CN112375695A - Copper ion induced saccharomyces cerevisiae engineering bacteria and construction method thereof - Google Patents
Copper ion induced saccharomyces cerevisiae engineering bacteria and construction method thereof Download PDFInfo
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Abstract
The invention relates to a copper ion induced saccharomyces cerevisiae engineering bacterium and a construction method thereof, wherein the construction method comprises the following steps: the Gal80 promoter in Saccharomyces cerevisiae was replaced with an endogenous CTR3 or CTR1 copper ion-inhibited promoter, and the Gal4 promoter in Saccharomyces cerevisiae was replaced with an endogenous CUP1 copper ion-induced promoter, so as to obtain a recombinant Saccharomyces cerevisiae. The saccharomyces cerevisiae engineering bacteria constructed by the method can utilize cheap copper ions as an inducer to realize controllable expression of proteins by saccharomyces cerevisiae, and has good strain stability.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a copper ion induced saccharomyces cerevisiae engineering bacterium and a construction method thereof.
Background
The saccharomyces cerevisiae expression system is one of exogenous protein expression systems, and a continuous expression system and an inducible expression system are common. In general, a sustained expression system employs a strong promoter of glycolytic pathway (e.g., GPD, PGK, etc.) and a transcription factor-related promoter (e.g., TEF1, TEF2, etc.), and since the target protein is continuously expressed, the load on the cells in the logarithmic growth phase is large, which causes problems such as the inhibition of cell growth and the low expression level of protein, and the sustained expression easily causes the instability of the strain, which is not favorable for industrial use. Inducible expression systems are more commonly used galactose inducible expression systems: the system is inhibited when glucose is used as a carbon source, and the target protein expression is induced only when the carbon source is converted into galactose, so that the system has the defects of high galactose cost and additional production cost.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a copper ion induced saccharomyces cerevisiae engineering bacterium and a construction method thereof. The engineering bacteria utilize cheap copper ions as an inducer, realize controllable expression of proteins by saccharomyces cerevisiae, and have good strain stability.
Therefore, in one aspect of the invention, the invention provides a construction method of a copper ion-induced saccharomyces cerevisiae engineering bacterium, which comprises the following steps:
the Gal80 promoter in Saccharomyces cerevisiae was replaced with an endogenous CTR3 or CTR1 copper ion-inhibited promoter, and the Gal4 promoter in Saccharomyces cerevisiae was replaced with an endogenous CUP1 copper ion-induced promoter, so as to obtain a recombinant Saccharomyces cerevisiae.
According to the construction method of the copper ion-induced saccharomyces cerevisiae engineering bacteria, the Gal80 promoter in saccharomyces cerevisiae is replaced by a CTR3 or CTR1 copper ion suppression promoter, the Gal4 promoter is replaced by a CUP1 copper ion induction promoter, and a conventional galactose induction expression system (such as a GAL1/10 expression system) is converted into a controllable copper ion induction system, so that the constructed expression system can express a target protein by taking a carbon source such as glucose as a raw material through induction of copper ions, and is used for purposes such as protein overexpression or biosynthesis.
In addition, the construction method of the copper ion-induced saccharomyces cerevisiae engineering bacteria provided by the embodiment of the invention can also have the following additional technical characteristics:
optionally, the CTR3 copper ion suppression promoter is obtained by PCR amplification by using a saccharomyces cerevisiae genome as a template, using CTR3_ fwd with a nucleotide sequence shown as SEQ ID NO. 1 and CTR3_ rev with a nucleotide sequence shown as SEQ ID NO. 2 as primers.
Optionally, the CTR1 copper ion suppression promoter is obtained by PCR amplification by using a saccharomyces cerevisiae genome as a template, using CTR1_ fwd with a nucleotide sequence shown as SEQ ID NO. 3 and CTR1_ rev with a nucleotide sequence shown as SEQ ID NO. 4 as primers.
Optionally, the CUP1 copper ion inducible promoter is obtained by PCR amplification by using a Saccharomyces cerevisiae genome as a template, and CUP1_ fwd with a nucleotide sequence shown as SEQ ID NO. 5 and CUP1_ rev with a nucleotide sequence shown as SEQ ID NO. 6 as primers.
Optionally, the CTR3 or CTR1 copper ion repression promoter is a Gal80 promoter in saccharomyces cerevisiae replaced by CRISPR/Cas9 traceless knock-in technology.
Optionally, the method further comprises transforming an expression vector of the foreign protein gene into the recombinant saccharomyces cerevisiae so as to obtain the recombinant engineered saccharomyces cerevisiae.
In a second aspect of the invention, the invention provides a copper ion-induced saccharomyces cerevisiae engineering bacterium constructed by the construction method of the engineering bacterium.
According to the saccharomyces cerevisiae engineering bacteria disclosed by the embodiment of the invention, cheap copper ions can be used as an inducer, controllable protein expression of saccharomyces cerevisiae can be realized, and the saccharomyces cerevisiae engineering bacteria have good strain stability.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a copper ion gradient induced green fluorescent protein expression according to an embodiment of the present invention;
FIG. 2 is a graph showing the effect of copper ions on carotene biosynthesis according to the embodiment of the present invention.
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market; the related experiments are all routine experimental methods if not specifically stated.
Sources of materials used: saccharomyces cerevisiae BY4741, CEN. PK2-1C, and TOP10 are commercially available, with TOP10 strain being used for vector construction. Conventional galactose-inducible expression vector pRS425GAL1 is commercially available. Phusion high fidelity DNA polymerase, restriction enzyme was purchased from Xiamen Lulong Biotech development Inc. Plasmid extraction kits, DNA purification kits, gel recovery kits and genomic DNA extraction kits were purchased from Shanghai bioengineering, Inc.
The LB medium consisted of: 10 g.L-1Peptone, 5 g. L-1Yeast powder, 5 g.L-1NaCl, the balance double distilled water, 0.1Mpa pressure 121 deg.C sterilization for 20 min.
YPD medium composition was: 10 g.L-1Yeast powder, 20 g.L-1Peptone, 20 g.L-1Sterilizing glucose and double distilled water in 0.1Mpa at 121 deg.C for 20 min.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Example 1
1. Preparing a promoter:
CTR3 promoter: a gene group of Saccharomyces cerevisiae CEN. PK2-1C is used as a template, a CTR3_ fwd with a nucleotide sequence shown as SEQ ID NO. 1 and a CTR3_ rev with a nucleotide sequence shown as SEQ ID NO. 2 are used as primers, a CTR3 promoter band is obtained through high-fidelity enzyme PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min, 98 deg.C for 10s, 56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2 min.
CTR1 promoter: a gene group of Saccharomyces cerevisiae CEN. PK2-1C is used as a template, a CTR1_ fwd with a nucleotide sequence shown as SEQ ID NO. 3 and a CTR1_ rev with a nucleotide sequence shown as SEQ ID NO. 4 are used as primers, a CTR1 promoter band is obtained through high-fidelity enzyme PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min, 98 deg.C for 10s, 56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2 min.
CUP1 promoter: a CUP1_ fwd with a nucleotide sequence shown as SEQ ID NO. 5 and a CUP1_ rev with a nucleotide sequence shown as SEQ ID NO. 6 are used as primers, a CUP1 promoter band is obtained through high fidelity enzyme PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min, 98 deg.C for 10s, 56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2 min.
Table 1: PCR amplification primer List
2. Construction of recombinant Saccharomyces cerevisiae
(1) The CTR3 promoter and the CUP1 promoter sequences prepared in the step 1 are respectively used for replacing a Gal80 promoter and a Gal4 promoter in a CEN.PK2-1C genome of saccharomyces cerevisiae by a CRISPR/Cas9 traceless knock-in technology. Wherein the CRISPR/Cas9 traceless knock-in procedure is operated by a reported method (DiCarlo JE, et al. nucleic Acids Res 41(7): 4336-43).
(2) The CTR1 promoter and the CUP1 promoter sequences prepared in the step 1 are respectively used for replacing a Gal80 promoter and a Gal4 promoter in the genome of Saccharomyces cerevisiae BY4741 BY a CRISPR/Cas9 traceless knock-in technology. Wherein the CRISPR/Cas9 traceless knock-in procedure is operated by a reported method (DiCarlo JE, et al. nucleic Acids Res 41(7): 4336-43).
Example 2
Competent cells of the recombinant s.cerevisiae constructed in step (1) of example 1, step 2 were prepared, and pRS425GAL1 expression vector containing EGFP green fluorescent protein gene (Sheff MA, et al. Yeast 21(8):661-70.) was introduced into competent cells of recombinant s.cerevisiae CEN. PK2-1C by electroporation. Wherein the preparation process and the electric shock method of the competent cells are both performed by the reported method (DiCarlo JE, et al. nucleic Acids Res 41(7): 4336-43).
Example 3
Competent cells are prepared from the recombinant saccharomyces cerevisiae constructed in the step (2) in the step 2 of the example 1, and then crtE, crtYB and crtI genes (Verwaal R, et al. apple Environ Microbiol 73(13):4342-50) of the phaffia rhodozyma dendron are respectively integrated into the BTS1, YPL062W and ROX1 sites of the genome of the competent cells BY4741 of the recombinant saccharomyces cerevisiae BY using a CRISPR/Cas9 traceless knock-in technology. Wherein the CRISPR/Cas9 traceless knock-in procedure is operated by a reported method (DiCarlo JE, et al. nucleic Acids Res 41(7): 4336-43).
Example 4
The recombinant saccharomyces cerevisiae containing the EGFP green fluorescent protein gene expression vector obtained in the embodiment 2 is inoculated in a leucine-deficient yeast nitrogen-containing culture solution, is inoculated into the leucine-deficient yeast nitrogen-containing culture solution containing copper ions with different concentrations for 24 hours of amplification culture according to the proportion of 1:100 after overnight culture, and then a green fluorescent protein signal is quantified through an enzyme labeling instrument.
Quantitative analysis of green fluorescent protein: and (3) detecting and analyzing by using a Biotek Synergy H1 enzyme-labeling instrument, wherein the excitation wavelength and the emission wavelength are respectively set to be 476/512 nm.
The results are shown in FIG. 1, where a copper ion gradient induced green fluorescent protein expression.
Example 5
The recombinant saccharomyces cerevisiae containing the carotene biosynthesis genes obtained in the embodiment 3 is inoculated in a yeast nitrogen-containing culture solution, the yeast nitrogen-containing culture solution is inoculated into yeast nitrogen-containing culture solutions containing copper ions with different concentrations according to the proportion of 1:100 after overnight culture, the yeast nitrogen-containing culture solutions are subjected to amplification culture for 24 hours, and then the copper ion induced synthesis of the carotene in the saccharomyces cerevisiae is qualitatively observed by naked eyes.
The results are shown in FIG. 2, which shows the effect of copper ion induced carotene biosynthesis, and the copper ion induced carotene production at 0-20 μ M.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (8)
1. A construction method of a copper ion induced saccharomyces cerevisiae engineering bacterium is characterized by comprising the following steps:
the Gal80 promoter in Saccharomyces cerevisiae was replaced with an endogenous CTR3 or CTR1 copper ion-inhibited promoter, and the Gal4 promoter in Saccharomyces cerevisiae was replaced with an endogenous CUP1 copper ion-induced promoter, so as to obtain a recombinant Saccharomyces cerevisiae.
2. The construction method of claim 1, wherein the CTR3 copper ion suppression promoter is obtained by PCR amplification using Saccharomyces cerevisiae genome as template, CTR3_ fwd with nucleotide sequence shown as SEQ ID NO. 1 and CTR3_ rev with nucleotide sequence shown as SEQ ID NO. 2 as primers.
3. The construction method of claim 1, wherein the CTR1 copper ion suppression promoter is obtained by PCR amplification using Saccharomyces cerevisiae genome as template, CTR1_ fwd with nucleotide sequence shown as SEQ ID NO. 3 and CTR1_ rev with nucleotide sequence shown as SEQ ID NO. 4 as primers.
4. The construction method according to claim 1, wherein the CUP1 copper ion inducible promoter is obtained by PCR amplification using Saccharomyces cerevisiae genome as template, CUP1_ fwd with nucleotide sequence as shown in SEQ ID NO. 5 and CUP1_ rev with nucleotide sequence as shown in SEQ ID NO. 6 as primers.
5. The construction method of claim 1, wherein the CTR3 or CTR1 copper ion repression promoter is Gal80 promoter in Saccharomyces cerevisiae replaced by CRISPR/Cas9 traceless knock-in technology.
6. The construction method of claim 1, wherein the CUP1 copper ion inducible promoter is a Gal4 promoter in Saccharomyces cerevisiae replaced by CRISPR/Cas9 traceless knock-in technology.
7. The construction method according to claim 1, further comprising transforming an expression vector of a foreign protein gene into the recombinant saccharomyces cerevisiae to obtain a recombinant engineered saccharomyces cerevisiae.
8. The copper ion-induced saccharomyces cerevisiae engineering bacteria are characterized by being constructed by the construction method of any one of claims 1 to 7.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114085785A (en) * | 2021-10-29 | 2022-02-25 | 湖北冠众通科技有限公司 | Saccharomyces cerevisiae gene engineering bacterium and construction method and application thereof |
| CN115820708A (en) * | 2022-09-06 | 2023-03-21 | 厦门大学 | Construction method of recombinant saccharomyces cerevisiae for sensing fungal pheromone and biosensor |
| CN116254286A (en) * | 2022-12-23 | 2023-06-13 | 厦门大学 | Cyanamide-induced saccharomyces cerevisiae engineering bacteria and construction method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982721A (en) * | 2019-12-09 | 2020-04-10 | 东莞市东阳光生物合成药有限公司 | Method for improving yield of saccharomyces cerevisiae metabolites |
| CN110982721B (en) * | 2019-12-09 | 2022-04-26 | 宜昌东阳光生化制药有限公司 | Method for improving yield of saccharomyces cerevisiae metabolites |
| CN114085785A (en) * | 2021-10-29 | 2022-02-25 | 湖北冠众通科技有限公司 | Saccharomyces cerevisiae gene engineering bacterium and construction method and application thereof |
| CN115820708A (en) * | 2022-09-06 | 2023-03-21 | 厦门大学 | Construction method of recombinant saccharomyces cerevisiae for sensing fungal pheromone and biosensor |
| CN115820708B (en) * | 2022-09-06 | 2024-10-11 | 厦门大学 | Construction method of recombinant saccharomyces cerevisiae for sensing fungal pheromone and biosensor |
| CN116254286A (en) * | 2022-12-23 | 2023-06-13 | 厦门大学 | Cyanamide-induced saccharomyces cerevisiae engineering bacteria and construction method thereof |
| CN116254286B (en) * | 2022-12-23 | 2024-07-12 | 厦门大学 | Cyanamide-induced saccharomyces cerevisiae engineering bacteria and construction method thereof |
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