Transcriptome analysis and reverse engineering verification of SNZ3 Val125Ile and Pho3 Asn134Asp revealed the mechanism of laboratory adaptive evolution to increase the yield of tyrosol in Saccharomyces cerevisiae S26

Transcriptome analysis and reverse engineering verification of SNZ3 Val125Ile and Pho3 Asn134Asp revealed the mechanism of laboratory adaptive evolution to increase the yield of tyrosol in Saccharomyces cerevisiae S26 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptome analysis and reverse engineering verification of SNZ3 Val125Ile and Pho3 Asn134Asp revealed the mechanism of laboratory adaptive evolution to increase the yield of tyrosol in Saccharomyces cerevisiae S26 Na Song, Huili Xia, Xiaoxue Yang, Siyao Liu, Linglong Xu, Kun Zhuang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5667010/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2025 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted 11 You are reading this latest preprint version Abstract Background Tyrosol is an important drug precursor, and Saccharomyces cerevisiae is one of the main microorganisms producing tyrosol. Although excessive metabolic modification increased the production of tyrosol, it also caused 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 under the condition of 100 g/L glucose which was 15.33% higher than that of S26, and its tyrosol production reached 817.83 mg/L. Transcriptome analysis showed that the strain S26-AE2 may through decreased expression of HXK2 reduce the transcriptional regulation of glucose repression and increase the expression of gene PGI1 to promote the utilization of glucose. The genes related to pyruvate synthesis were enhanced in strain S26-AE2. Under the 20 g/L glucose condition, the TCA cycle-related genes of the S26-AE2 were more active. Furthermore, the tyrosol production of S26 with SNZ3 Val125Ile mutation increased by 17.01% compared with the control strain S26 under the condition of 100 g/L glucose. Conclusions In this paper, a strain S26-AE2 with good growth and tyrosol production performance was obtained by adaptive evolution. The transcriptome reveals the differences in gene expression in metabolic pathways of adaptive evolutionary strains may be related to the growth of yeast and the production of tyrosol. Further reverse engineering verified the mutation of SNZ3 promoted the synthesis of tyrosol in S. cerevisiae in the glucose-rich medium. This study provides a theoretical basis for the metabolic engineering of S. cerevisiae to synthesize tyrosol and its derivatives. Tyrosol Adaptive laboratory evolution Transcriptome Genetic mutation Reverse engineering PHO3 SNZ3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Tyrosol is the precursor of salidroside and hydroxytyrosol and exhibits 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 – 5 ]. However, the olive leaf extraction method faces the problem of low extraction efficiency, and its production is highly subject to the supply of olive leaf resources. More importantly, both methods are not environmentally friendly, whether by extraction or chemical synthesis. In contrast, microbial cell factories exhibit many characteristics that are conducive to low-cost, environmentally friendly, and sustainable production of tyrosol [ 6 ]. In recent years, Saccharomyces cerevisiae and E. coil was successfully used to produce tyrosol [ 7 – 10 ]. Through these studies, titers of tyrosol achieved 1.3 g/L in S. cerevisiae [ 11 ] and 2.12 g/L in E. coil [ 12 ]. Although a certain amount of high yield has been achieved in S. cerevisiae , with the increase of modification, the robustness of yeast is reduced. In the early stage of our laboratory, a strain of S. cerevisiae S26 with a high yield of tyrosol was constructed. However, excessive modification caused the growth of yeast to be not vigorous, which greatly limited its application in fermentation tanks and industrial production. Adaptive laboratory evolution (ALE) is an efficient method to obtain strains with improved traits [ 13 ]. Through natural selection, abundant mutants are accumulated to quickly obtain strains with ideal characteristics. ALE has been applied to optimize 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 also can be combined with DNA sequence and bioinformatics analysis to understand the basic mechanism of molecular evolution and adaptive changes accumulated in the long-term selection process of microbial populations under specific growth conditions. Zongjie Dai et al. combining rational design, adaptive laboratory evolution, and reverse engineering, the specific growth rate of the best strain reaches 0.218 h − 1 , which is close to the maximum growth of S. cerevisiae with purely respiratory metabolism [ 15 ]. Henri Ingelman et al. used three different and independent adaptive laboratory evolution strategies to evolve the 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 by adaptive evolution [ 17 ]. Ryan A. LaCroix et al. used adaptive laboratory evolution to find 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 lowest glucose medium [ 19 ]. In this study, we used glucose as a single factor for adaptive evolution, and S.cerevisiae S26 was used as the starting strain for laboratory evolution [ 20 ]. Three strains were obtained in the evolutionary stage, and the strain S26-AE2 with better physiological characteristics was selected by experimental verification. Transcriptome analysis of the evolutionary strains revealed the changes in gene expression in the metabolic pathway of yeast after evolution. The increase of the TCA cycle and aromatic amino acid gene expression may act on the growth recovery of S. cerevisiae and the increase of tyrosol content. According to the screened potential single nucleotide polymorphism (SNP) mutations PHO3 Asn134Asp and SNZ3 Val125Ile , and reverse engineering verification were performed. The results showed that the SNZ3 Val125Ile mutation had a positive correlation with the production of yeast tyrosol. The research results will provide ideas for obtaining more robust industrial S. cerevisiae strains and provide 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 To improve the growth phenotype of S. cerevisiae S26, adaptive laboratory evolution was performed. As shown in Fig. 1 , the evolutionary process has gone through three stages. At stage 1, the concentration of glucose was increased to 40 g/L, and the strain was named S26-AE1. At this stage, the average value of the cycle was 16 hours. At stage 2, the concentration of glucose was increased from 40 g/L to 80 g/L. A total of 20 passages were carried out in this stage, and the strain S26-AE2 was obtained. The strain passaged once every 11.75 hours on average, and the time of the growth cycle was shortened. At stage 3, the glucose content increased from 80 g/L to 125 g/L and the strain was passaged once every 11.95 hours on average, the evolutionary strain was named S26-AE3. Then, the cellular morphology of yeast was observed by scanning electron microscopy (SEM). There was a small amount of cell damage in S26-AE1. S26-AE2 showed obvious cell damage, which may be attributed to the premature transition of cells into the stable phase or the increase of sugar concentration. However, with the further increase of evolutionary sugar concentration, the S26-AE3 strain did not appear more cell damage. In order to investigate the growth phenotype of strains after ALE, we performed the spot assay of strains S26-AE1, S26-AE2, and S26-AE3 were performed under 20 g/L and 100 g/L glucose, respectively. The growth of the evolutionary strains in 20 g/L and 100 g/L glucose medium was better than that of the original strain, indicating that adaptive laboratory evolution was conducive to the improvement of strain growth. Fermentation performance changes of evolutionary strains To determine the fermentation performance of evolutionary strains, the starting strain S26 and the evolutionary strains S26-AE1, S26-AE2, and S26-AE3 were cultured in a YEPD medium containing 100 g/L glucose. As shown in Fig. 3 A, the starting strain S26 entered a stable period in 48 hours, with an average OD 600 of 36.39 and a dry weight of 15.45 gDCW/L. The biomass of S26-AE1 was 8.99% higher than that of S26 at 48 hours (Fig. 3 B). S26-AE2 entered a stable phase after 24 hours, which was earlier than that of strain S26 (Fig. 3 C). The OD 600 in this period was 38.45 and the biomass of strain S26-AE2 was 17.82 gDCW/L at 48 hours, which was 15.33% higher than that of strain S26. Similarly, S26-AE3 also entered a stable period after 24 hours, with an OD 600 of 39.14 (Fig. 3 D). 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 utilization of glucose. In order to explore whether the adaptation of yeast to a high glucose environment is helpful to enhance the de novo synthesis of tyrosol, we further detected the content of tyrosol. As shown in Fig. 3 F, 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). The tyrosol production was no significant difference between strain S26-AE1 and strain S26. Surprisingly, the tyrosol production of S26-AE2 was significantly improved, especially at 48 hours, and its tyrosol production reached 817.83 mg/L, which was 69.20% higher than the highest yield of strain S26-AE1. However, the production of tyrosol in S26-AE3 reached 553.79 mg/L after 72 hours of fermentation (Fig. 3 H). It could be seen that the evolutionary S26-AE2 was beneficial to the production of tyrosol. To further explore the growth characteristics and physiological characteristics of the evolved strains. We determined 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 of the starting strain S26 in the fermentation medium for 12 hours was 89.30 g/L, and the glucose residue 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. 4 A and Fig. 4 B). The evolved strains S26-AE2 and S26-AE3 consumed glucose completely after 12 hours and produced 34.16 g/L and 36.12 g/L ethanol, respectively (Fig. 4 C and Fig. 4 D). In addition, strains S26 and S26-AE1 accumulated a certain amount of trehalose in the extracellular during the fermentation process (Fig. 4 E and Fig. 4 F). Compared with them, the trehalose production of strains S26-AE2 and S26-AE3 was significantly reduced (Fig. 4 G and Fig. 4 H). The intracellular glycerol content of S26 was 0.0378 mM at 24 hours of fermentation, and the glycerol contents of S26-AE1, S26-AE2 and S26-AE3 were 0.0103 mM, 0.0243 mM and 0.0083 mM, respectively (Fig. 4 I, Fig. 4 J, Fig. 4 K, and Fig. 4 L). Both trehalose and glycerol played an osmotic pressure protective role in cells, thereby reducing the damage caused by external stress on cells. In the process of evolution, the strains reduced the production of trehalose and glycerol, it could be speculated that the evolved strains have gradually adapted to grow on 100 g/L glucose medium. Transcriptome analysis of the mechanisms underlying adaptive evolution that leads to increased tyrosol production in S26-AE2 To explore the mechanism of S26-AE2 biomass increase and tyrosol increase after evolution, we used transcriptome to analyze the changes in gene expression of strains S26 and S26-AE2. Strains were cultured under 20 g/L glucose and 100 g/L glucose, respectively, and RNA was extracted for transcriptome determination after 24 hours of culture. According to the results of the transcriptome, the genes with significant differential expression were expressed in the form of volcanic maps, as shown in Fig. 4 . S26 had 807 up-regulated genes and 583 down-regulated genes under 100 g/L glucose compared to 20 g/L glucose (Fig. 5 A). Compared with S26, S26-AE2 had 36 up-regulated genes and 1 down-regulated gene under 20 g/L glucose (Fig. 5 B). Compared with S26-AE2 at 20 g/L glucose, S26-AE2 at 100 g/L glucose had 779 up-regulated genes and 621 down-regulated genes (Fig. 5 C). The evolutionary strain S26-AE2 had 9 up-regulated genes and 16 down-regulated genes at 100 g/L glucose compared to the original strain S26 (Fig. 5 D). This result indicated that the strains have significant differences in metabolism after laboratory evolution, and these differences may be due to the recovery of growth or the improvement of tyrosol. In order to further study the role of these differential genes in the metabolic pathway, we map these differential genes to the metabolic pathway to obtain a metabolic map of differentially expressed genes. As shown in Fig. 6 A, HXT5 is a low/medium affinity glucose transporter gene. the expression of gene HXT5 increased significantly in S26-AE2 at 20 g/L glucose conditions. According to the research of Stefan Buziol et al., the gene HXT5 was not expressed during the growth of the yeast cells in a rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation [ 21 ]. The expression levels of HXK1 and GLK1 were lower at 100 g/L glucose conditions than that at 20 g/L glucose conditions both in strain S26 and S26AE-2 which is similar to previous studies (Fig. 6 A) [ 22 ]. While the expression of HXK2 under 100 g/L glucose conditions was significantly higher than that under 20 g/L glucose conditions in strain. S26 and S26-AE2. According to the research of 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 HXK1 and GLK1 genes is automatically regulated by glycolytic products and themselves [ 23 ]. HXK2 is a dual-function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes [ 24 ]. HXK2 also plays an important regulatory role in glucose repression [ 25 – 27 ]. According to this information, we speculated that after adaptive evolution, the decreased expression of HXK2 in strain S26-AE2 reduced the transcriptional regulation of glucose repression in cells. The PGI1 (phosphoglucose isomerase 1) catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate, glucose 6-phosphate is entirely rerouted into the PP pathway. The gene expression of PGI1 in strain S26-AE2 increased slightly under 100 g/L glucose conditions (Fig. 6 A). Related research showed 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 of PGI1 gene expression may promote the utilization of glucose by evolutionary strain S26-AE2, which was consistent with the increase in glucose consumption rate (Fig. 4 C). Under the condition of 100 g/L glucose, the gene expression of GPD1 , GPD2 , and GPP2 increased both in S26 and S26-AE2 (Fig. 6 A). The gene GPD1 , one of the osmotic induction genes, encodes glycerol-3-phosphate dehydrogenase, the expression of GPD1 is the result of an interplay between different signaling pathways [ 29 ]. Deletion of structural genes like GPD1 and GPD2 leads to yeast osmosensitivity, which is not desirable for industrial strains [ 30 , 31 ]. GPP1 is an isoenzyme that catalyzes the formation of glycerol and is also involved in the formation of acetic acid. In the previous work of this paper, to reduce the production of acetic acid in the heterologous phosphoketolase (PHK) pathway, GPP1 was knocked out [ 20 ]. However, due to the presence of GPP2 , knockout of GPP1 does not seem to affect glycerol production. The gene expression of GPP2 in S26-AE2 was slightly lower than that of S26, which was consistent with the results of glycerol determination (Fig. 4 K). Glycerol enters the glycolytic pathway through the sequential action of two genes, namely cytosolic glycerol kinase GUT1 and glycerol-3-phosphate dehydrogenase GUT2 [ 32 ]. Interestingly, the expression of GUT2 in strain S26-AE2 was increased under 20 g/L glucose conditions. Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase [ 33 ]. The gene expression levels of PGK1 , GPM1 , GPM2 , ENO1 / ENO2 , and PYK1 involved in pyruvate production in the S26-AE2 were higher than those in strain S26 (Fig. 6 A). Therefore, the evolved strains S26-AE2 are likely to increase the production of pyruvate. In addition, the expression of PYK2 was low under 100 g/L glucose. Related studies have shown that the absence of PYK2 has no effect on the specific growth rate with glucose 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 way of glucose oxidative decomposition. Under the condition of 20 g/L glucose, the genes SOL 3, GND 1, and TKL 1 showed down-regulation in strain S26, and the genes SOL 4, GND 2, and TKL 2 showed up-regulation which is similar to the research of Bergman A et.al (Fig. 6 B) [ 34 ]. Under 100 g/L glucose conditions, the expressed genes SOL 3/4, GND 1/2, and TKL 1/2 were opposite to those under 20 g/L glucose conditions. However, after evolution, this rule was broken in strain S26-AE2. The SOL3 and SOL4 of strain S26-AE2 were down-regulated under 20 g/L glucose conditions and were up-regulated 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 the S26-AE2 (Fig. 6 B), which was significantly higher than that of S26, while the expression level of the gene was down-regulated under the condition of 100 g/L glucose, indicating that the gene was regulated by the external glucose content. ZWF1 is a NADP + -dependent dehydrogenase, and overexpression of ZWF1 can increase the supply of NADPH in yeast cells [ 35 ]. The TCA cycle is an important hub of energy metabolism. Transcriptome analysis showed that most of the genes involved in the TCA cycle were up-regulated under 20 g/L glucose conditions, and the gene expression under 100 g/L glucose conditions was down-regulated (Fig. 6 C). In addition, there is an obvious phenomenon that the gene expression of the S26-AE2 under 20 g/L glucose condition was significantly higher than that of the strain S26. It indicated that the TCA cycle-related genes of the evolutionary strains were more active. The higher expression levels include the SDH enzyme family (Succinate dehydrogenase complex), LSC enzyme (Succinyl-CoA ligase), and KGO enzyme. However, the difference was not obvious at 100 g/L glucose, suggesting that this difference may be related to the extracellular glucose content (Fig. 6 C). The enhancement of the TCA cycle promotes the consumption of pyruvate which mainly comes from the decomposition of glucose by cells. The TCA cycle has a promoting effect on cell growth and energy generation, which in a certain sense explains the recovery of strain growth through glucose evolution. Under the condition of 100 g/L glucose, the gene expression levels of MPC1 , PDB1 , LAT1 , PDX1 , SHH4 , and LSC1 in the evolutionary strains were slightly higher than those in strain 26, and the expression levels of ACO2 and MAE1 were 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, used to generate energy, or used as precursors for the biosynthesis of branched-chain amino acids [ 36 ]. Therefore, the increased gene expression of MPC1 in the evolutionary strain under 100 g/L glucose conditions might enhance 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 compensation pathway of the TCA cycle. Through the glyoxylic acid cycle, yeast can use fatty acids and acetic acid as the sole carbon source to synthesize carbohydrates, amino acids, and proteins to maintain normal cell growth. Under 20 g/L glucose, the gene expression levels of PYC1 , MDH2 , MDH3 , MLS1 , IDP2 , ACS1 , ACS2 , ALD6 , and PDC6 in the evolutionary strain were significantly higher than those in the original strain (Fig. 6 D). ACS1 and ACS2 are involved in acetic acid consumption. PCK1 catalyzes the conversion of oxaloacetate (OA) to phosphoenolpyruvate (PEP), and PYC1 catalyzes the conversion of pyruvate to OA. These two reactions are important steps in the gluconeogenesis pathway required to consume non-glucose carbon sources (such as ethanol or acetate) (Fig. 6 D). In the process of our previous strain construction, PDC1 was knocked out, and the expression of PDC2 increased after adaptive evolution [ 20 ]. This phenomenon proves that the metabolic capacity of evolved yeast is enhanced, but it 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. 6 E). The increased expression of ARO3 and ARO4 will be beneficial to the formation of DAHP and the increase of tyrosol production, which also explains the reason for the increase of tyrosol content in strain S26-AE2. Similarly, the increased expression of ARO2 , ARO7 , TYR1 , and ARO10 was beneficial to the synthesis of tyrosol. The results showed that the appropriate concentration of glucose could mobilize the 'enthusiasm' of aromatic amino acid synthesis. SNP mutation analysis of adaptive laboratory evolutionary strain S26-AE2 In order to find changes related to growth and tyrosol production at the genetic level, we performed gene sequencing on the evolutionary strain S26-AE2 to obtain SNPs-related information. By aligning all SNPs to chromosomes, it can be seen that the occurrence of SNPs is mainly in CM001525.1 and CM001529.1 (Fig. 7 A). Subsequently, the number of SNPs that occurred on all introns was statistically analyzed to obtain a petal diagram (Fig. 7 B), representing the number of SNPs genes after comparison between the two groups. The results showed that there were 19 SNP mutations in S100vsC20, S100vsS20, and C100vsC20. Then, 19 SNPs were screened and analyzed to obtain Table 1 . The results showed that there were five genes involved in SNP mutations, namely MAL32 , SNZ3 , DOT6 , HXT3 , and PHO3 . Table 1 Proteins with SNP mutations contained in strain S26-AE2. Gene description Chrom Genome position Ref Alt Anno Gene description Type Mutations MAL32 CM001523.1 800665 T C exonic synonymous SNV SNP Phe472Phe MAL32 CM001523.1 800863 G A exonic synonymous SNV SNP Leu538Leu SNZ3 CM001527.1 8368 C T exonic synonymous SNV SNP Gly93Gly SNZ3 CM001527.1 8416 C T exonic synonymous SNV SNP Ile109Ile SNZ3 CM001527.1 8443 C T exonic synonymous SNV SNP Asn118Asn SNZ3 CM001527.1 8455 A G exonic synonymous SNV SNP Leu122Leu SNZ3 CM001527.1 8462 G A exonic nonsynonymous SNV SNP Val125Ile DOT6 CM001526.1 332037 T G exonic nonsynonymous SNV SNP Lys355Asn DOT6 CM001526.1 332040 C A exonic nonsynonymous SNV SNP Arg354Ser HXT3 CM001525.1 1161695 A C exonic synonymous SNV SNP Val538Val PHO3 CM001523.1 422454 T C exonic nonsynonymous SNV SNP Asn134Asp PHO3 CM001523.1 422458 T C exonic synonymous SNV SNP Ser132Ser PHO3 CM001523.1 421594 A G exonic synonymous SNV SNP Asp420Asp Notes: 1. “Chrom” means chromosome. 2. “Ref” means Reference sequence. 3. “Alt” means Alternative sequence. 4. “Anno” means Annotation. 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 that harbours the MAL2 locus [ 37 ]. SNZ3 is the subunit of pyridoxal 5'-phosphate synthase and a member of a stationary phase-induced gene family. The transcription of SNZ3 is induced prior to diauxic shift, and also in the absence of thiamin in a Thi2p-dependent manner; 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, the expression is induced in low or high glucose conditions. HXT3 has a paralog, HXT5, that arose from the whole genome duplication. PHO3 is a constitutively expressed acid phosphatase similar to PHO5, brought to the cell surface by transport vesicles. It can hydrolyze thiamin phosphates in the periplasmic space and increase cellular thiamin uptake. The expression of PHO3 is repressed by thiamin. As shown in Table 1 , SNZ3 had mutations at 118, 122, and 125 amino acid residues, 118 and 122 were synonymous mutations, and 125 had non-synonymous mutations. PHO3 was mutated at 132, 134, and 420 sites, and only 420 sites had non-synonymous mutations. DOT6 also had non-synonymous mutations at 335 and 354 sites. Next, we first analyzed the mutations of SNZ3 and PHO3 . Molecular docking analysis of SNZ3 and PHO3 mutations on the binding of small molecules. In order to compare the potential effects of PHO3 and SNZ3 mutations on the catalysis of substrates thiamine phosphate and d-ribose 5-phosphate and the formation of thiamine and pyridoxine, we simulated the catalytic activity pockets of the proteins before and after the mutation and docked the proteins before and after the mutation with small molecules. The docking results showed that after the mutation of PHO3 Asn134Asp , its vina score with the small molecule substance thiamine phosphate decreased to-6.6, which indicated that the affinity between the receptor and the ligand was improved (Table S3). At the same time, the interaction of PRO51, PRO363, MET365, and GLY366 residues with small molecules was reduced, and the binding of ALA348, ILE398, and CYS408 residues was increased (Fig. 8 E 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 THR81, ASP231, THR258, PHE260, SER288, TYR289, and VAL375 residues to small molecule thiamine was reduced and the binding of SER253 and LYS254 residues was increased (Fig. 8 F and Table S4). After SNZ3 mutation, its vina score with the small molecule d-ribose 5-phosphate decreased to -5.7 (Table S3), but the residue sites before and after mutation did not change (Fig. 8 K and Table S4). After SNZ3 mutation, its vina score with pyridoxal was still − 5.6, but increased the binding of THR4 and ALA69 residues (Fig. 8 L). Reverse engineering verification improves yeast growth and robustness We performed reverse engineering verification based on the screened mutation information to determine the function of these gene mutations and their effects on yeast. Through Crsispr-cas9 site-directed mutagenesis technology, we obtained three mutant strains S26-PHO3 Mut , S26-SNZ3 Mut , and S26-PHO3 Mut + SNZ3 Mut based on strain S26. The strains were cultured in a YEPD medium containing 20 g/L and 100 g/L glucose, respectively. The growth index and tyrosol production were measured after 72 hours of culture. Under 20 g/L culture conditions, the tyrosol yield of mutant strain S26-SNZ3 Mut increased by 5.61% compared with the control strain S26 at 60 h (Fig. 9 A and Fig. 9 C). Under the condition of 100 g/L glucose culture, the tyrosol production of SNZ3 mutant increased by 17.01% compared with the control strain S26 at 72 hours (Fig. 9 E and Fig. 9 G). The results showed that the mutation of SNZ3 promoted the production of tyrosol in yeast in a 100 g/L glucose medium. Under 100 g/L culture conditions, the tyrosol production of SNZ3 and PHO3 co-mutants increased by 11.37% compared to the control strain S26 at 72 hours. Studies have shown that pyridoxine can promote the growth of yeast and help yeast produce aromatic amino acids [ 39 , 40 ]. Combined with the function of SNZ3, we speculated that the mutation promoted the utilization and transport of pyridoxine. The increased utilization 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. The reverse engineering demonstrated that the single-nucleotide mutation of SNZ3 could increase the titer of tyrosol. Discussion In this paper, in order to restore the growth of the high-yield tyrosol strain S26, we carried out adaptive laboratory evolution. By 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 production strain. In the evolutionary experiment, we obtained three strains, after evaluating the growth and tyrosol production of the three strains. We selected the S26-AE2 strain for transcriptome analysis and genome analysis, trying to clarify the mechanism of evolution on strain growth and tyrosol production through the above analysis. In transcriptome analysis, we analyzed the expression levels of key genes from glucose metabolism to tyrosol production in yeast. The analysis of the expression of these genes showed the metabolic changes of the evolutionary strains S26-AE2, among which the key metabolic changes were the glycolysis pathway, tricarboxylic acid cycle pathway, and tyrosol synthesis pathway. We speculate that this metabolic change may contribute to the recovery of yeast growth and the production of tyrosol. In the genome analysis of the evolutionary strains, we found mutations in the key genes PHO3 and SNZ3 . This gene has the effect of promoting yeast growth in previous studies. S. cerevisiae itself can produce a certain amount of thiamine [ 41 ]. The biosynthesis of thiamine is regulated by NAD + -dependent histone deacetylase Hst1 [ 42 , 43 ]. Studies have shown that exogenous addition of thiamine can promote the production of ethanol by yeast [ 39 , 40 ]. In addition, regulating the synthesis of thiamine in S. cerevisiae can increase the yield of pyruvate [ 44 ]. On this basis, we carried out reverse engineering verification, trying to explore the effect of this gene mutation on yeast growth tyrosol by directed mutation. According to our molecular docking results, we found that gene mutations enhance the interaction between proteins and small molecules. After the mutation of S26, we determined the growth of yeast and found that the mutation of PHO3 and SNZ3 genes was indeed beneficial to the production of growth substrate tyrosol in yeast. However, not all mutations are positive mutations, studies have shown a PHO6 mutant of S cerevisiae , lacking a regulatory gene for the synthesis of periplasmic thiamine-repressible acid phosphatase activity, was found to be 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 ]. The lack of thiamine will affect the activity of phosphoketolase, leading to metabolic disorders. Pyridoxine is the precursor of thiamine (Fig. 10 ). PHO3 Asn134Asp and SNZ3 Val125Ile might act on the increase in the synthesis of pyridoxine and thiamine. Adequate thiamine will theoretically help phosphoketolase increase the production of E4P, the precursor of tyrosol. Therefore, we hypothesized that the mutation of PHO3 and SNZ3 may be one of the reasons for the increase of tyrosol production in the S26-AE2 strain by affecting the activity of phosphoketolase. Conclusion In this work, the growth of the high-yield tyrosol strain S26 was restore though the laboratory adaptive evolution. Transcriptome analysis revealed differences in gene expression between the evolutionary strain S26-AE2 and the original strain S26 in the glycolysis, TCA cycle, and tyrosol synthesis pathways. Molecular docking and reverse engineering verified the effectiveness of SNZ3 Val125Ile mutation in improving tyrosol production. This study provides a theoretical basis for the metabolic engineering of S. cerevisiae to synthesize tyrosol and its derivatives. Materials and methods Strains construction and cultivation The S. cerevisiae 26 was used in this study. YEPD medium (20 g/L peptone, 10 g/L yeast extract, 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 sulfate, without Amion acids), 1.29 g/L Do supplement-Ura was used for yeast transformation. The yeast strains were cultured at 30°C, shaking at 200 rpm for 72 h. E. coli JM110 and E. coli DH5 α were used for the construction of plasmids and cultured in an 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, shaking at 200 rpm. The plasmids used in this study and the primers are listed in Table S1 and Table S2. Adaptive laboratory evolution S. cerevisiae S26 was used as the parental strain and cultured in a shake flask with YEPD medium. The strain S26 suspension was seeded into the EVOL cell (Luoyang Huaqing Tianmu Biotechnology Co., Ltd., Luoyang, China) [ 48 ], which enabled automatically passaged and monitored biomass (OD 600 ). The initial glucose concentration of the evolution medium was 20 g/L and the amount of glucose supplementation gradually increased in the process of evolution. When the biomass reached 15, it was automatically passed to the next generation. Samples were taken in each stage and cultured in a YEPD medium with corresponding glucose concentration. The strains were preserved in a glycerol tube. RNA extraction The S. cerevisiae S26 and S26-AE2 strains were cultured at 30 ℃, 200 rpm for 12 h, the cultures were inoculated into YEPD or YEPD containing 100 g/L glucose with an initial OD 600 of 0.5. Three biological replicates were set up. The cultures were collected, and total RNA was extracted. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD 260/280 = 1.8–2.2, OD 260/230 ≥ 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 Bio-pharm Biotechnology Co., Ltd. (Shanghai, China). Different expression analysis To identify differential expression genes (DEGs) between different samples, differential expression analysis was performed using the DESeq2 or DEGseq. DEGs with |log2FC|≧1 and FDR ≤ 0.05 (DESeq2) or FDR ≤ 0.001 (DEGseq) were considered to be significantly different expressed genes. In addition, the DEGs were mapped to KEGG and the expression level was expressed in the form of heat map. Analytical methods The cell density (OD 600 ) was measured by a spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd., China). The concentration of tyrosol and trehalose was determined by high-performance liquid chromatography (HPLC) (Thermo Fisher Scientific, Massachusetts, USA). Thermo-C18 column (4.6 mm×250 mm, 5 µm) was used. Detection conditions: Mobile phase containing 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 min ~ 50 min 10% B), column temperature was 30 ℃, flow rate was 1 mL/min, detection wavelength was 224 nm. Trehalose was determined by a refractive index detector with a mobile phase of 0.05 mmol/L H 2 SO 4 at a flow rate of 1 mL/min, with a column temperature of 50 ℃. Glucose and ethanol were measured using a biosensor (Sieman Technology Co., Ltd., Shenzhen, China). Glycerin determination with the tissue cell triglyceride (TG) assay kit (Applygen Technologies Inc., Beijing, China). Morphological assays of yeast cells were analyzed by a scanning electron microscope (JEOL JSM6390LV, Tokyo, Japan). Spot assay S. cerevisiae was cultured in YEPD medium to log phase growth, and the cells were harvested by centrifugation (4000 ×g, 5 min) and suspended with sterile water. After that, the cells were diluted to an OD 600 of 1 with sterile water. The yeast cells in aliquots of tenfold serial dilutions were spotted on YEPD plates and YEPD plates with 100 g/L of glucose supplementation and cultured at 30°C for 24 h. SNP analysis The SNP density map is created using CMplot in the R language pack. ( https://www.bioinformatics.com.cn/ ) [ 49 ]. The raw data has been uploaded to the SRA database. Molecular docking The protein structural model of PHO3 and SNZ3 was constructed by SWISS-MODEL ( https://swissmodel.expasy.org/ ). Pumchem obtains the small molecule file for docking ( https://pubchem.ncbi.nlm.nih.gov/ ). Molecular docking was carried out through CB-Dock2 ( https://cadd.labshare.cn/cb-dock2/php/index.php ) [ 50 ]. DNA manipulations The CRISPR-Cas9 system was applied to modify the genome. 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 ]. To construct homologous recombination fragments, the plasmid pUC57 was firstly linearized using restriction enzymes, and the upstream and downstream homologous arms with mutation were ligated together through ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing, China). The gRNA expression vectors and homologous recombination fragments were co-converted into yeast by the LiAc/ssDNA/PEG method [ 53 ]. The corrected yeast clones were selected from the SC-Ura medium. Statistics and reproducibility The significance of groups of data was determined with a t-test by using GraphPad Prism 8 (GraphPad Software, Massachusetts, USA). The data analysis and graphing were performed by GraphPad Prism 8. All experiments were conducted with three biological replicates. The heatmap package in R language is used to make heat maps and volcano maps. Abbreviations Abbreviation Full name Abbreviation Full name ACO2 Aconitate hydratase LSC Succinyl-CoA ligase ACS1/ACS2 Acetyl-CoA ligase MAE1 Malate dehydrogenase ALD6 Aldehyde dehydrogenase MAL32 Alpha-glucosidase ALE Adaptive laboratory evolution MDH2/MDH3 Malate dehydrogenase ARO10 Phenylpyruvate decarboxylase MLS1 Malate synthase ARO2 Bifunctional chorismate synthase/riboflavin reductase MPC1 Mitochondrial pyruvate carrier ARO3/ARO4 3-deoxy-7-phosphoheptulonate synthase OA Oxaloacetate ARO7 Chorismate mutase PDB1 Pyruvate dehydrogenase DAHP 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate PDC6 Indolepyruvate decarboxylase 6 DOT6 Transcriptional regulatory protein PDX1 Pyridoxine biosynthesis protein 1 ENO1/ENO2 Phosphopyruvate hydratase PEP Phosphoenolpyruvate FBA1 Fructose-bisphosphate aldolase PFK Phosphofructokinase FBP Fructose-1,6-bisphosphatase PGI1 phosphoglucose isomerase 1 GAP Type I glyceraldehyde-3-phosphate dehydrogenase PGK1 Phosphoglycerate kinase 1 GLK1 Glucokinase PHK Peterologous phosphoketolase pathway GND1/GND2 Phosphogluconate dehydrogenase PHO3/PHO5/PHO6 Acid phosphatase GPD1/GPD2 Glycerol-3-phosphate dehydrogenase PPP Pentose phosphate pathway GPM1/GPM2 Phosphoglycerate mutase PYC1 Pyruvate carboxylase 1 GPP2 Glycerol-3-phosphate dehydrogenase PYK1 Pyruvate kinase GUT1 Glycerol kinase RKl1 Ribose-5-phosphate isomerase GUT2 Glycerol-3-phosphate dehydrogenase SDH Succinate dehydrogenase membrane anchor subunit HXK1/HXK2 Hexokinase 1 SHH4 Succinate dehydrogenase HXT2/HXT10 High-affinity glucose transporter genes SNP Single nucleotide polymorphism HXT3 Low affinity glucose transporter SNZ3 Pyridoxal 5'-phosphate synthase HXT4 hexose transporter SOL3/SOL4 6-phosphogluconolactonase HXT5 Low/medium affinity glucose transporter TCA Tricarboxylic acid cycle IDP2 Isocitrate dehydrogenase TKL1/TKL2 Transketolase KGO - TYR1 Prephenate dehydrogenase (NADP+) LAT1 Dihydrolipoyllysine-residue acetyltransferase ZWF1 NADP+-dependent dehydrogenase Declarations Author 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 editing. 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 (T2022011), the Natural Science Foundation of Hubei Province (No. 2024AFB803). Availability of data and materials The datasets presented in this study can be found in online repositories. Transcriptome data has been uploaded to the SRA database. The accession numbers are: SRR30733273, SRR30733275, SRR30733276, SRR30733274. The data can be found below: https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1163055. Ethics, Consent to Participate, and Consent to Publish declarations: not applicable. Competing interests Authors declare that this manuscript is original and authors have no known conflict of interest associated with this manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5667010","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392537185,"identity":"7adb8d59-68a8-492f-9357-6202273f7940","order_by":0,"name":"Na Song","email":"","orcid":"","institution":"Hubei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Song","suffix":""},{"id":392537186,"identity":"abf3f1df-ee59-436b-a1a7-f9efc13237d9","order_by":1,"name":"Huili Xia","email":"","orcid":"","institution":"Huanghuai University","correspondingAuthor":false,"prefix":"","firstName":"Huili","middleName":"","lastName":"Xia","suffix":""},{"id":392537187,"identity":"619abe2f-b391-436c-8540-de3364a12e80","order_by":2,"name":"Xiaoxue Yang","email":"","orcid":"","institution":"Hubei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxue","middleName":"","lastName":"Yang","suffix":""},{"id":392537188,"identity":"1e14a2e8-53b4-4e62-818d-e409021da23d","order_by":3,"name":"Siyao Liu","email":"","orcid":"","institution":"Hubei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Siyao","middleName":"","lastName":"Liu","suffix":""},{"id":392537189,"identity":"dc133d28-bac8-4756-92ed-a5c1e34712ae","order_by":4,"name":"Linglong Xu","email":"","orcid":"","institution":"Hubei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Linglong","middleName":"","lastName":"Xu","suffix":""},{"id":392537190,"identity":"d2f8bfbd-dc4a-4aba-a58d-f2a9f045d3ca","order_by":5,"name":"Kun Zhuang","email":"","orcid":"","institution":"Hubei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Zhuang","suffix":""},{"id":392537191,"identity":"f2de036c-b6c5-484e-b0e2-ccf319908620","order_by":6,"name":"Lan Yao","email":"","orcid":"","institution":"Hubei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Lan","middleName":"","lastName":"Yao","suffix":""},{"id":392537192,"identity":"88f0e82f-6224-433d-936f-41cd70b2ce6a","order_by":7,"name":"Shihui Yang","email":"","orcid":"","institution":"Hubei University","correspondingAuthor":false,"prefix":"","firstName":"Shihui","middleName":"","lastName":"Yang","suffix":""},{"id":392537193,"identity":"4cfb10a0-5089-491d-b157-6eaee6fb6550","order_by":8,"name":"Xiong Chen","email":"","orcid":"","institution":"Hubei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiong","middleName":"","lastName":"Chen","suffix":""},{"id":392537194,"identity":"78d294b9-9b7c-4b05-979f-b85104958acc","order_by":9,"name":"Jun Dai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBACxgYGBgkGA5sEAzCXjXgtaUAtzERqAQEJBobDJGhhnpF78MaPgvN55hL5Bxg+lB1m4J/dQMBhM/KSLXsMbhdbzkgGcs4dZpC4c4CQlhwzCR6D24kbbiQzMPO2HWYwkEggrEXyj8E5iJa/xGqR5jE4ANHCSJSWnjfG1jIGyYk7ex4bHOw5l84jcYOAFsP2HMObb/7YJW5nT3z44EeZtRz/DEJaGpA4B4CYB796IJAnqGIUjIJRMApGAQAjukKvH0MnugAAAABJRU5ErkJggg==","orcid":"","institution":"Hubei University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Dai","suffix":""}],"badges":[],"createdAt":"2024-12-18 07:23:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5667010/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5667010/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13068-025-02627-4","type":"published","date":"2025-03-05T15:56:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72025506,"identity":"1f25b29b-eabd-4915-8386-9358cd06d518","added_by":"auto","created_at":"2024-12-20 18:31:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":174469,"visible":true,"origin":"","legend":"\u003cp\u003eLaboratory adaptive evolution diagram of \u003cem\u003eS. cerevisiae \u003c/em\u003eS26. The total evolution time was 529 hours, and the evolution process was divided into three stages.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/ad13f353a604576624e27ccb.png"},{"id":72025474,"identity":"ced1567f-4d1d-4712-a464-4cc434c9c148","added_by":"auto","created_at":"2024-12-20 18:31:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3076330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eThe cellular morphology of the original strain and the evolved strains cultured in 100 g/L glucose medium for 24 hours was observed by scanning electron microscope. \u003cstrong\u003eB \u003c/strong\u003eThe picture of spot assay. After the original strain and the evolved strain were cultured on a solid YEPD medium for 24 hours, which contained 20 g/L and 100 g/L glucose, a picture was taken.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/8a43c312ff20a95ad4e90d2e.png"},{"id":72026088,"identity":"78839900-6965-4122-b9d2-ee65d487061c","added_by":"auto","created_at":"2024-12-20 18:39:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":165237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e, and \u003cstrong\u003eD\u003c/strong\u003e are the OD\u003csub\u003e600 \u003c/sub\u003eand dry weight of the strain S26, S26-AE1, S26-AE2, and S26-AE3 which were cultured in YEPD liquid medium containing 100 g/L glucose. \u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e, and \u003cstrong\u003eH\u003c/strong\u003e are the tyrosol production of the strain S26, S26-AE1, S26-AE2, and S26-AE3 which were cultured in a YEPD liquid medium containing 100 g/L glucose.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/f942324da9edf6bab3747df7.png"},{"id":72025516,"identity":"c2d82617-577c-4f0e-9231-8cfa9d8ebbd7","added_by":"auto","created_at":"2024-12-20 18:31:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":164483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e, and \u003cstrong\u003eD\u003c/strong\u003e are the glucose consumption and ethanol production of the strain S26, S26-AE1, S26-AE2, and S26-AE3 which were cultured in YEPD liquid medium containing 100 g/L glucose. \u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e, and \u003cstrong\u003eH\u003c/strong\u003e are the trehalose changes of the strain S26, S26-AE1, S26-AE2, and S26-AE3 which were cultured in a YEPD liquid medium containing 100 g/L glucose. \u003cstrong\u003eI\u003c/strong\u003e, \u003cstrong\u003eJ\u003c/strong\u003e, \u003cstrong\u003eK\u003c/strong\u003e, and \u003cstrong\u003eL\u003c/strong\u003e are the glycerol changes of the strain S26, S26-AE1, S26-AE2, and S26-AE3 which were cultured in a YEPD liquid medium containing 100 g/L glucose.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/18f9c6b2c587250265dd535f.png"},{"id":72025482,"identity":"3a091854-2fc1-4e24-b36e-01117927facd","added_by":"auto","created_at":"2024-12-20 18:31:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":351773,"visible":true,"origin":"","legend":"\u003cp\u003eThe volcano plots of gene expression levels of strains S26 and S26-AE2 under 20 g/L and 100 g/L glucose. \u003cstrong\u003eA \u003c/strong\u003eThe number of differential genes of group C100 compared with group C20. \u003cstrong\u003eB \u003c/strong\u003eThe number of differential genes of group S20 compared with group C20, \u003cstrong\u003eC \u003c/strong\u003eThe number of differential genes of group S100 compared with group S20, \u003cstrong\u003eD\u003c/strong\u003e The number of differential genes of group S100 compared with group C100. Group C20 represents the strain S26 cultured under 20 g/L glucose conditions, the group C100 represents the strain S26 cultured under 100 g/L glucose conditions. The group S20 represents the strain S26-AE2 cultured under 20 g/L glucose conditions, and the group S100 represents the strain S26-AE2 cultured under 100 g/L glucose conditions.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/123ca12c1450184467004132.png"},{"id":72025477,"identity":"3c086f63-a9b5-4cb0-93a4-bceb9ef2bb1b","added_by":"auto","created_at":"2024-12-20 18:31:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":763714,"visible":true,"origin":"","legend":"\u003cp\u003eThe metabolic pathway map of S26 and S26-AE2 gene expression level under 20 g/L and 100 g/L glucose conditions. \u003cstrong\u003eA\u003c/strong\u003eGlycolysis pathway. \u003cstrong\u003eB\u003c/strong\u003e The pentose phosphate pathway. \u003cstrong\u003eC\u003c/strong\u003eTricarboxylic acid (TCA) cycle pathway. \u003cstrong\u003eD\u003c/strong\u003e Glyoxylic acid pathway. \u003cstrong\u003eE\u003c/strong\u003eThe tyrosol synthesis pathway. Group C20 (C20-1, C20-2, and C20-3) represents the gene expression levels of strain S26 cultured under 20 g/L glucose conditions. Group S20 (S20-1, S20-2, and S20-3) represents the gene expression levels of strain S26 cultured under 100 g/L glucose conditions. Group C100 (C100-1, C100-2, and C100-3) represents the gene expression levels of strain S26-AE2 cultured under 20 g/L glucose conditions. Group S100 (S100-1, S100-2, and S100-3) represents the gene expression levels of strain S26-AE2 cultured under 100 g/L glucose conditions.\u003c/p\u003e","description":"","filename":"Fig.6A6B.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/8b12868d689b816b283a6b83.png"},{"id":72025519,"identity":"42743d24-9722-4af8-8c2a-a799395a645a","added_by":"auto","created_at":"2024-12-20 18:31:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":140104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e The SNP density map of strain S26AE-2 represents the distribution of SNPs on chromosomes. \u003cstrong\u003eB\u003c/strong\u003e The petal map of SNPs event represents the statistical analysis of SNPs on introns of group S100vsC20, S100vsC100, S20vsC20, C100vsC20, and S100vsS20.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/e95fc0be23313972e50493db.png"},{"id":72025490,"identity":"f88cc2bf-1b69-4ce2-9b7e-cf766d5068b4","added_by":"auto","created_at":"2024-12-20 18:31:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3442475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e, and \u003cstrong\u003eJ\u003c/strong\u003e represent the catalytic centers of PHO3 and SNZ3 proteins before and after mutation. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e represent the molecular docking of PHO3\u003csup\u003eWT\u003c/sup\u003e with thiamine phosphate and thiamine. \u003cstrong\u003eE\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e represent the molecular docking of PHO3\u003csup\u003eAsn134Asp\u003c/sup\u003e with thiamine phosphate and thiamine. \u003cstrong\u003eH\u003c/strong\u003e, \u003cstrong\u003eI\u003c/strong\u003e represent the molecular docking of SNZ\u003csup\u003eWT\u003c/sup\u003e with D-Ribose 5-phosphate and pyridoxal. \u003cstrong\u003eK \u003c/strong\u003eand \u003cstrong\u003eL\u003c/strong\u003e represent the molecular docking of SNZ\u003csup\u003eVal125Ile \u003c/sup\u003ewith D-Ribose 5-phosphate and pyridoxal.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/603618b3a05a9cd5b24b4738.png"},{"id":72025503,"identity":"9fe181be-49e3-40fb-a9ea-ed59584908cb","added_by":"auto","created_at":"2024-12-20 18:31:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":318049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e, and \u003cstrong\u003eD\u003c/strong\u003e represent the OD\u003csub\u003e600\u003c/sub\u003e and tyrosol production of strain S26, S26-PHO3\u003csup\u003eMut\u003c/sup\u003e, S26-SNZ3\u003csup\u003eMut\u003c/sup\u003e, and S26-PHO3\u003csup\u003eMut\u003c/sup\u003e+SNZ3\u003csup\u003eMut \u003c/sup\u003ewhich cultured on YEPD containing 20 g/L glucose. \u0026nbsp;\u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e, and \u003cstrong\u003eH\u003c/strong\u003e represent the OD\u003csub\u003e600\u003c/sub\u003e and tyrosol production of strain S26, S26-PHO3\u003csup\u003eMut\u003c/sup\u003e, S26-SNZ3\u003csup\u003eMut\u003c/sup\u003e, and S26-PHO3\u003csup\u003eMut\u003c/sup\u003e+SNZ3\u003csup\u003eMut \u003c/sup\u003ewhich cultured on YEPD containing 100 g/L glucose.\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/faed85664ebc9b08a459ec4c.png"},{"id":72026098,"identity":"eb37286f-a3c8-4b5c-839a-61ea43c9bda7","added_by":"auto","created_at":"2024-12-20 18:39:50","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":448679,"visible":true,"origin":"","legend":"\u003cp\u003eRegulatory mechanism of thiamine-dependent enzyme. Xfpk is an important phosphoketolase involved in the synthesis of tyrosol.\u003c/p\u003e","description":"","filename":"Fig.10.png","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/d969567d9a99af108b7687ed.png"},{"id":78190447,"identity":"c692000b-a54c-49b7-b42d-0ec77f935845","added_by":"auto","created_at":"2025-03-10 19:49:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9425875,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/80234a8f-9561-4410-8c5c-b60374f23a36.pdf"},{"id":72025491,"identity":"be247b7a-3278-47b6-a4d5-97d4260fc746","added_by":"auto","created_at":"2024-12-20 18:31:49","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27132812,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/980ddafa407baf733b69a8b2.tif"},{"id":72025479,"identity":"65db0494-2e2e-493f-aaaf-2af17970a08f","added_by":"auto","created_at":"2024-12-20 18:31:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20604,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-5667010/v1/701eb3d05ffbab083639e943.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptome analysis and reverse engineering verification of SNZ3 Val125Ile and Pho3 Asn134Asp revealed the mechanism of laboratory adaptive evolution to increase the yield of tyrosol in Saccharomyces cerevisiae S26","fulltext":[{"header":"Background","content":"\u003cp\u003eTyrosol is the precursor of salidroside and hydroxytyrosol and exhibits various pharmaceutical activities such as anticancer, antioxidant, and anti-inflammatory activities [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the olive leaf extraction method faces the problem of low extraction efficiency, and its production is highly subject to the supply of olive leaf resources. More importantly, both methods are not environmentally friendly, whether by extraction or chemical synthesis. In contrast, microbial cell factories exhibit many characteristics that are conducive to low-cost, environmentally friendly, and sustainable production of tyrosol [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In recent years, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eE. coil\u003c/em\u003e was successfully used to produce tyrosol [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Through these studies, titers of tyrosol achieved 1.3 g/L in \u003cem\u003eS. cerevisiae\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and 2.12 g/L in \u003cem\u003eE. coil\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although a certain amount of high yield has been achieved in \u003cem\u003eS. cerevisiae\u003c/em\u003e, with the increase of modification, the robustness of yeast is reduced. In the early stage of our laboratory, a strain of \u003cem\u003eS. cerevisiae\u003c/em\u003e S26 with a high yield of tyrosol was constructed. However, excessive modification caused the growth of yeast to be not vigorous, which greatly limited its application in fermentation tanks and industrial production.\u003c/p\u003e \u003cp\u003eAdaptive laboratory evolution (ALE) is an efficient method to obtain strains with improved traits [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Through natural selection, abundant mutants are accumulated to quickly obtain strains with ideal characteristics. ALE has been applied to optimize 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 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. ALE also can be combined with DNA sequence and bioinformatics analysis to understand the basic mechanism of molecular evolution and adaptive changes accumulated in the long-term selection process of microbial populations under specific growth conditions. Zongjie Dai et al. combining rational design, adaptive laboratory evolution, and reverse engineering, the specific growth rate of the best strain reaches 0.218 h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which is close to the maximum growth of \u003cem\u003eS. cerevisiae\u003c/em\u003e with purely respiratory metabolism [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Henri Ingelman et al. used three different and independent adaptive laboratory evolution strategies to evolve the wild-type \u003cem\u003eClostridium autoethanogenum\u003c/em\u003e to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Kuk-Ki Hong et al. improved the growth rate of \u003cem\u003eS. cerevisiae\u003c/em\u003e under specific conditions of galactose by adaptive evolution [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Ryan A. LaCroix et al. used adaptive laboratory evolution to find that key mutations caused \u003cem\u003eE. coli\u003c/em\u003e K-12 MG1655 to grow rapidly on glucose-based media [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Eugen Pfeifer et al. performed a comparative adaptive laboratory evolution experiment on \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e ATCC 13032 and its phage-free variant MB001, thereby increasing the growth rate on the lowest glucose medium [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we used glucose as a single factor for adaptive evolution, and \u003cem\u003eS.cerevisiae\u003c/em\u003e S26 was used as the starting strain for laboratory evolution [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Three strains were obtained in the evolutionary stage, and the strain S26-AE2 with better physiological characteristics was selected by experimental verification. Transcriptome analysis of the evolutionary strains revealed the changes in gene expression in the metabolic pathway of yeast after evolution. The increase of the TCA cycle and aromatic amino acid gene expression may act on the growth recovery of \u003cem\u003eS. cerevisiae\u003c/em\u003e and the increase of tyrosol content. According to the screened potential single nucleotide polymorphism (SNP) mutations PHO3\u003csup\u003eAsn134Asp\u003c/sup\u003e and SNZ3\u003csup\u003eVal125Ile\u003c/sup\u003e, and reverse engineering verification were performed. The results showed that the SNZ3\u003csup\u003eVal125Ile\u003c/sup\u003e mutation had a positive correlation with the production of yeast tyrosol. The research results will provide ideas for obtaining more robust industrial \u003cem\u003eS. cerevisiae\u003c/em\u003e strains and provide a theoretical basis for further modification of the strain to increase tyrosol production.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAdaptive laboratory evolution of\u003c/b\u003e \u003cb\u003eS. cerevisiae\u003c/b\u003e \u003cb\u003eS26 for improved growth phenotypes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo improve the growth phenotype of \u003cem\u003eS. cerevisiae\u003c/em\u003e S26, adaptive laboratory evolution was performed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the evolutionary process has gone through three stages. At stage 1, the concentration of glucose was increased to 40 g/L, and the strain was named S26-AE1. At this stage, the average value of the cycle was 16 hours. At stage 2, the concentration of glucose was increased from 40 g/L to 80 g/L. A total of 20 passages were carried out in this stage, and the strain S26-AE2 was obtained. The strain passaged once every 11.75 hours on average, and the time of the growth cycle was shortened. At stage 3, the glucose content increased from 80 g/L to 125 g/L and the strain was passaged once every 11.95 hours on average, the evolutionary strain was named S26-AE3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, the cellular morphology of yeast was observed by scanning electron microscopy (SEM). There was a small amount of cell damage in S26-AE1. S26-AE2 showed obvious cell damage, which may be attributed to the premature transition of cells into the stable phase or the increase of sugar concentration. However, with the further increase of evolutionary sugar concentration, the S26-AE3 strain did not appear more cell damage. In order to investigate the growth phenotype of strains after ALE, we performed the spot assay of strains S26-AE1, S26-AE2, and S26-AE3 were performed under 20 g/L and 100 g/L glucose, respectively. The growth of the evolutionary strains in 20 g/L and 100 g/L glucose medium was better than that of the original strain, indicating that adaptive laboratory evolution was conducive to the improvement of strain growth.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFermentation performance changes of evolutionary strains\u003c/h2\u003e \u003cp\u003eTo determine the fermentation performance of evolutionary strains, the starting strain S26 and the evolutionary strains S26-AE1, S26-AE2, and S26-AE3 were cultured in a YEPD medium containing 100 g/L glucose. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the starting strain S26 entered a stable period in 48 hours, with an average OD\u003csub\u003e600\u003c/sub\u003e of 36.39 and a dry weight of 15.45 gDCW/L. The biomass of S26-AE1 was 8.99% higher than that of S26 at 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). S26-AE2 entered a stable phase after 24 hours, which was earlier than that of strain S26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The OD\u003csub\u003e600\u003c/sub\u003e in this period was 38.45 and the biomass of strain S26-AE2 was 17.82 gDCW/L at 48 hours, which was 15.33% higher than that of strain S26. Similarly, S26-AE3 also entered a stable period after 24 hours, with an OD\u003csub\u003e600\u003c/sub\u003e of 39.14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). 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 utilization of glucose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to explore whether the adaptation of yeast to a high glucose environment is helpful to enhance the de novo synthesis of tyrosol, we further detected the content of tyrosol. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, 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). The tyrosol production was no significant difference between strain S26-AE1 and strain S26. Surprisingly, the tyrosol production of S26-AE2 was significantly improved, especially at 48 hours, and its tyrosol production reached 817.83 mg/L, which was 69.20% higher than the highest yield of strain S26-AE1. However, the production of tyrosol in S26-AE3 reached 553.79 mg/L after 72 hours of fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). It could be seen that the evolutionary S26-AE2 was beneficial to the production of tyrosol.\u003c/p\u003e \u003cp\u003eTo further explore the growth characteristics and physiological characteristics of the evolved strains. We determined 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 of the starting strain S26 in the fermentation medium for 12 hours was 89.30 g/L, and the glucose residue 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The evolved strains S26-AE2 and S26-AE3 consumed glucose completely after 12 hours and produced 34.16 g/L and 36.12 g/L ethanol, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, strains S26 and S26-AE1 accumulated a certain amount of trehalose in the extracellular during the fermentation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Compared with them, the trehalose production of strains S26-AE2 and S26-AE3 was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). The intracellular glycerol content of S26 was 0.0378 mM at 24 hours of fermentation, and the glycerol contents of S26-AE1, S26-AE2 and S26-AE3 were 0.0103 mM, 0.0243 mM and 0.0083 mM, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). Both trehalose and glycerol played an osmotic pressure protective role in cells, thereby reducing the damage caused by external stress on cells. In the process of evolution, the strains reduced the production of trehalose and glycerol, it could be speculated that the evolved strains have gradually adapted to grow on 100 g/L glucose medium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptome analysis of the mechanisms underlying adaptive evolution that leads to increased tyrosol production in S26-AE2\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the mechanism of S26-AE2 biomass increase and tyrosol increase after evolution, we used transcriptome to analyze the changes in gene expression of strains S26 and S26-AE2. Strains were cultured under 20 g/L glucose and 100 g/L glucose, respectively, and RNA was extracted for transcriptome determination after 24 hours of culture. According to the results of the transcriptome, the genes with significant differential expression were expressed in the form of volcanic maps, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. S26 had 807 up-regulated genes and 583 down-regulated genes under 100 g/L glucose compared to 20 g/L glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Compared with S26, S26-AE2 had 36 up-regulated genes and 1 down-regulated gene under 20 g/L glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Compared with S26-AE2 at 20 g/L glucose, S26-AE2 at 100 g/L glucose had 779 up-regulated genes and 621 down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The evolutionary strain S26-AE2 had 9 up-regulated genes and 16 down-regulated genes at 100 g/L glucose compared to the original strain S26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). This result indicated that the strains have significant differences in metabolism after laboratory evolution, and these differences may be due to the recovery of growth or the improvement of tyrosol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to further study the role of these differential genes in the metabolic pathway, we map these differential genes to the metabolic pathway to obtain a metabolic map of differentially expressed genes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cem\u003eHXT5\u003c/em\u003e is a low/medium affinity glucose transporter gene. the expression of gene \u003cem\u003eHXT5\u003c/em\u003e increased significantly in S26-AE2 at 20 g/L glucose conditions. According to the research of Stefan Buziol et al., the gene \u003cem\u003eHXT5\u003c/em\u003e was not expressed during the growth of the yeast cells in a rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The expression levels of \u003cem\u003eHXK1\u003c/em\u003e and \u003cem\u003eGLK1\u003c/em\u003e were lower at 100 g/L glucose conditions than that at 20 g/L glucose conditions both in strain S26 and S26AE-2 which is similar to previous studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While the expression of \u003cem\u003eHXK2\u003c/em\u003e under 100 g/L glucose conditions was significantly higher than that under 20 g/L glucose conditions in strain. S26 and S26-AE2. According to the research of Rodr\u0026iacute;guez A et.al, when glucose is present in the culture medium the \u003cem\u003eGLK1\u003c/em\u003e gene is either not expressed or expressed at a very low level and the transcription of \u003cem\u003eHXK1\u003c/em\u003e and \u003cem\u003eGLK1\u003c/em\u003e genes is automatically regulated by glycolytic products and themselves [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eHXK2\u003c/em\u003e is a dual-function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eHXK2\u003c/em\u003e also plays an important regulatory role in glucose repression [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. According to this information, we speculated that after adaptive evolution, the decreased expression of \u003cem\u003eHXK2\u003c/em\u003e in strain S26-AE2 reduced the transcriptional regulation of glucose repression in cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PGI1 (phosphoglucose isomerase 1) catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate, glucose 6-phosphate is entirely rerouted into the PP pathway. The gene expression of \u003cem\u003ePGI1\u003c/em\u003e in strain S26-AE2 increased slightly under 100 g/L glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Related research showed that \u003cem\u003ePGI1\u003c/em\u003e-deletion mutants of \u003cem\u003eS. cerevisiae\u003c/em\u003e cannot grow on glucose as the sole carbon source and are even inhibited by glucose [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, the increase of \u003cem\u003ePGI1\u003c/em\u003e gene expression may promote the utilization of glucose by evolutionary strain S26-AE2, which was consistent with the increase in glucose consumption rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eUnder the condition of 100 g/L glucose, the gene expression of \u003cem\u003eGPD1\u003c/em\u003e, \u003cem\u003eGPD2\u003c/em\u003e, and \u003cem\u003eGPP2\u003c/em\u003e increased both in S26 and S26-AE2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The gene \u003cem\u003eGPD1\u003c/em\u003e, one of the osmotic induction genes, encodes glycerol-3-phosphate dehydrogenase, the expression of \u003cem\u003eGPD1\u003c/em\u003e is the result of an interplay between different signaling pathways [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Deletion of structural genes like \u003cem\u003eGPD1\u003c/em\u003e and \u003cem\u003eGPD2\u003c/em\u003e leads to yeast osmosensitivity, which is not desirable for industrial strains [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. \u003cem\u003eGPP1\u003c/em\u003e is an isoenzyme that catalyzes the formation of glycerol and is also involved in the formation of acetic acid. In the previous work of this paper, to reduce the production of acetic acid in the heterologous phosphoketolase (PHK) pathway, \u003cem\u003eGPP1\u003c/em\u003e was knocked out [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, due to the presence of \u003cem\u003eGPP2\u003c/em\u003e, knockout of \u003cem\u003eGPP1\u003c/em\u003e does not seem to affect glycerol production. The gene expression of \u003cem\u003eGPP2\u003c/em\u003e in S26-AE2 was slightly lower than that of S26, which was consistent with the results of glycerol determination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Glycerol enters the glycolytic pathway through the sequential action of two genes, namely cytosolic glycerol kinase GUT1 and glycerol-3-phosphate dehydrogenase GUT2 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Interestingly, the expression of \u003cem\u003eGUT2\u003c/em\u003e in strain S26-AE2 was increased under 20 g/L glucose conditions.\u003c/p\u003e \u003cp\u003ePyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The gene expression levels of \u003cem\u003ePGK1\u003c/em\u003e, \u003cem\u003eGPM1\u003c/em\u003e, \u003cem\u003eGPM2\u003c/em\u003e, \u003cem\u003eENO1\u003c/em\u003e/\u003cem\u003eENO2\u003c/em\u003e, and \u003cem\u003ePYK1\u003c/em\u003e involved in pyruvate production in the S26-AE2 were higher than those in strain S26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Therefore, the evolved strains S26-AE2 are likely to increase the production of pyruvate. In addition, the expression of \u003cem\u003ePYK2\u003c/em\u003e was low under 100 g/L glucose. Related studies have shown that the absence of \u003cem\u003ePYK2\u003c/em\u003e has no effect on the specific growth rate with glucose as a carbon source, which may also explain why the expression of \u003cem\u003ePYK2\u003c/em\u003e is low under 100 g/L glucose conditions [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pentose phosphate pathway (PPP) is a way of glucose oxidative decomposition. Under the condition of 20 g/L glucose, the genes \u003cem\u003eSOL\u003c/em\u003e3, \u003cem\u003eGND\u003c/em\u003e1, and \u003cem\u003eTKL\u003c/em\u003e1 showed down-regulation in strain S26, and the genes \u003cem\u003eSOL\u003c/em\u003e4, \u003cem\u003eGND\u003c/em\u003e2, and \u003cem\u003eTKL\u003c/em\u003e2 showed up-regulation which is similar to the research of Bergman A et.al (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Under 100 g/L glucose conditions, the expressed genes \u003cem\u003eSOL\u003c/em\u003e3/4, \u003cem\u003eGND\u003c/em\u003e1/2, and \u003cem\u003eTKL\u003c/em\u003e1/2 were opposite to those under 20 g/L glucose conditions. However, after evolution, this rule was broken in strain S26-AE2. The \u003cem\u003eSOL3\u003c/em\u003e and \u003cem\u003eSOL4\u003c/em\u003e of strain S26-AE2 were down-regulated under 20 g/L glucose conditions and were up-regulated under 100 g/L glucose conditions. In addition, under the condition of 20 g/L glucose, the expression levels of \u003cem\u003eZWF1\u003c/em\u003e and \u003cem\u003eRKl1\u003c/em\u003e increased in the S26-AE2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), which was significantly higher than that of S26, while the expression level of the gene was down-regulated under the condition of 100 g/L glucose, indicating that the gene was regulated by the external glucose content. \u003cem\u003eZWF1\u003c/em\u003e is a NADP\u003csup\u003e+\u003c/sup\u003e-dependent dehydrogenase, and overexpression of \u003cem\u003eZWF1\u003c/em\u003e can increase the supply of NADPH in yeast cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe TCA cycle is an important hub of energy metabolism. Transcriptome analysis showed that most of the genes involved in the TCA cycle were up-regulated under 20 g/L glucose conditions, and the gene expression under 100 g/L glucose conditions was down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In addition, there is an obvious phenomenon that the gene expression of the S26-AE2 under 20 g/L glucose condition was significantly higher than that of the strain S26. It indicated that the TCA cycle-related genes of the evolutionary strains were more active. The higher expression levels include the SDH enzyme family (Succinate dehydrogenase complex), LSC enzyme (Succinyl-CoA ligase), and KGO enzyme. However, the difference was not obvious at 100 g/L glucose, suggesting that this difference may be related to the extracellular glucose content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The enhancement of the TCA cycle promotes the consumption of pyruvate which mainly comes from the decomposition of glucose by cells. The TCA cycle has a promoting effect on cell growth and energy generation, which in a certain sense explains the recovery of strain growth through glucose evolution. Under the condition of 100 g/L glucose, the gene expression levels of \u003cem\u003eMPC1\u003c/em\u003e, \u003cem\u003ePDB1\u003c/em\u003e, \u003cem\u003eLAT1\u003c/em\u003e, \u003cem\u003ePDX1\u003c/em\u003e, \u003cem\u003eSHH4\u003c/em\u003e, and \u003cem\u003eLSC1\u003c/em\u003e in the evolutionary strains were slightly higher than those in strain 26, and the expression levels of \u003cem\u003eACO2\u003c/em\u003e and \u003cem\u003eMAE1\u003c/em\u003e were 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, used to generate energy, or used as precursors for the biosynthesis of branched-chain amino acids [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, the increased gene expression of \u003cem\u003eMPC1\u003c/em\u003e in the evolutionary strain under 100 g/L glucose conditions might enhance the intake of pyruvate and reduce the accumulation of pyruvate produced by glycolysis.\u003c/p\u003e \u003cp\u003eThe glyoxylic acid cycle is particularly important for plants and some microorganisms. This pathway is the compensation pathway of the TCA cycle. Through the glyoxylic acid cycle, yeast can use fatty acids and acetic acid as the sole carbon source to synthesize carbohydrates, amino acids, and proteins to maintain normal cell growth. Under 20 g/L glucose, the gene expression levels of \u003cem\u003ePYC1\u003c/em\u003e, \u003cem\u003eMDH2\u003c/em\u003e, \u003cem\u003eMDH3\u003c/em\u003e, \u003cem\u003eMLS1\u003c/em\u003e, \u003cem\u003eIDP2\u003c/em\u003e, \u003cem\u003eACS1\u003c/em\u003e, \u003cem\u003eACS2\u003c/em\u003e, \u003cem\u003eALD6\u003c/em\u003e, and \u003cem\u003ePDC6\u003c/em\u003e in the evolutionary strain were significantly higher than those in the original strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). \u003cem\u003eACS1\u003c/em\u003e and \u003cem\u003eACS2\u003c/em\u003e are involved in acetic acid consumption. \u003cem\u003ePCK1\u003c/em\u003e catalyzes the conversion of oxaloacetate (OA) to phosphoenolpyruvate (PEP), and \u003cem\u003ePYC1\u003c/em\u003e catalyzes the conversion of pyruvate to OA. These two reactions are important steps in the gluconeogenesis pathway required to consume non-glucose carbon sources (such as ethanol or acetate) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In the process of our previous strain construction, \u003cem\u003ePDC1\u003c/em\u003e was knocked out, and the expression of \u003cem\u003ePDC2\u003c/em\u003e increased after adaptive evolution [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This phenomenon proves that the metabolic capacity of evolved yeast is enhanced, but it is not conducive to the production of tyrosol.\u003c/p\u003e \u003cp\u003eThe expression levels of \u003cem\u003eARO3\u003c/em\u003e and \u003cem\u003eARO4\u003c/em\u003e under 100 g/L glucose conditions were higher than those under 20 g/L glucose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The increased expression of \u003cem\u003eARO3\u003c/em\u003e and \u003cem\u003eARO4\u003c/em\u003e will be beneficial to the formation of DAHP and the increase of tyrosol production, which also explains the reason for the increase of tyrosol content in strain S26-AE2. Similarly, the increased expression of \u003cem\u003eARO2\u003c/em\u003e, \u003cem\u003eARO7\u003c/em\u003e, \u003cem\u003eTYR1\u003c/em\u003e, and \u003cem\u003eARO10\u003c/em\u003e was beneficial to the synthesis of tyrosol. The results showed that the appropriate concentration of glucose could mobilize the 'enthusiasm' of aromatic amino acid synthesis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSNP mutation analysis of adaptive laboratory evolutionary strain S26-AE2\u003c/h3\u003e\n\u003cp\u003eIn order to find changes related to growth and tyrosol production at the genetic level, we performed gene sequencing on the evolutionary strain S26-AE2 to obtain SNPs-related information. By aligning all SNPs to chromosomes, it can be seen that the occurrence of SNPs is mainly in CM001525.1 and CM001529.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Subsequently, the number of SNPs that occurred on all introns was statistically analyzed to obtain a petal diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), representing the number of SNPs genes after comparison between the two groups. The results showed that there were 19 SNP mutations in S100vsC20, S100vsS20, and C100vsC20. Then, 19 SNPs were screened and analyzed to obtain Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results showed that there were five genes involved in SNP mutations, namely \u003cem\u003eMAL32\u003c/em\u003e, \u003cem\u003eSNZ3\u003c/em\u003e, \u003cem\u003eDOT6\u003c/em\u003e, \u003cem\u003eHXT3\u003c/em\u003e, and \u003cem\u003ePHO3\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProteins with SNP mutations contained in strain S26-AE2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u0026nbsp;description\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChrom\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenome\u0026nbsp;position\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlt\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAnno\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGene\u0026nbsp;description\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMutations\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMAL32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001523.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e800665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePhe472Phe\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMAL32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001523.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e800863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLeu538Leu\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSNZ3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001527.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eGly93Gly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSNZ3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001527.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eIle109Ile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSNZ3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001527.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8443\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAsn118Asn\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSNZ3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001527.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLeu122Leu\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSNZ3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001527.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003enonsynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eVal125Ile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDOT6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001526.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e332037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003enonsynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLys355Asn\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDOT6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001526.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e332040\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003enonsynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eArg354Ser\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHXT3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001525.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1161695\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eVal538Val\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePHO3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001523.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e422454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003enonsynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAsn134Asp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePHO3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001523.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e422458\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSer132Ser\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePHO3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM001523.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e421594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eexonic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esynonymous\u0026nbsp;SNV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAsp420Asp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNotes:\u0026nbsp;\u003c/strong\u003e1. \u0026ldquo;Chrom\u0026rdquo; means chromosome. 2. \u0026ldquo;Ref\u0026rdquo; means Reference sequence. 3. \u0026ldquo;Alt\u0026rdquo; means Alternative sequence. 4. \u0026ldquo;Anno\u0026rdquo; means Annotation.\u003c/p\u003e \u003cp\u003eMAL32 encodes alpha-glucosidase which is involved in carbohydrate transport and metabolism. The deletion of \u003cem\u003eMAL31\u003c/em\u003e and \u003cem\u003eMAL32\u003c/em\u003e may have led to the aneuploidy of CHRIII that harbours the \u003cem\u003eMAL2\u003c/em\u003e locus [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. SNZ3 is the subunit of pyridoxal 5'-phosphate synthase and a member of a stationary phase-induced gene family. The transcription of \u003cem\u003eSNZ3\u003c/em\u003e is induced prior to diauxic shift, and also in the absence of thiamin in a Thi2p-dependent manner; forms a coregulated gene pair with \u003cem\u003eSNO3\u003c/em\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. 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, the expression is induced in low or high glucose conditions. HXT3 has a paralog, HXT5, that arose from the whole genome duplication. PHO3 is a constitutively expressed acid phosphatase similar to PHO5, brought to the cell surface by transport vesicles. It can hydrolyze thiamin phosphates in the periplasmic space and increase cellular thiamin uptake. The expression of \u003cem\u003ePHO3\u003c/em\u003e is repressed by thiamin.\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, SNZ3 had mutations at 118, 122, and 125 amino acid residues, 118 and 122 were synonymous mutations, and 125 had non-synonymous mutations. PHO3 was mutated at 132, 134, and 420 sites, and only 420 sites had non-synonymous mutations. DOT6 also had non-synonymous mutations at 335 and 354 sites. Next, we first analyzed the mutations of \u003cem\u003eSNZ3\u003c/em\u003e and \u003cem\u003ePHO3\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular docking analysis of\u003c/b\u003e \u003cb\u003eSNZ3\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePHO3\u003c/b\u003e \u003cb\u003emutations on the binding of small molecules.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to compare the potential effects of \u003cem\u003ePHO3\u003c/em\u003e and \u003cem\u003eSNZ3\u003c/em\u003e mutations on the catalysis of substrates thiamine phosphate and d-ribose 5-phosphate and the formation of thiamine and pyridoxine, we simulated the catalytic activity pockets of the proteins before and after the mutation and docked the proteins before and after the mutation with small molecules. The docking results showed that after the mutation of PHO3\u003csup\u003eAsn134Asp\u003c/sup\u003e, its vina score with the small molecule substance thiamine phosphate decreased to-6.6, which indicated that the affinity between the receptor and the ligand was improved (Table S3). At the same time, the interaction of PRO51, PRO363, MET365, and GLY366 residues with small molecules was reduced, and the binding of ALA348, ILE398, and CYS408 residues was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE 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 THR81, ASP231, THR258, PHE260, SER288, TYR289, and VAL375 residues to small molecule thiamine was reduced and the binding of SER253 and LYS254 residues was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF and Table S4). After SNZ3 mutation, its vina score with the small molecule d-ribose 5-phosphate decreased to -5.7 (Table S3), but the residue sites before and after mutation did not change (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK and Table S4). After SNZ3 mutation, its vina score with pyridoxal was still \u0026minus;\u0026thinsp;5.6, but increased the binding of THR4 and ALA69 residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eReverse engineering verification improves yeast growth and robustness\u003c/h3\u003e\n\u003cp\u003eWe performed reverse engineering verification based on the screened mutation information to determine the function of these gene mutations and their effects on yeast. Through Crsispr-cas9 site-directed mutagenesis technology, we obtained three mutant strains S26-PHO3\u003csup\u003eMut\u003c/sup\u003e, S26-SNZ3\u003csup\u003eMut\u003c/sup\u003e\u003csub\u003e,\u003c/sub\u003e and S26-PHO3\u003csup\u003eMut\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;SNZ3\u003csup\u003eMut\u003c/sup\u003e based on strain S26. The strains were cultured in a YEPD medium containing 20 g/L and 100 g/L glucose, respectively. The growth index and tyrosol production were measured after 72 hours of culture. Under 20 g/L culture conditions, the tyrosol yield of mutant strain S26-SNZ3\u003csup\u003eMut\u003c/sup\u003e increased by 5.61% compared with the control strain S26 at 60 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). Under the condition of 100 g/L glucose culture, the tyrosol production of SNZ3 mutant increased by 17.01% compared with the control strain S26 at 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG). The results showed that the mutation of SNZ3 promoted the production of tyrosol in yeast in a 100 g/L glucose medium. Under 100 g/L culture conditions, the tyrosol production of SNZ3 and PHO3 co-mutants increased by 11.37% compared to the control strain S26 at 72 hours. Studies have shown that pyridoxine can promote the growth of yeast and help yeast produce aromatic amino acids [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Combined with the function of SNZ3, we speculated that the mutation promoted the utilization and transport of pyridoxine. The increased utilization 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. The reverse engineering demonstrated that the single-nucleotide mutation of SNZ3 could increase the titer of tyrosol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this paper, in order to restore the growth of the high-yield tyrosol strain S26, we carried out adaptive laboratory evolution. By 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 production strain. In the evolutionary experiment, we obtained three strains, after evaluating the growth and tyrosol production of the three strains. We selected the S26-AE2 strain for transcriptome analysis and genome analysis, trying to clarify the mechanism of evolution on strain growth and tyrosol production through the above analysis.\u003c/p\u003e \u003cp\u003eIn transcriptome analysis, we analyzed the expression levels of key genes from glucose metabolism to tyrosol production in yeast. The analysis of the expression of these genes showed the metabolic changes of the evolutionary strains S26-AE2, among which the key metabolic changes were the glycolysis pathway, tricarboxylic acid cycle pathway, and tyrosol synthesis pathway. We speculate that this metabolic change may contribute to the recovery of yeast growth and the production of tyrosol.\u003c/p\u003e \u003cp\u003eIn the genome analysis of the evolutionary strains, we found mutations in the key genes \u003cem\u003ePHO3\u003c/em\u003e and \u003cem\u003eSNZ3\u003c/em\u003e. This gene has the effect of promoting yeast growth in previous studies. \u003cem\u003eS. cerevisiae\u003c/em\u003e itself can produce a certain amount of thiamine [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The biosynthesis of thiamine is regulated by NAD\u003csup\u003e+\u003c/sup\u003e-dependent histone deacetylase Hst1 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Studies have shown that exogenous addition of thiamine can promote the production of ethanol by yeast [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition, regulating the synthesis of thiamine in \u003cem\u003eS. cerevisiae\u003c/em\u003e can increase the yield of pyruvate [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. On this basis, we carried out reverse engineering verification, trying to explore the effect of this gene mutation on yeast growth tyrosol by directed mutation. According to our molecular docking results, we found that gene mutations enhance the interaction between proteins and small molecules. After the mutation of S26, we determined the growth of yeast and found that the mutation of \u003cem\u003ePHO3\u003c/em\u003e and \u003cem\u003eSNZ3\u003c/em\u003e genes was indeed beneficial to the production of growth substrate tyrosol in yeast. However, not all mutations are positive mutations, studies have shown a \u003cem\u003ePHO6\u003c/em\u003e mutant of \u003cem\u003eS cerevisiae\u003c/em\u003e, lacking a regulatory gene for the synthesis of periplasmic thiamine-repressible acid phosphatase activity, was found to be auxotrophic for thiamine [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThiamine 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 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The lack of thiamine will affect the activity of phosphoketolase, leading to metabolic disorders. Pyridoxine is the precursor of thiamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). PHO3\u003csup\u003eAsn134Asp\u003c/sup\u003e and SNZ3\u003csup\u003eVal125Ile\u003c/sup\u003e might act on the increase in the synthesis of pyridoxine and thiamine. Adequate thiamine will theoretically help phosphoketolase increase the production of E4P, the precursor of tyrosol. Therefore, we hypothesized that the mutation of \u003cem\u003ePHO3\u003c/em\u003e and \u003cem\u003eSNZ3\u003c/em\u003e may be one of the reasons for the increase of tyrosol production in the S26-AE2 strain by affecting the activity of phosphoketolase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this work, the growth of the high-yield tyrosol strain S26 was restore though the laboratory adaptive evolution. Transcriptome analysis revealed differences in gene expression between the evolutionary strain S26-AE2 and the original strain S26 in the glycolysis, TCA cycle, and tyrosol synthesis pathways. Molecular docking and reverse engineering verified the effectiveness of SNZ3\u003csup\u003eVal125Ile\u003c/sup\u003e mutation in improving tyrosol production. This study provides a theoretical basis for the metabolic engineering of \u003cem\u003eS. cerevisiae\u003c/em\u003e to synthesize tyrosol and its derivatives.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStrains construction and cultivation\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eS. cerevisiae\u003c/em\u003e 26 was used in this study. YEPD medium (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose) was used to cultivate \u003cem\u003eS. cerevisiae\u003c/em\u003e, 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 sulfate, without Amion acids), 1.29 g/L Do supplement-Ura was used for yeast transformation. The yeast strains were cultured at 30\u0026deg;C, shaking at 200 rpm for 72 h. \u003cem\u003eE. coli\u003c/em\u003e JM110 and \u003cem\u003eE. coli\u003c/em\u003e DH5 α were used for the construction of plasmids and cultured in an LB medium containing 5 g/L Yeast extract, 10 g/L Peptone, and 10 g/L NaCl, pH 7.2. The \u003cem\u003eE. coli\u003c/em\u003e were cultured at 37\u0026deg;C, shaking at 200 rpm. The plasmids used in this study and the primers are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table S2.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAdaptive laboratory evolution\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eS. cerevisiae\u003c/em\u003e S26 was used as the parental strain and cultured in a shake flask with YEPD medium. The strain S26 suspension was seeded into the EVOL cell (Luoyang Huaqing Tianmu Biotechnology Co., Ltd., Luoyang, China) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], which enabled automatically passaged and monitored biomass (OD\u003csub\u003e600\u003c/sub\u003e). The initial glucose concentration of the evolution medium was 20 g/L and the amount of glucose supplementation gradually increased in the process of evolution. When the biomass reached 15, it was automatically passed to the next generation. Samples were taken in each stage and cultured in a YEPD medium with corresponding glucose concentration. The strains were preserved in a glycerol tube.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eS. cerevisiae\u003c/em\u003e S26 and S26-AE2 strains were cultured at 30 ℃, 200 rpm for 12 h, the cultures were inoculated into YEPD or YEPD containing 100 g/L glucose with an initial OD\u003csub\u003e600\u003c/sub\u003e of 0.5. Three biological replicates were set up. The cultures were collected, and total RNA was extracted. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD\u003csub\u003e260/280\u003c/sub\u003e = 1.8\u0026ndash;2.2, OD\u003csub\u003e260/230\u003c/sub\u003e \u0026ge; 2.0, RIN\u0026thinsp;\u0026ge;\u0026thinsp;6.5, 28S:18S\u0026thinsp;\u0026ge;\u0026thinsp;1.0, \u0026gt; 1 \u0026micro;g) were used to construct the sequencing library. RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDifferent expression analysis\u003c/h2\u003e \u003cp\u003eTo identify differential expression genes (DEGs) between different samples, differential expression analysis was performed using the DESeq2 or DEGseq.\u0026nbsp;DEGs with |log2FC|≧1 and FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05 (DESeq2) or FDR\u0026thinsp;\u0026le;\u0026thinsp;0.001 (DEGseq) were considered to be significantly different expressed genes. In addition, the DEGs were mapped to KEGG and the expression level was expressed in the form of heat map.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical methods\u003c/h2\u003e \u003cp\u003eThe cell density (OD\u003csub\u003e600\u003c/sub\u003e) was measured by a spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd., China). The concentration of tyrosol and trehalose was determined by high-performance liquid chromatography (HPLC) (Thermo Fisher Scientific,\u0026ensp;Massachusetts, USA). Thermo-C18 column (4.6 mm\u0026times;250 mm, 5 \u0026micro;m) was used. Detection conditions: Mobile phase containing 0.05% (v/v) formic acid aqueous solution (A) and acetonitrile (B), gradient elution (0\u0026thinsp;~\u0026thinsp;20 min, 20% B; 20\u0026thinsp;~\u0026thinsp;25 min, 95% B; 25\u0026thinsp;~\u0026thinsp;35 min, 95% B; 35\u0026thinsp;~\u0026thinsp;40 min 95% B; 40 min\u0026thinsp;~\u0026thinsp;50 min 10% B), column temperature was 30 ℃, flow rate was 1 mL/min, detection wavelength was 224 nm. Trehalose was determined by a refractive index detector with a mobile phase of 0.05 mmol/L H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e at a flow rate of 1 mL/min, with a column temperature of 50 ℃. Glucose and ethanol were measured using a biosensor (Sieman Technology Co., Ltd., Shenzhen, China). Glycerin determination with the tissue cell triglyceride (TG) assay kit (Applygen Technologies Inc., Beijing, China). Morphological assays of yeast cells were analyzed by a scanning electron microscope (JEOL JSM6390LV, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSpot assay\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. cerevisiae\u003c/em\u003e was cultured in YEPD medium to log phase growth, and the cells were harvested by centrifugation (4000 \u0026times;g, 5 min) and suspended with sterile water. After that, the cells were diluted to an OD\u003csub\u003e600\u003c/sub\u003e of 1 with sterile water. The yeast cells in aliquots of tenfold serial dilutions were spotted on YEPD plates and YEPD plates with 100 g/L of glucose supplementation and cultured at 30\u0026deg;C for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSNP analysis\u003c/h2\u003e \u003cp\u003eThe SNP density map is created using CMplot in the R language pack. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The raw data has been uploaded to the SRA database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eThe protein structural model of PHO3 and SNZ3 was constructed by SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Pumchem obtains the small molecule file for docking (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Molecular docking was carried out through CB-Dock2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cadd.labshare.cn/cb-dock2/php/index.php\u003c/span\u003e\u003cspan address=\"https://cadd.labshare.cn/cb-dock2/php/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDNA manipulations\u003c/h2\u003e \u003cp\u003eThe CRISPR-Cas9 system was applied to modify the genome. Plasmid pML104 was used to construct gRNA expression vectors [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The specific guide RNA sequences were designed using the CHOPCHOP web tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispor.tefor.net\u003c/span\u003e\u003cspan address=\"http://crispor.tefor.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. To construct homologous recombination fragments, the plasmid pUC57 was firstly linearized using restriction enzymes, and the upstream and downstream homologous arms with mutation were ligated together through ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing, China). The gRNA expression vectors and homologous recombination fragments were co-converted into yeast by the LiAc/ssDNA/PEG method [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The corrected yeast clones were selected from the SC-Ura medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistics and reproducibility\u003c/h2\u003e \u003cp\u003eThe significance of groups of data was determined with a \u003cem\u003et-test\u003c/em\u003e by using GraphPad Prism 8 (GraphPad Software, Massachusetts, USA). The data analysis and graphing were performed by GraphPad Prism 8. All experiments were conducted with three biological replicates. The heatmap package in R language is used to make heat maps and volcano maps.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"593\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFull name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFull name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eACO2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eAconitate hydratase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eLSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eSuccinyl-CoA ligase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eACS1/ACS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eAcetyl-CoA ligase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eMAE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eMalate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eALD6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eAldehyde dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eMAL32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eAlpha-glucosidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eALE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eAdaptive laboratory evolution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eMDH2/MDH3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eMalate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eARO10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003ePhenylpyruvate decarboxylase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eMLS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eMalate synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eARO2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eBifunctional chorismate synthase/riboflavin reductase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eMPC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eMitochondrial pyruvate carrier\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eARO3/ARO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003e3-deoxy-7-phosphoheptulonate synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eOA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eOxaloacetate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eARO7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eChorismate mutase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePDB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePyruvate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eDAHP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003e3-Deoxy-D-arabino-heptulosonic acid 7-phosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePDC6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eIndolepyruvate decarboxylase 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eDOT6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eTranscriptional regulatory protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePDX1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePyridoxine biosynthesis protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eENO1/ENO2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003ePhosphopyruvate hydratase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePEP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePhosphoenolpyruvate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eFBA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eFructose-bisphosphate aldolase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePFK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePhosphofructokinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eFBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eFructose-1,6-bisphosphatase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePGI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ephosphoglucose isomerase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eGAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eType I glyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePGK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePhosphoglycerate kinase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eGLK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eGlucokinase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePHK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePeterologous phosphoketolase pathway\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eGND1/GND2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003ePhosphogluconate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePHO3/PHO5/PHO6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eAcid phosphatase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eGPD1/GPD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eGlycerol-3-phosphate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePentose phosphate pathway\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eGPM1/GPM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003ePhosphoglycerate mutase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePYC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePyruvate carboxylase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eGPP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eGlycerol-3-phosphate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003ePYK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePyruvate kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eGUT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eGlycerol kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eRKl1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eRibose-5-phosphate isomerase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eGUT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eGlycerol-3-phosphate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eSDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eSuccinate dehydrogenase membrane anchor subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eHXK1/HXK2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eHexokinase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eSHH4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eSuccinate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eHXT2/HXT10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eHigh-affinity glucose transporter genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eSNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eSingle nucleotide polymorphism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eHXT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eLow affinity glucose transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eSNZ3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePyridoxal 5\u0026apos;-phosphate synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eHXT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003ehexose transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eSOL3/SOL4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003e6-phosphogluconolactonase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eHXT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eLow/medium affinity glucose transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eTCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eTricarboxylic acid cycle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eIDP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eIsocitrate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eTKL1/TKL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eTransketolase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eKGO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eTYR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003ePrephenate dehydrogenase (NADP+)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eLAT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185px;\"\u003e\n \u003cp\u003eDihydrolipoyllysine-residue acetyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eZWF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eNADP+-dependent dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN 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,\u0026nbsp;and\u0026nbsp;S\u0026nbsp;H\u0026nbsp;Y\u0026nbsp;participated in data analysis and manuscript editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis 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 (T2022011), the Natural Science Foundation of Hubei Province (No. 2024AFB803).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets presented in this study can be found in online repositories. Transcriptome data has been uploaded to the SRA database. The accession numbers are: SRR30733273, SRR30733275, SRR30733276, SRR30733274. The data can be found below: https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1163055.\u003c/p\u003e\n\u003cp\u003eEthics, Consent to Participate, and Consent to Publish declarations: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that this manuscript is original and authors have no known conflict of interest associated with this manuscript. We have not excluded any individual who satisfied the authorship criteria. We confirm that all of the listed authors have read and approved the content of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKim YY, Lee S, Kim MJ, Kang BC, Dhakal H, Choi YA, et al. Tyrosol attenuates lipopolysaccharide-induced acute lung injury by inhibiting the inflammatory response and maintaining the alveolar capillary barrier. 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Glucose repression can be alleviated by reducing glucose phosphorylation rate in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Sci Rep 2018;81:2613.\u003c/li\u003e\n\u003cli\u003eMoreno F, Herrero P. The hexokinase 2-dependent glucose signal transduction pathway of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. FEMS Microbiol Rev 2002;261:83-90.\u003c/li\u003e\n\u003cli\u003eHeux S, Cadiere A, Dequin S. Glucose utilization of strains lacking PGI1 and expressing a transhydrogenase suggests differences in the pentose phosphate capacity among \u003cem\u003eSaccharomyces cerevisiae \u003c/em\u003estrains. FEMS Yeast Res 2008;82:217-224.\u003c/li\u003e\n\u003cli\u003eRep M, Albertyn J, Thevelein JM, Prior BA, Hohmann S. Different signalling pathways contribute to the control of GPD1 gene expression by osmotic stress in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Microbiology (Reading) 1999;1453:715-727.\u003c/li\u003e\n\u003cli\u003eJagtap RS, Mahajan DM, Mistry SR, Bilaiya M, Singh RK, Jain R. Improving ethanol yields in sugarcane molasses fermentation by engineering the high osmolarity glycerol pathway while maintaining osmotolerance in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Appl Microbiol Biotechnol 2019;1032:1031-1042.\u003c/li\u003e\n\u003cli\u003eAwasthy C, Hefny ZA, Van Genechten W, Himmelreich U, Van Dijck P. Involvement of 2-deoxyglucose-6-phosphate phosphatases in facilitating resilience against ionic and osmotic stress in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Microbiol Spectr 2024;128:13624.\u003c/li\u003e\n\u003cli\u003eGrauslund M, R\u0026oslash;nnow B. Carbon source-dependent transcriptional regulation of the mitochondrial glycerol-3-phosphate dehydrogenase gene, GUT2, from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. 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Int J Food Microbiol 2021;354:109206.\u003c/li\u003e\n\u003cli\u003eXu G, Hua Q, Duan N, Liu L, Chen J. Regulation of thiamine synthesis in\u003cem\u003e Saccharomyces cerevisiae\u003c/em\u003e for improved pyruvate production. Yeast 2012;296:209-217.\u003c/li\u003e\n\u003cli\u003eKawasaki Y, Nosaka K, Kaneko Y, Nishimura H, Iwashima A. Regulation of thiamine biosynthesis in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. J Bacteriol 1990;17210:6145-6147.\u003c/li\u003e\n\u003cli\u003eEhrmann AK, Wronska AK, Perli T, de Hulster EAF, Luttik MAH, van den Broek M, et al. Engineering \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e for fast vitamin-independent aerobic growth. Metab Eng 2024;82:201-215.\u003c/li\u003e\n\u003cli\u003eLibuda F, Phosphoketolase - A mechanistic update, in: Department of Molecular Structural Biology, University G\u0026ouml;ttingen, 2018, pp. 141.\u003c/li\u003e\n\u003cli\u003eLi L, Zhang Q, Shi R, Yao M, Tian K, Lu F, et al. Multidimensional combinatorial screening for high-level production of erythritol in\u003cem\u003e Yarrowia lipolytica\u003c/em\u003e. Bioresour Technol 2024;406\u003c/li\u003e\n\u003cli\u003eTang D, Chen M, Huang X, Zhang G, Zeng L, Zhang G, et al. SRplot: A free online platform for data visualization and graphing. PLoS One 2023;1811:e0294236.\u003c/li\u003e\n\u003cli\u003eLiu Y, Yang X, Gan J, Chen S, Xiao ZX, Cao Y. CB-Dock2: improved protein-ligand blind docking by integrating cavity detection, docking and homologous template fitting. Nucleic Acids Res 2022;501:159-164.\u003c/li\u003e\n\u003cli\u003eLaughery MF, Wyrick JJ. Simple CRISPR-Cas9 Genome Editing in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Curr Protoc Mol Biol 2019;1291:110.\u003c/li\u003e\n\u003cli\u003eLabun K, Montague TG, Gagnon JA, Thyme SB, Valen E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 2016;441:272-276.\u003c/li\u003e\n\u003cli\u003eGietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2007;21:31-34.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tyrosol, Adaptive laboratory evolution, Transcriptome, Genetic mutation, Reverse engineering, PHO3, SNZ3","lastPublishedDoi":"10.21203/rs.3.rs-5667010/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5667010/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTyrosol is an important drug precursor, and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e is one of the main microorganisms producing tyrosol. Although excessive metabolic modification increased the production of tyrosol, it also caused a decrease in the growth rate of yeast. Therefore, this study attempted to restore the growth of \u003cem\u003eS. cerevisiae\u003c/em\u003e through adaptive evolution and further improve tyrosol production.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAfter the adaptive laboratory evolution of \u003cem\u003eS. cerevisiae\u003c/em\u003e S26, three evolutionary strains were obtained. The biomass of strain S26-AE2 reached 17.82 under the condition of 100 g/L glucose which was 15.33% higher than that of S26, and its tyrosol production reached 817.83 mg/L. Transcriptome analysis showed that the strain S26-AE2 may through decreased expression of \u003cem\u003eHXK2\u003c/em\u003e reduce the transcriptional regulation of glucose repression and increase the expression of gene \u003cem\u003ePGI1\u003c/em\u003e to promote the utilization of glucose. The genes related to pyruvate synthesis were enhanced in strain S26-AE2. Under the 20 g/L glucose condition, the TCA cycle-related genes of the S26-AE2 were more active. Furthermore, the tyrosol production of S26 with SNZ3\u003csup\u003eVal125Ile\u003c/sup\u003e mutation increased by 17.01% compared with the control strain S26 under the condition of 100 g/L glucose.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn this paper, a strain S26-AE2 with good growth and tyrosol production performance was obtained by adaptive evolution. The transcriptome reveals the differences in gene expression in metabolic pathways of adaptive evolutionary strains may be related to the growth of yeast and the production of tyrosol. Further reverse engineering verified the mutation of \u003cem\u003eSNZ3\u003c/em\u003e promoted the synthesis of tyrosol in \u003cem\u003eS. cerevisiae\u003c/em\u003e in the glucose-rich medium. This study provides a theoretical basis for the metabolic engineering of \u003cem\u003eS. cerevisiae\u003c/em\u003e to synthesize tyrosol and its derivatives.\u003c/p\u003e","manuscriptTitle":"Transcriptome analysis and reverse engineering verification of SNZ3 Val125Ile and Pho3 Asn134Asp revealed the mechanism of laboratory adaptive evolution to increase the yield of tyrosol in Saccharomyces cerevisiae S26","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-20 18:31:43","doi":"10.21203/rs.3.rs-5667010/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-29T11:31:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-21T18:00:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-21T11:38:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152414356774854654909939732568515803606","date":"2025-01-14T10:44:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1011143819417646333625814476089758467","date":"2025-01-13T19:19:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111800024715665273927964668545785954375","date":"2025-01-12T18:14:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294744389998490454414797214827369861081","date":"2025-01-11T17:03:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-22T16:20:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-19T19:47:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-18T12:20:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels and Bioproducts","date":"2024-12-18T07:21:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"812c93ca-8850-4ab7-af71-fe714af8cf8e","owner":[],"postedDate":"December 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-10T19:45:29+00:00","versionOfRecord":{"articleIdentity":"rs-5667010","link":"https://doi.org/10.1186/s13068-025-02627-4","journal":{"identity":"biotechnology-for-biofuels-and-bioproducts","isVorOnly":false,"title":"Biotechnology for Biofuels and Bioproducts"},"publishedOn":"2025-03-05 15:56:52","publishedOnDateReadable":"March 5th, 2025"},"versionCreatedAt":"2024-12-20 18:31:43","video":"","vorDoi":"10.1186/s13068-025-02627-4","vorDoiUrl":"https://doi.org/10.1186/s13068-025-02627-4","workflowStages":[]},"version":"v1","identity":"rs-5667010","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5667010","identity":"rs-5667010","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}