Saccharomyces cerevisiae galactose inducible promoter, expression vector and application thereof

Abstract

The invention relates to a saccharomyces cerevisiae galactose inducible promoter, an expression vector and application thereof, and belongs to the technical field of biology. The invention provides a method for improving the expression intensity of a galactose inducible promoter in saccharomyces cerevisiae, 1-7 upstream activation sequences are inserted into the galactose inducible promoter PGAL1, and the related upstream activation sequences are derived from different types of yeast galactose inducible promoters. Galactose inducible unidirectional and bidirectional promoter libraries and expression vectors are respectively constructed on the basis, the optimal unidirectional inducible promoter UIP-A2 is obtained through screening, the strength of the optimal unidirectional inducible promoter UIP-A2 is 2.37 times that of PGAL1, and the induction strength of the optimal bidirectional inducible promoter in two directions is 6.02 times and 3.87 times that of the optimal bidirectional inducible promoter before modification respectively; by using the method provided by the invention, the expression intensity of the galactose inducible promoter in the saccharomyces cerevisiae can be remarkably improved, a powerful tool is provided for efficiently expressing exogenous genes by the saccharomyces cerevisiae, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a galactose-inducible promoter of Saccharomyces cerevisiae, an expression vector and applications thereof. Background Art

[0002] As an important industrial microorganism, Saccharomyces cerevisiae is widely used in the field of biotechnology. Its excellent fermentation ability, easy cultivation, and strong genetic modification ability make it have important applications in food, beverage, drug production, and metabolic engineering. Saccharomyces cerevisiae is used in the fermentation industry to produce alcohol, ethanol, and other fermentation products, and plays an important role in traditional industries such as brewing and bread fermentation. In addition, with the development of synthetic biology, Saccharomyces cerevisiae has been used as an expression system for the production of recombinant proteins, enzymes, drug molecules, and biofuels. Its small genome and precise genome editing make it widely used in biopharmaceutical production and industrial microbial engineering. The potential of Saccharomyces cerevisiae in increasing the yield of metabolites, optimizing process flows, and developing new biosynthetic pathways has also been continuously explored, showing broad development prospects.

[0003] Saccharomyces cerevisiae galactose-inducible promoter (such as P GAL1 , P GAL10 and P GAL7 Promoters are important tools for regulating gene expression, and their research is of great significance in the fields of genetic engineering and metabolic engineering. These promoters can precisely regulate gene expression through the presence or absence of galactose, thereby achieving spatiotemporal specific expression regulation of target genes. In recent years, research on galactose-inducible promoters has mainly focused on the following aspects: First, through site-directed mutagenesis and promoter modification technology, the regulatory ability of these promoters has been optimized, and the induction efficiency and dynamic response range have been improved. Secondly, combined with modern synthetic biology technology, integrated and modular promoter systems have been developed, which can work in conjunction with other regulatory elements to achieve more complex gene circuit design. In addition, in-depth research on the galactose metabolic pathway has revealed the association between metabolites and promoter activity, providing new targets for promoter optimization. At present, the research has also further explored the industrialization potential of promoters, such as the production of recombinant proteins and metabolites through a galactose-inducible system. With the deepening of research, the application prospects of galactose-inducible promoters of Saccharomyces cerevisiae in precise regulation and efficient expression will be broader. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for improving the expression intensity of a galactose-inducible promoter in Saccharomyces cerevisiae. GAL11-7 upstream activation sequences were inserted, and the upstream activation sequences involved were derived from different types of yeast galactose-inducible promoters, and on this basis, galactose-inducible unidirectional and bidirectional promoter libraries were constructed respectively.

[0005] The first object of the present invention is to provide a method for increasing the expression intensity of a galactose-inducible promoter in Saccharomyces cerevisiae. GAL1 1-7 upstream activation sequences are inserted upstream, and the upstream activation sequence is shown in any one of SEQ ID NO.1-7.

[0006] Furthermore, the UAS sequence shown in SEQ ID NO.1 is derived from the Saccharomyces cerevisiae promoter P ScGAL1 The UAS sequence shown in SEQ ID NO.2 is derived from the Saccharomyces cerevisiae promoter P ScGAL2 The UAS sequence shown in SEQ ID NO.3 is derived from the Saccharomyces cerevisiae promoter P ScGAL7 , the UAS sequence shown in SEQ ID NO.4 is derived from the promoter P of Saccharomyces kudriavzevii SkGAL1 The UAS sequence shown in SEQ ID NO.5 is derived from the promoter P of Saccharomyces cerevisiae SeGAL2 The UAS sequence shown in SEQ ID NO.6 is derived from the Bayesian yeast promoter P SbGAL2 , the UAS sequence shown in SEQ ID NO.7 is derived from the promoter P of Saccharomyces kudriavzevii SkGAL2 .

[0007] Furthermore, the P ScGAL1 The sequence of the promoter is shown in SEQ ID NO.8. ScGAL2 The sequence of the promoter is shown in SEQ ID NO.9, and the P ScGAL7 The sequence of the promoter is shown in SEQ ID NO.10. SkGAL1 The sequence of the promoter is shown in SEQ ID NO.11, and the P SeGAL2 The sequence of the promoter is shown in SEQ ID NO.12. SbGAL2 The sequence of the promoter is shown in SEQ ID NO.13. SkGAL2 The sequence of the promoter is shown in SEQ ID NO.14.

[0008] The second object of the present invention is to provide a galactose-inducible expression vector, wherein the galactose-inducible expression vector uses PGAL1 as an initiator, an upstream activation sequence is inserted upstream of the initiator, and the upstream activation sequence is shown in SEQ ID NO.7.

[0009] Furthermore, the galactose-inducible expression vector is TSPG5 For the terminator.

[0010] In one embodiment of the present invention, the galactose-inducible expression vector further comprises a URA3 tag, and the sequence of the URA3 tag is shown in SEQ ID NO.15.

[0011] The third object of the present invention is to provide cells containing the above galactose-inducible expression vector.

[0012] Furthermore, the cell is derived from Saccharomyces cerevisiae.

[0013] In one embodiment of the present invention, the Saccharomyces cerevisiae is Saccharomyces cerevisiae CEN.PK113-5D.

[0014] The fourth object of the present invention is to provide a galactose-inducible bidirectional promoter, the bidirectional promoter comprising a first promoter unit and a second promoter unit, wherein the first promoter unit and the second promoter unit have opposite transcription directions;

[0015] The first startup unit is P GAL7 is the promoter, in the P GAL7 A first upstream activation sequence is inserted upstream, wherein the first upstream activation sequence is shown as SEQ ID NO.2;

[0016] The second startup unit is P GAL1 is the promoter, in the P GAL1 A second upstream activation sequence is inserted upstream, and the second upstream activation sequence is shown as SEQ ID NO.5.

[0017] In one embodiment of the present invention, the first starting unit is T CYC1 As a terminator, the second promoter unit is T SPG5 For the terminator.

[0018] The fifth object of the present invention is to provide an expression vector containing the above-mentioned galactose-inducible bidirectional promoter.

[0019] In one embodiment of the present invention, the expression vector further comprises a URA3 tag, and the sequence of the URA3 tag is shown in SEQ ID NO.15.

[0020] The sixth object of the present invention is to provide a cell comprising the above-mentioned galactose-inducible bidirectional promoter or the above-mentioned expression vector.

[0021] Furthermore, the cell is derived from Saccharomyces cerevisiae.

[0022] In one embodiment of the present invention, the Saccharomyces cerevisiae is Saccharomyces cerevisiae CEN.PK113-5D.

[0023] The seventh object of the present invention is to provide the above-mentioned galactose-inducible bidirectional promoter or the above-mentioned use in reporting dual fluorescence in Saccharomyces cerevisiae.

[0024] In one embodiment of the present invention, the dual fluorescence is obtained by stimulating green fluorescent protein GFP and red fluorescent protein mScarlet-I.

[0025] In one embodiment of the present invention, the nucleotide sequence of the green fluorescent protein GFP is shown as SEQ ID NO.16, and the nucleotide sequence of the red fluorescent protein mScarlet-I is shown as SEQ ID NO.17.

[0026] Beneficial effects of the present invention:

[0027] The present invention is carried out by galactose inducible promoter P GAL1 Inserting 1-7 specific upstream activation sequences upstream can increase the expression intensity of the promoter in Saccharomyces cerevisiae and help enhance the expression effect of related genes. GAL1 The invention provides a suitable vector basis for realizing efficient expression of target gene in Saccharomyces cerevisiae by using a galactose-inducible bidirectional promoter and a galactose-inducible bidirectional expression vector based thereon, which comprises a first promoter unit and a second promoter unit with opposite transcription directions, respectively with P GAL1 and P GAL7 By inserting different upstream activation sequences in the same direction into the starting promoter, the tool for simultaneously regulating the transcriptional expression of genes in different directions in Saccharomyces cerevisiae has been expanded, and the means of gene expression regulation have been enriched. It can be applied to the dual fluorescence reporter of Saccharomyces cerevisiae, and provides a powerful application approach for related detection and analysis, which will help promote the research on gene expression related to Saccharomyces cerevisiae and its application development in the fields of fluorescence detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0029] Figure 1 This is a schematic diagram of the screening process of a unidirectional inducible promoter in Example 1 of the present invention;

[0030] Figure 2 This is a schematic diagram of the construction of a unidirectional inducible promoter library in Example 1 of the present invention;

[0031] Figure 3 The results of screening for unidirectional inducible promoters in Example 1 of the present invention are as follows;

[0032] Figure 4 This is a schematic diagram of the sequencing results of the unidirectional inducible promoter in Example 1 of the present invention;

[0033] Figure 5 This is a schematic diagram of the construction of a bidirectional inducible promoter library in Example 2 of the present invention;

[0034] Figure 6 The results of screening for bidirectional inducible promoters in Example 2 of the present invention;

[0035] Figure 7 This is a schematic diagram of the sequencing results of the bidirectional inducible promoter BIP-B11 in Example 2 of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0037] The strains and vectors involved in the examples are as follows:

[0038] The plasmid was constructed in E. coli Top10, and the plasmid library was assembled in series using the Golden Gate technology. The constructed unidirectional inducible promoter library plasmid was transferred into Saccharomyces cerevisiae CEN.PK113-5D.

[0039] The culture medium involved in the embodiment is as follows:

[0040] Escherichia coli LB medium: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L.

[0041] SDG-U medium: glucose 20 g / L, galactose 20 g / L, ammonium sulfate 5 g / L, amino-free yeast nitrogen source YNB 6.7 g / L.

[0042] The fluorescence determination method involved in the embodiment is as follows:

[0043] The fluorescence data of promoter characterization is expressed as fluorescence value / OD 600 Based on the results, the excitation wavelength of mScarlet-I is 570 nm, the emission wavelength is 605 nm, and the gain is 80. The excitation wavelength of GFP is 488 nm, the emission wavelength is 523 nm, and the gain is 60. 600 Detected by UV spectrophotometer.

[0044] Example 1: Construction and characterization of a unidirectional inducible promoter library

[0045] Galactose-inducible promoters have been found in different yeast strains. The main factor affecting their induction ability is UAS. Galactose-activated UAS sequences from different sources all contain binding sites for the GAL4 protein of that strain. Therefore, by screening galactose-inducible promoters from different yeast sources and annotating G4BS using JASPAR, a database for annotation of eukaryotic transcription factor binding sites, the galactose-activated UAS sequence of the promoter in that strain can be obtained. By concatenating UAS sequences, galactose-inducible promoters with different induction strengths can be obtained.

[0046] The GAL4 protein binding sites of galactose-inducible promoters from different sources were predicted using the JASPAR database. Taking the G4BS sequence as the basic site, a certain length was derived on the left and right sides to obtain 7 upstream activation sequences UAS corresponding to galactose-inducible promoters, all of which were about 100-200 bp in length, namely:

[0047] Saccharomyces cerevisiae promoter P ScGAL1 Source, nucleotide sequence is shown in SEQ ID NO.1;

[0048] Saccharomyces cerevisiae promoter P ScGAL2 Source, nucleotide sequence is shown in SEQ ID NO.2;

[0049] Saccharomyces cerevisiae promoter P ScGAL7 Source, nucleotide sequence is shown in SEQ ID NO.3;

[0050] Saccharomyces kudriavzevii promoter P SkGAL1 Source, nucleotide sequence is shown in SEQ ID NO.4;

[0051] Yeast promoter P SeGAL2 Source, nucleotide sequence is shown in SEQ ID NO.5;

[0052] Bayle's yeast promoter P SbGAL2 Source, nucleotide sequence is shown in SEQ ID NO.6;

[0053] Saccharomyces kudriavzevii promoter P SkGAL2 Source, the nucleotide sequence is shown in SEQ ID NO.7.

[0054] Insert the above UAS1-7 sequence into P ScGAL1 The 5' end of the cascade inducible promoter library was constructed and assembled by GoldenGate. The specific construction method is shown in Figure 2 The UAS sequences were synthesized by GenScript Biotechnology, and overhangs were introduced at both ends of each UAS using PCR. A total of seven libraries with different numbers of UAS were constructed, with a theoretical library size of 71 +7 2 +7 3 +7 4 +7 5 +7 6 +7 7 , for specific connection system and connection procedure, please refer to The Golden Gate assembly kit (BsaI-HF v2) instruction manual was used to transform the completed system into E. coli. After the post-culture, the culture was expanded. All the post-culture liquid was transferred to a 500 mL flask containing 50 mL of LB liquid culture medium containing kanamycin for culture. After overnight culture, the bacterial liquid was collected and the promoter library plasmid was extracted using the UNlQ-200 column plasmid DNA midi extraction kit.

[0055] The promoter library plasmid was transformed into Saccharomyces cerevisiae CEN.PK113-5D, plated and sorted by flow cytometry. 600 The ratio was used as the initial screening condition to screen single cells with a higher ratio. The single cells enriched by screening were cultured for 12 hours and then coated on a plate, and four rounds of shallow well plate rescreening were performed. 600 As a screening criterion, its rigor has been verified in three rounds of screening, so in the fourth round of rescreening, 31 tandem inducible promoters from different libraries were finally selected, such as Figure 3 As shown, the best one is UIP-A02, whose strength is P ScGAL1 2.37 times of the promoter. Thirteen of the best promoters were selected for sequencing. The sequencing results are shown in the figure below. Figure 4 As shown, it can be seen that UIP-A02 is in P ScGAL1 Upstream insertion P SkGAL2 The UAS sequence of the source.

[0056] Example 2: Construction and characterization of a bidirectional inducible promoter

[0057] like Figure 5 The bidirectional inducible promoter library is constructed as shown in Figure 1. Unlike the unidirectional inducible promoter library, the UAS sequence has different directions when constructing the bidirectional inducible promoter. The UAS sequence inserted upstream of the original promoter has two directions: the same direction as the original promoter and the opposite direction to the original promoter. The theoretical number of libraries is 196. Figure 5 As shown, three rounds of screening were performed, and each round was performed by fermentation of a single colony to detect mScarlet-I / OD 600 and GFP / OD 600 Finally, a better bidirectional inducible promoter BIP-B11 was obtained. ScGAL1 The induction intensity in the direction was 6.02 times that of the control, PScGAL7 The induction intensity in the direction is 3.87 times that of the control. The schematic diagram of the sequencing results is shown in Figure 7 As shown, in P ScGAL1 The upstream insertion is in the same direction, originating from P ScGAL2 The UAS sequence of the promoter is ScGAL7 Upstream upstream is inserted in the same direction as it, derived from P SeGAL2 UAS sequence of the promoter.

[0058] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for increasing the expression intensity of a galactose-inducible promoter in Saccharomyces cerevisiae, characterized in that: In the galactose-inducible promoter P GAL1 1-7 upstream activation sequences are inserted upstream, and the upstream activation sequence is shown in any one of SEQ ID NO.1-7.

2. A galactose-inducible expression vector, characterized in that: The galactose-inducible expression vector is GAL1 is an initiator, an upstream activation sequence is inserted upstream of the initiator, and the upstream activation sequence is shown in SEQ ID NO.

7.

3. The galactose-inducible expression vector according to claim 2, characterized in that: The galactose-inducible expression vector is T SPG5 For the terminator.

4. A cell containing the galactose-inducible expression vector according to claim 2 or 3.

5. The cell according to claim 4, characterized in that: The cells are derived from Saccharomyces cerevisiae.

6. A galactose-inducible bidirectional promoter, characterized in that: The bidirectional promoter comprises a first promoter unit and a second promoter unit, and the transcription direction of the first promoter unit is opposite to that of the second promoter unit; The first startup unit is P GAL7 is the promoter, in the P GAL7 A first upstream activation sequence is inserted upstream, wherein the first upstream activation sequence is shown as SEQ ID NO.2; The second startup unit is P GAL1 is the promoter, in the P GAL1 A second upstream activation sequence is inserted upstream, and the second upstream activation sequence is shown as SEQ ID NO.

5.

7. An expression vector containing the galactose-inducible bidirectional promoter according to claim 6.

8. A cell comprising the galactose-inducible bidirectional promoter according to claim 6 or the expression vector according to claim 7.

9. The cell according to claim 8, characterized in that: The cells are derived from Saccharomyces cerevisiae.

10. Use of the galactose-inducible bidirectional promoter according to claim 6 or the expression vector according to claim 7 in reporting dual fluorescence of Saccharomyces cerevisiae.

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