Enhanced Heat Tolerance and Ethanol Production of Industrial Saccharomyces cerevisiae via Replacement of the CDC15 Gene

Enhanced Heat Tolerance and Ethanol Production of Industrial Saccharomyces cerevisiae via Replacement of the CDC15 Gene | 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 Enhanced Heat Tolerance and Ethanol Production of Industrial Saccharomyces cerevisiae via Replacement of the CDC15 Gene Xiaoling Chen, Dong Chen, Qi Lu, Ying Chen, Zhilong Lu, Suisheng Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8993691/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background CDC15 is an important gene involved in mitotic exit and cytokinesis in S.cerevisiae . However, its additional functions remain unclear. Comparative genomics analysis between the fast-growing strain MC15 and the high-ethanol-producing strain MF01 revealed CDC15 as a significant sequence-divergent gene. To validate the hypothesis that replacing this gene could enhance MF01's growth rate, this study engineered the recombinant strain MF01- CDC15 (MT) by substituting the CDC15 in MF01 (WT) with that from MC15. Rapid growth is one of the desirable traits required for production strains. Achieving sufficient yeast cell density not only reduces contamination by contaminating microbes but also shortens the ethanol production cycle and improves production efficiency. Results The CDC15 mutation significantly improved cell viability on solid medium at 50–54°C. In liquid culture, the MT entered the stationary phase 8 h earlier than WT. Furthermore, the maximum cell densities achieved by MT at 30°C, 37°C and 41°C increased by 37%, 57%, 155%, respectively, compared to WT. Unexpectedly, CDC15 mutation caused ethanol yields to increase by 26.41% using sucrose and 29.56% using molasses as carbon sources at 30°C. Notably, MT cells maintained normal morphology at 41°C, whereas WT displayed cell adhesion. The intracellular trehalose content in MT was 4.8 times higher than that in WT at 41°C ( p = 0.014). Homology modeling indicated that the Phe626→Leu626 mutation in Cdc15p of MT may contribute to structural stability. The combination of this structural change and the trehalose accumulation likely contributes to MT’s superior heat resistance. Conclusions This study provides more detailed information about the function of CDC15 and suggest some clues to efficiently improve the performance of industrial yeast strains. CDC15 gene S. cerevisiae ethanol tolerance trehalose Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Although Saccharomyces cerevisiae is a famous cell factory for producing chemicals [ 1 , 2 ] , it remains the most widely used microbial host for ethanol production and the dominant organism in fuel ethanol manufacturing [ 3 , 4 ] . Bioethanol is regarded as a promising renewable liquid fuel alternative to mitigate energy crises [ 5 , 6 ] . However, production costs are a major issue for the production of fuel ethanol compared with fossil fuels [ 7 ] . Sugarcane molasses, a low-cost byproduct of sugar production containing approximately 45% (w/w) [ 4 , 8 ] , even 50–65% [ 9 ] , fermentable sugars, has particular potential as feedstock in southern China's sugarcane-growing regions. In Brazil, one of the largest ethanol biofuel producers and exporters in the world [ 5 , 10 ] , fuel ethanol is produced mainly from sugarcane juice and, in some cases, from molasses [ 7 ] . Molasses is diluted prior to fermentation [ 5 ] , since molasses’s high osmotic pressure (derived from ~ 50% total sugars [ 8 , 9 ] ) causes intracellular water loss in cells, leading to slow or even halted cell growth [ 11 ] . Such hypertonic stress not only impedes yeast growth but also disrupts metabolic pathways, ultimately diminishing ethanol production efficiency and extending fermentation duration [ 12 ] . Therefore, strains capable of withstanding high osmotic pressure for low-dilution or undiluted fermentation are particularly valuable. Rapidly growing cells enable efficient acquisition of sufficient cell biomass to initiate ethanol production, and thereby reduce production costs. Alternatively, recycled cells can be employed [ 5 ] , which is not applicable in China. In Brazil, the fermentation process generally employs high cell densities to rapidly achieve sufficient cell numbers for efficient fermentation, with certain aspects depending on the growth performance of the strain under current manufacturing practices. The number of recycling cycles can be increased if the cells exhibit high ethanol tolerance, since ethanol is toxic to yeast at high concentrations. Ethanol accumulation in later stages inhibits cellular growth at 9% (v/v) [ 4 ] or completely halts it at 12% (v/v) [ 13 ] , resulting in incomplete sugar utilization and reduced ethanol yield, thereby increasing production costs. Some pathways required for ethanol tolerance overlap with those for thermotolerance [ 14 ] . Fermentation is an exothermic process, leading to an increase in fermentation temperature. While temperature control is essential for optimal fermentation, cooling systems significantly increase production costs [ 15 ] . High-temperature fermentation using thermotolerant strains offers a cost-effective alternative, since a 5°C increase in the fermentation temperature could drastically reduce expenses [ 7 ] . The United States produces fuel ethanol primarily from corn through saccharification using α-amylase and glucoamylase, followed by fermentation with S.cerevisiae . Under these conditions, employing thermotolerant yeasts can more effectively reduce costs [ 7 ] . Besides, when producing fuel ethanol from corn through saccharification using α-amylase and glucoamylase, followed by fermentation with S.cerevisiae —a process primarily employed in the United States—employing thermotolerant yeasts can more effectively reduce costs [ 7 ] . Unfortunately, S. cerevisiae strains face a critical limitation: their optimal growth range (25–30°C) and failure to proliferate above 40°C [ 13 ] . Temperature exerts significant effects on cellular molecular composition, volume expansion, and protein folding [ 16 ] . Heat shock induces the expression of genes associated with RNA processing/modification, protein degradation, protein folding/transport [ 17 ] , and also alters phosphorylation states of proteins involved in transcription, protein folding and degradation, cell cycle regulation and morphogenesis [ 18 ] . Inappropriate temperature results in slower cell growth rates [ 19 ] , which affects fermentation rate during ethanol production. Additionally, temperature influences sugar utilization efficiency, and cell inactivation rate in ethanol-containing environments [ 19 ] . Heat shock induces the expression of genes associated with respiration, and alternative carbon source utilization [ 20 ] . Although thermotolerant mutants that could proliferate at 40°C [ 21 – 23 ] , 41°C [ 13 , 15 ] or 42°C [ 14 , 22 , 24 ] have been obtained, their ethanol productivity remains unknown or low [ 22 ] . Some existing thermotolerant mutants either rely on plasmid-borne gene responsible for the Htg + phenotype, or derive from laboratory strains [ 13 , 24 ] , rendering them unsuitable for industrial applications due to genetic instability, as plasmid carrying a resistance selection marker is easily lost in the absence of selective pressure [ 25 , 26 ] . Besides, laboratory strains lack the tolerance [ 27 ] , genetic, and phenotypic diversity [ 28 ] of industrial strains, and do not exhibit the positive correlation between tolerance and fermentation capacity seen in industrial strains [ 29 ] . We previously isolated three wild-type industrial strains, MF01, MC15, and ME13, from samples derived from the same environment (Table 1 ). Although MF01 exhibits the highest ethanol yield and has been adopted for industrial-scale ethanol production [ 4 , 30 ] , its growth rate is lower than that of ME13 and MC15. In contrast, MC15, with the lowest ethanol yield, has the highest growth rate among them. Comparative genomics analysis of these strains identified the cell cycle key gene CDC15 as a sequence-divergent gene, suggesting it may be a critical gene underlying differences in growth rate. Given CDC15 is an important gene involved in cell cycle [ 31 ] . In theory, replacing the CDC15 gene in MF01 with CDC15 from MC15 may enhance the growth rate of MF01, which could positively impact ethanol fermentation as biomass yield critically affects fermentation efficiency [ 32 ] . In this study, the CDC15 gene of MF01 was modified by substituting it with the homologous CDC15 gene from MC15, generating the engineered strain MF01- CDC15 . We hypothesize that homologous recombination of this cell cycle-associated differential gene derived from MC15 may accelerate the growth rate of MF01. As predicted, MF01- CDC15 exhibited accelerated growth. Unexpectedly, CDC15 replacement not only increased ethanol yield but also conferred enhanced thermotolerance—a critical trait for industrial fermentation where cooling costs and thermal stress are major bottlenecks. These findings first identifies CDC15 as a previously uncharacterized key target for enhancing ethanol productivity in industrial yeast strains, thereby enabling the rational design of robust cell factories for scalable biofuel and bio-based chemical production. Table 1 Strains used in this study Strain Genotype Characteristics Origin Reference MF01 Wild-type,MATa/MATa Higher ethanol yield and exogenous gene integration rate compared with MC15 and ME13 Isolated by our lab from sugar mill waste in 2010 [ 4 , 30 ] MC15 Wild-type,MATa/MATa Lower ethanol yield and higher growth rate compared with MF01 and ME13 Isolated by our lab from sugar mill waste in 2010 [ 4 ] ME13 Wild-type,MATa/MATa Intermediate ethanol production and growth rate among MF01, MC15 and ME13 Isolated by our lab from sugar mill waste in 2010 / MF01- CDC15 Derived from MF01,MATa/MATa Higher ethanol yield, growth rate and heat resistance compared with MF01 This work / 2. Methods 2.1 Strain and Culture Conditions Strain MF01 (MATa/MATα) is a wild-type (WT) diploid S. cerevisiae . Strain MF01- CDC15 (MATa/MATα), which is a diploid derived from MF01, was prepared by replacing the CDC15 gene of MF01 with that from a fast-growing strain MC15. The yeast cells were incubated in YPD medium, as previously described [ 4 ] , in 250 ml Erlenmeyer flasks, unless specified otherwise. 2.2 Construction and Screening of the Mutant Strain 2.2.1 Preparation and Transformation of CDC15 Homologous Donor The CDC15 donor (Fig.SS1 in Miscellaneous File Not for Publication) used for homologous recombination was amplified by PCR with chromosomal DNA of the MC15 strain serving as the template. The donor was amplified with primers CDC15 -F and CDC15 -R described in Table 2 , using the following PCR protocol: 94°C for 5 min; 94°C for 40 s, 56°C for 40 s, 72°C for 4 min for 35 cycles; and a final step of 72°C for 10 min. The PCR products were purified before transformation. Preparation of haploid strains was carried out as previously described [ 4 ] . Both types a and α protoplasts of MF01 were subjected to electroporation to introduce the CDC15 gene from MC15. Table 2 Primers used in this study 2.2.2 Screening for Recombinant Srains The transformed mixed solution was transferred into 1 mL cold solution containing 18% sucrose and incubated for 1 h at 30°C without shaking. After addition of 1 mL YPD, the cells were cultivated again in a shaker and then plated onto YPD agar. The endogenous CDC15 locus in MF01 could be precisely replaced with the MC15-sourced CDC15 sequence, owing to the high homologous recombination efficiency of S. cerevisiae [ 26 , 33 , 34 ] . Selection of recombinant mutants was based on phenotypic variations in growth rate. The largest colony on the YPD plate was picked and cultured to exponential phase in liquid YPD. Equal numbers of cells were transferred to fresh YPD and cultured for 8 ~ 10 h followed by detection of OD 600 . Cells with an OD 600 roughly 10% higher than that of the WT were selected, and then cultured and selected again as described above. Following serial screening, the potential mutant strains were obtained. After validation by PCR with the DNA of potential mutant as template and sequencing, haploid recombinant strains were obtained. Diploid recombinant strain, named MF01- CDC15 (MT), was constructed from mating types a and α of recombinant strains by mating as previously described [ 4 ] . To assess the genetic stability of MT, the strain was serially passaged for 30 generations, and the CDC15 locus was PCR-amplified using chromosomal DNA from MT as template. The PCR product was verified by sequencing, and its sequence was identical to that in MC15 (data not shown), indicating successful replacement of CDC15 and its genetic stability over generations. 2.3 Growth Curve Determination A single colony was isolated and inoculated into 5 ml preculture overnight. Equal numbers of cells were harvested by centrifugation at 4°C, resuspended in sterile water and shifted to 50 ml fresh YPD, at a final concentration of roughly 1×10 6 cells/ml, and cultured for 48 h at 30°C, 37°C, 41°C, respectively. Samples were taken at 8-h intervals and stored at 4°C until the number of cells in each sample was determined by Countstar BioFerm IY1200 (Shanghai Ruiyu Biotech Co.Ltd, shanghai, China). 2.4 Analysis of Yeast Cells for Trehalose Quantification and Enzyme Activity Assays About 1× 10 8 overnight cells were inoculated into 100 mL YPD and grown for 8-8.5 h. Cells from shake flask cultures were harvested by centrifugation (6000 rpm, 5 min, 4°C), washed once with sterile deionized water and twice with 0.1 M PBS. About 38 mg (wet weight) of the resulting pellet was transfered to a 1.5 ml sterile EP tube for trehalose content determination, and the remaining pellet was resuspended in 2 ml 0.1 M PBS, and then quickly frozen (− 80 ℃) until use for enzyme activities assayed. 2.4.1 Trehalose Content Determination The above cell pellet that was washed twice with PBS was dried to a constant weight at 55°C, then the rehalose was extracted with trichloroacetic acid followed by anthrone-based detection as before [ 4 ] . 2.4.2 Enzyme Assays After thawing the samples in room temperature, the crude enzyme solution prepared as the previous method [ 4 ] . Protein determination was done using Bradford Protein Assay Kit(Order No. C503031, Sangon Biotech, shanghai,China). 2.4.2.1 Assay of SOD Activity The intracellular total superoxide dismutase (SOD) activity was determined by hydroxylamine method as described previously [ 35 ] with some modifications. (1) Preparation of solutions. The Xanthine oxidase (XOD) solution was prepared as follows: 0.5 mg of xanthine oxidase (50 U/mg, purchased from MACKLIN, China) was dissolved in 0.5 ml of buffer solution B (containing104 mM KH 2 PO 4 and 78 mM Na 2 B 4 O 7 in deionized water) to obtain a concentration of 50 U/ml (XOD solution I). The working XOD solution was then prepared by adding 125 µl of XOD solution I to 975 µl of buffer solution B. All reagents for the color developer were stored in bottles at 0–4°C until use. (2) Assay Procedure. The procedure was as follows: 0.5 ml of water, 0.1 ml of crude enzyme solution, and 0.2 ml of reagent A (consisting of equal volumes of 1 mM hydroxylamine and 1 mM hypoxanthine, resulting in final concentrations of 0.2 mM for each) were mixed. The reaction was initiated by adding 0.2 ml of xanthine oxidase (XOD) solution (final concentration of 1.25 mU/ml XOD). This mixture (1.0 ml total) was incubated for 30 min at 37°C. Then, 1.25 ml of reagent III (prepared by mixing 1.5 volumes of 1.2 g/L sulfanilic acid with 1 volume of acetic acid just before use) was added, and the mixture was allowed to stand for 5 min at room temperature. Subsequently, 0.75 ml of reagent II (278.3 mg/L N-1-naphthylethylenediamine dihydrochloride dissolved in deionized water) was added. Reagents III and II were added in two separate steps (to final concentration of 300 µg/ml sulfanilic acid, 5 µg/ml N-1-naphthylethylenediamine, and 16.7% acetic acid), taking into account the color development process, which differed from the previous method. A control for each sample (the crude enzyme solution of each sample, boiled for 5 min to inactivate the enzyme) was prepared and added after the color developer to avoid incomplete enzyme inactivation leading to inaccurate data. The final mixture (3.0 ml) was allowed to stand for 20 min at room temperature, and the optical absorption was measured at 550 nm. The principle of the assay is as follows: Xanthine oxidase catalyzes the reaction of hypoxanthine, generating superoxide anion radicals (O₂⁻•⁻, which then oxidize hydroxylamine to form nitrite. The nitrite exhibits a purplish-red color in the presence of a chromogenic agent, and its absorbance is measured using a microplate reader. Due to the specific inhibitory effect of SOD in yeast cells on O₂⁻•, the formation of nitrite is reduced, resulting in a decrease in absorbance during colorimetric measurement. 2.4.2.2 Alcohol dehydrogenase assay Alcohol dehydrogenase (ADH) activity was assayed by the NAD + reduction method. ADH catalyzes the dehydrogenation of ethanol, and the removed hydrogen is received by oxidized coenzyme I (NAD + ), converting it to reduced coenzyme I (NADH). The amount of NADH generated is measured by monitoring the absorbance change at 340 nm, since NAD + has no absorption at 340 nm but NADH absorbs at this wavelength. The assay system contained 108.8 µl of 0.1 M PBS (prepared by dissolving 14.2 g Na 2 HPO 4 , 2.38 g KH 2 PO 4 , 80 g NaCl, 2 g KCl, in 1L distilled water), 40 µl of 5 mM NAD + (prepared by dissolving 38.9 mg of NAD + in 10 ml of distilled water), and 1.2µl of anhydrous ethanol as the substrate. The reaction was initiated by the addition of 50 µl of cell extract to the well of a 96-well microtiter plate. A control for each sample was included, prepared by replacing the cell extract with its inactivated enzyme (prepared by boiling for 5 min). After 20 min of incubation at 30°C, the activity was assayed by measuring the increase in absorbance at 340 nm. Activity is expressed as the increase in absorbance per milligram of protein. 2.5 Ethanol Fermentation Sucrose fermentation Fermentation with sucrose was performed as previously described [ 4 ] with minor modification. Briefly, 0.4×l0 8 overnight cells were inoculated into 40 ml yeast extract peptone sucrose medium (10 g/L yeast extract, 20 g/L peptone, 25 g/L sucrose, pH unadjusted) in a 250 mL Erlenmeyer flask and incubated with shaking for 38 h, followed by static incubation at 30 and 37 ℃ under separate conditions. Molasses fermentation Fermentation with molasses was conducted according to the prior method [ 4 ] with slight modification. 0.4×10 8 overnight cells were grown in 30 ml molasses medium (containing 20.6 °Bx molasses) in a 250 mL Erlenmeyer flask and incubated for approximately 9 h. An additional molasses medium (replacing 20.6 °BX molasses with 50.3 °BX molasses) was supplemented, after which the cells were grown for another 30 h, followed by static conditions for 40 h at 30 and 37 ℃in separate experiments. 2.6 Sugar Content Determination The reducing sugars and total sugars in the fermented liquid were determined using the 3,5-dinitrosalicylic acid (DNS) reagent. Prior to analysis, the total sugars were hydrolyzed to reducing sugars following a previously described method [ 4 ] . 2.7 Statistical Analysis Statistical analysis was performed with the Student’s t -test using Microsoft Excel (Tails: two-tail,Type: paired t-test). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p 0.05) indicates non-significance. 3. Results 3.1 Sequence Comparison of Cdc15p among MT, WT and S288C Reveals Convergent Adaptation Comparison of the predicted Cdc15p amino acid sequence in MT with the known sequences of WT and S288C revealed one residue difference between WT and MT (F 626 L), two differences between WT and S288C (T 851 S, D 902 V), and three differences between MT and S288C (F 626 L, T 851 S, D 902 V) (Fig. 1 ). The substitution at residue 626 (Phe→Leu) between WT and MT is caused by a single nucleotide polymorphism (SNP) at position 1878 of the ORF (T →A in MT, Fig.SS3 in Miscellaneous File Not for Publication). The higher protein sequence similarity between MT and WT may reflect their convergent adaptation to identical growth environments, as both strains were isolated from the same geographic site. 3.2 CDC15 Mutation Does Not Alter Cellular Morphology at Normal Temperature but Causes Nonadherent Growth Cdc15p plays a crucial and multifaceted role in the cell cycle, specifically in mitotic exit [ 36 ] . While its role in cell cycle progression [ 37 ] is well established and CDC15 overexpression does not affect mitosis [ 38 ] , potential effects of CDC15 mutations on cellular morphogenesis remain unexplored. To evaluate this phenotype, we compared the WT and MT strains under both solid and liquid culture conditions. On YPD agar plates, both strains formed colonies with white, viscous, normally smooth, and lustrous surfaces (Fig.SS5 in Miscellaneous File Not for Publication), indicating that their colony size and morphology were normal at 30 ℃. These results indicate that CDC15 mutation does not seem to have any adverse effects on morphology. Unexpectedly, some WT cells exhibited adherent growth at 37°C, with more pronounced effects observed at 41°C, but the MT did not (Fig. 2 , Fig. S1 ). 3.3 The CDC15 Mutation Improves Cell Viability under Thermal Stress (50°C to 54°C) In practical ethanol production, localized temperatures within fermentation tanks may transiently exceed 50°C due to metabolic heat accumulation compounded by ambient thermal variations. Such transient thermal stresses, when exceeding the strain's tolerance threshold, invariably result in suboptimal fermentation performance ranging from diminished ethanol yield to complete process failure. Therefore, thermotolerant strains demonstrating high viability under production conditions is one of the desirable characteristics for strains. High cell viability is one of the critical criteria for strain selection in Brazil's industrial ethanol production [ 5 ] . To evaluate the heat shock tolerance of WT and MT strains and investigate the impact of the CDC15 mutation on thermotolerance, we analyzed the post-thermal stress viability of both strains. Following a 10-min heat shock at 42°C to 48°C, cell growth was delayed in both solid and liquid cultures, with no obvious distinction observed between the WT and MT. However, at temperatures between 50°C and 54°C, significant differences emerged between the WT and MT. On plates incubated at 50°C, the MT exhibited significantly higher colony counts.than that of WT, and this difference became more pronounced at 52°C. A small number of mutant colonies survived at 54°C, whereas no surviving WT colonies were detected (Fig.SS7 in Miscellaneous File Not for Publication). These results indicate that after heat shock at 50°C to 54°C, the mutant strain exhibited a higher cell viability than the WT strain, suggesting enhanced thermal tolerance in the mutants. No cell growth was observed at temperatures between 56°C and 62°C (data not shown). It is reported that protein translation ceased after 10 min of exposure to 46°C [ 39 ] , and that only rare thermotolerant strains survive following a 5-min heat shock at 54°C [ 13 ] . Strikingly, however, the MT strain in our study still exhibited survival after a 10-min heat shock at 54°C, but the WT did not. This not only demonstrates the superior thermotolerance of the MT strain, but also confirms that the CDC15 mutant exhibits a significantly higher cell viability than the WT following heat shock. 3.4 The CDC15 Mutation Promotes Cellular Proliferation under both Normal Conditions (30°C) and Thermal Stress (37°C, 41°C) Previous studies suggest that although protein translation did not decrease significantly within the first 30 min of exposure to 42°C, the synthesis of de novo proteins was affected under prolonged thermal stress at 42°C [ 39 ] . To further investigate temperature-dependent phenotypic differences, we analyzed the proliferation capacity of the WT and MT under varying temperature conditions. The MT entered the stationary phase 8 h earlier (at 16 h ) and achieved a higher maximum cell density (4.37×10 8 cells/mL) than the WT (3.18×10 8 cells/mL) at 30°C. As the temperature increases, both strains show decreased growth rates. However, the cell number of MT consistently exceeded that of the WT at all measured time points, and the fold difference increased with rising temperatures (Fig.S 2). The maximum cell densities of the MT at 30°C, 37°C and 41°C increased by 37%, 57%, 155%, respectively. Additionally, the cell number of MT cultured at 37°C was higher than that of WT at 30°C, and the cell number of MT at 41°C was comparable to that of WT at 37°C. In brief, the hierarchical distribution of cell counts is MT30 (MT at 30°C, similarly hereinafter) > MT37 > WT30 > WT37 ≈ MT41 > WT41 (Fig. 3 ). Collectively, this suggests that the CDC15 mutation enhances the strain's ability to grow under both normal and heat stress conditions, consistent with observations in post-thermal stress viability. 3.5 CDC15 Mutation Enhances Ethanol Production The ability of MT to grow rapidly at 37°C and 41°C holds enormous potential for applications in ethanol production [ 23 ] . To evaluate the effect of the mutation on ethanol production, we tested its fermentation capability. Upon fermentation with sucrose as the sole carbon source, MT at 30°C showed the highest ethanol production among all samples after 35 h (Fig. 4 A). When molasses was used as the sole carbon source, MT37 displayed the best ethanol productivity during the first 38 h, after which MT at 30°C showed higher productivity until 70 h when MT at 37°C again became highest (Fig. 4 B). When sucrose was used as the carbon source, MT strain achieved maximum ethanol concentrations of 12.03% ± 0.01% at 30°C and 11.14% ± 0.01% at 37°C, representing 26.41% and 3.79% increases over WT, respectively. Similarly, with molasses, MT produced maximum concentrations of 11.02% ± 0.07% at 30°C and 10.54% ± 0.06% at 37°C, corresponding to 29.56% and 3.84% improvements over WT, respectively (Fig. 4 ). These results suggest that the CDC15 mutation enhances ethanol production, and ethanol fermentation from molasses by S. cerevisiae is more difficult, resulting from the complex composition, compared with that from sucrose. Unexpectedly, both MT and WT strains showed higher ethanol production at 37°C than that of WT at 30°C, regardless of carbon source, except with sucrose at 54–62 h and with molasses at 70 h (Fig. 4 ). These results may result from the genetic background of the strains and suggest that both strains, especially MT, hold potential for ethanol production under high temperatures (37°C) conditions. 3.6 Trehalose Content Explains Higher Heat Resistance of MT Compared to WT High temperature induces the accumulation of trehalose in S.cerevisiae cells [ 40 , 41 ] . Trehalose accumulation accumulated in response to heat shock may maintain cell viability [ 32 ] and promote growth under high-temperature conditions [ 15 ] by preventing heat-induced damage to the cytoplasm [ 40 ] . Cell viability declines dramatically when trehalose levels are reduced below 2mg/g dry cell weight (DCW) [ 5 ] . High intracellular trehalose content serves as an indicator for ethanol-producing industrial strain screening in Brazil [ 5 ] . Therefore, we investigated the trehalose content in MT and WT strains. At 30°C and 37°C, MT showed slightly higher trehalose content than WT (67 vs. 34 mg/g DCW; 63 vs. 23 mg/g DCW) without statistical significance. However, at 41°C, both strains exhibited pronounced trehalose accumulation, consistent with the literature report by Hottiger [ 40 ] , with MT displaying significantly higher content compared with WT (MT: 972 mg/g DCW; WT: 202 mg/g DCW; p = 0.014). These results may suggest that the elevated trehalose content in MT contributes to its enhanced heat resistance relative to WT (Fig. 5 ). Reported intracellular trehalose concentrations in S. cerevisiae vary widely across studies [ 5 , 40 , 41 ] , with limited datasets showing comparable values [ 5 , 41 ] . For instance, Hottiger et al. observed that S. cerevisiae accumulated 1 g trehalose per g protein under 40°C stress [ 40 ] , while the industrial strain PE-2 and a baker’s yeast strain exhibited 12.5 mg/g and 6.3 mg/g trehalose, respectively [ 5 ] . Notably, PE-2’s elevated trehalose levels correlate with its industrial robustness. Although both the present study and Hottiger's work [ 40 ] employed trichloroacetic acid extraction followed by anthrone-based detection of trehalose content, direct comparisons between these datasets are not feasible due to differences in strain cultivation conditions and normalization methods. Direct comparisons between MT (this study) and PE-2 are also confounded by divergent cultivation and extraction protocols: PE-2 was grown under harsh fermentation conditions (33°C, molasses medium, 9.1% ethanol) until the mid-fermentation phase [ 5 ] , whereas MT was cultured in YPD medium for 8–8.5 h. In this study, the trehalose content of WT cultured at 30°C was 34 mg/g DCW, which is close to the range of 31–40 mg/g DCW reported by Alexandre [ 42 ] . However, Alexandre's protocol differed from both this study and existing literature [ 5 , 40 , 41 ] . Specifically, after 1 h stress treatment at 40°C, cells were incubated in 0.25 M Na₂CO₃ for 2 h at 90°C, and the trehalose content was determined by measuring the glucose liberated by trehalase digestion [ 42 ] . There are also discrepancies in the trends of trehalose content variation under temperature stress across different studies [ 40 , 41 ] . Asada [ 41 ] reported trehalose yields of 14.88, 13.94, and 4.41 mg/g at 40°C, 45°C, and 50°C, respectively, which contrasts with Hottiger’s observation of higher levels at 45°C versus 40°C and complete synthesis cessation at 50°C [ 40 ] . Asada treated the cells by boiling and measured the trehalose content using a detection kit [ 41 ] . It remains unclear whether different processing and assay methods influence observed trend. Overall, variations in stress conditions, extraction methods, and quantification assays preclude direct cross-study comparisons. Nonetheless, MT demonstrated significantly higher trehalose content than WT at 41°C, which is consistent with MT’s superior heat tolerance over WT, indicating better industrial adaptability compared to WT, despite the latter’s current industrial application [ 4 ] . 3.7 Non-Dominant Roles of SOD and ADH in MT/WT Thermotolerance Divergence It has been reported that superoxide dismutase (SOD) enhance the heat resistance of strains [ 43 ] , while ADH activity can serve as a critical indicator of cellular ethanol tolerance [ 44 ] . ADH1 overexpression can improve tolerance to glycolaldehyde [ 45 ] and co-expression of ADH1 and TAL1 improves ethanol production in the presence of furfural [ 46 ] . Hybrids of thermotolerant Kluyveromyces marxianus and thermosensitive S. cerevisiae produce more than 6% (v/v) ethanol at 45°C, and these hybrids contain ADH components derived from S. cerevisiae [ 47 ] . To determine whether other factors apart from trehalose contribute to the improved thermotolerance of MT, we examined the activities of these tolerance-related enzymes. As the temperature increased from 30°C to 37°C and then to 41°C, both SOD and ADH activities of both strains increased almost universally. At all temperatures, the SOD activity of MT was higher than that of WT, but the difference was not significant (Fig. S3 ). As the temperature increased, both strains exhibited an elevation in ADH activity. Nonetheless, the ADH activity of MT did not consistently surpass that of WT at identical temperatures, and no significant disparity was observed between them (Fig. S4). This suggests that SOD and ADH do not play a decisive role in the differential high-temperature tolerance between WT and MT under equivalent temperature stress conditions. 3.8 Effect of Trehalose Supplementation on Thermotolerance and Adherent Growth Since strains with higher trehalose levels exhibit increased cell viability under heat stress [ 42 ] and exogenous trehalose supplementation enhances freeze tolerance of yeast [ 48 ] , we added trehalose to the culture medium to evaluate growth differences between WT and MT under different temperatures with/without trehalose. At all temperatures, MT consistently exhibited higher cell counts than WT regardless of trehalose supplementation. At 37°C and 41°C, both strains generally showed increased cell density with trehalose supplementation compared to unsupplemented controls, except for MT at 48 h (37°C), WT at 40 h and 48 h (37°C), MT at 16 h and WT at 48 h (41°C). No significant differences were observed in cell growth between trehalose-supplemented and unsupplemented cultures at 30°C for either strain (Fig. 6 ). These results indicate that trehalose supplementation improves high-temperature (37°C and 41°C) tolerance in both strains. Without trehalose supplementation, MT cells exhibited higher numbers than WT at all tested temperatures and both strains showed a decline in cell counts with increasing stress intensity (Fig. 6 ), aligning with prior observations (Fig. 3 ). Trehalose supplementation did not mitigate the adherent growth of WT cells under heat stress (Fig.S11 in Miscellaneous File Not for Publication). 4. Discussion 4.1 Mechanistic Hypothesis for Enhanced Thermotolerance in CDC15 Mutant Strain Our study identified a single residue difference between WT and MT (F 626 L, Fig. 1 ), and homology modeling of Cdc15p indicates that residue 626 is located within an α-helical region on the protein's outer surface, spatially proximate to an area enriched with charged residues (residues 281–305) [ 37 ] (Fig. 7 ). The fragment encompassing residues 281–362, which contains this charged cluster, is able to interact individually with Tem1p, a protein known to interact with Cdc15p [ 37 ] . Although both Leu and Phe are hydrophobic amino acids, the benzene ring of Phe displays significant rotational mobility, resulting in pronounced steric hindrance. In contrast, while Leu's side chain retains conformational flexibility, its spatial constraints are substantially reduced. The Phe626→Leu626 substitution may improve protein folding efficiency, stabilize the tertiary structure. Since changes in amino acid sequences can alter the thermostability of proteins, thereby affecting the heat resistance of yeast strains [ 49 ] .We hypothesize that the mutation of Cdc15p optimizes its interaction with protein partners such as Tem1p, thereby enhancing the heat resistance performance of the MT (Fig. 8 ). This is supported by the following evidence: First, TEM1 is closely related to CDC15 . For example, the lethality associated with TEM1 deficiency can be rescued by overexpression of CDC15 [ 50 ] , and other CDC15 mutations (Leu356 and Phe357) weaken the interaction between Tem1p and Cdc15p, leading to temperature-sensitive growth, a defect that was efficiently suppressed by the overexpression of TEM1 [ 37 ] . Second, the mutated residue at position 626 is spatially adjacent to the region in Cdc15p that can independently interact with Tem1p (Fig. 7 ). However, these hypotheses require further validation. 4.2 Differences between the Aggregation of WT under Heat Stress and Classical Pseudohyphal Growth It was observed that partial WT cells exhibited adhesion at 37°C and nearly all WT cells showed adhesion at 41°C, in contrast, MT cells remained dispersed (Fig. 2 ). To the best of our knowledge, this is the first reported adhesion growth trait associated with the cell cycle-related gene CDC15 . Similarly, the deletion of another cell cycle-related gene MBP1 leads to pseudohyphae formation [ 4 ] , whereas it was previously widely accepted that starvation is a prerequisite for pseudohyphae formation in S. cerevisiae [ 51 , 52 ] . However, in this study, under high temperature condition, WT cells formed clusters through adhesion aggregation, distinct from the chains of elongated cells aggregated together during typical pseudohyphae formation [ 53 , 54 ] . We speculated that the two different growth states associated with CDC15 and MBP1 arise from distinct mechanisms causing variations in "pseudohyphae" morphology and extent, which aligns with previous findings [ 53 ] . The cell cycle-related genes CDC15 [ 36 ] and MBP1 [ 4 ] have been implicated in tolerance to heat stress (this study) and ethanol [ 4 ] , respectively, and MBP1 is a known transcriptional regulator of numerous genes [ 55 ] . Furthermore, the deletion of MBP1 or mutation of CDC15 results in cell elongation and aggregation [ 4 ] and cell non-adhesion, different from WT, (this study), respectively, but their cooperative regulation of growth remains unknown. 4.3 Effects of Trehalose on Thermotolerance and Ethanol associated with CDC15 Intracellular accumulation of trehalose is believed to increase a yeast’s tolerance to freezing, dehydration [ 56 ] . Protection against freezing in yeast cells by trehalose requires its presence on both sides of the plasma membrane [ 48 ] . Under high temperature conditions, the intracellular trehalose content of MT was higher than that of WT (Fig. 5 ), and MT exhibited superior growth capacity compared to WT (Fig. 3 ). Although the addition of trehalose increased cell counts of both strains, MT's count still exceeded WT's under stress (Fig. 6 ). The potential reasons for these differences may be found in the previous studies, which indicates that the degradation of intracellular trehalose facilitates cellular recovery from heat stress, and that intracellular trehalose is associated with cell cycle regulation, while extracellular trehalose can act as a carbon source [ 57 ] . The higher intracellular trehalose content of MT may result from the mutation of the cell cycle-related gene CDC15 affecting trehalose synthesis and/or degradation, since trehalose and its degradation are associated with cell cycle regulation [ 57 ] . However, further verification is needed. Trehalose can be hydrolyzed by trehalase into glucose, potentially providing more substrate for glycolysis [ 57 ] . This means cells can mobilize more glucose to participate in glycolysis reactions, thereby generating more pyruvate, which is converted into ethanol under anaerobic conditions. This may result in the higher ethanol yield in MT, as MT shows higher trehalose content than WT (Fig. 5 ). Additionally, during the late stages of fermentation, cells utilize ethanol and/or glucose derived from trehalose hydrolysis to sustain growth due to the lack of available carbon sources. We speculate that WT likely consumes more ethanol because its trehalose content is lower than MT, which could also contribute to WT's lower ethanol yield compared to MT. 5. Conclusions CDC15 mutation (Phe626 →Leu626) enhanced thermotolerance and suppressed heat-induced cell aggregation under high-temperature stress. This is likely mediated by trehalose-mediated stress protection and Cdc15p conformational flexibility. Furthermore, CDC15 mutation improved ethanol production efficiency. Although the molecular mechanisms require further investigation, this unexpectedly discovery of enhanced thermotolerance and fermentation traits highlights CDC15 's multifaceted functionality in stress adaptation and metabolic regulation. This finding positions CDC15 as a promising genomic editing target for developing robust industrial yeast strains, and fermentation efficiency are paramount. Abbreviations ADH Alcohol dehydrogenase SOD superoxide dismutase WT MF01,wild-type MT MF01- CDC15 XOD Xanthine oxidase NAD+ oxidized coenzyme I NADH reduced coenzyme I DNS 3,5-dinitrosalicylic acid SNP single nucleotide polymorphism DCW dry cell weight MT30 MT grows at 30°C MT37 MT grows at 37°C MT41 MT grows at 41°C WT30 WT grows at 30°C WT37 WT grows at 37°C WT41 WT grows at 41°C Declarations Authors' contributions X.C.: Funding acquisition, Investigation, Validation, Writing – original draft, Writing – review & editing. D.C.: Methodology, Resources. Q.L.: Data curation, Investigation. Y. C.: Investigation, Funding acquisition. Z.L.: Validation. S.Z.: Methodology. All authors reviewed the manuscript. Availability of data and materials The original contributions presented in this study are included in the article/Supplementary Material/Miscellaneous File Not for Publication. Further inquiries can be directed to the corresponding authors. Competing interests The authors declare no competing interests. Funding This project was supported by the Natural Science Foundation of China (32360018), the Natural Science Foundation of Guangxi, China (2023GXNSFAA026462), Science and Technology Major Project of Guangxi (GuikeAA24206052) and the Natural Science Foundation of Guangxi, China (2024GXNSFAA010494). References Walls LEMJ, Rios-Solis L. Enhancing Saccharomyces cerevisiae taxane biosynthesis and overcoming nutritional stress-induced pseudohyphal growth. Microorganisms. 2022;10(1):163. Kwak S, Jin YS. Production of fuels and chemicals from xylose by engineered Saccharomyces cerevisiae : A review and perspective. Microb Cell Fact. 2017;16(1):82. Park YR, Yang JW, Sunwoo IY, et al. Enhancement of catabolite regulatory genes in Saccharomyces cerevisiae to increase ethanol production using hydrolysate from red seaweed gloiopeltis furcata. J Biotechnol. 2021;333:1–9. Chen X, Lu Z, Chen Y, et al. Deletion of the MBP1 gene, involved in the cell cycle, affects respiration and pseudohyphal differentiation in Saccharomyces cerevisiae . Microbiol Spectr. 2021;9(1):e0008821. Basso LC, de Amorim HV, de Oliveira AJ, et al. Yeast selection for fuel ethanol production in brazil. FEMS Yeast Res. 2008;8(7):1155–63. Ghazali MFSM, Mustafa M. Bioethanol as an alternative fuels: A review on production strategies and technique for analysis. Energy Convers Management: X. 2025;26:100933. Abdel-Banat BM, Hoshida H, Ano A, et al. High-temperature fermentation: How can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol. 2010;85(4):861–7. Meishan F. Shuaishuai, Integrating sugarcane molasses into sequential cellulosic biofuel production based on ssf process of high solid loading. Biotechnol Biofuels, 2017. Fernández-López CLT-SB, Salgado-Cervantes MA, García PG, Aguilar-Uscanga MG. Use of sugarcane molasses b as an alternative for ethanol production with wild-type yeast Saccharomyces cerevisiae itv-01 at high sugar concentrations. Bioprocess Biosyst Eng. 2012;35(4):605–14. Richard C, Brazil. A world leader. Sugar Journal: Covering World's Sugar Ind, 2006(7):69. Gemma Bellí EG, Martí A, Herrero. E Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/cdc28 activity in Saccharomyces cerevisiae . Mol Microbiol. 2001;39(4):1022–35. Thatipamala R, Rohani S, Hill GA. Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation. Biotechnol Bioeng,40(2):289–97. Shahsavarani H, Sugiyama M, Kaneko Y, et al. Superior thermotolerance of Saccharomyces cerevisiae for efficient bioethanol fermentation can be achieved by overexpression of rsp5 ubiquitin ligase. Biotechnol Adv. 2012;30(6):1289–300. Kim HS, Kim NR, Yang J, et al. Identification of novel genes responsible for ethanol and/or thermotolerance by transposon mutagenesis in Saccharomyces cerevisiae . Appl Microbiol Biotechnol. 2011;91(4):1159–72. Suthee Benjaphokee PK. Choowong Auesukaree,Thipa Asvarak, Minetaka Sugiyama1 Yoshinobu Kaneko, Harashima CBaS. Cdc19 encoding pyruvate kinase is important for high-temperature tolerance. Saccharomyces cerevisiae New Biotechnol. 2012;29(2):166–76. Knapp BD, Huang KC. The effects of temperature on cellular physiology. Annual Rev Biophys. 2022;51:499–526. Causton HC, Ren B, Koh SS, et al. Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell. 2001;12(2):323–37. Kanshin E, Kubiniok P, Thattikota Y, et al. Phosphoproteome dynamics of Saccharomyces cerevisiae under heat shock and cold stress. Mol Syst Biol. 2015;11(6):813. Coleman MC, Fish R, Block DE. Temperature-dependent kinetic model for nitrogen-limited wine fermentations. Appl Environ Microbiol. 2007;73(18):5875–84. Ulane R, Ogur M. Genetic and physiological control of serine and glycine biosynthesis in Saccharomyces. J Bacteriol. 1972;109(1):34–43. Shimoda C, Itadani A, Sugino A, et al. Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae . Genes Genet Syst. 2006;81(6):391–7. Sridhar M, Sree NK, Rao LV. Effect of uv radiation on thermotolerance, ethanol tolerance and osmotolerance of Saccharomyces cerevisiae vs1 and vs3 strains. Bioresour Technol. 2002;83(3):199–202. Pattanakittivorakul S, Lertwattanasakul N, Yamada M, et al. Selection of thermotolerant Saccharomyces cerevisiae for high temperature ethanol production from molasses and increasing ethanol production by strain improvement. Antonie Van Leeuwenhoek. 2019;112(7):975–90. Sun H, Jia H, Li J et al. Rational synthetic combination genetic devices boosting high temperature ethanol fermentation. Synth Syst Biotechnol, 2017,2(2):121–9. Fukuda N, Kaishima M, Ishii J, et al. Continuous crossbreeding of sake yeasts using growth selection systems for a-type and α-type cells. AMB Express. 2016;6(1):45. Generoso WC, Gottardi M, Oreb M, et al. Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae . J Microbiol Methods. 2016;127:203–5. Jagtap RS, Mahajan DM, Mistry SR, et al. Improving ethanol yields in sugarcane molasses fermentation by engineering the high osmolarity glycerol pathway while maintaining osmotolerance in Saccharomyces cerevisiae . Appl Microbiol Biotechnol. 2019;103(2):1031–42. Molinet J, Cubillos FA. Wild yeast for the future: Exploring the use of wild strains for wine and beer fermentation. Front Genet. 2020;11(589350):2020. Li-li Li Mei-li, Ye Yan-rui, Ye. Ying Lin. Correlation Between Stress Tolerance and Fermentation Behavior of Saccharomyces cerevisiae . J South China Univ Technol (Natural Sci Edition). 2011;39(9):134–9. Lu DC, Zhang Sui-sheng, Chen Y, Huang Ri-bo. High gravity alcoholic fermentation from sugarcane molasses by a high-yield Saccharomyces cerevisiae strains MF1001 in industrial scale, Guangxi Sciences,2011,18(4):385–391. Shirayama M, Matsui Y, Toh-e A. Dominant mutant alleles of yeast protein kinase gene cdc15 suppress the lte1 defect in termination of m phase and genetically interact with cdc14. Mol Gen genetics: MGG. 1996;251(2):176–85. Varela C, Pizarro F, Agosin E. Biomass content governs fermentation rate in nitrogen-deficient wine musts. Appl Environ Microbiol. 2004;70(6):3392–400. Zhang Y, Wang J, Wang Z et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae . Nat Commun, 1053,10(1):019–09005. DiCarlo JE, Norville JE, Mali P, et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 2020;41(7):4336–43. Oyanagui Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Anal Biochem. 1984;142(2):290–6. Rock JM, Angelika A. Cdc15 integrates tem1 gtpase-mediated spatial signals with polo kinase-mediated temporal cues to activate mitotic exit. Genes Dev,25(18):1943–54. Asakawa K, Yoshida S, Otake F, et al. A novel functional domain of cdc15 kinase is required for its interaction with tem1 gtpase in Saccharomyces cerevisiae . Genetics. 2001;157(4):1437–50. Schweitzer B, Philippsen P. Cdc15, an essential cell cycle gene in Saccharomyces cerevisiae , encodes a protein kinase domain. Yeast. 1991;7(3):265–73. Mühlhofer M, Berchtold E, Stratil CG et al. The heat shock response in yeast maintains protein homeostasis by chaperoning and replenishing proteins. Cell reports, 2019,29(13):4593–4607 e4598. Hottiger T, Schmutz P, Wiemken A. Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae . J Bacteriol. 1987;169(12):5518–22. Asada R, Watanabe T, Tanaka Y, et al. Trehalose accumulation and radiation resistance due to prior heat stress in Saccharomyces cerevisiae . Arch Microbiol. 2022;204(5):275. Alexandre H, Plourde L, Charpentier C, et al. Lack of correlation between trehalose accumulation, cell viability and intracellular acidification as induced by various stresses in Saccharomyces cerevisiae . Microbiology. 1998;144(Pt 4):1103–11. Davidson JF, Whyte B, Bissinger PH, et al. Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae . Proc Natl Acad Sci U S A. 1996;93(10):5116–21. Brown SD, GAM, Karpinets TV, Parks JM, Smolin N, Yang S, Land ML, Klingeman DM, Bhandiwad A, Rodriguez M, Jr, Raman B, Shao X, Mielenz JR, Smith JC, Keller M. Lynd L.R. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in clostridium thermocellum Proceedings of the National Academy of Sciences, 2011,108(33):13752–13757. Jayakody LN, Horie K, Hayashi N, et al. Improvement of tolerance of Saccharomyces cerevisiae to hot-compressed water-treated cellulose by expression of adh1. Appl Microbiol Biotechnol. 2012;94(1):273–83. Hasunuma T, Ismail KSK, Nambu Y, et al. Co-expression of tal1 and adh1 in recombinant xylose-fermenting Saccharomyces cerevisiae improves ethanol production from lignocellulosic hydrolysates in the presence of furfural. J Biosci Bioeng. 2014;117(2):165–9. Dhamija SS, Gera R, Singh DK, et al. Physiological and biochemical characterization of intergeneric hybrids of thermotolerant and non-thermotolerant yeasts. J Basic Microbiol. 1997;37(5):307–12. Diniz-Mendes L, Bernardes E, de Araujo PS, et al. Preservation of frozen yeast cells by trehalose. Biotechnol Bioeng. 1999;65(5):572–8. Walunjkar N, Lai TY, Akhter N et al. Pervasive divergence in protein thermostability is mediated by both structural changes and cellular environments. Mol Biol Evol, 2025,42(7). Shirayama M, Matsui Y, Toh EA. The yeast tem1 gene, which encodes a gtp-binding protein, is involved in termination of m phase. Mol Cell Biol. 1994;14(11):7476–82. Miled C, Mann C, Faye G. Xbp1-mediated repression of clb gene expression contributes to the modifications of yeast cell morphology and cell cycle seen during nitrogen-limited growth. Mol Cell Biol. 2001;21(11):3714–24. Gimeno CJ, Ljungdahl PO, Styles CA, et al. Unipolar cell divisions in the yeast s. Cerevisiae lead to filamentous growth: Regulation by starvation and RAS. Cell. 1992;68(6):1077–90. Kim J, Rose MD. Stable pseudohyphal growth in budding yeast induced by synergism between septin defects and altered map-kinase signaling. PLoS Genet,11(12):e1005684. Mutlu N, Sheidy DT, Hsu A, et al. A stress-responsive signaling network regulating pseudohyphal growth and ribonucleoprotein granule abundance in Saccharomyces cerevisiae . Genetics. 2019;213(2):705–20. Hendler A, Medina EM, Kishkevich A, et al. Gene duplication and co-evolution of G1/s transcription factor specificity in fungi are essential for optimizing cell fitness. PLoS Genet. 2017;13(5):e1006778. Shima J, Hino A, Yamada-Iyo C, et al. Stress tolerance in doughs of Saccharomyces cerevisiae trehalase mutants derived from commercial baker's yeast. Appl Environ Microbiol. 1999;65(7):2841–6. Chen A, Stadulis SE, deLeuze K et al. Evaluating cellular roles and phenotypes associated with trehalose degradation genes in Saccharomyces cerevisiae . G3 (Bethesda), 2024,14(11). Hanks SK, Quinn AM, Hunter T. The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science. 1988;241(4861):42–52. Jiménez J, Cid VJ, Cenamor R, et al. Morphogenesis beyond cytokinetic arrest in Saccharomyces cerevisiae . J Cell Biol. 1998;143(6):1617–34. Additional Declarations No competing interests reported. Supplementary Files MiscellaneousFileNotforPublication.docx OpenwithSPDBVStructuralmodel1SuperimposedStructuresofCdc15pfromMutantMTandWildTypeWT.pdb SupplementaryFile.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviews received at journal 29 Mar, 2026 Reviews received at journal 27 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers invited by journal 17 Mar, 2026 Editor assigned by journal 05 Mar, 2026 Submission checks completed at journal 05 Mar, 2026 First submitted to journal 28 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-8993691","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608369036,"identity":"eeac83b0-8934-44b0-8ad0-fc5c8b433bc1","order_by":0,"name":"Xiaoling Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYDACZiB+YAAkJIA4wUBCjp+Z+fADgloSYFoeFFgYS7azpRkQtCmBAaKF8cGHikSD8zwKEvhUGxxnfvggoeCOXf/s5mMPgA5LMD7Mw2DAUGMTjUuLZDObsUGCwbPkGXeOpQMZEnlmh3kPPGA4lpbbgEMLPzODmUSCweFkA4kcEEOi2OwwX4IBY8NhnFrYmNm/QbXkgxgSiZubeQwk8GnhZ+YB22IHtIUNrGUDMwEtks08xUAvHE6QuJEGdpixxGFgICfg8YvB+eMbH3z4c9ief0byM8kff+rk+PsPH37wocYGpxYYSERVkEBAOQjYE6FmFIyCUTAKRioAALX/VkZfhQecAAAAAElFTkSuQmCC","orcid":"","institution":"Guangxi Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Chen","suffix":""},{"id":608369039,"identity":"07d6ebca-b9f2-4fe8-9ea7-3bec1ca32bb9","order_by":1,"name":"Dong Chen","email":"","orcid":"","institution":"Guangxi Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Chen","suffix":""},{"id":608369040,"identity":"8481cff1-05d1-4f26-a8f2-97eb2458d71d","order_by":2,"name":"Qi Lu","email":"","orcid":"","institution":"Guangxi Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Lu","suffix":""},{"id":608369041,"identity":"3e3334f7-20de-444f-abde-1d18b245a0f1","order_by":3,"name":"Ying Chen","email":"","orcid":"","institution":"Guangxi Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Chen","suffix":""},{"id":608369043,"identity":"991a12ae-5d1e-4f13-ab46-44c252a1b5ce","order_by":4,"name":"Zhilong Lu","email":"","orcid":"","institution":"Guangxi Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhilong","middleName":"","lastName":"Lu","suffix":""},{"id":608369045,"identity":"4291907d-bf10-460c-9298-104041f77b96","order_by":5,"name":"Suisheng Zhang","email":"","orcid":"","institution":"Guangxi Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Suisheng","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-02-28 09:10:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8993691/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8993691/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104987373,"identity":"4b73204a-b617-43a1-8f8f-f3a64d5c3631","added_by":"auto","created_at":"2026-03-19 14:41:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":168732,"visible":true,"origin":"","legend":"\u003cp\u003eCdc15p sequence alignment of MT, WT and S288C (NCBI Reference Sequence: NP_009411.2\u003csup\u003e[38]\u003c/sup\u003e, NC_001133.9)\u003c/p\u003e\n\u003cp\u003eNote: The differential amino acids were indicated in shadow.\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/24b7c464ff55890ea02530c6.png"},{"id":104987420,"identity":"fca23987-6fbb-4ae1-98fb-38f6059a67c9","added_by":"auto","created_at":"2026-03-19 14:42:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2426607,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characteristics of cells cultured at varying temperatures. Representative micrographs were captured during growth curves determination using Countstar BioFerm IY1200 (one was randomly selected from two parallel cultures). Strain (WT, MT) and culture temperature (30 °C, 37 °C, 41°C) are labeled above and on the left of the figure, respectively. This image only displays cells at 8 h \u0026nbsp;of cultivation. Further details are available in Fig. S1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/68e7758c0b5f51f5f1007ddb.png"},{"id":104987412,"identity":"50608ec3-765c-4f6b-914a-b07641a710f0","added_by":"auto","created_at":"2026-03-19 14:42:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":244357,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of the strains under different temperature conditions.\u003c/p\u003e\n\u003cp\u003eThe data obtained from the Countstar BioFerm IY1200 systemrepresent the mean ± SD of two independent biological replicates. The asterisk adjacent to the temperature value denotes statistically significant differences observed at the corresponding time points under that specific temperature.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/ab2bec60e0117dfc3e6dd3c2.png"},{"id":104987389,"identity":"36727376-334a-4fe9-91ae-90ceb7aeefdf","added_by":"auto","created_at":"2026-03-19 14:41:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":332924,"visible":true,"origin":"","legend":"\u003cp\u003eEthanol production with sucrose (A) or molasses(B)\u003c/p\u003e\n\u003cp\u003eEthanol content is determined by gas chromatography. The data shown represent the mean ± SD of three independent biological replicates.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/bec895b4afadbecb3230e5a9.png"},{"id":104987369,"identity":"8fe5657e-d81f-4da6-8ea3-8ef8a68f0af2","added_by":"auto","created_at":"2026-03-19 14:41:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":60359,"visible":true,"origin":"","legend":"\u003cp\u003eTrehalose content (per mg of DCW)\u003c/p\u003e\n\u003cp\u003eThe data shown represent the mean ± SD of two or three independent biological replicates.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/f882284c152fd22766722cd5.png"},{"id":104987374,"identity":"383df704-bc21-4534-80e9-6b9fe727cf74","added_by":"auto","created_at":"2026-03-19 14:41:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":380091,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of cultures with and without trehalose.\u003c/p\u003e\n\u003cp\u003eAfter two rounds of overnight culture, cells were harvested by centrifugation at 4°C. A total of 2 × 10\u003csup\u003e7\u003c/sup\u003e cells were inoculated into 10 mL of YPD medium in 50 mL centrifuge tubes, with or without trehalose supplementation (final concentration: 0.3 g/L). The cultures were incubated at 30 °C (A), 37 °C (B), and 41 °C (C). Samples were collected at 8-h intervals and subsequently subjected to cell counting. Data are presented as the mean ± SD of three independent biological replicates. Trehalose-supplemented cultures are denoted by \"-T\".\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/93d9bc9ac1794e8e4009fc5d.png"},{"id":104987370,"identity":"6f205627-8b2f-4e92-afb0-fcb49ea1d3d1","added_by":"auto","created_at":"2026-03-19 14:41:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1773030,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of Cdc15p Structure.\u003c/p\u003e\n\u003cp\u003eThe SwissModel platform (http://swissmodel.expasy.org/) was used to perform homology modeling with the highest similarity sequence. Swiss-PdbViewer 4.1.0 was used to superimpose the Cdc15p structure of MT and WT. The α-helix encompassing residue 626 is highlighted in yellow. The region enriched with charged residues (residues 281-305)\u003csup\u003e[37]\u003c/sup\u003e is shown in cyan(). Lower right panel: Rotational and zoom views of the mutated residue (Phe626, Leu626) in the α-helical. The side chains of Phe626 (WT) and Leu626 (MT) are colored green and red, respectively. For further details, see Fig.S5 and Structural model 1.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/c64ca6bd18008df3f9c9e09a.png"},{"id":104987383,"identity":"6a04e4c3-7838-4a20-8ae2-e5becbd812f0","added_by":"auto","created_at":"2026-03-19 14:41:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":253813,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/684e80a3396e39137c95e241.png"},{"id":104987513,"identity":"0a5fe8d0-cd1d-4a3b-8362-355e8bc6828b","added_by":"auto","created_at":"2026-03-19 14:42:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8354047,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/cd365ea1-fed6-41bf-abb7-712a24ce1f86.pdf"},{"id":104987368,"identity":"02e7fddf-e9fd-44eb-a4c3-7d35383ff57a","added_by":"auto","created_at":"2026-03-19 14:41:39","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5539121,"visible":true,"origin":"","legend":"","description":"","filename":"MiscellaneousFileNotforPublication.docx","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/9df56facf755921e0b2edd99.docx"},{"id":104987378,"identity":"66c7f529-9dfd-4db3-a3d5-8f05d57126a1","added_by":"auto","created_at":"2026-03-19 14:41:47","extension":"pdb","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1226685,"visible":true,"origin":"","legend":"","description":"","filename":"OpenwithSPDBVStructuralmodel1SuperimposedStructuresofCdc15pfromMutantMTandWildTypeWT.pdb","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/cb5c57d0f95dd95d3f4ad457.pdb"},{"id":104987382,"identity":"1924dc08-0946-4247-908c-9258048ca99e","added_by":"auto","created_at":"2026-03-19 14:41:50","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10930922,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8993691/v1/1252f7a3605bd43864a0d8a3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced Heat Tolerance and Ethanol Production of Industrial Saccharomyces cerevisiae via Replacement of the CDC15 Gene","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlthough \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e is a famous cell factory for producing chemicals\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, it remains the most widely used microbial host for ethanol production and the dominant organism in fuel ethanol manufacturing \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Bioethanol is regarded as a promising renewable liquid fuel alternative to mitigate energy crises\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, production costs are a major issue for the production of fuel ethanol compared with fossil fuels \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Sugarcane molasses, a low-cost byproduct of sugar production containing approximately 45% (w/w) \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, even 50\u0026ndash;65% \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, fermentable sugars, has particular potential as feedstock in southern China's sugarcane-growing regions.\u003c/p\u003e \u003cp\u003eIn Brazil, one of the largest ethanol biofuel producers and exporters in the world\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, fuel ethanol is produced mainly from sugarcane juice and, in some cases, from molasses \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Molasses is diluted prior to fermentation \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, since molasses\u0026rsquo;s high osmotic pressure (derived from ~\u0026thinsp;50% total sugars\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e) causes intracellular water loss in cells, leading to slow or even halted cell growth\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Such hypertonic stress not only impedes yeast growth but also disrupts metabolic pathways, ultimately diminishing ethanol production efficiency and extending fermentation duration\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Therefore, strains capable of withstanding high osmotic pressure for low-dilution or undiluted fermentation are particularly valuable.\u003c/p\u003e \u003cp\u003eRapidly growing cells enable efficient acquisition of sufficient cell biomass to initiate ethanol production, and thereby reduce production costs. Alternatively, recycled cells can be employed \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, which is not applicable in China. In Brazil, the fermentation process generally employs high cell densities to rapidly achieve sufficient cell numbers for efficient fermentation, with certain aspects depending on the growth performance of the strain under current manufacturing practices. The number of recycling cycles can be increased if the cells exhibit high ethanol tolerance, since ethanol is toxic to yeast at high concentrations. Ethanol accumulation in later stages inhibits cellular growth at 9% (v/v)\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e or completely halts it at 12% (v/v)\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, resulting in incomplete sugar utilization and reduced ethanol yield, thereby increasing production costs. Some pathways required for ethanol tolerance overlap with those for thermotolerance\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFermentation is an exothermic process, leading to an increase in fermentation temperature. While temperature control is essential for optimal fermentation, cooling systems significantly increase production costs\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. High-temperature fermentation using thermotolerant strains offers a cost-effective alternative, since a 5\u0026deg;C increase in the fermentation temperature could drastically reduce expenses\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The United States produces fuel ethanol primarily from corn through saccharification using α-amylase and glucoamylase, followed by fermentation with \u003cem\u003eS.cerevisiae\u003c/em\u003e. Under these conditions, employing thermotolerant yeasts can more effectively reduce costs \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBesides, when producing fuel ethanol from corn through saccharification using α-amylase and glucoamylase, followed by fermentation with \u003cem\u003eS.cerevisiae\u003c/em\u003e\u0026mdash;a process primarily employed in the United States\u0026mdash;employing thermotolerant yeasts can more effectively reduce costs \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnfortunately, \u003cem\u003eS. cerevisiae\u003c/em\u003e strains face a critical limitation: their optimal growth range (25\u0026ndash;30\u0026deg;C) and failure to proliferate above 40\u0026deg;C\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Temperature exerts significant effects on cellular molecular composition, volume expansion, and protein folding \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Heat shock induces the expression of genes associated with RNA processing/modification, protein degradation, protein folding/transport\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, and also alters phosphorylation states of proteins involved in transcription, protein folding and degradation, cell cycle regulation and morphogenesis \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Inappropriate temperature results in slower cell growth rates\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, which affects fermentation rate during ethanol production. Additionally, temperature influences sugar utilization efficiency, and cell inactivation rate in ethanol-containing environments\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Heat shock induces the expression of genes associated with respiration, and alternative carbon source utilization\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough thermotolerant mutants that could proliferate at 40\u0026deg;C\u003csup\u003e[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, 41\u0026deg;C\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e or 42\u0026deg;C \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e have been obtained, their ethanol productivity remains unknown or low \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Some existing thermotolerant mutants either rely on plasmid-borne gene responsible for the Htg\u003csup\u003e+\u003c/sup\u003e phenotype, or derive from laboratory strains\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, rendering them unsuitable for industrial applications due to genetic instability, as plasmid carrying a resistance selection marker is easily lost in the absence of selective pressure \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Besides, laboratory strains lack the tolerance\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, genetic, and phenotypic diversity\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e of industrial strains, and do not exhibit the positive correlation between tolerance and fermentation capacity seen in industrial strains\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe previously isolated three wild-type industrial strains, MF01, MC15, and ME13, from samples derived from the same environment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although MF01 exhibits the highest ethanol yield and has been adopted for industrial-scale ethanol production\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, its growth rate is lower than that of ME13 and MC15. In contrast, MC15, with the lowest ethanol yield, has the highest growth rate among them. Comparative genomics analysis of these strains identified the cell cycle key gene \u003cem\u003eCDC15\u003c/em\u003e as a sequence-divergent gene, suggesting it may be a critical gene underlying differences in growth rate. Given \u003cem\u003eCDC15\u003c/em\u003e is an important gene involved in cell cycle\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In theory, replacing the \u003cem\u003eCDC15\u003c/em\u003e gene in MF01 with \u003cem\u003eCDC15\u003c/em\u003e from MC15 may enhance the growth rate of MF01, which could positively impact ethanol fermentation as biomass yield critically affects fermentation efficiency\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In this study, the \u003cem\u003eCDC15\u003c/em\u003e gene of MF01 was modified by substituting it with the homologous \u003cem\u003eCDC15\u003c/em\u003e gene from MC15, generating the engineered strain MF01-\u003cem\u003eCDC15\u003c/em\u003e. We hypothesize that homologous recombination of this cell cycle-associated differential gene derived from MC15 may accelerate the growth rate of MF01. As predicted, MF01-\u003cem\u003eCDC15\u003c/em\u003e exhibited accelerated growth. Unexpectedly, \u003cem\u003eCDC15\u003c/em\u003e replacement not only increased ethanol yield but also conferred enhanced thermotolerance\u0026mdash;a critical trait for industrial fermentation where cooling costs and thermal stress are major bottlenecks. These findings first identifies \u003cem\u003eCDC15\u003c/em\u003e as a previously uncharacterized key target for enhancing ethanol productivity in industrial yeast strains, thereby enabling the rational design of robust cell factories for scalable biofuel and bio-based chemical production.\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\u003eStrains used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"left\" 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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eOrigin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMF01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild-type,MATa/MATa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigher ethanol yield and exogenous gene integration rate compared with MC15 and ME13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eIsolated by our lab from sugar mill waste in 2010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMC15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild-type,MATa/MATa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLower ethanol yield and higher growth rate compared with MF01 and ME13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eIsolated by our lab from sugar mill waste in 2010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eME13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild-type,MATa/MATa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntermediate ethanol production and growth rate among MF01, MC15 and ME13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eIsolated by our lab from sugar mill waste in 2010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMF01-\u003cem\u003eCDC15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDerived from MF01,MATa/MATa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eHigher ethanol yield, growth rate and heat resistance compared with MF01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Strain and Culture Conditions\u003c/h2\u003e \u003cp\u003eStrain MF01 (MATa/MATα) is a wild-type (WT) diploid \u003cem\u003eS. cerevisiae\u003c/em\u003e. Strain MF01-\u003cem\u003eCDC15\u003c/em\u003e (MATa/MATα), which is a diploid derived from MF01, was prepared by replacing the \u003cem\u003eCDC15\u003c/em\u003e gene of MF01 with that from a fast-growing strain MC15. The yeast cells were incubated in YPD medium, as previously described \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, in 250 ml Erlenmeyer flasks, unless specified otherwise.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.2 Construction and Screening of the Mutant Strain\u003c/b\u003e\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Preparation and Transformation of \u003cem\u003eCDC15\u003c/em\u003e Homologous Donor\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eCDC15\u003c/em\u003e donor (Fig.SS1 in Miscellaneous File Not for Publication) used for homologous recombination was amplified by PCR with chromosomal DNA of the MC15 strain serving as the template. The donor was amplified with primers \u003cem\u003eCDC15\u003c/em\u003e-F and \u003cem\u003eCDC15\u003c/em\u003e-R described in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, using the following PCR protocol: 94\u0026deg;C for 5 min; 94\u0026deg;C for 40 s, 56\u0026deg;C for 40 s, 72\u0026deg;C for 4 min for 35 cycles; and a final step of 72\u0026deg;C for 10 min. The PCR products were purified before transformation. Preparation of haploid strains was carried out as previously described\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Both types a and α protoplasts of MF01 were subjected to electroporation to introduce the \u003cem\u003eCDC15\u003c/em\u003e gene from MC15.\u003c/p\u003e \u003cp\u003eTable 2 Primers used in this study\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Screening for Recombinant Srains\u003c/h2\u003e \u003cp\u003eThe transformed mixed solution was transferred into 1 mL cold solution containing 18% sucrose and incubated for 1 h at 30\u0026deg;C without shaking. After addition of 1 mL YPD, the cells were cultivated again in a shaker and then plated onto YPD agar.\u003c/p\u003e \u003cp\u003eThe endogenous \u003cem\u003eCDC15\u003c/em\u003e locus in MF01 could be precisely replaced with the MC15-sourced \u003cem\u003eCDC15\u003c/em\u003e sequence, owing to the high homologous recombination efficiency of \u003cem\u003eS. cerevisiae\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Selection of recombinant mutants was based on phenotypic variations in growth rate. The largest colony on the YPD plate was picked and cultured to exponential phase in liquid YPD. Equal numbers of cells were transferred to fresh YPD and cultured for 8\u0026thinsp;~\u0026thinsp;10 h followed by detection of OD\u003csub\u003e600\u003c/sub\u003e. Cells with an OD\u003csub\u003e600\u003c/sub\u003e roughly 10% higher than that of the WT were selected, and then cultured and selected again as described above. Following serial screening, the potential mutant strains were obtained. After validation by PCR with the DNA of potential mutant as template and sequencing, haploid recombinant strains were obtained. Diploid recombinant strain, named MF01-\u003cem\u003eCDC15\u003c/em\u003e (MT), was constructed from mating types a and α of recombinant strains by mating as previously described\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. To assess the genetic stability of MT, the strain was serially passaged for 30 generations, and the \u003cem\u003eCDC15\u003c/em\u003e locus was PCR-amplified using chromosomal DNA from MT as template. The PCR product was verified by sequencing, and its sequence was identical to that in MC15 (data not shown), indicating successful replacement of \u003cem\u003eCDC15\u003c/em\u003e and its genetic stability over generations.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Growth Curve Determination\u003c/h2\u003e \u003cp\u003eA single colony was isolated and inoculated into 5 ml preculture overnight. Equal numbers of cells were harvested by centrifugation at 4\u0026deg;C, resuspended in sterile water and shifted to 50 ml fresh YPD, at a final concentration of roughly 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml, and cultured for 48 h at 30\u0026deg;C, 37\u0026deg;C, 41\u0026deg;C, respectively. Samples were taken at 8-h intervals and stored at 4\u0026deg;C until the number of cells in each sample was determined by Countstar BioFerm IY1200 (Shanghai Ruiyu Biotech Co.Ltd, shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analysis of Yeast Cells for Trehalose Quantification and Enzyme Activity Assays\u003c/h2\u003e \u003cp\u003eAbout 1\u0026times; 10\u003csup\u003e8\u003c/sup\u003e overnight cells were inoculated into 100 mL YPD and grown for 8-8.5 h. Cells from shake flask cultures were harvested by centrifugation (6000 rpm, 5 min, 4\u0026deg;C), washed once with sterile deionized water and twice with 0.1 M PBS. About 38 mg (wet weight) of the resulting pellet was transfered to a 1.5 ml sterile EP tube for trehalose content determination, and the remaining pellet was resuspended in 2 ml 0.1 M PBS, and then quickly frozen (\u0026minus;\u0026thinsp;80 ℃) until use for enzyme activities assayed.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Trehalose Content Determination\u003c/h2\u003e \u003cp\u003eThe above cell pellet that was washed twice with PBS was dried to a constant weight at 55\u0026deg;C, then the rehalose was extracted with trichloroacetic acid followed by anthrone-based detection as before\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Enzyme Assays\u003c/h2\u003e \u003cp\u003eAfter thawing the samples in room temperature, the crude enzyme solution prepared as the previous method\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Protein determination was done using Bradford Protein Assay Kit(Order No. C503031, Sangon Biotech, shanghai,China).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section4\"\u003e \u003ch2\u003e2.4.2.1 Assay of SOD Activity\u003c/h2\u003e \u003cp\u003eThe intracellular total superoxide dismutase (SOD) activity was determined by hydroxylamine method as described previously\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e with some modifications. (1) Preparation of solutions. The Xanthine oxidase (XOD) solution was prepared as follows: 0.5 mg of xanthine oxidase (50 U/mg, purchased from MACKLIN, China) was dissolved in 0.5 ml of buffer solution B (containing104 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and 78 mM Na\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e in deionized water) to obtain a concentration of 50 U/ml (XOD solution I). The working XOD solution was then prepared by adding 125 \u0026micro;l of XOD solution I to 975 \u0026micro;l of buffer solution B. All reagents for the color developer were stored in bottles at 0\u0026ndash;4\u0026deg;C until use. (2) Assay Procedure. The procedure was as follows: 0.5 ml of water, 0.1 ml of crude enzyme solution, and 0.2 ml of reagent A (consisting of equal volumes of 1 mM hydroxylamine and 1 mM hypoxanthine, resulting in final concentrations of 0.2 mM for each) were mixed. The reaction was initiated by adding 0.2 ml of xanthine oxidase (XOD) solution (final concentration of 1.25 mU/ml XOD). This mixture (1.0 ml total) was incubated for 30 min at 37\u0026deg;C. Then, 1.25 ml of reagent III (prepared by mixing 1.5 volumes of 1.2 g/L sulfanilic acid with 1 volume of acetic acid just before use) was added, and the mixture was allowed to stand for 5 min at room temperature. Subsequently, 0.75 ml of reagent II (278.3 mg/L N-1-naphthylethylenediamine dihydrochloride dissolved in deionized water) was added. Reagents III and II were added in two separate steps (to final concentration of 300 \u0026micro;g/ml sulfanilic acid, 5 \u0026micro;g/ml N-1-naphthylethylenediamine, and 16.7% acetic acid), taking into account the color development process, which differed from the previous method. A control for each sample (the crude enzyme solution of each sample, boiled for 5 min to inactivate the enzyme) was prepared and added after the color developer to avoid incomplete enzyme inactivation leading to inaccurate data. The final mixture (3.0 ml) was allowed to stand for 20 min at room temperature, and the optical absorption was measured at 550 nm.\u003c/p\u003e \u003cp\u003eThe principle of the assay is as follows: Xanthine oxidase catalyzes the reaction of hypoxanthine, generating superoxide anion radicals (O₂⁻\u0026bull;⁻, which then oxidize hydroxylamine to form nitrite. The nitrite exhibits a purplish-red color in the presence of a chromogenic agent, and its absorbance is measured using a microplate reader. Due to the specific inhibitory effect of SOD in yeast cells on O₂⁻\u0026bull;, the formation of nitrite is reduced, resulting in a decrease in absorbance during colorimetric measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e2.4.2.2 Alcohol dehydrogenase assay\u003c/h2\u003e \u003cp\u003eAlcohol dehydrogenase (ADH) activity was assayed by the NAD\u003csup\u003e+\u003c/sup\u003e reduction method. ADH catalyzes the dehydrogenation of ethanol, and the removed hydrogen is received by oxidized coenzyme I (NAD\u003csup\u003e+\u003c/sup\u003e), converting it to reduced coenzyme I (NADH). The amount of NADH generated is measured by monitoring the absorbance change at 340 nm, since NAD\u003csup\u003e+\u003c/sup\u003e has no absorption at 340 nm but NADH absorbs at this wavelength. The assay system contained 108.8 \u0026micro;l of 0.1 M PBS (prepared by dissolving 14.2 g Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 2.38 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 80 g NaCl, 2 g KCl, in 1L distilled water), 40 \u0026micro;l of 5 mM NAD\u003csup\u003e+\u003c/sup\u003e (prepared by dissolving 38.9 mg of NAD\u003csup\u003e+\u003c/sup\u003e in 10 ml of distilled water), and 1.2\u0026micro;l of anhydrous ethanol as the substrate. The reaction was initiated by the addition of 50 \u0026micro;l of cell extract to the well of a 96-well microtiter plate. A control for each sample was included, prepared by replacing the cell extract with its inactivated enzyme (prepared by boiling for 5 min). After 20 min of incubation at 30\u0026deg;C, the activity was assayed by measuring the increase in absorbance at 340 nm. Activity is expressed as the increase in absorbance per milligram of protein.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Ethanol Fermentation\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eSucrose fermentation\u003c/strong\u003e \u003cp\u003eFermentation with sucrose was performed as previously described\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e with minor modification. Briefly, 0.4\u0026times;l0\u003csup\u003e8\u003c/sup\u003e overnight cells were inoculated into 40 ml yeast extract peptone sucrose medium (10 g/L yeast extract, 20 g/L peptone, 25 g/L sucrose, pH unadjusted) in a 250 mL Erlenmeyer flask and incubated with shaking for 38 h, followed by static incubation at 30 and 37 ℃ under separate conditions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMolasses fermentation\u003c/strong\u003e \u003cp\u003eFermentation with molasses was conducted according to the prior method\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e with slight modification. 0.4\u0026times;10\u003csup\u003e8\u003c/sup\u003e overnight cells were grown in 30 ml molasses medium (containing 20.6 \u0026deg;Bx molasses) in a 250 mL Erlenmeyer flask and incubated for approximately 9 h. An additional molasses medium (replacing 20.6 \u0026deg;BX molasses with 50.3 \u0026deg;BX molasses) was supplemented, after which the cells were grown for another 30 h, followed by static conditions for 40 h at 30 and 37 ℃in separate experiments.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Sugar Content Determination\u003c/h2\u003e \u003cp\u003eThe reducing sugars and total sugars in the fermented liquid were determined using the 3,5-dinitrosalicylic acid (DNS) reagent. Prior to analysis, the total sugars were hydrolyzed to reducing sugars following a previously described method\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed with the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test using Microsoft Excel (Tails: two-tail,Type: paired t-test). * \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and **** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 indicate statistical significance, and ns (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) indicates non-significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Sequence Comparison of Cdc15p among MT, WT and S288C Reveals Convergent Adaptation\u003c/h2\u003e \u003cp\u003eComparison of the predicted Cdc15p amino acid sequence in MT with the known sequences of WT and S288C revealed one residue difference between WT and MT (F \u003csup\u003e626\u003c/sup\u003e L), two differences between WT and S288C (T\u003csup\u003e851\u003c/sup\u003e S, D\u003csup\u003e902\u003c/sup\u003e V), and three differences between MT and S288C (F \u003csup\u003e626\u003c/sup\u003e L, T\u003csup\u003e851\u003c/sup\u003e S, D\u003csup\u003e902\u003c/sup\u003e V) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The substitution at residue 626 (Phe\u0026rarr;Leu) between WT and MT is caused by a single nucleotide polymorphism (SNP) at position 1878 of the ORF (T \u0026rarr;A in MT, Fig.SS3 in Miscellaneous File Not for Publication). The higher protein sequence similarity between MT and WT may reflect their convergent adaptation to identical growth environments, as both strains were isolated from the same geographic site.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2 \u003cem\u003eCDC15\u003c/em\u003e Mutation Does Not Alter Cellular Morphology at Normal Temperature but Causes Nonadherent Growth\u003c/h2\u003e \u003cp\u003eCdc15p plays a crucial and multifaceted role in the cell cycle, specifically in mitotic exit\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. While its role in cell cycle progression\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e is well established and \u003cem\u003eCDC15\u003c/em\u003e overexpression does not affect mitosis\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, potential effects of \u003cem\u003eCDC15\u003c/em\u003e mutations on cellular morphogenesis remain unexplored. To evaluate this phenotype, we compared the WT and MT strains under both solid and liquid culture conditions. On YPD agar plates, both strains formed colonies with white, viscous, normally smooth, and lustrous surfaces (Fig.SS5 in Miscellaneous File Not for Publication), indicating that their colony size and morphology were normal at 30 ℃. These results indicate that \u003cem\u003eCDC15\u003c/em\u003e mutation does not seem to have any adverse effects on morphology. Unexpectedly, some WT cells exhibited adherent growth at 37\u0026deg;C, with more pronounced effects observed at 41\u0026deg;C, but the MT did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The \u003cem\u003eCDC15\u003c/em\u003e Mutation Improves Cell Viability under Thermal Stress (50\u0026deg;C to 54\u0026deg;C)\u003c/h2\u003e \u003cp\u003eIn practical ethanol production, localized temperatures within fermentation tanks may transiently exceed 50\u0026deg;C due to metabolic heat accumulation compounded by ambient thermal variations. Such transient thermal stresses, when exceeding the strain's tolerance threshold, invariably result in suboptimal fermentation performance ranging from diminished ethanol yield to complete process failure. Therefore, thermotolerant strains demonstrating high viability under production conditions is one of the desirable characteristics for strains. High cell viability is one of the critical criteria for strain selection in Brazil's industrial ethanol production\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo evaluate the heat shock tolerance of WT and MT strains and investigate the impact of the \u003cem\u003eCDC15\u003c/em\u003e mutation on thermotolerance, we analyzed the post-thermal stress viability of both strains. Following a 10-min heat shock at 42\u0026deg;C to 48\u0026deg;C, cell growth was delayed in both solid and liquid cultures, with no obvious distinction observed between the WT and MT. However, at temperatures between 50\u0026deg;C and 54\u0026deg;C, significant differences emerged between the WT and MT. On plates incubated at 50\u0026deg;C, the MT exhibited significantly higher colony counts.than that of WT, and this difference became more pronounced at 52\u0026deg;C. A small number of mutant colonies survived at 54\u0026deg;C, whereas no surviving WT colonies were detected (Fig.SS7 in Miscellaneous File Not for Publication). These results indicate that after heat shock at 50\u0026deg;C to 54\u0026deg;C, the mutant strain exhibited a higher cell viability than the WT strain, suggesting enhanced thermal tolerance in the mutants. No cell growth was observed at temperatures between 56\u0026deg;C and 62\u0026deg;C (data not shown).\u003c/p\u003e \u003cp\u003eIt is reported that protein translation ceased after 10 min of exposure to 46\u0026deg;C \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, and that only rare thermotolerant strains survive following a 5-min heat shock at 54\u0026deg;C \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Strikingly, however, the MT strain in our study still exhibited survival after a 10-min heat shock at 54\u0026deg;C, but the WT did not. This not only demonstrates the superior thermotolerance of the MT strain, but also confirms that the \u003cem\u003eCDC15\u003c/em\u003e mutant exhibits a significantly higher cell viability than the WT following heat shock.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 The\u003c/b\u003e \u003cb\u003eCDC15\u003c/b\u003e \u003cb\u003eMutation Promotes Cellular Proliferation under both Normal Conditions (30\u0026deg;C) and Thermal Stress (37\u0026deg;C, 41\u0026deg;C)\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious studies suggest that although protein translation did not decrease significantly within the first 30 min of exposure to 42\u0026deg;C, the synthesis of de novo proteins was affected under prolonged thermal stress at 42\u0026deg;C \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. To further investigate temperature-dependent phenotypic differences, we analyzed the proliferation capacity of the WT and MT under varying temperature conditions. The MT entered the stationary phase 8 h earlier (at 16 h ) and achieved a higher maximum cell density (4.37\u0026times;10\u003csup\u003e8\u003c/sup\u003e cells/mL) than the WT (3.18\u0026times;10\u003csup\u003e8\u003c/sup\u003e cells/mL) at 30\u0026deg;C. As the temperature increases, both strains show decreased growth rates. However, the cell number of MT consistently exceeded that of the WT at all measured time points, and the fold difference increased with rising temperatures (Fig.S 2). The maximum cell densities of the MT at 30\u0026deg;C, 37\u0026deg;C and 41\u0026deg;C increased by 37%, 57%, 155%, respectively. Additionally, the cell number of MT cultured at 37\u0026deg;C was higher than that of WT at 30\u0026deg;C, and the cell number of MT at 41\u0026deg;C was comparable to that of WT at 37\u0026deg;C. In brief, the hierarchical distribution of cell counts is MT30 (MT at 30\u0026deg;C, similarly hereinafter) \u0026gt; MT37\u0026thinsp;\u0026gt;\u0026thinsp;WT30\u0026thinsp;\u0026gt;\u0026thinsp;WT37\u0026thinsp;\u0026asymp;\u0026thinsp;MT41\u0026thinsp;\u0026gt;\u0026thinsp;WT41 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Collectively, this suggests that the \u003cem\u003eCDC15\u003c/em\u003e mutation enhances the strain's ability to grow under both normal and heat stress conditions, consistent with observations in post-thermal stress viability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 \u003cem\u003eCDC15\u003c/em\u003e Mutation Enhances Ethanol Production\u003c/h2\u003e \u003cp\u003eThe ability of MT to grow rapidly at 37\u0026deg;C and 41\u0026deg;C holds enormous potential for applications in ethanol production \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. To evaluate the effect of the mutation on ethanol production, we tested its fermentation capability. Upon fermentation with sucrose as the sole carbon source, MT at 30\u0026deg;C showed the highest ethanol production among all samples after 35 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). When molasses was used as the sole carbon source, MT37 displayed the best ethanol productivity during the first 38 h, after which MT at 30\u0026deg;C showed higher productivity until 70 h when MT at 37\u0026deg;C again became highest (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). When sucrose was used as the carbon source, MT strain achieved maximum ethanol concentrations of 12.03% \u0026plusmn; 0.01% at 30\u0026deg;C and 11.14% \u0026plusmn; 0.01% at 37\u0026deg;C, representing 26.41% and 3.79% increases over WT, respectively. Similarly, with molasses, MT produced maximum concentrations of 11.02% \u0026plusmn; 0.07% at 30\u0026deg;C and 10.54% \u0026plusmn; 0.06% at 37\u0026deg;C, corresponding to 29.56% and 3.84% improvements over WT, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results suggest that the \u003cem\u003eCDC15\u003c/em\u003e mutation enhances ethanol production, and ethanol fermentation from molasses by \u003cem\u003eS. cerevisiae\u003c/em\u003e is more difficult, resulting from the complex composition, compared with that from sucrose. Unexpectedly, both MT and WT strains showed higher ethanol production at 37\u0026deg;C than that of WT at 30\u0026deg;C, regardless of carbon source, except with sucrose at 54\u0026ndash;62 h and with molasses at 70 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results may result from the genetic background of the strains and suggest that both strains, especially MT, hold potential for ethanol production under high temperatures (37\u0026deg;C) conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Trehalose Content Explains Higher Heat Resistance of MT Compared to WT\u003c/h2\u003e \u003cp\u003eHigh temperature induces the accumulation of trehalose in \u003cem\u003eS.cerevisiae\u003c/em\u003e cells \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Trehalose accumulation accumulated in response to heat shock may maintain cell viability \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e and promote growth under high-temperature conditions \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e by preventing heat-induced damage to the cytoplasm \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Cell viability declines dramatically when trehalose levels are reduced below 2mg/g dry cell weight (DCW) \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. High intracellular trehalose content serves as an indicator for ethanol-producing industrial strain screening in Brazil \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, we investigated the trehalose content in MT and WT strains.\u003c/p\u003e \u003cp\u003eAt 30\u0026deg;C and 37\u0026deg;C, MT showed slightly higher trehalose content than WT (67 vs. 34 mg/g DCW; 63 vs. 23 mg/g DCW) without statistical significance. However, at 41\u0026deg;C, both strains exhibited pronounced trehalose accumulation, consistent with the literature report by Hottiger \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, with MT displaying significantly higher content compared with WT (MT: 972 mg/g DCW; WT: 202 mg/g DCW; p\u0026thinsp;=\u0026thinsp;0.014). These results may suggest that the elevated trehalose content in MT contributes to its enhanced heat resistance relative to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eReported intracellular trehalose concentrations in \u003cem\u003eS. cerevisiae\u003c/em\u003e vary widely across studies\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, with limited datasets showing comparable values \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. For instance, Hottiger et al. observed that \u003cem\u003eS. cerevisiae\u003c/em\u003e accumulated 1 g trehalose per g protein under 40\u0026deg;C stress\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, while the industrial strain PE-2 and a baker\u0026rsquo;s yeast strain exhibited 12.5 mg/g and 6.3 mg/g trehalose, respectively\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Notably, PE-2\u0026rsquo;s elevated trehalose levels correlate with its industrial robustness. Although both the present study and Hottiger's work\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e employed trichloroacetic acid extraction followed by anthrone-based detection of trehalose content, direct comparisons between these datasets are not feasible due to differences in strain cultivation conditions and normalization methods. Direct comparisons between MT (this study) and PE-2 are also confounded by divergent cultivation and extraction protocols: PE-2 was grown under harsh fermentation conditions (33\u0026deg;C, molasses medium, 9.1% ethanol) until the mid-fermentation phase \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, whereas MT was cultured in YPD medium for 8\u0026ndash;8.5 h. In this study, the trehalose content of WT cultured at 30\u0026deg;C was 34 mg/g DCW, which is close to the range of 31\u0026ndash;40 mg/g DCW reported by Alexandre \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. However, Alexandre's protocol differed from both this study and existing literature \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Specifically, after 1 h stress treatment at 40\u0026deg;C, cells were incubated in 0.25 M Na₂CO₃ for 2 h at 90\u0026deg;C, and the trehalose content was determined by measuring the glucose liberated by trehalase digestion\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere are also discrepancies in the trends of trehalose content variation under temperature stress across different studies \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Asada \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e reported trehalose yields of 14.88, 13.94, and 4.41 mg/g at 40\u0026deg;C, 45\u0026deg;C, and 50\u0026deg;C, respectively, which contrasts with Hottiger\u0026rsquo;s observation of higher levels at 45\u0026deg;C versus 40\u0026deg;C and complete synthesis cessation at 50\u0026deg;C \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Asada treated the cells by boiling and measured the trehalose content using a detection kit \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. It remains unclear whether different processing and assay methods influence observed trend.\u003c/p\u003e \u003cp\u003eOverall, variations in stress conditions, extraction methods, and quantification assays preclude direct cross-study comparisons. Nonetheless, MT demonstrated significantly higher trehalose content than WT at 41\u0026deg;C, which is consistent with MT\u0026rsquo;s superior heat tolerance over WT, indicating better industrial adaptability compared to WT, despite the latter\u0026rsquo;s current industrial application\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Non-Dominant Roles of SOD and ADH in MT/WT Thermotolerance Divergence\u003c/h2\u003e \u003cp\u003eIt has been reported that superoxide dismutase (SOD) enhance the heat resistance of strains \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e, while ADH activity can serve as a critical indicator of cellular ethanol tolerance \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eADH1\u003c/em\u003e overexpression can improve tolerance to glycolaldehyde \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e and co-expression of \u003cem\u003eADH1\u003c/em\u003e and \u003cem\u003eTAL1\u003c/em\u003e improves ethanol production in the presence of furfural \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Hybrids of thermotolerant \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e and thermosensitive \u003cem\u003eS. cerevisiae\u003c/em\u003e produce more than 6% (v/v) ethanol at 45\u0026deg;C, and these hybrids contain ADH components derived from \u003cem\u003eS. cerevisiae\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. To determine whether other factors apart from trehalose contribute to the improved thermotolerance of MT, we examined the activities of these tolerance-related enzymes.\u003c/p\u003e \u003cp\u003eAs the temperature increased from 30\u0026deg;C to 37\u0026deg;C and then to 41\u0026deg;C, both SOD and ADH activities of both strains increased almost universally. At all temperatures, the SOD activity of MT was higher than that of WT, but the difference was not significant (Fig.\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). As the temperature increased, both strains exhibited an elevation in ADH activity. Nonetheless, the ADH activity of MT did not consistently surpass that of WT at identical temperatures, and no significant disparity was observed between them (Fig. S4). This suggests that SOD and ADH do not play a decisive role in the differential high-temperature tolerance between WT and MT under equivalent temperature stress conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Effect of Trehalose Supplementation on Thermotolerance and Adherent Growth\u003c/h2\u003e \u003cp\u003eSince strains with higher trehalose levels exhibit increased cell viability under heat stress \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e and exogenous trehalose supplementation enhances freeze tolerance of yeast \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e, we added trehalose to the culture medium to evaluate growth differences between WT and MT under different temperatures with/without trehalose. At all temperatures, MT consistently exhibited higher cell counts than WT regardless of trehalose supplementation. At 37\u0026deg;C and 41\u0026deg;C, both strains generally showed increased cell density with trehalose supplementation compared to unsupplemented controls, except for MT at 48 h (37\u0026deg;C), WT at 40 h and 48 h (37\u0026deg;C), MT at 16 h and WT at 48 h (41\u0026deg;C). No significant differences were observed in cell growth between trehalose-supplemented and unsupplemented cultures at 30\u0026deg;C for either strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results indicate that trehalose supplementation improves high-temperature (37\u0026deg;C and 41\u0026deg;C) tolerance in both strains. Without trehalose supplementation, MT cells exhibited higher numbers than WT at all tested temperatures and both strains showed a decline in cell counts with increasing stress intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003e), aligning with prior observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Trehalose supplementation did not mitigate the adherent growth of WT cells under heat stress (Fig.S11 in Miscellaneous File Not for Publication).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Mechanistic Hypothesis for Enhanced Thermotolerance in \u003cem\u003eCDC15\u003c/em\u003e Mutant Strain\u003c/h2\u003e \u003cp\u003eOur study identified a single residue difference between WT and MT (F\u003csup\u003e626\u003c/sup\u003eL, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and homology modeling of Cdc15p indicates that residue 626 is located within an α-helical region on the protein's outer surface, spatially proximate to an area enriched with charged residues (residues 281\u0026ndash;305)\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The fragment encompassing residues 281\u0026ndash;362, which contains this charged cluster, is able to interact individually with Tem1p, a protein known to interact with Cdc15p \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Although both Leu and Phe are hydrophobic amino acids, the benzene ring of Phe displays significant rotational mobility, resulting in pronounced steric hindrance. In contrast, while Leu's side chain retains conformational flexibility, its spatial constraints are substantially reduced. The Phe626\u0026rarr;Leu626 substitution may improve protein folding efficiency, stabilize the tertiary structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince changes in amino acid sequences can alter the thermostability of proteins, thereby affecting the heat resistance of yeast strains \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e.We hypothesize that the mutation of Cdc15p optimizes its interaction with protein partners such as Tem1p, thereby enhancing the heat resistance performance of the MT (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This is supported by the following evidence: First, \u003cem\u003eTEM1\u003c/em\u003e is closely related to \u003cem\u003eCDC15\u003c/em\u003e. For example, the lethality associated with \u003cem\u003eTEM1\u003c/em\u003e deficiency can be rescued by overexpression of \u003cem\u003eCDC15\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e, and other \u003cem\u003eCDC15\u003c/em\u003e mutations (Leu356 and Phe357) weaken the interaction between Tem1p and Cdc15p, leading to temperature-sensitive growth, a defect that was efficiently suppressed by the overexpression of \u003cem\u003eTEM1\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Second, the mutated residue at position 626 is spatially adjacent to the region in Cdc15p that can independently interact with Tem1p (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, these hypotheses require further validation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Differences between the Aggregation of WT under Heat Stress and Classical Pseudohyphal Growth\u003c/h2\u003e \u003cp\u003eIt was observed that partial WT cells exhibited adhesion at 37\u0026deg;C and nearly all WT cells showed adhesion at 41\u0026deg;C, in contrast, MT cells remained dispersed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To the best of our knowledge, this is the first reported adhesion growth trait associated with the cell cycle-related gene \u003cem\u003eCDC15\u003c/em\u003e. Similarly, the deletion of another cell cycle-related gene \u003cem\u003eMBP1\u003c/em\u003e leads to pseudohyphae formation \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, whereas it was previously widely accepted that starvation is a prerequisite for pseudohyphae formation in \u003cem\u003eS. cerevisiae\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. However, in this study, under high temperature condition, WT cells formed clusters through adhesion aggregation, distinct from the chains of elongated cells aggregated together during typical pseudohyphae formation \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. We speculated that the two different growth states associated with \u003cem\u003eCDC15\u003c/em\u003e and \u003cem\u003eMBP1\u003c/em\u003e arise from distinct mechanisms causing variations in \"pseudohyphae\" morphology and extent, which aligns with previous findings \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe cell cycle-related genes \u003cem\u003eCDC15\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e and \u003cem\u003eMBP1\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e have been implicated in tolerance to heat stress (this study) and ethanol \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, respectively, and \u003cem\u003eMBP1\u003c/em\u003e is a known transcriptional regulator of numerous genes \u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Furthermore, the deletion of \u003cem\u003eMBP1\u003c/em\u003e or mutation of \u003cem\u003eCDC15\u003c/em\u003e results in cell elongation and aggregation \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e and cell non-adhesion, different from WT, (this study), respectively, but their cooperative regulation of growth remains unknown.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Effects of Trehalose on Thermotolerance and Ethanol associated with \u003cem\u003eCDC15\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eIntracellular accumulation of trehalose is believed to increase a yeast\u0026rsquo;s tolerance to freezing, dehydration \u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. Protection against freezing in yeast cells by trehalose requires its presence on both sides of the plasma membrane \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Under high temperature conditions, the intracellular trehalose content of MT was higher than that of WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and MT exhibited superior growth capacity compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although the addition of trehalose increased cell counts of both strains, MT's count still exceeded WT's under stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The potential reasons for these differences may be found in the previous studies, which indicates that the degradation of intracellular trehalose facilitates cellular recovery from heat stress, and that intracellular trehalose is associated with cell cycle regulation, while extracellular trehalose can act as a carbon source\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe higher intracellular trehalose content of MT may result from the mutation of the cell cycle-related gene \u003cem\u003eCDC15\u003c/em\u003e affecting trehalose synthesis and/or degradation, since trehalose and its degradation are associated with cell cycle regulation\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. However, further verification is needed.\u003c/p\u003e \u003cp\u003eTrehalose can be hydrolyzed by trehalase into glucose, potentially providing more substrate for glycolysis \u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. This means cells can mobilize more glucose to participate in glycolysis reactions, thereby generating more pyruvate, which is converted into ethanol under anaerobic conditions. This may result in the higher ethanol yield in MT, as MT shows higher trehalose content than WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Additionally, during the late stages of fermentation, cells utilize ethanol and/or glucose derived from trehalose hydrolysis to sustain growth due to the lack of available carbon sources. We speculate that WT likely consumes more ethanol because its trehalose content is lower than MT, which could also contribute to WT's lower ethanol yield compared to MT.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cem\u003eCDC15\u003c/em\u003e mutation (Phe626 \u0026rarr;Leu626) enhanced thermotolerance and suppressed heat-induced cell aggregation under high-temperature stress. This is likely mediated by trehalose-mediated stress protection and Cdc15p conformational flexibility. Furthermore, \u003cem\u003eCDC15\u003c/em\u003e mutation improved ethanol production efficiency. Although the molecular mechanisms require further investigation, this unexpectedly discovery of enhanced thermotolerance and fermentation traits highlights \u003cem\u003eCDC15\u003c/em\u003e's multifaceted functionality in stress adaptation and metabolic regulation. This finding positions \u003cem\u003eCDC15\u003c/em\u003e as a promising genomic editing target for developing robust industrial yeast strains, and fermentation efficiency are paramount.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eADH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlcohol dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esuperoxide dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMF01,wild-type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMF01-\u003cem\u003eCDC15\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eXOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eXanthine oxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAD+\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoxidized coenzyme I\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNADH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereduced coenzyme I\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3,5-dinitrosalicylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSNP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esingle nucleotide polymorphism\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edry cell weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMT30\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMT grows at 30\u0026deg;C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMT37\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMT grows at 37\u0026deg;C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMT41\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMT grows at 41\u0026deg;C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT30\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWT grows at 30\u0026deg;C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT37\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWT grows at 37\u0026deg;C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT41\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWT grows at 41\u0026deg;C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.C.: Funding acquisition, Investigation, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. D.C.: Methodology, Resources. Q.L.: Data curation, Investigation. Y. C.: Investigation, Funding acquisition. Z.L.: Validation. S.Z.: Methodology. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in this study are included in the article/Supplementary Material/Miscellaneous File Not for Publication. Further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by the Natural Science Foundation of China (32360018), the Natural Science Foundation of Guangxi, China (2023GXNSFAA026462), Science and Technology Major Project of Guangxi (GuikeAA24206052) and the Natural Science Foundation of Guangxi, China (2024GXNSFAA010494).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWalls LEMJ, Rios-Solis L. Enhancing \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e taxane biosynthesis and overcoming nutritional stress-induced pseudohyphal growth. Microorganisms. 2022;10(1):163.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwak S, Jin YS. Production of fuels and chemicals from xylose by engineered \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e: A review and perspective. Microb Cell Fact. 2017;16(1):82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark YR, Yang JW, Sunwoo IY, et al. Enhancement of catabolite regulatory genes in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e to increase ethanol production using hydrolysate from red seaweed gloiopeltis furcata. J Biotechnol. 2021;333:1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Lu Z, Chen Y, et al. Deletion of the \u003cem\u003eMBP1\u003c/em\u003e gene, involved in the cell cycle, affects respiration and pseudohyphal differentiation in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Microbiol Spectr. 2021;9(1):e0008821.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasso LC, de Amorim HV, de Oliveira AJ, et al. Yeast selection for fuel ethanol production in brazil. FEMS Yeast Res. 2008;8(7):1155\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhazali MFSM, Mustafa M. Bioethanol as an alternative fuels: A review on production strategies and technique for analysis. Energy Convers Management: X. 2025;26:100933.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdel-Banat BM, Hoshida H, Ano A, et al. High-temperature fermentation: How can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol. 2010;85(4):861\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeishan F. Shuaishuai, Integrating sugarcane molasses into sequential cellulosic biofuel production based on ssf process of high solid loading. Biotechnol Biofuels, 2017.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFern\u0026aacute;ndez-L\u0026oacute;pez CLT-SB, Salgado-Cervantes MA, Garc\u0026iacute;a PG, Aguilar-Uscanga MG. Use of sugarcane molasses b as an alternative for ethanol production with wild-type yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e itv-01 at high sugar concentrations. Bioprocess Biosyst Eng. 2012;35(4):605\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichard C, Brazil. A world leader. Sugar Journal: Covering World's Sugar Ind, 2006(7):69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGemma Bell\u0026iacute; EG, Mart\u0026iacute; A, Herrero. E Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/cdc28 activity in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Mol Microbiol. 2001;39(4):1022\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThatipamala R, Rohani S, Hill GA. Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation. Biotechnol Bioeng,40(2):289\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahsavarani H, Sugiyama M, Kaneko Y, et al. Superior thermotolerance of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e for efficient bioethanol fermentation can be achieved by overexpression of rsp5 ubiquitin ligase. Biotechnol Adv. 2012;30(6):1289\u0026ndash;300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HS, Kim NR, Yang J, et al. Identification of novel genes responsible for ethanol and/or thermotolerance by transposon mutagenesis in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Appl Microbiol Biotechnol. 2011;91(4):1159\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuthee Benjaphokee PK. Choowong Auesukaree,Thipa Asvarak, Minetaka Sugiyama1 Yoshinobu Kaneko, Harashima CBaS. Cdc19 encoding pyruvate kinase is important for high-temperature tolerance. Saccharomyces cerevisiae New Biotechnol. 2012;29(2):166\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnapp BD, Huang KC. The effects of temperature on cellular physiology. Annual Rev Biophys. 2022;51:499\u0026ndash;526.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCauston HC, Ren B, Koh SS, et al. Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell. 2001;12(2):323\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanshin E, Kubiniok P, Thattikota Y, et al. Phosphoproteome dynamics of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e under heat shock and cold stress. Mol Syst Biol. 2015;11(6):813.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColeman MC, Fish R, Block DE. Temperature-dependent kinetic model for nitrogen-limited wine fermentations. Appl Environ Microbiol. 2007;73(18):5875\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUlane R, Ogur M. Genetic and physiological control of serine and glycine biosynthesis in Saccharomyces. J Bacteriol. 1972;109(1):34\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimoda C, Itadani A, Sugino A, et al. Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Genes Genet Syst. 2006;81(6):391\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSridhar M, Sree NK, Rao LV. Effect of uv radiation on thermotolerance, ethanol tolerance and osmotolerance of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e vs1 and vs3 strains. Bioresour Technol. 2002;83(3):199\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePattanakittivorakul S, Lertwattanasakul N, Yamada M, et al. Selection of thermotolerant \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e for high temperature ethanol production from molasses and increasing ethanol production by strain improvement. Antonie Van Leeuwenhoek. 2019;112(7):975\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun H, Jia H, Li J et al. Rational synthetic combination genetic devices boosting high temperature ethanol fermentation. Synth Syst Biotechnol, 2017,2(2):121\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFukuda N, Kaishima M, Ishii J, et al. Continuous crossbreeding of sake yeasts using growth selection systems for a-type and α-type cells. AMB Express. 2016;6(1):45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeneroso WC, Gottardi M, Oreb M, et al. Simplified CRISPR-Cas genome editing for \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. J Microbiol Methods. 2016;127:203\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJagtap RS, Mahajan DM, Mistry SR, et al. 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;103(2):1031\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolinet J, Cubillos FA. Wild yeast for the future: Exploring the use of wild strains for wine and beer fermentation. Front Genet. 2020;11(589350):2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi-li Li Mei-li, Ye Yan-rui, Ye. Ying Lin. Correlation Between Stress Tolerance and Fermentation Behavior of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. J South China Univ Technol (Natural Sci Edition). 2011;39(9):134\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu DC, Zhang Sui-sheng, Chen Y, Huang Ri-bo. High gravity alcoholic fermentation from sugarcane molasses by a high-yield \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strains MF1001 in industrial scale, Guangxi Sciences,2011,18(4):385\u0026ndash;391.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShirayama M, Matsui Y, Toh-e A. Dominant mutant alleles of yeast protein kinase gene cdc15 suppress the lte1 defect in termination of m phase and genetically interact with cdc14. Mol Gen genetics: MGG. 1996;251(2):176\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarela C, Pizarro F, Agosin E. Biomass content governs fermentation rate in nitrogen-deficient wine musts. Appl Environ Microbiol. 2004;70(6):3392\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang J, Wang Z et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Nat Commun, 1053,10(1):019\u0026ndash;09005.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiCarlo JE, Norville JE, Mali P, et al. Genome engineering in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e using CRISPR-Cas systems. Nucleic Acids Res. 2020;41(7):4336\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyanagui Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Anal Biochem. 1984;142(2):290\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRock JM, Angelika A. Cdc15 integrates tem1 gtpase-mediated spatial signals with polo kinase-mediated temporal cues to activate mitotic exit. Genes Dev,25(18):1943\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsakawa K, Yoshida S, Otake F, et al. A novel functional domain of cdc15 kinase is required for its interaction with tem1 gtpase in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Genetics. 2001;157(4):1437\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchweitzer B, Philippsen P. Cdc15, an essential cell cycle gene in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, encodes a protein kinase domain. Yeast. 1991;7(3):265\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;hlhofer M, Berchtold E, Stratil CG et al. The heat shock response in yeast maintains protein homeostasis by chaperoning and replenishing proteins. Cell reports, 2019,29(13):4593\u0026ndash;4607 e4598.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHottiger T, Schmutz P, Wiemken A. Heat-induced accumulation and futile cycling of trehalose in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. J Bacteriol. 1987;169(12):5518\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsada R, Watanabe T, Tanaka Y, et al. Trehalose accumulation and radiation resistance due to prior heat stress in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Arch Microbiol. 2022;204(5):275.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexandre H, Plourde L, Charpentier C, et al. Lack of correlation between trehalose accumulation, cell viability and intracellular acidification as induced by various stresses in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Microbiology. 1998;144(Pt 4):1103\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavidson JF, Whyte B, Bissinger PH, et al. Oxidative stress is involved in heat-induced cell death in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Proc Natl Acad Sci U S A. 1996;93(10):5116\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown SD, GAM, Karpinets TV, Parks JM, Smolin N, Yang S, Land ML, Klingeman DM, Bhandiwad A, Rodriguez M, Jr, Raman B, Shao X, Mielenz JR, Smith JC, Keller M. Lynd L.R. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in clostridium thermocellum Proceedings of the National Academy of Sciences, 2011,108(33):13752\u0026ndash;13757.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJayakody LN, Horie K, Hayashi N, et al. Improvement of tolerance of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e to hot-compressed water-treated cellulose by expression of adh1. Appl Microbiol Biotechnol. 2012;94(1):273\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHasunuma T, Ismail KSK, Nambu Y, et al. Co-expression of tal1 and adh1 in recombinant xylose-fermenting \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e improves ethanol production from lignocellulosic hydrolysates in the presence of furfural. J Biosci Bioeng. 2014;117(2):165\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhamija SS, Gera R, Singh DK, et al. Physiological and biochemical characterization of intergeneric hybrids of thermotolerant and non-thermotolerant yeasts. J Basic Microbiol. 1997;37(5):307\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiniz-Mendes L, Bernardes E, de Araujo PS, et al. Preservation of frozen yeast cells by trehalose. Biotechnol Bioeng. 1999;65(5):572\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalunjkar N, Lai TY, Akhter N et al. Pervasive divergence in protein thermostability is mediated by both structural changes and cellular environments. Mol Biol Evol, 2025,42(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShirayama M, Matsui Y, Toh EA. The yeast tem1 gene, which encodes a gtp-binding protein, is involved in termination of m phase. Mol Cell Biol. 1994;14(11):7476\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiled C, Mann C, Faye G. Xbp1-mediated repression of clb gene expression contributes to the modifications of yeast cell morphology and cell cycle seen during nitrogen-limited growth. Mol Cell Biol. 2001;21(11):3714\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGimeno CJ, Ljungdahl PO, Styles CA, et al. Unipolar cell divisions in the yeast s. Cerevisiae lead to filamentous growth: Regulation by starvation and RAS. Cell. 1992;68(6):1077\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim J, Rose MD. Stable pseudohyphal growth in budding yeast induced by synergism between septin defects and altered map-kinase signaling. PLoS Genet,11(12):e1005684.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMutlu N, Sheidy DT, Hsu A, et al. A stress-responsive signaling network regulating pseudohyphal growth and ribonucleoprotein granule abundance in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Genetics. 2019;213(2):705\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHendler A, Medina EM, Kishkevich A, et al. Gene duplication and co-evolution of G1/s transcription factor specificity in fungi are essential for optimizing cell fitness. PLoS Genet. 2017;13(5):e1006778.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShima J, Hino A, Yamada-Iyo C, et al. Stress tolerance in doughs of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e trehalase mutants derived from commercial baker's yeast. Appl Environ Microbiol. 1999;65(7):2841\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen A, Stadulis SE, deLeuze K et al. Evaluating cellular roles and phenotypes associated with trehalose degradation genes in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. G3 (Bethesda), 2024,14(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanks SK, Quinn AM, Hunter T. The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science. 1988;241(4861):42\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJim\u0026eacute;nez J, Cid VJ, Cenamor R, et al. Morphogenesis beyond cytokinetic arrest in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. J Cell Biol. 1998;143(6):1617\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CDC15 gene, S. cerevisiae, ethanol, tolerance, trehalose","lastPublishedDoi":"10.21203/rs.3.rs-8993691/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8993691/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCDC15\u003c/em\u003e is an important gene involved in mitotic exit and cytokinesis in \u003cem\u003eS.cerevisiae\u003c/em\u003e. However, its additional functions remain unclear. Comparative genomics analysis between the fast-growing strain MC15 and the high-ethanol-producing strain MF01 revealed \u003cem\u003eCDC15\u003c/em\u003e as a significant sequence-divergent gene. To validate the hypothesis that replacing this gene could enhance MF01's growth rate, this study engineered the recombinant strain MF01-\u003cem\u003eCDC15\u003c/em\u003e (MT) by substituting the \u003cem\u003eCDC15\u003c/em\u003e in MF01 (WT) with that from MC15. Rapid growth is one of the desirable traits required for production strains. Achieving sufficient yeast cell density not only reduces contamination by contaminating microbes but also shortens the ethanol production cycle and improves production efficiency.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eCDC15\u003c/em\u003e mutation significantly improved cell viability on solid medium at 50\u0026ndash;54\u0026deg;C. In liquid culture, the MT entered the stationary phase 8 h earlier than WT. Furthermore, the maximum cell densities achieved by MT at 30\u0026deg;C, 37\u0026deg;C and 41\u0026deg;C increased by 37%, 57%, 155%, respectively, compared to WT. Unexpectedly, \u003cem\u003eCDC15\u003c/em\u003e mutation caused ethanol yields to increase by 26.41% using sucrose and 29.56% using molasses as carbon sources at 30\u0026deg;C. Notably, MT cells maintained normal morphology at 41\u0026deg;C, whereas WT displayed cell adhesion. The intracellular trehalose content in MT was 4.8 times higher than that in WT at 41\u0026deg;C (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014). Homology modeling indicated that the Phe626\u0026rarr;Leu626 mutation in Cdc15p of MT may contribute to structural stability. The combination of this structural change and the trehalose accumulation likely contributes to MT\u0026rsquo;s superior heat resistance.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study provides more detailed information about the function of \u003cem\u003eCDC15\u003c/em\u003e and suggest some clues to efficiently improve the performance of industrial yeast strains.\u003c/p\u003e","manuscriptTitle":"Enhanced Heat Tolerance and Ethanol Production of Industrial Saccharomyces cerevisiae via Replacement of the CDC15 Gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 14:39:01","doi":"10.21203/rs.3.rs-8993691/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-19T12:36:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-16T15:21:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T03:26:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-27T14:22:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76651967398812163585895167951396361769","date":"2026-03-19T11:53:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100573278175247797809053446605236134579","date":"2026-03-19T06:52:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3967777338617724425817209734514115835","date":"2026-03-18T15:12:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-17T13:10:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-05T05:54:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-05T05:51:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2026-02-28T09:05:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f942f999-2c33-4e59-91ad-c7709c5a1c44","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-10T21:23:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 14:39:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8993691","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8993691","identity":"rs-8993691","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}