Gene dose effect target for improving tolerance of yeast and application of gene dose effect target

Abstract

The invention relates to a gene dose effect target for improving the tolerance of yeast and application, and particularly provides a group of gene combinations YCL051W-YCL047C capable of enhancing the heat resistance of the yeast, and the gene combinations include YCL051W, YCL050C, YCL049C, YCL048W and YCL047C. A yeast engineering bacterium with heat resistance can be constructed by overexpressing a gene combination YCL051W-YCL047C in a yeast strain, the target combination is suitable for various different types of industrial strains, and the modified strain shows enhanced heat resistance and can accelerate microbial metabolism, improve the production efficiency and remarkably reduce the industrial fermentation cost; meanwhile, the production process efficiency and stability are improved, and the contamination risk is reduced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to gene dose-effect targets and applications for enhancing yeast tolerance. Background Technology

[0002] The challenges faced by yeast strains in terms of tolerance are a major bottleneck for achieving efficient and economical production in practical applications. Currently, industrial yeast strains face numerous pressures, including substrate and product inhibition and environmental stress. The tolerance of the strain directly determines the economic benefits and process stability of production; therefore, improving the tolerance of the strain is an effective way to reduce production costs and increase fermentation efficiency. Yeast, as a traditional model microorganism and industrial "cell factory," plays a core role in food, fuel, and chemical production. However, during fermentation, cells are subjected to various environmental stresses, such as changes in high temperature, pH, and osmotic pressure. These long-term, continuous stress combinations often inhibit cell growth and lead to cell death. For example, the temperature sensitivity of yeast has always been a major limiting factor in industrial fermentation. There is a significant difference between the optimal temperature for enzymatic hydrolysis (45-50℃) and the optimal temperature for fermentation (30-35℃), resulting in high cooling costs and low fermentation efficiency. Caspeta et al. reported that some protein-coding genes in Saccharomyces cerevisiae are often overexpressed when the temperature rises. However, when the temperature exceeds 35℃, yeast faces high-temperature stress.

[0003] Currently, methods to enhance yeast tolerance include adaptive evolution, interspecies hybridization with naturally thermotolerant yeasts, or precise editing of specific genes through metabolic and genetic engineering techniques. Examples include using genetic engineering to overexpress the ubiquitin ligase gene RSP5 and knocking out the glycosylphosphatidylinositol-anchored membrane protein gene DFG5 to enhance strain-related tolerance phenotypes. Despite some progress, research on tolerance is still insufficient. Solving the problem of strain tolerance is key to achieving economic viability in biomanufacturing and is currently a core research focus in the field of microbial manufacturing. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides gene dose-effect targets and applications for enhancing yeast tolerance.

[0005] The technical solution adopted in this invention is: to enhance the gene dose-effect target of yeast tolerance and to enhance the gene combination of yeast heat resistance, including... YCL051W, YCL050C, YCL049C, YCL048W and YCL047C .

[0006] Preferably, YCL051W Gene ID: 850306 YCL050C Gene ID: 850307 YCL049C GeneID: 850308 YCL048WGene ID: 850309 YCL047C Gene ID: 850310.

[0007] Preferably, yeast trehalose biosynthesis is enhanced through a gene dose-dependent emergent effect, thereby enhancing yeast heat resistance.

[0008] A thermostable engineered yeast strain, simultaneously overexpressing YCL051W, YCL050C, YCL049C, YCL048W and YCL047C Yeast.

[0009] Preferably, the yeast is Saccharomyces cerevisiae BY4741, 273614N, YJM981, UWOPS05-217.3, SK1 or Y55.

[0010] Application of gene dose-effect targets for enhancing yeast tolerance in yeast strain modification.

[0011] Preferably, the modified yeast strain can grow in an environment of 40-42℃.

[0012] Preferably, the modified yeast strain is used in the production of food, fuel, or chemicals.

[0013] The advantages and positive effects of this invention are: it provides a set of target combinations that can improve the heat resistance of yeast, including... YCL051W, YCL050C, YCL049C, YCL048W and YCL047C This combination of targets can accelerate microbial metabolism, improve production efficiency, and significantly reduce industrial fermentation costs; at the same time, it can enhance the efficiency and stability of the production process and reduce the risk of contamination.

[0014] Furthermore, this invention deepens the understanding of heat resistance mechanisms and provides new insights into other types of strains and the genetic mechanisms of heat resistance. Attached Figure Description

[0015] Figure 1 Dilution plate experiments of simultaneous overexpression and individual overexpression of the five genes;

[0016] Figure 2 The procedure for fully functionally validating small chromosome regions in strain BY4741; where arrows represent genes, diamonds represent LoxP sites, gray yeast models represent euploid phenotypes, and pink yeast models represent aneuploid phenotypes.

[0017] Figure 3 Dilution plate experiments were conducted using strains that overexpressed any four gene combinations through rearrangement.

[0018] Figure 4 Dilution plate experiment of strains overexpressing any three gene combinations constructed by rearrangement;

[0019] Figure 5 Dilution plate experiment of strains overexpressing any two gene combinations constructed by rearrangement;

[0020] Figure 6 In other yeast strains YCL051W–YCL047C Functional verification of gene loci;

[0021] Figure 7 RNA-seq analysis of strains overexpressing all five genes and strains overexpressing any combination of four genes;

[0022] Figure 8 qPCR quantification of TPS1 and TPS2 in the strain;

[0023] Figure 9 LC-MS / MS quantification of trehalose per unit biomass in the strain. Detailed Implementation

[0024] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0025] This invention relates to gene dose-effect targets and applications for enhancing yeast heat tolerance. Specifically, it provides a set of gene combinations capable of enhancing yeast heat tolerance. YCL051W - YCL047C ,include YCL051W, YCL050C, YCL049C, YCL048W and YCL047C .in, YCL051W Gene ID: 850306 YCL050C Gene ID: 850307 YCL049C GeneID: 850308 YCL048W Gene ID: 850309 YCL047C Gene ID: 850310.

[0026] Focusing on fundamental research into yeast tolerance, this study systematically validated the necessity and sufficiency of five genes in enhancing yeast thermotolerance through individual and combined overexpression of five genes, as well as random overexpression of four / three / two genes. Furthermore, by precisely locating thermotolerance genes based on both necessity and sufficiency, and analyzing the dose-response effects between genes, it was discovered that gene combinations containing these five targets enhance yeast trehalose biosynthesis through a dose-dependent emergent effect, thereby enhancing yeast thermotolerance. This more precisely elucidates the genetic mechanism of yeast thermotolerance and identifies tolerance targets for strain modification.

[0027] Simultaneous overexpression in Saccharomyces cerevisiae YCL051W - YCL047C The constructed engineered yeast strain can maintain high growth activity under heat stress (40-42℃), giving the yeast strain heat resistance. YCL051W - YCL047CSimultaneous overexpression of *Saccharomyces cerevisiae* BY4741, 273614N, YJM981, UWOPS05-217.3, SK1, or Y55 resulted in better heat resistance, demonstrating... YCL051W - YCL047C The heat resistance effect exhibits high functional conservation, remaining functionally conserved across different strains, and thus possesses potential industrial application value.

[0028] This target combination can accelerate microbial metabolism, improve production efficiency, and significantly reduce industrial fermentation costs; it also enhances production process efficiency and stability while reducing the risk of contamination. Based on these characteristics, gene combinations that enhance yeast heat resistance can be developed. YCL051W - YCL047C It is used in yeast modification, and is especially suitable for the modification of industrial yeast strains. The modified yeast strains are used in the production of food, fuel or chemicals. For example, in ethanol production, heat-resistant yeast is a key factor in improving conversion efficiency.

[0029] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described are performed according to the corresponding product instructions. Unless otherwise specified, all instruments, reagents, and consumables used in the examples can be purchased from commercial companies. The genes screened in the following examples can all have their specific sequence information obtained by searching for the gene name on NCBI.

[0030] Example 1: Validation of genes that enhance yeast tolerance

[0031] YCL051W - YCL047C The region contains 5 genes. YCL051W, YCL050C, YCL049C, YCL048W, YCL047C Among them, existing literature has been published. YCL051W It is related to the regulation of cell wall structure and stress response, and YCL051W- YCL047C The other four genes within the locus have not yet been characterized.

[0032] Will YCL051W, YCL050C, YCL049C, YCL048W, YCL047C Five genes were overexpressed individually, in combination, and randomly to verify their effects on yeast tolerance.

[0033] (1) Construction of co-overexpression strains

[0034] Will YCL051W, YCL050C, YCL049C, YCL048W and YCL047C The recombinant plasmid was constructed by ligating it into the vector PRS413 using Gibson assembly. The recombinant plasmid was then transformed into BY4741 and cultured in SC selective medium at 30°C. Single clones were picked for sequencing verification. The correctly sequenced strain was identified as BY4741+YCL051W-YCL047C, and dilution plate experiments were performed.

[0035] SC medium: 20 g / L glucose, 6.7 g / L deaminized yeast nitrogen source (YNB), 2 g / L drop-out (deficient in L-leucine (Leu), L-histidine (His), L-tryptophan (Trp), and uracil (Ura); adjust the pH to 6.0 based on the amount of amino acid stock solution added). For solid medium, add an additional 20 g / L agar powder and adjust the pH to 6.5. Incubate at 40°C.

[0036] Yeast cells were cultured in selective medium at 30°C with shaking at 220 rpm overnight. Then, 200 μL of the culture was transferred to 5 mL of selective medium and cultured at 30°C until OD600 = 1. The solution was serially diluted 5 times in distilled water in 10-fold increments, and 3-4 μL of the diluted solution was dropped onto plates sequentially from the lowest to the highest concentration. The culture was incubated at 40°C for 2–6 days. Images were acquired using a PhenoBooth + Colony Counter instrument (SingerInstruments, UK).

[0037] The dilution spot test results of strain BY4741+YCL051W-YCL047C are as follows: Figure 1 As shown, the engineered strain that simultaneously overexpressed five genes exhibited significantly improved heat resistance.

[0038] (2) Construction of single-gene overexpression strains

[0039] To further determine the relationship between these five genes, each of the five candidate genes in this region was overexpressed individually. YCL051W, YCL050C, YCL049C, YCL048W, YCL047C ).Will YCL051W, YCL050C, YCL049C, YCL048W or YCL047C Recombinant plasmids were constructed by ligating them into the vector PRS413 using Gibson assembly, following the method in step (1). These plasmids were then transformed into BY4741 to construct strains BY4741+YCL051W, BY4741+YCL050C, BY4741+YCL049C, BY4741+YCL048W, and BY4741+YCL047C, respectively. The results are as follows: Figure 1 As shown, none of the strains that overexpressed the five candidate genes individually acquired heat resistance in haploid strains; heat resistance is considered to be a cooperative effect among multiple genes.

[0040] (3) Construction of random overexpression strains

[0041] according to Figure 2The procedure involved designing a local rearrangement system and introducing LoxP sites flanking five genes on a plasmid to achieve rearrangement and generate different gene combinations. The recombinant plasmid containing the rearrangement system was transformed into BY4741, and strains were constructed as shown in Table 1 through random rearrangement.

[0042] Table 1

[0043]

[0044] For yYHY030 (overexpressing) containing rearrangeable plasmids YCL051W, YCL050C, YCL049C, YCL048W and YCL047C The strains were rearranged, and strains yYHY031-yYHY045 with different genotype combinations were selected for dilution and plate testing at 40℃. The results are as follows... Figure 3 , 4 As shown in Figure 5, it was found that genotype combinations overexpressing four, three, or two genes did not confer heat resistance to the strain. Only when all five genes were overexpressed simultaneously did the strain exhibit heat resistance; this indicates that these five genes collectively conferred heat resistance to strain BY4741 through a dose-dependent emergent effect.

[0045] Example 2: Verification of the conservation of yeast tolerance gene function

[0046] To assess the functional conservation of these genes, the carriers were respectively... YCL051W-YCL047C The plasmid at the locus was introduced into five industrial or laboratory Saccharomyces cerevisiae (273614N, YJM981, UWOPS05-217.3, SK1, and Y55), and dilution plate experiments were performed. The results are as follows: Figure 6 As shown, all five strains exhibited enhanced heat resistance after plasmid transfer, indicating that the dose-response effect of this gene is conserved across different strains and its potential industrial applications.

[0047] Example 3: Exploring the gene mechanism for enhancing yeast tolerance

[0048] RNA-seq analysis was performed on strain yYHY030, which overexpresses all five genes, and strains yYHY031, yYHY032, yYHY033, yYHY034, and yYHY035, which overexpress any combination of four genes. The Z-scores of the YCL051W–YCL047C gene loci and the trehalose biosynthesis gene (…) were analyzed. TPS1, TPS2, NTH1, ATH1 The expression of [symptom] was analyzed, with three independent biological replicates for each strain. Results are as follows: Figure 7 As shown, when the copy number of all five genes is increased, the RNA expression level is significantly higher than that of the combination of increasing the copy number of only four genes.

[0049] Further GO enrichment analysis revealed that this region was significantly associated with genes related to cell wall components, particularly those involved in trehalose biosynthesis. It was found that when five genes were simultaneously overexpressed, genes involved in trehalose biosynthesis, such as... TPS1, TPS2, NTH1 and ATH1 It showed significant upregulation at the RNA level, such as Figure 8 As shown in the figure. The trehalose content per unit biomass in each strain was quantified by LC-MS / MS, and the results are shown below. Figure 9 As shown in the figure. Further analysis using qPCR and LC-MS / MS confirmed that the trehalose content in the strain simultaneously overexpressing five genes (yYHY030) was significantly higher than that in the strain overexpressing four gene combinations. This further demonstrates... YCL051W–YCL047C Yeast heat resistance is enhanced by a gene dose-dependent emergent effect that enhances trehalose biosynthesis.

[0050] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A gene dosage effect target for enhancing yeast tolerance, characterized in that: A combination of genes to enhance heat tolerance of yeast, comprising YCL051W, YCL050C, YCL049C, YCL048W and YCL047C .

2. The gene dose effect target for improving yeast tolerance according to claim 1, characterized in that: YCL051W Gene ID: 850306, YCL050C Gene ID: 850307, YCL049C Gene ID: 850308, YCL048W Gene ID: 850309, YCL047C Gene ID: 850310.

3. The gene dose effect target for improving yeast tolerance according to claim 1 or 2, characterized in that: Yeast heat resistance is enhanced by a gene dose-dependent emergent effect that enhances trehalose biosynthesis.

4. A thermoresistant engineered yeast strain, characterized in that: Simultaneous overexpression YCL051W, YCL050C, YCL049C, YCL048W and YCL047C of the yeast fungus.

5. The heat-resistant yeast engineering bacteria according to claim 4, characterized in that: The yeast strains are Saccharomyces cerevisiae BY4741, 273614N, YJM981, UWOPS05-217.3, SK1, or Y55.

6. The application of the gene dose-effect target for enhancing yeast tolerance as described in any one of claims 1-3 in yeast strain modification.

7. Use according to claim 6, characterized in that: The modified yeast strain can grow in an environment of 40-42℃.

8. Use according to claim 6, characterized in that: Modified yeast strains are used in the production of food, fuel, or chemicals.

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