosphate, and glucose 6-phosphate is entirely rerouted into the PP pathway. The expression of the PGI1 gene in strain S26-AE2 increased slightly under 100 g/L glucose conditions (Fig. 6A). Related research has shown that PGI1 deletion mutants of S. cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose [28]. Therefore, the increase in PGI1 gene expression may promote the utilisation of glucose by the evolutionary strain S26-AE2, which was consistent with the increase in the glucose consumption rate (Fig. 4C).

In the presence of 100 g/L glucose, the expression of the GPD1, GPD2, and GPP2 genes increased in both S26 and S26-AE2 (Fig. 6A). The GPD1 gene, an osmotic induction-related gene, encodes glycerol-3-phosphate dehydrogenase, and the expression of GPD1 is the result of the interplay between different signalling pathways [29]. The deletion of structural genes such as GPD1 and GPD2 leads to osmosensitivity in yeast, which is not desirable for industrial strains [30, 31]. GPP1 is an isoenzyme that catalyses the formation of glycerol and is also involved in the formation of acetic acid. In the previous work, GPP1 was knocked out to reduce the production of acetic acid by the heterologous phosphoketolase (PHK) pathway [20]. However, due to the presence of GPP2, the knockout of GPP1 does not seem to affect glycerol production. The expression of the GPP2 gene in S26-AE2 was slightly lower than that in S26, which was consistent with the results of the glycerol determination (Fig. 4K). Glycerol enters the glycolytic pathway through the sequential actions of two genes, namely, the cytosolic glycerol kinase GUT1 and the glycerol-3-phosphate dehydrogenase GUT2 [32]. Interestingly, the expression of GUT2 in strain S26-AE2 increased upon stimulation with 20 g/L glucose.

Pyruvate, a precursor for several amino acids, can be synthesised from phosphoenolpyruvate by pyruvate kinase [33]. The gene expression levels of PGK1, GPM1, GPM2, ENO1/ENO2, and PYK1, which are involved in pyruvate production, in strain S26-AE2 were higher than those in strain S26 (Fig. 6A). Therefore, the evolved strain S26-AE2 is likely to increase the production of pyruvate. In addition, the expression of PYK2 was low in the presence of 100 g/L glucose. Related studies have shown that the absence of PYK2 has no effect on the specific growth rate when glucose is used as a carbon source, which may also explain why the expression of PYK2 is low under 100 g/L glucose conditions [33].

The pentose phosphate pathway (PPP) is a mechanism of the oxidative decomposition of glucose. In the presence of 20 g/L glucose, the expression of the SOL3, GND1, and TKL1 genes was downregulated in strain S26, and the expression of the SOL4, GND2, and TKL2 genes was upregulated, similar to the findings of Bergman A et al. (Fig. 6B) [34]. Under 100 g/L glucose conditions, the expression pattern of the SOL3/4, GND1/2, and TKL1/2 genes was opposite that under 20 g/L glucose conditions. However, after evolution, these phenomena were not very obvious in strain S26-AE2. SOL3 and SOL4 expression was downregulated in strain S26-AE2 under 20 g/L glucose conditions and upregulated under 100 g/L glucose conditions. In addition, under the condition of 20 g/L glucose, the expression levels of ZWF1 and RKl1 increased in strain S26-AE2 (Fig. 6B), and were significantly higher than those in strain S26, whereas the expression levels of these genes were downregulated under the condition of 100 g/L glucose, indicating that the genes were regulated by the external glucose content. ZWF1 is an NADP+-dependent dehydrogenase, and the overexpression of ZWF1 can increase the supply of NADPH in yeast cells [35].

The TCA cycle is an important hub of energy metabolism. The transcriptome analysis showed that most of the genes involved in the TCA cycle were upregulated under 20 g/L glucose conditions and that gene expression was downregulated under 100 g/L glucose conditions (Fig. 6C). In addition, gene expression in strain S26-AE2 under 20 g/L glucose conditions was clearly significantly higher than that in strain S26. These findings indicated that the TCA cycle-related genes of the evolutionary strains were more active. The genes with relatively high expression levels included the SDH (succinate dehydrogenase) enzyme family, the LSC (succinyl-CoA ligase) enzyme, and the KGO enzyme. However, the difference was not obvious in the presence of 100 g/L glucose, suggesting that this difference may be related to the extracellular glucose content (Fig. 6C). An increase in TCA cycle activity promotes the consumption of pyruvate, which is derived mainly from the decomposition of glucose by cells. The TCA cycle promotes cell growth and energy generation, which in a certain sense explains the recovery of strain growth through glucose evolution. In the presence of 100 g/L glucose, the expression levels of the MPC1, PDB1, LAT1, PDX1, SHH4, and LSC1 genes in the evolutionary strains were slightly higher than those in strain 26, and the expression levels of ACO2 and MAE1 decreased. Mitochondrial pyruvate carrier (MPC) complexes are responsible for the uptake of cytoplasmic pyruvate into mitochondria and are dissimilated by the pyruvate dehydrogenase complex and the oxidative TCA cycle; these complexes are used to generate energy or as precursors for the biosynthesis of branched-chain amino acids [36]. Therefore, the increased expression of the MPC1 gene in the evolutionary strain under 100 g/L glucose conditions might increase the intake of pyruvate and reduce the accumulation of pyruvate produced by glycolysis.

The glyoxylic acid cycle is particularly important for plants and some microorganisms. This pathway is the compensatory pathway of the TCA cycle. Through the glyoxylic acid cycle, yeast can use fatty acids and acetic acid as the sole carbon sources to synthesise carbohydrates, amino acids, and proteins to maintain normal cell growth. In the presence of 20 g/L glucose, the expression levels of the PYC1, MDH2, MDH3, MLS1, IDP2, ACS1, ACS2, ALD6, and PDC6 genes in the evolutionary strain were significantly higher than those in the original strain (Fig. 6D). ACS1 and ACS2 are involved in acetic acid consumption. PCK1 catalyses the conversion of oxaloacetate (OA) to phosphoenolpyruvate (PEP), and PYC1 catalyses the conversion of pyruvate to OA. These two reactions are important steps in the gluconeogenesis pathway and are required to consume nonglucose carbon sources (such as ethanol or acetate) (Fig. 6D). During the construction of our previous strain, PDC1 was knocked out, and the expression of PDC2 increased after adaptive evolution [20]. This phenomenon shows that the metabolic capacity of evolved yeast has increased, but is not conducive to the production of tyrosol.

The expression levels of ARO3 and ARO4 under 100 g/L glucose conditions were higher than those under 20 g/L glucose conditions (Fig. 6E). The increased expression of ARO3 and ARO4 promotes the formation of DAHP and increases tyrosol production, which also explains the increase in the tyrosol content in strain S26-AE2. Similarly, the increased expression of ARO2, ARO7, TYR1, and ARO10 promoted the synthesis of tyrosol. The results showed that the appropriate concentration of glucose could mobilise aromatic amino acid synthesis.

SNP mutation analysis of the adaptive laboratory evolutionary strain S26-AE2

We performed gene sequencing on the evolutionary strain S26-AE2 to obtain SNP-related information and identify changes related to growth and tyrosol production at the genetic level. Aligning all the SNPs to chromosomes revealed that SNPs mainly occurred in CM001525.1 and CM001529.1 (Fig. 7A). The number of SNPs that occurred on all introns was subsequently statistically analysed to obtain a petal diagram (Fig. 7B), which represents the number of SNP-containing genes after a comparison between the two groups. The results revealed 19 SNP mutations in S26-AE2-100vsS26-20, S26-AE2-100vsS26-AE2-20, and S26-100vsS26-20. Next, 19 SNPs were screened and analysed to obtain the data presented in Table 1. The results revealed five genes with SNP mutations, namely, MAL32, SNZ3, DOT6, HXT3, and PHO3.

Fig. 7
figure 7

A SNP density map of strain S26AE-2 showing the distribution of SNPs across chromosomes. B Petal map of SNP events representing the statistical analysis of SNPs in introns of groups S26-AE2-100vsS26-20, S26-AE2-100vsS26-100, S26-AE2-20vsS26-20, S26-100vsS26-20, and S26-AE2-100vsS26-AE2-20

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Table 1 Proteins with SNP mutations contained in strain S26-AE2
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MAL32 encodes alpha-glucosidase, which is involved in carbohydrate transport and metabolism. The deletion of MAL31 and MAL32 may have led to the aneuploidy of CHRIII, which harbours the MAL2 locus [37]. SNZ3 is a subunit of pyridoxal 5'-phosphate synthase and a member of a stationary phase-induced gene family. The transcription of SNZ3 is induced prior to a diauxic shift and in the absence of thiamine in a Thi2p-dependent manner; SNZ3 forms a coregulated gene pair with SNO3 [38]. DOT6 is a transcriptional regulatory protein and is involved in rRNA and ribosome biogenesis. HXT3 is a low-affinity glucose transporter of the major facilitator superfamily, and its expression is induced under low- or high-glucose conditions. HXT3 has a paralogue, HXT5, that arose from whole-genome duplication. PHO3 is a constitutively expressed acid phosphatase similar to PHO5 that is transported to the cell surface via vesicles. It can hydrolyse thiamine phosphates in the periplasmic space and increase cellular thiamine uptake. The expression of PHO3 is repressed by thiamine.

As shown in Table 1, SNZ3 contained mutations at amino acid residues 118, 122, and 125; synonymous mutations were identified at amino acids 118 and 122, and a nonsynonymous mutation was identified at amino acid residues 125. PHO3 was mutated at residues 132, 134, and 420, and only amino acid residues 420 exhibited a nonsynonymous mutation. DOT6 also had nonsynonymous mutations at residues sites 335 and 354. Next, we analysed the mutations in SNZ3 and PHO3.

Molecular docking analysis of the SNZ3 and PHO3 mutants binding to small molecules

We simulated the catalytic pockets of the proteins before and after mutation and docked the original and mutated proteins with small molecules to compare the potential effects of PHO3 and SNZ3 mutations on the catalysis of the substrates thiamine phosphate and D-ribose 5-phosphate and the formation of thiamine and pyridoxine. The docking results showed that after the introduction of the PHO3Asn134Asp mutation, its Vina score for the interaction with the small-molecule substance thiamine phosphate decreased to −6.6, which indicated that the affinity between the receptor and the ligand improved (Table S3). Moreover, the interaction of the PRO51, PRO363, MET365, and GLY366 residues with small molecules was reduced, and the binding of the ALA348, ILE398, and CYS408 residues was increased (Fig. 8E and Table S4). After the PHO3 mutation, its Vina score decreased to -5.4 after docking with the small-molecule thiamine (Table S3). The binding of the THR81, ASP231, THR258, PHE260, SER288, TYR289, and VAL375 residues to the small-molecule thiamine was reduced, and the binding of the SER253 and LYS254 residues was increased (Fig. 8F and Table S4). After SNZ3 mutation, the Vina score for the interaction with the small-molecule D-ribose 5-phosphate decreased to −5.7 (Table S3), but the residues before and after mutation did not change (Fig. 8K and Table S4). After SNZ3 mutation, its Vina score for the interaction with pyridoxal was still −5.6, but the binding of the THR4 and ALA69 residues increased (Fig. 8L).

Fig. 8
figure 8

A, D, G, and J Catalytic centres of the PHO3 and SNZ3 proteins before and after mutation. B and C Molecular docking of PHO3WT with thiamine phosphate and thiamine, respectively. E and F Molecular docking of PHO3Asn134Asp with thiamine phosphate and thiamine, respectively. H, I Molecular docking of SNZWT with D-ribose 5-phosphate and pyridoxal. K and L Molecular docking of SNZVal125Ile with D-ribose 5-phosphate and pyridoxal

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Reverse engineering verification improves yeast growth and robustness

We performed reverse engineering verification based on the screened mutation information to determine the functions of these mutant genes and their effects on yeast. Using CRISPR-Cas9 site-directed mutagenesis technology, we obtained three mutant strains, S26-PHO3Mut, S26-SNZ3Mut, and S26-PHO3Mut + SNZ3Mut, with strain S26. The strains were cultured in YEPD medium supplemented with 20 g/L or 100 g/L glucose. The growth index and tyrosol production were measured after 72 h of culture. Under 20 g/L culture conditions, the tyrosol yield of the mutant strain S26-SNZ3Mut increased by 5.61% compared with that of the control strain S26 at 60 h (Fig. 9A, C). Under 100 g/L glucose culture, tyrosol production by the SNZ3 mutant increased by 17.01% compared with that of the control strain S26 at 72 h (Fig. 9E, E). The results showed that the mutation of SNZ3 promoted tyrosol production in yeast in 100 g/L glucose medium. Under 100 g/L culture conditions, the tyrosol production of the SNZ3 and PHO3 comutants increased by 11.37% compared with that of the control strain S26 at 72 h. Studies have shown that pyridoxine can promote yeast growth and help yeast produce aromatic amino acids [39, 40]. Given the function of SNZ3, we speculated that the mutation promoted the utilisation and transport of pyridoxine. The increased utilisation of pyridoxine by yeast promoted the growth of yeast in 100 g/L glucose medium. The recovery of yeast growth further promoted the production of tyrosol. Reverse engineering demonstrated that a single-nucleotide mutation in SNZ3 could increase the concentration of tyrosol.

Fig. 9
figure 9

A, B, C, and D The OD600 and tyrosol production of strains S26, S26-PHO3Mut, S26-SNZ3Mut, and S26-PHO3Mut + SNZ3Mut, which were cultured on YEPD medium supplemented with 20 g/L glucose. E, F, G, and H The OD600 and tyrosol production of strains S26, S26-PHO3Mut, S26-SNZ3Mut, and S26-PHO3Mut + SNZ3Mut, which were cultured on YEPD medium supplemented with 100 g/L glucose

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Discussion

In this work, we performed adaptive laboratory evolution to restore the growth of the high-yield tyrosol-producing strain S26. Using glucose as a variable, we tried to balance the relationship between yeast growth and tyrosol production and then obtained a more robust industrial tyrosol-producing strain. In the evolutionary experiment, we obtained three strains after evaluating their growth and tyrosol production. We selected the S26-AE2 strain for the transcriptome analysis and genome analysis to clarify the mechanism of evolution of strain growth and tyrosol production through the above analysis.

For transcriptome analysis, we analysed the expression levels of key genes related to processes ranging from glucose metabolism to tyrosol production in yeast. The analysis of the expression of these genes revealed metabolic changes in the evolutionary strain S26-AE2, amongst which the key metabolic changes were those in the glycolysis pathway, tricarboxylic acid cycle, and tyrosol synthesis pathway. We speculate that these metabolic changes may contribute to the recovery of yeast growth and the production of tyrosol.

In the genome analysis of the evolutionary strains, we detected mutations in the key genes PHO3 and SNZ3. These genes promoted yeast growth in the previous studies. S. cerevisiae itself can produce a certain amount of thiamine [41]. Thiamine biosynthesis is regulated by the NAD+-dependent histone deacetylase Hst1 [42, 43]. Studies have shown that the addition of exogenous thiamine can promote ethanol production by yeast [39, 40]. In addition, regulating thiamine synthesis in S. cerevisiae can increase the yield of pyruvate [44]. Based on this information, we conducted reverse engineering verification to explore the effect of mutations in these genes on the growth of yeast and tyrosol production via direct mutation. Our molecular docking results revealed that mutations in these genes enhance the interaction between proteins and small molecules. After the mutations were introduced into strain S26, we determined yeast growth and found that the mutation of the PHO3 and SNZ3 genes was indeed beneficial for the production of the growth substrate tyrosol in yeast. However, not all mutations have beneficial effects, and studies have shown that a PHO6 mutant of S cerevisiae, which lack a regulatory gene required for periplasmic thiamine synthesis and repressible acid phosphatase activity, is auxotrophic for thiamine [45, 46].

Thiamine is an essential coenzyme of phosphoketolase, which is involved in substrate activation and carbon‒carbon-bond cleavage. After the combination of thiamine and phosphoketolase, a stable active complex is formed [47]. A lack of thiamine affects the activity of phosphoketolase, leading to metabolic disorders. Pyridoxine is the precursor of thiamine (Fig. 10). PHO3Asn134Asp and SNZ3Val125Ile might increase the synthesis of pyridoxine and thiamine. Adequate thiamine will theoretically help phosphoketolase increase the production of E4P, the precursor of tyrosol. Therefore, we hypothesised that the mutations of PHO3 and SNZ3 may be some of the reasons for the increase in tyrosol production in the S26-AE2 strain, because they alter the activity of phosphoketolase.

Fig. 10
figure 10

Regulatory mechanism of thiamine-dependent enzymes. Xfpk is an important phosphoketolase involved in tyrosol synthesis

Full size image

Conclusions

In this work, the growth of the high-yield tyrosol-producing strain S26 was restored through adaptive evolution in the laboratory. The transcriptome analysis revealed differences in gene expression between the evolutionary strain S26-AE2 and the original strain S26 in terms of glycolysis, the TCA cycle, and tyrosol synthesis pathways. Molecular docking and reverse engineering verified the effectiveness of the SNZ3Val125Ile mutation in increasing tyrosol production. This study provides a theoretical basis for the metabolic engineering of S. cerevisiae to synthesise tyrosol and its derivatives.

Materials and methods

Strain construction and cultivation

S. cerevisiae strain 26 was used in this study. YEPD medium (20 g/L peptone, 10 g/L yeast extract, and 20 g/L glucose) was used to cultivate S. cerevisiae, and glucose was added at a concentration of 100 g/L if needed. SC-Ura medium containing 20 g/L glucose, 6.7 g/L yeast nitrogen base (with ammonium sulphate, without ammonium acids), and 1.29 g/L Do supplement-Ura was used for yeast transformation. The yeast strains were cultured at 30 °C and shaken at 200 rpm for 72 h. E. coli JM110 and E. coli DH5α were used for the construction of plasmids and were cultured in LB medium containing 5 g/L yeast extract, 10 g/L peptone, and 10 g/L NaCl, pH 7.2. The E. coli were cultured at 37 °C with shaking at 200 rpm. All reagents were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The plasmids used in this study and the primers used are listed in Table S1 and Table S2.

Adaptive laboratory evolution

S. cerevisiae S26 was used as the parental strain and cultured in a shaker flask with YEPD medium. The S26 strain suspension was seeded into EVOL cells (Luoyang Huaqing Tianmu Biotechnology Co., Ltd., Luoyang, China) [48], which enabled automatic passaging, and the OD600 was monitored. The initial glucose concentration of the evolution medium was 20 g/L, and the amount of added glucose gradually increased during the process of evolution. When the OD600 reached 15, the yeast was automatically passaged to the next generation. Samples were collected at each stage and cultured in YEPD medium with the corresponding glucose concentration. The strains were preserved in glycerol in a tube. The average passage time was calculated by dividing the total duration of each stage by the number of passages.

RNA extraction

The S. cerevisiae S26 and S26-AE2 strains were cultured at 30 ℃ and 200 rpm for 12 h, and the cultures were inoculated into YEPD medium or YEPD medium containing 100 g/L glucose at an initial OD600 of 0.5. Three biological replicates were established. The cultures were collected, and total RNA was extracted. The RNA quality was subsequently determined using a 5300 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified using an ND-2000 (NanoDrop Technologies, Wilmington, DE, USA). Only high-quality RNA samples (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, > 1 μg) were used to construct the sequencing library. RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Biopharm Biotechnology Co., Ltd. (Shanghai, China).

Differential expression analysis

A differential expression analysis was performed using DESeq2 or DEGseq to identified differentially expressed genes (DEGs) between different samples. DEGs with |log2FC|≥ 1 and FDR ≤ 0.05 (DESeq2) or FDR ≤ 0.001 (DEGseq) were considered significantly differentially expressed genes. In addition, the DEGs were mapped to KEGG pathways, and the expression levels are presented in a heatmap.

Analytical methods

The cell density (OD600) was measured with a spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd., China). The concentrations of tyrosol and trehalose were determined by high-performance liquid chromatography (HPLC) (Thermo Fisher Scientific, Waltham, MA, USA). A Thermo-C18 column (4.6 mm × 250 mm, 5 μm) was used. The detection conditions were as follows: mobile phase containing a 0.05% (v/v) formic acid aqueous solution (A) and acetonitrile (B); gradient elution (0–20 min, 20% B; 20–25 min, 95% B; 25–35 min, 95% B; 35–40 min 95% B; 40–50 min, 10% B); column temperature of 30 ℃; flow rate of 1 mL/min; and detection wavelength of 224 nm. The trehalose content was determined using a refractive index detector with a mobile phase of 0.05 mmol/L H2SO4 at a flow rate of 1 mL/min and a column temperature of 50 ℃. Glucose and ethanol contents were measured using a biosensor (Sieman Technology Co., Ltd., Shenzhen, China). The glycerine determination was performed with a tissue cell triglyceride (TG) assay kit (Applygen Technologies Inc., Beijing, China). The morphology of the yeast cells was analysed with a scanning electron microscope (JEOL JSM6390LV, Tokyo, Japan).

Spot assay

S. cerevisiae was cultured in YEPD medium to the log phase of growth, and the cells were harvested by centrifugation (4000 × g, 5 min) and suspended in sterile water. Afterwards, the cells were diluted to an OD600 of 1 with sterile water. The yeast cells were prepared as aliquots in tenfold serial dilutions, spotted on YEPD plates and YEPD plates supplemented with 100 g/L glucose, and cultured at 30 °C for 24 h.

SNP analysis

The SNP density map was created using CMplot in the R language package (https://www.bioinformatics.com.cn/) [49]. The raw data have been uploaded to the SRA database.

Molecular docking

The structural models of the PHO3 and SNZ3 proteins were constructed with SWISS-MODEL (https://swissmodel.expasy.org/). The structures of small molecules were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) for docking. Molecular docking was performed using CB-Dock2 (https://cadd.labshare.cn/cb-dock2/php/index.php) [50].

DNA manipulations

The CRISPR-Cas9 system was used to modify the genome. The plasmid pML104 was used to construct gRNA expression vectors [51]. The specific guide RNA sequences were designed using the CHOPCHOP web tool (http://crispor.tefor.net) [52]. The pUC57 plasmid was first linearized with restriction enzymes, and the upstream and downstream homologous arms with mutations were ligated together using the ClonExpress MultiS One-Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China) to construct homologous recombination fragments. The gRNA expression vectors and homologous recombination fragments were cotransformed into yeast by the LiAc/ssDNA/PEG method [53]. The correct yeast clones were selected from the SC-Ura media.

Statistics and reproducibility

The significance of differences in data between groups was determined with a t test using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). The data were analysed and graphed with GraphPad Prism 8. All the experiments were conducted with three biological replicates. The heatmap package in R was used to generate heatmaps and volcano plots.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

ACO2:

Aconitate hydratase

ACS1/ACS2:

Acetyl-CoA ligase

ALD6:

Aldehyde dehydrogenase

ALE:

Adaptive laboratory evolution

ARO10:

Phenylpyruvate decarboxylase

ARO2:

Bifunctional chorismate synthase/riboflavin reductase

ARO3/ARO4:

3-Deoxy-7-phosphoheptulonate synthase

ARO7:

Chorismate mutase

DAHP:

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate

DOT6:

Transcriptional regulatory protein

ENO1/ENO2:

Phosphopyruvate hydratase

FBA1:

Fructose-bisphosphate aldolase

FBP:

Fructose-1,6-bisphosphatase

GAP:

Type I glyceraldehyde-3-phosphate dehydrogenase

GLK1:

Glucokinase

GND1/GND2:

Phosphogluconate dehydrogenase

GPD1/GPD2:

Glycerol-3-phosphate dehydrogenase

GPM1/GPM2:

Phosphoglycerate mutase

GPP2:

Glycerol-3-phosphate dehydrogenase

GUT1:

Glycerol kinase

GUT2:

Glycerol-3-phosphate dehydrogenase

HXK1/HXK2:

Hexokinase 1

HXT2/HXT10:

High-affinity glucose transporter genes

HXT3:

Low affinity glucose transporter

HXT4:

Hexose transporter

HXT5:

Low/medium-affinity glucose transporter

IDP2:

Isocitrate dehydrogenase

LAT1:

Dihydrolipoyllysine-residue acetyltransferase

LSC:

Succinyl-CoA ligase

MAE1:

Malate dehydrogenase

MAL32:

A lpha-glucosidase

MDH2/MDH3:

Malate dehydrogenase

MLS1:

Malate synthase

MPC1:

Mitochondrial pyruvate carrier

OA:

Oxaloacetate

PDB1:

Pyruvate dehydrogenase

PDC6:

Indolepyruvate decarboxylase 6

PDX1:

Pyridoxine biosynthesis protein 1

PEP:

Phosphoenolpyruvate

PFK:

Phosphofructokinase

PGI1:

Phosphoglucose isomerase 1

PGK1:

Phosphoglycerate kinase 1

PHK:

Peterologous phosphoketolase pathway

PHO3/PHO5/PHO6:

Acid phosphatase

PPP:

Pentose phosphate pathway

PYC1:

Pyruvate carboxylase 1

PYK1:

Pyruvate kinase

RKl1:

Ribose-5-phosphate isomerase

SDH:

Succinate dehydrogenase membrane anchor subunit

SHH4:

Succinate dehydrogenase

SNP:

Single nucleotide polymorphism

SNZ3:

Pyridoxal 5'-phosphate synthase

SOL3/SOL4:

6-Phosphogluconolactonase

TCA:

Tricarboxylic acid cycle

TKL1/TKL2:

Transketolase

TYR1:

Prephenate dehydrogenase (NADP+)

ZWF1:

NADP+-dependent dehydrogenase

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Funding

This work was supported by the National Natural Science Foundations of China (Grant Nos. 31871789 and 41876114), the key project of the Hubei Provincial Department of Education (Grant No. T2022011), and the Natural Science Foundation of Hubei Province (Grant No. 2024AFB803).

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Authors and Affiliations

  1. Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Key Laboratory of Fermentation Engineering (Ministry of Education), National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Hubei Key Laboratory of Industrial Microbiology, School of Life and Health Sciences, Hubei University of Technology, No. 28, Nanli Road, Hongshan District, Wuhan, 430068, Hubei, People’s Republic of China

    Na Song, Xiaoxue Yang, Siyao Liu, Linglong Xu, Kun Zhuang, Lan Yao, Xiong Chen & Jun Dai

  2. College of Biological and Food Engineering, Huanghuai University, Zhumadian, 463000, People’s Republic of China

    Huili Xia

  3. School of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023, People’s Republic of China

    Kun Zhuang

  4. State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, 430062, Hubei, People’s Republic of China

    Shihui Yang & Jun Dai

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  1. Na Song
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Contributions

N S, HL X, J D, and X C designed the whole and wrote the final manuscript. XX Y, SY L, and LL X carried out the part experiments and data collection. K Z, L Y, and S H Y participated in data analysis and manuscript.

Corresponding authors

Correspondence to Xiong Chen or Jun Dai.

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Song, N., Xia, H., Yang, X. et al. Transcriptome analysis and reverse engineering verification of SNZ3Val125Ile and Pho3Asn134Asp revealed the mechanism of adaptive laboratory evolution to increase the yield of tyrosol in Saccharomyces cerevisiae strain S26-AE2. Biotechnol. Biofuels Bioprod. 18, 29 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13068-025-02627-4

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13068-025-02627-4

Biotechnology for Biofuels and Bioproducts

ISSN: 2731-3654

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