Cyberlindnera fabianii is a Baijiu-associated non-Saccharomyces yeast that may encounter transient heat stress during fermentation, but the mechanisms underlying its thermotolerance remain poorly understood. Here, we evaluated heat survival, culture turbidity, culturability, JC-1-based relative mitochondrial polarization, and transcriptomic responses of C. fabianii at 45 °C under different glucose concentrations. C. fabianii showed higher CFU-based survival than Saccharomyces cerevisiae after 2 h of heat exposure. During prolonged incubation, 30% glucose improved culturability and was accompanied by higher relative mitochondrial membrane potential (MMP), less disrupted JC-1 staining patterns, weaker repression of glycolysis- and TCA-cycle-related transcripts, and increased PPP-related transcript levels together with upregulation of ROS-scavenging genes. These findings indicate a glucose-associated improvement in heat-stress tolerance, particularly involving early maintenance of mitochondrial polarization and metabolic/redox-related transcriptional responses; however, the current data do not determine whether this effect is transient or long-lasting. Under glucose-limited conditions, glycerol or xylose co-feeding increased turbidity and partially supported CFU retention, with substrate-dependent differences in extracellular glucose consumption. Overall, these findings suggest that glucose availability is closely associated with thermotolerance and mitochondrial polarization in C. fabianii, and that, in liquid culture, selected co-substrates may partially reproduce aspects of the high-glucose-associated phenotype without requiring comparable glucose supplementation.

Recently, renewable biopolymers have gained growing attention as an alternative to petroleum-based materials in the packaging industry due to their eco-friendliness, biodegradability, and biocompatibility. This study introduces an innovative method for producing active films, which uses natural bacterial cellulose (BC) films as the matrix and incorporates (-)-Epigallocatechin-3-gallate (EGCG) through an immersion process. The incorporation of EGCG improves the barrier performance against oxygen and UV of the BC-based active films while preserving their tensile strength without compromising their opacity. More importantly, the active films exhibited significant antibacterial effects, with the efficacy increasing with the concentration of EGCG. Specifically, the diameters of the inhibition zones enlarged progressively against both S. aureus (from 13.88 to 16.25 mm, p < 0.05) and E. coli (from 12.38 to 14.13 mm). Correspondingly, the antibacterial rate of the active films increased from 61.4% to 80.61% (p < 0.05) against S. aureus and from 57.38% to 60.38% against E. coli. Additionally, the BC-based active films developed in this work exhibit excellent biodegradability, being capable of achieving complete biodegradation within 21 days of soil burial. This breakthrough exhibits considerable potential of BC-based active films as eco-friendly packaging materials, showing exceptional promise for sustainable active food packaging applications.

Polyethylene terephthalate (PET) hydrolases efficiently hydrolyze the ester bonds in PET, converting it into valuable monomers or oligomers, offering a sustainable biological solution to global PET plastic pollution. However, the large-scale development of high-performance PET hydrolases remains challenging due to limitations in traditional enzyme resource mining methods, including low throughput and lengthy cycles. Recent advances in artificial intelligence (AI) provide novel methodologies to overcome these challenges. This review systematically summarizes how AI empowers the high-throughput screening of PET hydrolases from massive biological databases, while allowing the accurate prediction of enzyme structures and functions. Furthermore, it critically analyzes AI-driven strategies for enzyme molecular engineering and highlights the emerging frontier of AI-assisted de novo enzyme design. By systematically evaluating the advantages and challenges of AI models in the research of PET hydrolases, this review provides an integrated technical framework and theoretical foundation to guide future innovation in enzyme mining and plastic biodegradation.

Chinese Baijiu, a globally renowned distilled spirit, is transitioning from traditional spontaneous fermentation to mechanized production. This review examines microbial community succession, flavor science challenges, and Synthetic Microbial Consortia (SMC) applications during this transformation. Traditional brewing occurs in open environments, yielding diverse microbiota and distinctive flavors, but suffers from inefficiency and inconsistent quality. Mechanized production improves efficiency and standardization, yet alters fermentation equipment and environmental conditions, restructuring the microbial ecosystem. These changes impact core microbial communities and disrupt synthesis pathways of key volatiles (e.g., esters, alcohols, acids) and health-related metabolites like phenolics. Complex microbial interactions underpinning traditional flavors are often simplified in mechanized systems. SMC-based precision fermentation has emerged to address this, using defined microbial combinations to enable targeted flavor regulation and beneficial metabolite enrichment. However, SMC still struggles to fully replicate traditional flavor complexity and precisely control microbial succession. Future research should integrate multi-omics with AI and real-time monitoring to develop intelligent facilities enabling precise microenvironment control. This review provides a scientific framework for the modernizational upgrade of the Baijiu industry, while offering valuable references and methodological insights that may support the scientific advancement of other traditional fermented food systems.

Through evolutionary engineering strategies, scientists have successfully cultivated multiple strains of Saccharomyces cerevisiae with enhanced tolerance, demonstrating significant potential in improving S. cerevisiae resistance. In this study, S. cerevisiae CEN.PK113-7D was continuously cultured for 80 days in a medium containing lignocellulosic inhibitors (furfural, acetic acid, and vanillin). The evolved strain, S. cerevisiae CEN.PK113-AL80-4, exhibited 12 h reduction in lag phase under multiple stress conditions and 17% increase in the ethanol conversion rate. The double mutant strain RG was constructed by mutating genes such as Rad18 and Gcn1 using CRISPR/Cas9 gene editing technology. Under the stress of 2 g/L furfural, 3 g/L acetic acid, and 1.5 g/L vanillin, ethanol yield reached 5.88 ± 0.28 g/L (the conversion rate was 0.29 ± 0.01 g/g). However, the original strain cannot grow. Mechanism studies have shown that Rad18 and Gcn1 significantly enhance stress tolerance by increasing the activities of catalase (CAT) (75%) and superoxide dismutase (SOD) (27.6%), increasing intracellular glycerol content, and strengthening carbon metabolism and oxidative stress responses. This study lays a solid theoretical foundation for developing more robust strains and advancing efficient utilization of lignocellulosic biomass.

Tyrosol is an important drug precursor, and Saccharomyces cerevisiae is one of the main microorganisms that produces tyrosol. Although excessive metabolic modification increases the production of tyrosol, it also causes a decrease in the growth rate of yeast. Therefore, this study attempted to restore the growth of S. cerevisiae through adaptive evolution and further improve tyrosol production. After the adaptive laboratory evolution of S. cerevisiae S26, three evolutionary strains were obtained. The biomass of strain S26-AE2 reached 17.82 g DCW/L in the presence of 100 g/L glucose, which was 15.33

The distinctive flavor and aroma of Chinese baijiu are closely linked to the microorganisms involved in the fermentation process. Lactiplantibacillus plantarum, a dominant species in the fermentation of Chinese baijiu, has become a prominent research focus. In this study, we selected well-characterized pure cultures of microorganisms to construct diverse chassis microflora. The primary objective was to investigate the effects of L. plantarum on the fermentation process of Chinese baijiu and its association with metabolites produced by different chassis microflora. Our results demonstrated that the concentrations of ethyl lactate and other volatile aromatic compounds increased in all fermentation protocols where L. plantarum was added. The addition of L. plantarum also significantly increased the concentration of total organic acids, particularly lactic acid, which rose by 17 to 123 times. Furthermore, L. plantarum helped maintain the stability of ethanol concentration during the middle and late stages of fermentation. Notably, among the three different chassis microbial fermentation protocols involving L. plantarum, the protocol with the highest microbial diversity exhibited a greater capacity to produce lactic acid (1.56 ± 0.19 mg/g), ethanol (5.74 ± 0.47 mg/g), and reducing sugars (6.39 ± 0.31 mg/g). These findings provide valuable insights into the potential of L. plantarum for modulating the flavor of Chinese baijiu.

The enzymatic hydrolysis of lignocellulose continues to be encumbered by elevated production costs and diminished cellulase efficiency. In this work, modified DES recovered lignin was obtained by grafting acrylamide and acryloyl chloride to enhance glucose release. At a cellulase dosage of 5 FPU/g-cellulose and pH of 5.5, modified lignin promoted glucose yield of dilute-acid-pretreated wheat straw by 158 % compared with control. The mechanism by which modified lignin promotes enzymatic hydrolysis was further explored. The binding constant was reduced from (3.3510 ± 0.8361)* 104 to (2.7600 ± 0.6027)* 103 L•mol-1 after modification. Modified lignin could make α-helix content enhancement so that cellulase had a compact and stable spatial structure. Lignin binds within the catalytic tunnel of cellulase and that the modified lignin interacts with cellulase with increased hydrogen bonding, resulting in a more compact cellulase structure. The modified lignin might reduce the unproductive adsorption of cellulase, and increase stability and cellulose accessibility to reduce cellulase cost.

IntroductionStaphylococcus has garnered increasing attention for its role in improving fermentation results and promoting the biosynthesis of aromatic compounds.MethodsThis study investigated the effects of exogenously introduced Staphylococcus saprophyticus on the microbial community structure, functional gene expression, and volatile aroma profiles during the fermentation of cigar filler leaves.ResultsThe results demonstrated that S. saprophyticus significantly enhanced the accumulation of alcohols and ketones. LEfSe analysis identified Bacillus as a key differential genus in the inoculated group. Spearman correlation analysis revealed positive associations between Staphylococcus and Bacillus, as well as with key aroma compounds such as 1-methyl-4-(2-methyloxiranyl)-7-oxabicyclo [4.1.0] heptane and cis-6-nonenal. EGGNOG functional annotation indicated upregulation of carbohydrate and amino acid metabolism pathways. Additionally, CAZy analysis revealed increased abundance of glycosyltransferases and carbohydrate-binding modules, which may facilitate sugar conversion and utilization.DiscussionThese findings provide a theoretical basis for the application of exogenous microorganisms in cigar fermentation and offer insights into the regulation of microbial community structure for quality improvement.

The cigar wrapper leaves (CWLs), as a symbol of the intrinsic quality and appearance of cigars, reflects the overall quality of the cigar. The Shaoxing-flavored T3 Jiuqu used in Shaoxing wine production contains a large number of high-quality microorganisms, such as molds and yeasts, which play a significant role in enhancing flavor and quality. Among these microorganisms, several positively promote the fermentation of CWLs. A dominant strain, S1, was isolated and identified from the T3 Jiuqu and inoculated into the fermentation of CWLs. Gas Chromatography-Mass Spectrometry (GC-MS) was employed to analyze the volatile aroma components in the CWLs. The results showed that the contents of substances such as Phenethyl alcohol, Dihydroactinidiolide, Sclareol, and Farnesyl acetone were significantly increased compared to pre-fermentation (NF) and the natural fermentation with only water (WF) group. Specifically, Phenethyl alcohol content increased by 261.63% compared to WF group during the same turning-over period, while Farnesyl acetone content increased by 144.99%. The proportions of sugars and nicotine also increased significantly. Metagenomic analysis of the microbial samples on the surface of CWLs revealed that inoculating S1 significantly improved and altered microbial community structure. At the phylum level, the proportion of Pseudomonadota increased dramatically to 17%, while the proportion of Uroviricotasharply decreased sharply from 9% to 0. At the genus level, the previously dominant Staphylococcus genus was replaced by a balanced coexistence of Pantoea, Enterobacter, Cronobacter, and Aspergillus. This balanced microbial distribution significantly improved the quality of the CWLs.

Tyrosol is an important component of pharmaceuticals, nutraceuticals, and cosmetics, and their biosynthetic pathways are currently a hot research topic. D-Erythrose 4-phosphate is a key precursor for the biosynthesis of tyrosol in Saccharomyces cerevisiae. Hence, the flux of D-Erythrose 4-phosphate determined the yield of tyrosol synthesis. In this study, we first obtained an S. cerevisiae strain S19 with a tyrosol yield of 247.66 mg/L by metabolic engineering strategy. To increase the production of D-Erythrose 4-phosphate, highly active phosphoketolase BA-C was obtained by bioinformatics combined with tyrosol yield assay. The key residue sites 183, 217, and 320 were obtained by molecular docking, kinetic simulation, and tyrosol yield verification. After mutation, the highly efficient phosphoketolase BA-CHis320Met was obtained, with a 37.32% increase in enzyme activity. The tyrosol production of strain S26 with BA-CHis320Arg increased by 43.05% than strain S25 with BA-C and increased by 151.19% compared with the strain S19 without phosphoketolase in a 20 L fermenter. The mining and modification of phosphoketolase will provide strong support for the de novo synthesis of aromatic compounds.

During microbial industrial production, microorganisms often face diverse stressors, including organic solvents, high salinity, and high sugar levels. Enhancing microorganism tolerance to such stresses is crucial for producing high-value-added products. Previous studies on the mechanisms of 2-phenylethanol (2-PE) tolerance in Saccharomyces cerevisiae revealed a potential connection between the sugar transporter-like protein (Stl1) mutation (F427L) and increased tolerance to high sugar and salt stress, suggesting a broader role in multistress tolerance. Herein, we showed that the Stl1(F427L) mutant strain (STL) exhibits significantly improved multistress tolerance in the presence of glycerol. Molecular dynamics simulations indicated that Stl1(F427L) may enhance glycerol molecular binding, resulting in a significant increase in the intracellular glycerol content of the mutant strain STL. Additionally, under multistress conditions, pyruvate and ergosterol levels and catalase (CAT) and superoxide dismutase (SOD) activities were significantly increased in the mutant strain STL compared with the control strain 5D. This resulted in a notable increase in cell membrane toughness and a decrease in intracellular reactive oxygen species levels. These findings highlight the mechanism by which Stl1(F427L) enhances S. cerevisiae tolerance to multistress. Importantly, they provide novel insights into and methodologies for improving the resilience of industrial microorganisms. IMPORTANCE Stl1(F427L) exhibits improved strain tolerance to multistress when adding glycerol, may enhance glycerol molecular binding, and can make a significant increase in intracellular glycerol content. It can reduce reactive oxygen species levels and increase ergosterol content. This paper provides novel insights and methods to get robust industrial microorganisms.

This study explores the multifaceted regulatory mechanisms of Thermophilic actinomycetes MC-34 (TA) in solidstate fermentation of Chinese Baijiu, focusing on their synergistic enhancement of flavor quality. Through monoand co-culture fermentation experiments, we revealed that the introduction of thermophilic TA significantly enhances ethanol production (up to 269.56 % increase) and sugar-alcohol conversion efficiency (up to 164.41 % increase). The activation of interspecies metabolic interaction networks via hydrolase secretion promotes the biosynthesis of 23 characteristic flavor compounds (e.g., ethyl acetate and isoamyl alcohol), resulting in 20 %- 30 % greater flavor diversity in experimental groups compared to controls. First, TA were found to enrich organic acid profiles (e.g., isobutyric acid) via cooperative metabolic pathways, concurrently stabilizing environmental pH (50 % reduction in pH fluctuation during the late fermentation phase). Furthermore, a novel mechanism was identified: TA inhibited alpha-amylase and glucoamylase activities (maximum inhibition rate: 98.9 %), thereby optimizing substrate utilization efficiency through enzymatic balance regulation. This study challenges the conventional view that TA only participates in terpenoid synthesis. Instead, it innovatively constructs a synergistic metabolic network model involving actinomycetes, yeast, and mold. These findings provide theoretical foundations for synthetic microbial consortia development and modernization of traditional brewing processes, offering critical references for enhancing Baijiu flavor quality and industrial production optimization.

Introduction:Tobacco flower buds play a crucial role in enhancing the aroma quality of cigar tobacco leaves (CTLs). By incorporating tobacco flower bud extract into the fermentation process, this study investigates its effects on microbial community dynamics and the volatile aroma compounds in CTLs, aiming to improve cigar flavor and quality during fermentation. Methods:To investigate the effects of tobacco flower bud extract on microbial communities and aroma quality during the fermentation of cigar tobacco leaves, volatile aroma components were evaluated using gas chromatography-mass spectrometry (GC-MS). The microbial community dynamics across different fermentation stages were analyzed using metagenomic sequencing. Results and Discussion:Results revealed that tobacco flower buds contain 23 characteristic aroma compounds, including β-ionone and phenylethanal. Notably, the extract induced a pronounced microbial shift, enriching Aspergillus in unfermented leaves and promoting Staphylococcus dominance (97%-98%) during fermentation. This shift facilitated carbohydrate and protein degradation, significantly reducing nicotine content (P < 0.001), increased total sugar (12.5%-18.75%) and reducing sugar levels (13.04%-27.27%), and optimized the potassium-to-chloride ratio. Aroma analysis demonstrated significant enrichment of carotenoid degradation products (farnesyl acetone, citronellal) and Maillard reaction products (5-methyl-2-furaldehyde) in the FE group, with total aroma content increasing by 11.9% compared to control (FW). Metagenomic functional analysis further indicated that the extract inhibited pathways related to harmful metabolite synthesis (47.0% reduction) and enhanced carbohydrate metabolism (30.6% increasing). This study confirms that tobacco flower bud extract reshapes microbial communities and metabolic networks by simultaneously suppressing harmful microbes and enhancing aroma, providing theoretical support for optimizing cigar fermentation and agricultural waste utilization.

The 2-phenylethanol (2-PE) tolerance phenotype is crucial to the production of 2-PE, and Pdr1p mutation can significantly increase the tolerance of 2-PE in Saccharomyces cerevisiae. However, its underlying molecular mechanisms are still unclear, hindering the rational design of superior 2-PE tolerance performance. Here, the physiology and biochemistry of the PDR1_862 and 5D strains were analyzed. At 3.5 g/L 2-PE, the ethanol concentration of PDR1_862 decreased by 21

Zygosaccharomyces rouxii used in soy sauce brewing is an osmotolerant and halotolerant yeast, but it is not tolerant to high temperatures and the underlying mechanisms remain poorly understood. Using a synthetic medium containing only Pro as a nitrogen source, the response of Z. rouxii in protein level to high-temperature stress (40 °C, HTS) during the lag phase was investigated. Within the first two h, the total intracellular protein concentration was significantly decreased from 220.99 ± 6.58 μg/mg DCW to 152.63 ± 10.49 μg/mg DCW. The analysis of the amino acid composition of the total protein through vacuum proteolysis technology and HPLC showed that new amino acids (Thr, Tyr, Ser, and His) were added to newborn protein over time during the lag phase under HTS. The nutritional conditions used in this study determined that the main source of amino acid supply for protein synthesis was through amino acid biosynthesis and ubiquitination-mediated protein degradation. Differential expression analysis of the amino acid biosynthesis-related genes in the transcriptome showed that most genes were upregulated under HTS, excluding ARO8, which was consistently repressed during the lag phase. RT-qPCR results showed that high-temperature stress significantly increased the upregulation of proteolysis genes, especially PSH1 (E3 ubiquitin ligase) by 13.23 ± 1.44 fold (p < 0.0001) within 4 h. Overall, these results indicated that Z. rouxii adapt to prolonged high temperatures stress by altering its basal protein composition. This protein renewal was related to the regulation of proteolysis and the biosynthesis of amino acids.

To improve the growth and sporulation efficiency of Bacillus subtilis,the effect of malate on the cell growth and metabolism of B.subtilis DF was investigated in a 30 L bioreactor.At 22 h of fermentation,a biomass of 3.68 × 1010 CFU/mL was obtained by malate addition to a final concentration of 1.5％during 16-27 h after the pH reached 8.0 at 16 h,which was 71.9％higher than that of the control group.Meanwhile,the number of spores was 3.63 × 1010 CFU/mL,which was 92.1％higher than that of the control group.Differential transcriptomic analysis showed that malate increased the metabolic efficiency of the gluconeogenesis and pentose phosphate pathways with 1.38 to 2.51-fold up-regulation of the zwf,pckA and fbaA genes,respectively and the metabolic efficiency of the tricarboxylic acid(TCA)cycle with 3.10-fold up-regulation of the citZ gene,reduced cellular oxidative stress with a 72.9％down-regulation of the poxL gene,a 3.88-fold up-regulation of the trxA gene,and thus a 45.9％-51.3％reduction in H2O2 concentration during 19-24 h,and enhanced the phosphorylation efficiency of Spo0A with down-regulation of the kipL,rapAD and abrB genes by 57.5％-75.9％,and up-regulation of the clpX,phrCF,spo0B and sigA genes by 1.73-13.76 folds,significantly improving the growth and sporulation efficiency of B.subtilis DF.These findings provide theoretical supports for the industrial application of B.subtilis.

IntroductionMicrobial succession and metabolic adjustment during cigar tobacco leaf (CTL) fermentation are key factors to improve the quality and flavor of CTLs. However, the interactions in the above processes remain to be further elucidated.MethodsBacillus altitudinis inoculants were added to the CTLs, and metagenomics and metabolomics were used to analyze the effects of the inoculants on regulating microbial succession, metabolic shift, and aroma production during fermentation.Results and discussionThe addition of the inoculants reinforced the CTL macromolecule transformation and facilitated the aroma production efficiently, and the total aroma production was increased by 43% compared with natural fermentation. The omics analysis showed that Staphylococcus was a main contributor to fatty acid degradation, inositol phosphate metabolism, energy supply (oxidative phosphorylation), nutrient transport (ABC transporter and phosphotransferase system [PTS]), and aroma production (terpenoid backbone biosynthesis, phenylalanine metabolism, and degradation of aromatic compounds). Furthermore, Staphylococcus was positively correlated with TCA cycle intermediates (citric acid, fumaric acid, and aconitic acid), cell wall components, peptidoglycan intermediates (GlcNAc-1-P and UDP-GlcNAc), and phytic acid degradation products (inositol). The characteristics collectively showed Staphylococcus to be the most dominant in the microbial community at the genus level during microflora succession. The addition of the inoculants supplemented the nutritional components of the CTLs, enhanced the metabolic activity and diversity of bacteria such as Corynebacterium, improved their competitive advantages in the microflora succession, and facilitated the richness of microbial communities. Additionally, a metabolic shift in nicotine degradation and NAD + anabolism from Staphylococcus to Corynebacterium in fermentation with inoculants was first observed. Meanwhile, the significantly correlative differential metabolites with Staphylococcus and Corynebacterium were a metabolic complement, thus forming a completely dynamic fermentation ecosystem. The results provided evidence for CTL fermentation optimization.

Complete synthetic minimal media for Zygosaccharomyces rouxii growth were designed for high temperature (40 ℃, HTS) and high salt stress (18% NaCl, HSS) in this study, and the difference in the nutritional requirements of Z. rouxii cells under long-term adverse environmental conditions was analyzed. The differences in the metabolism and gene expression of organic acids, amino acids and sugars during the period from the growth adaptation stage to the early logarithmic stage were highlighted between HSS and HTS conditions. The results showed that Z. rouxii cells exposed to HSS needed more exogenous amino acids, vitamin and amino acid supplementation alleviated HTS-induced damage in yeast cells. The adversity transcription gene MSN4 and the hypertonic regulatory protein gene HOG1 responded to high salt, while the heat shock regulatory protein gene HSF1 and the superoxide dismutase gene SOD1 responded to high temperature. In summary, different strategies for organic acid, amino acid and sugar metabolism were adopted by Z. rouxii in response to HSS and HTS. This study deepens the understanding of the mechanism of temperature tolerance in salt-tolerant Z. rouxii, which will contribute to the development of new brewing yeast cells with tolerance to both high salt and temperature.

IntroductionFurfural, a main inhibitor produced during pretreatment of lignocellulose, has shown inhibitory effects on S. cerevisiae.MethodIn the present study, new strains named 12–1 with enhanced resistance to furfural were obtained through adaptive laboratory evolution, which exhibited a shortened lag phase by 36 h, and an increased ethanol conversion rate by 6.67% under 4 g/L furfural.Results and DiscussionTo further explore the mechanism of enhanced furfural tolerance, ADR1_1802 mutant was constructed by CRISPR/Cas9 technology, based on whole genome re-sequencing data. The results indicated that the time when ADR1_1802 begin to grow was shortened by 20 h compared with reference strain (S. cerevisiae CEN.PK113-5D) when furfural was 4 g/L. Additionally, the transcription levels of GRE2 and ADH6 in ADR1_ 1802 mutant were increased by 53.69 and 44.95%, respectively, according to real-time fluorescence quantitative PCR analysis. These findings suggest that the enhanced furfural tolerance of mutant is due to accelerated furfural degradation. Importance: Renewable carbon worldwide is vital to achieve “zero carbon” target. Bioethanol obtained from biomass is one of them. To make bioethanol price competitive to fossil fuel, higher ethanol yield is necessary, therefore, monosaccharide produced during biomass pretreatment should be effectively converted to ethanol by Saccharomyces cerevisiae. However, inhibitors formed by glucose or xylose oxidation could make ethanol yield lower. Thus, inhibitor tolerant Saccharomyces cerevisiae is important to this process. As one of the main component of pretreatment hydrolysate, furfural shows obvious impact on growth and ethanol production of Saccharomyces cerevisiae. To get furfural tolerant Saccharomyces cerevisiae and find the underlying mechanism, adaptive laboratory evolution and CRISPR/Cas9 technology were applied in the present study