Gene and method for improving stress resistance of yeast

Abstract

The invention discloses a gene and a method for improving stress resistance of yeast, and belongs to the field of bioengineering and technology. The invention relates to a gene and a method for improving the stress resistance of yeast, which are characterized in that the gene capable of improving the stress resistance of the yeast is a dolichol phosphate mannose glycoprotein mannosyl transferase gene, and the gene is expressed in the yeast, so that the global protein mannosylation modification of cells can be improved, the thickness of cell membranes is increased, and the stress resistance of the yeast is improved. Therefore, the ability of yeast to resist high temperature, hyperosmosis and other stress environments is improved, and the growth performance of yeast cells is improved.

Description

Technical Field

[0001] The present invention relates to a gene and method for improving the stress resistance of yeast, and belongs to the field of bioengineering and technology. Background Art

[0002] Driven by the green and sustainable economy, biomanufacturing has become an important technology for promoting industrialization in the 21st century. Microbial cell factories have been used for the biosynthesis of a series of chemicals such as bulk chemicals, pharmaceutical products, plant natural products, and biofuels. However, during the fermentation process of cell factories, various "stress factors" are often encountered, such as high temperature, acid-base, high osmotic pressure, high salt, etc. These stress factors will inhibit the growth metabolism and production performance of strains. The traditional method to eliminate stress is to control the fermentation process, but the process optimization consumes too much energy and is not conducive to the low-carbon and low-cost green development model. In order to achieve the production characteristics of energy conservation, emission reduction, efficiency improvement, and quality improvement in the actual biotransformation process, in addition to having a strong metabolic level, cell factories should also have stress-resistant physiological characteristics; however, due to the different stress resistance abilities of industrial strains, the production costs vary greatly. Strains with strong stress resistance are undoubtedly more cost-effective. Therefore, improving the stress resistance of strains is the key to improving production efficiency and reducing economic costs.

[0003] The methods for improving the stress resistance of strains include non-rational methods and rational methods. Non-rational methods include random mutation of genes, physical and chemical mutagenesis, and laboratory adaptive evolution of strains. With the continuous development of synthetic biology technology, stress-resistant strains are also obtained through rational methods such as expressing stress-resistant functional genes and dynamic regulation. Currently, the reported stress-resistant functional genes mainly include genes related to the regulation of cell wall and cell membrane, DNA repair, oxidative stress, energy production, and signal transduction, as well as efflux pumps, heat shock proteins, and global transcription factors.

[0004] The discovery and application of new stress-resistant genes are effective means to improve the stress resistance of strains and production performance, and are one of the hotspots continuously concerned by those skilled in the art. Summary of the Invention

[0005] The purpose of the present invention is to provide a gene and method for improving the stress resistance of yeast.

[0006] Through the analysis of the genome and transcriptome sequencing data of Yarrowia lipolytica, the present invention discovers that the expression of a class of enzymes - dolichol phosphate mannose protein mannosyltransferase can improve the growth performance and stress resistance of Yarrowia lipolytica. The function of this class of enzymes is to perform mannosylation modification on proteins, which can improve the stability of free enzymes in cells on the one hand and increase the thickness of the cell wall composed of glycoproteins on the other hand.

[0007] The dolichol phosphate mannose protein mannosyltransferase gene is assembled with a promoter and a terminator to obtain an expression cassette. An expression cassette refers to a DNA sequence that can effectively express a target gene in a target strain. The expression cassette includes a promoter (Promoter) that initiates the transcription of the target gene, the target gene (GOI), a terminator (Terminator) that terminates the transcription of the target gene, and a selection marker gene. The selection marker gene is used to test whether the target gene has been successfully constructed into the chromosome of the strain. Subsequently, the successfully assembled expression cassette is transformed and expressed in the target strain. By screening positive transformants, a target strain capable of expressing the target gene is obtained. The target strain is Yarrowia lipolytica or Saccharomyces cerevisiae. The promoters and terminators used are constitutive promoters commonly used. The materials and methods used are all conventional technical means in the art, and the present invention will not elaborate on them here. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 It is a diagram showing the intracellular global glycosylated protein abundances of the strain YL8 overexpressing the YALI0_C23364g gene and the parental strain Yarrowia lipolytica CLIB89.

[0009] Figure 2 It is a diagram showing the differences in cell section structures between the strain YL8 overexpressing the YALI0_C23364g gene and the parental strain Yarrowia lipolytica CLIB89 at 35°C. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0010] The following are specific examples to further illustrate the present invention. The purpose of the specific examples is only for illustration and not for limiting the scope of the present invention. Without departing from the scope of the claims, those skilled in the art can modify various aspects of the present invention, but these modifications also fall within the protection scope of the present invention. For example, replacing the cell expression system and vector plasmid used in this example with other commonly used expression systems and vectors in the art can be understood and achieved by other technicians in the art.

[0011] In addition, it should be noted that unless otherwise specified, all materials and reagents used in the following specific examples are commonly used materials and reagents in the art and can be obtained through regular commercial channels; the methods used are all conventional methods well-known to those skilled in the art.

[0012] Example 1: Acquisition and construction of stress-resistant gene expression cassette

[0013] According to the sequence described in the claims, design PCR primers to amplify genes YALI0_D10549g, YALI0_C23364g, YALI0_E05929g, YALI1_E18121g, YALI1_D13201g, YALI1_C32184g, P GAP , T TEF . Amplify P GAP and T TEF1 from Saccharomyces cerevisiae.

[0014] Construct the following genetic circuits on the Yarrowia lipolytica expression plasmid pYL15 (GenBank accession: KU378202) using Gibson assembly method: (1) P GAP -YALI0_D10549g-T TEF -URA; (2) P GAP -YALI0_C23364g-T TEF -URA; (3) P GAP -YALI0_E05929g-T TEF -URA; (4) P GAP -YALI1_E18121g-T TEF -URA; (5) P GAP -YALI1_D13201g-T TEF -URA; (6) P GAP -YALI1_C32184g-T TEF -URA.

[0015] Construct the following genetic circuits on the Saccharomyces cerevisiae expression plasmid pYES2 (www.snapgene.com / plasmids / yeast_plasmids / pYES2) using Gibson assembly method: (7) HOL-P GAP -YALI0_D10549g-T TEF1 -URA3-HOR; (8) HOL-P GAP -YALI0_C23364g-T TEF1 -URA3-HOR; (9) HOL-P GAP -YALI0_E05929g-T TEF1 -URA3-HOR; (10) HOL-P GAP -YALI1_E18121g-T TEF1 -URA3-HOR; (11) HOL-P GAP -YALI1_D13201g-T TEF1 -URA3-HOR; (12) HOL-PGAP -YALI1_C32184g-T TEF1 -URA3-HOR。

[0016] Transform the above Gibson assembly product into Escherichia coli DH5α competent cells, culture overnight on the corresponding resistant solid medium, and pick single colonies for PCR verification of the plasmid. Extract the plasmid from Escherichia coli, and the plasmid is verified to be successfully ligated by sequencing for subsequent yeast transformation.

[0017] Example 2: Yeast transformation of stress-resistant gene circuits

[0018] Transform the above plasmids (1)-(6) into strains Yarrowia lipolytica CLIB122 and Yarrowia lipolytica CLIB89 by electroporation respectively. Spread the transformed bacteria on SD-URA solid medium and culture at 30 °C for 3 days. Verify positive clones by colony PCR. Name the strains YL1-YL12.

[0019] Transform the above plasmids (7)-(12) into strain Saccharomyces cerevisiae S288c by the method of lithium acetate transformation. Spread the transformed bacteria on SD-URA solid medium and culture at 30 °C for 3 days. Verify positive clones by colony PCR. Name the strains SC1-SC6.

[0020] Example 3: Characterization of yeast stress resistance

[0021] Pick single colonies of the above constructed strains and inoculate them into 2 mL of YPD medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) respectively, and culture in a shaker at 30 °C and 200 rpm for 24 h. Take 0.5 mL of the culture of each strain and inoculate it into fresh 5 mL of YPD medium, and continue to culture in a shaker at 30 °C and 200 rpm for 18 h to obtain seed liquid.

[0022] Use the seed liquid of the strain to inoculate into 20 mL of YPD at an initial OD 600 = 0.2 ratio respectively, and culture under different stress environments: (1) high temperature stress (35 °C); (2) high osmotic pressure (200 g / L glucose, 30 °C); (3) high salt (60 g / L NaCl, 30 °C). After culturing for 5 days, sample to detect the biomass of the cells (OD 600 ). The detection results are as follows in the table, and the results show that all 6 dolichol phosphate mannosyltransferases can improve the growth of cells under stress.

[0023] Strain / Condition (1) (2) (3) Yarrowia lipolytica CLIB122 13.6 29.3 20.6 YL1 30.3 33.9 28.9 YL2 32.8 35.7 29.6 YL3 31.6 34.2 29.0 YL4 33.0 35.4 30.3 YL5 32.1 33.4 28.4 YL6 31.4 34.5 29.7 Yarrowia lipolytica CLIB89 14.3 30.2 21.2 YL7 30.6 34.9 29.5 YL8 33.0 36.2 30.6 YL9 31.9 35.0 29.9 YL10 33.2 36.1 30.8 YL11 32.5 33.9 28.9 YL12 32.4 34.7 29.9 Saccharomyces cerevisiae S288c 28.3 14.7 24.2 SC1 33.1 20.6 28.3 SC2 35.9 21.6 27.6 SC3 34.2 18.3 26.9 SC4 35.6 22.5 28.4 SC5 33.8 19.6 27.3 SC6 34.2 20.1 28.7

[0024] Example 4: Characterization of the global protein glycosylation level in yeast

[0025] Samples of the engineered strain YL8 and the parental strain Yarrowia lipolytica CLIB89 cultured at 35°C for 5 days were selected to detect the differences in the protein glycosylation levels in the cells. After the cells were disrupted at low temperature, the supernatant protein solution was collected. After reduction alkylation, enzymatic digestion, and desalting, hydrophilic interaction materials were used to enrich the glycosylated peptides in the sample to improve the detection rate of glycopeptides. After enrichment, the glycopeptides were detected by LC-MS / MS, and software was used for glycopeptide matching and qualitative identification, and relative quantitative analysis was completed based on the peak area. From Figure 1 It can be seen that in the strain YL8 overexpressing the YALI0_C23364g gene, the abundance of glycosylated proteins was significantly increased compared with the control strain. The glycosylation modifications of 59 proteins changed significantly. Among them, the glycosylation levels of 13 proteins decreased, and the glycosylation levels of 46 proteins increased. The proteins with increased glycosylation levels include cell structural proteins, substances storage and osmotic pressure regulation, heat shock response and other proteins.

[0026] Example 5: Characterization of the properties of yeast cell wall

[0027] The engineered strain YL8 and the parental strain Yarrowia lipolytica CLIB89 were observed by transmission electron microscopy to measure the thickness of their cell walls. The cell wall structure of the 64p strain was observed and analyzed by transmission electron microscopy. From Figure 2 it can be known that the average thickness of the cell wall of the engineered strain YL8 increased by 54% compared with the control strain, and the number of intracellular vacuoles increased significantly, and it maintained the growth and production performance changes at high temperature by regulating osmotic pressure and so on.

[0028] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

[0029] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0030] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention, but all belong to the content scope of the present invention.

Claims

1. A gene and method for improving the stress resistance of yeast, characterized in that, Expression of the dolichol phosphate mannosylprotein mannosyltransferase gene in yeast can improve the stress resistance of yeast cells.

2. The dolichol phosphate mannose-protein mannosyltransferase gene according to claim 1, characterized in that, The dolichol phosphate mannosylprotein mannosyltransferase gene is one or more of the genes YALI0_D10549g (GenBank: CAG80848.1), YALI0_C23364g (GenBank: CAG82498.1), YALI0_E05929g (GenBank: CAG79188.1), YALI1_E18121g (GenBank: XP_503966.3), YALI1_D13201g (GenBank: XP_502660.3), YALI1_C32184g (GenBank: XP_502178.1) from Yarrowia lipolytica.

3. The yeast according to claim 1, characterized in that, The yeast is Yarrowia lipolytica CLIB122 (GenBank: GCA_000002525.1), Yarrowia lipolytica CLIB89 (GenBank: GCA_001761485.1), or Saccharomyces cerevisiae S288c (GenBank: GCA_000146045.2).

4. The gene expression pattern according to claim 1, characterized in that, The expression method in yeast is plasmid expression or integrated genomic expression.

5. The improvement of stress resistance according to claim 1, wherein The stress resistance is resistance to high temperature, high osmotic pressure, and salt.

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