Method for regulating expression intensity of ydgI gene promoter

Abstract

The invention provides a method for regulating and controlling the expression intensity of a ydgI gene promoter, and particularly, betaine is added into an expression system containing the ydgI gene promoter, so that the expression intensity of the ydgI gene promoter can be regulated and controlled to be reduced in a logarithmic phase and / or improved in a stable phase of microbial growth. By utilizing the method, the expression level of a target gene in an expression system can be accurately regulated and controlled.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a method for regulating the expression intensity of a ydgI gene promoter by adding betaine. Background Art

[0002] The promoter is a DNA sequence that RNA polymerase specifically recognizes and binds to. It is a component of the gene and controls the start time of gene transcription and the degree of transcription expression like a "switch". The promoter is a DNA sequence located upstream of the 5' end of the structural gene, which can guide the holoenzyme (Holoenzyme) to correctly bind to the template, activate RNA polymerase, and start gene transcription. The promoter itself cannot directly control gene expression, but controls structural gene expression by binding to transcription factors.

[0003] Currently, commonly used promoter types include constitutive promoters (promoters that can activate gene expression in all cells) and inducible promoters (specific inducible promoters can activate the transcriptional expression of downstream structural genes only under the stimulation of certain physical signals, chemical signals or small molecule signals). The regulation of constitutive promoters is not affected by external conditions, and the expression of the activated genes is continuous, but it does not show time specificity, cannot be artificially regulated, and is not operable in practical applications; while inducible promoters can artificially regulate the expression of structural genes through special external signals. At present, commonly used induction signals include chemical substances such as IPTG and lactose, which face problems such as high costs in actual production processes.

[0004] Betaine (Betaine, molecular formula is C5H 12 NO2) has multiple physiological functions such as improving the tolerance of cells to the osmotic pressure of the extracellular environment, providing methyl donors for the synthesis of target products, and protecting the activity of key enzymes in organisms, and is widely used in microbial fermentation production processes. CN106636023A announced that the expression intensity of the ZWF gene promoter was enhanced by 0.2-0.98 times by adding betaine to the expression system or fermentation system. In addition, the induction function of betaine has not yet been recognized, developed and utilized. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for regulating the expression intensity of the ydgI gene promoter, specifically by adding betaine to the microbial expression system to achieve the purpose of reducing and / or increasing the expression intensity of the ydgI gene promoter.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for regulating the expression intensity of the ydgI gene promoter, wherein betaine is added to an expression system containing the ydgI gene promoter, and the expression intensity of the ydgI gene promoter can be regulated to decrease in the logarithmic phase of microbial growth and / or increase in the stable phase.

[0008] As described above in the method provided by the present invention in the first aspect, the expression intensity of the ydgI gene promoter is reduced by 60%-70% during the logarithmic phase of microbial growth.

[0009] As described above in the method provided by the present invention in the first aspect, the expression intensity of the ydgI gene promoter is increased by 110%-160% during the stable period of microbial growth.

[0010] In a second aspect, the present invention provides a method for regulating the expression level of a target gene, the method comprising adding betaine to an expression system containing the target gene and its upstream ydgI gene promoter, thereby regulating the expression level of the target gene to decrease during the logarithmic phase of microbial growth and / or increase during the stable phase.

[0011] The beneficial effects of the present invention are as follows:

[0012] The betaine added to the microbial fermentation system in the present invention is different from the common methyl donor, but is used as a new inducer to regulate the expression intensity of the ydgI gene promoter in the expression system to significantly reduce the logarithmic phase and / or significantly increase the stable phase; and then the method can be used to achieve precise regulation of the expression level of the target gene in the expression system. This regulatory mechanism is of great significance for cell growth, development and response to environmental changes. Realizing this precise regulation has the following advantages or effects:

[0013] Optimal allocation of resources: During the logarithmic growth phase, cells proliferate rapidly. Reducing promoter strength can reduce unnecessary gene expression, save energy and resources, and prepare for the stable period.

[0014] Adapting to environmental changes: As cells enter the stable phase, environmental conditions and cell requirements change. At this time, increasing promoter strength can upregulate the expression of key genes, helping cells better adapt to environmental changes and maintain cell function and survival.

[0015] Precise regulation of gene expression: By dynamically regulating promoter strength, cells can precisely control the timing and level of gene expression, ensuring that specific genes are expressed at specific growth stages, thereby optimizing the overall function and adaptability of the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Comparison of ydgI gene transcription levels under the action of betaine in Example 1.

[0017] Figure 2 : Comparison of strain growth curves under the action of betaine in Example 1. DETAILED DESCRIPTION

[0018] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0019] In a first aspect, the present invention provides a method for regulating the expression intensity of the ydgI gene promoter, wherein betaine is added to an expression system containing the ydgI gene promoter, and the expression intensity of the ydgI gene promoter can be regulated to decrease in the logarithmic phase of microbial growth and / or increase in the stable phase.

[0020] According to the method provided by the present invention, the expression intensity of the ydgI gene promoter is regulated to decrease during the logarithmic phase of microbial growth, for example, by 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0021] According to a preferred embodiment of the present invention, the expression intensity of the ydgI gene promoter is reduced by 60%-70% during the stable period of microbial growth.

[0022] According to the method provided by the present invention, the expression intensity of the ydgI gene promoter is regulated to increase during the stable phase of microbial growth, for example, by 100% or more, 120% or more, 140% or more, 160% or more, 180% or more, or 200% or more.

[0023] According to a preferred embodiment of the present invention, the expression intensity of the ydgI gene promoter is increased by 110%-160% during the logarithmic phase of microbial growth.

[0024] According to a preferred embodiment of the present invention, the amount of betaine added is a working concentration of 1 g / L in the fermentation system.

[0025] According to the present invention, the expression system can be a common bacterial expression system, for example, an Escherichia coli expression system or a Bacillus subtilis expression system.

[0026] According to a preferred embodiment of the present invention, the expression system is an Escherichia coli expression system.

[0027] According to a preferred embodiment of the present invention, in the Escherichia coli expression system to which betaine is added, the expression intensity of the ydgI gene promoter is significantly reduced from the beginning of fermentation to 12h, especially by 60%-70% at 8-12h; and significantly increased after 16h of fermentation, especially by 110%-160% at 16-20h.

[0028] According to a preferred embodiment of the present invention, the nucleotide sequence of the ydgI gene promoter is shown as SEQ ID No.1.

[0029] According to the present invention, the ydgI gene encodes an arginine:ornithine antiporter.

[0030] In a second aspect, the present invention provides a method for regulating the expression level of a target gene, the method comprising adding betaine to an expression system containing the target gene and its upstream ydgI gene promoter, thereby regulating the expression level of the target gene to decrease during the logarithmic phase of microbial growth and / or increase during the stable phase.

[0031] The term "logarithmic phase" or "logarithmic growth phase", also called exponential growth phase, refers to a period of time when the number of microbial cells increases exponentially after the delay period. During this period, the nutrition is sufficient, the cells grow rapidly, the number of viable bacteria increases at a constant geometric progression, and the logarithm of the number of bacteria on the growth curve rises linearly and reaches a peak state. The morphology, staining, physiological activity, etc. of the bacteria are relatively typical and sensitive to the effects of environmental factors.

[0032] The term "stable phase" or "stable growth phase" refers to the phase after the logarithmic phase and before the decay phase, during the growth of the microbial population, when the number of newly propagated cells and the number of dead cells are basically in a stable equilibrium, the number of live cells reaches a dynamic equilibrium, and the growth rate of the population is equal to zero. During this phase, nutrients are gradually depleted and harmful metabolites accumulate, resulting in a slowdown in the reproduction rate of microorganisms and a slow increase in the number of deaths, but the total number of live bacteria remains relatively stable and reaches the highest level.

[0033] The present invention will be described in more detail below through specific examples. The specific operation means of molecular biology, genetic engineering, etc. involved in the embodiments of the present invention can be implemented according to technical manuals, textbooks or literature reports easily available to those skilled in the art, and it is not necessary to describe the operation process in detail here.

[0034] Embodiment 1:

[0035] This embodiment provides a method for regulating the expression intensity of the ydgI gene promoter in an Escherichia coli expression system, comprising the following experimental steps:

[0036] 1) Use an inoculating loop to take a loop of E. coli MG1655 bacterial solution from the glycerol tube and transfer it to the antibiotic-free activation slant medium, streak it densely, and culture it at 37°C for 12 hours (first generation slant);

[0037] 2) Take a loopful of bacteria from the first generation slant and streak it onto another slant, and culture it at 37°C for 8 hours (second generation slant);

[0038] 3) Take a loopful of bacteria from the second generation slant and transfer it to 50 mL of seed medium. Culture at 37°C and 200 rpm for 8 h.

[0039] 4) Inoculate 20% of the inoculum into 30 mL of fermentation medium, culture at 37°C, 200 rpm, and adjust the pH to 6.7-7.0 with ammonia water. No betaine was added to the control medium, and betaine was added to the experimental medium at a final concentration of 1 g / L. Samples were taken at 8 h, 12 h, 16 h, and 20 h of fermentation to determine the transcription amount, and the transcription level of the prpB gene, a dynamic significant difference gene under the action of betaine, was verified.

[0040] 5) Culture medium formula:

[0041] Activated slant medium (g / L): glucose 1, peptone 10, beef extract 5, yeast powder 5, NaCl 2.5, agar powder 20, adjust pH to 7.0-7.2, sterilize at 121℃ for 20min.

[0042] Seed culture medium (g / L): sucrose 25, yeast powder 10, peptone 6, KH2PO4 1.2, MgSO4·7H2O 0.5, FeSO4·7H2O 0.01, MnSO4·3H2O 0.01, V B1 1.3mg / L,V H 0.3mg / L, adjust pH to 7.0-7.2, add 1-2 drops of defoaming agent, sterilize at 115℃ for 15min.

[0043] Fermentation medium (g / L): glucose 30, yeast powder 2, peptone 4, sodium citrate 1, KH2PO42, MgSO4·7H2O0.7, FeSO4·7H2O 0.1, MnSO4·3H2O 0.1, V B1 0.0008, V H 0.0002, adjust pH to 7.0-7.2, and sterilize at 115℃ for 15min.

[0044] Experimental results:

[0045] Comparison of transcription levels of ydgI gene promoter at different time periods Figure 1As shown. At 8h and 12h of fermentation, the transcription level of the ydgI gene promoter in the experimental group (1g / L betaine added) was reduced by 60% and 70% respectively compared with that in the control group (no betaine added); at 16h and 20h of fermentation, the transcription level of the ydgI gene promoter in the experimental group (1g / L betaine added) was increased by 110% and 160% respectively compared with that in the control group (no betaine added). The results show that the addition of betaine in the microbial expression system can significantly regulate the expression intensity of the ydgI gene promoter, and the regulation includes positive regulation and negative regulation, which is specifically manifested in that the expression intensity of the ydgI gene promoter can be significantly reduced in the logarithmic growth period (especially 8-12h) and / or significantly increased in the stable period (especially 16-20h). In addition, according to the strain OD 600 Growth curve Figure 2 As shown, there was no significant difference in the growth of strains between the experimental group and the control group.

[0046] Embodiment 2:

[0047] This embodiment provides a method for regulating the expression level of a target gene in an expression system containing a ydgI gene promoter and a green fluorescent protein gene gfp, comprising the following experimental steps:

[0048] 1) Amplification of ydgI gene promoter and gfp gene and construction of overlapping fragments:

[0049] The primers P were designed based on the promoter sequence of the ydgI gene in the genome of E. coli MG1655. ydgI -up and P ydgI -down; using the gfp gene sequence as a template, the amplification primers gfp-up and gfp-down were designed, and the primer P ydgI -down and gfp-up contain 20 bp overlapping sequence, primer P ydgI The 5' ends of -up and gfp-down contain homologous sequences upstream and downstream of the vector to be cloned. The primers and promoter sequences involved are as follows:

[0050]

[0051]

[0052] PrimeSTAR HSDNA Polymerase was used to amplify the promoter region of the ydgI gene. ydgI and gfp genes, and the PCR products were recovered using a kit to obtain P ydgI and GFP fragments.

[0053] The PCR system is as follows:

[0054]

[0055] PrimeSTAR HSDNA Polymerase was then used to ydgI The fragment and gfp fragment were overlapped by PCR amplification, and the P ydgI -gfp is for standby use.

[0056] The overlapping PCR system is as follows:

[0057]

[0058] 2) Construction of vector pSTV28-P ydgI -gfp:

[0059] The vector pSTV28 and the overlapping fragment P were cloned using the ClonExpress-II One Step Cloning Kit. ydgI -gfp for recombination ligation.

[0060] The recombinant connection system is as follows:

[0061]

[0062] 3) Transformation of recombination ligation products:

[0063] ① Place the recombinant ligation product in a 37°C water bath for 30 minutes, then place it in an ice bath for 5 minutes, add it to the competent E. coli DH5α medium, mix it with a pipette, and continue to place it in an ice bath for 20 minutes;

[0064] ② Place the EP tube in a 42℃ water bath and heat shock for 60s;

[0065] ③Incubate on ice for 2 minutes, add 800 μL of SOC recovery solution, and incubate at 37°C, 200 rpm for 1 hour;

[0066] ④ Centrifuge at 8000rpm for 2min, retain 100μL of liquid, resuspend the bacteria, spread on LB plates containing ampicillin resistance, and culture at 37℃ overnight.

[0067] 4) Verification of positive clones:

[0068] The colony PCR system is as follows:

[0069]

[0070] Use tweezers to hold the toothpick in the clean bench, quickly pass the tip of the toothpick through the flame 2-3 times, and accurately pick a single colony on the plate with a single colony with the tip of the toothpick. First, gently draw a line on the plate with grid lines, and then put the toothpick into the corresponding PCR tube and rinse it several times. After electrophoresis verification, the positive strain was obtained, and the strain with the correct number on the plate was inoculated into the LB shake tube, cultured at 37℃ overnight, and stored in the glycerol tube at -80℃, named E.coliDH5α-pSTV28-P ydgI -gfp.

[0071] Determination of E.coli DH5α-pSTV28-P under the condition of exogenous addition of betaine ydgI The fluorescence intensity of the -gfp strain is shown in Table 1. The results show that when betaine is added to the fermentation system at a final concentration of 1 g / L, E. coli DH5α-pSTV28-P ydgI Compared with the control group (without betaine), the fluorescence intensity of the -gfp strain was significantly weakened at 8-12h of fermentation, indicating that the expression level of the gfp gene was significantly reduced; the fluorescence intensity was significantly enhanced at 16-20h of fermentation, indicating that the expression level of the gfp gene was significantly increased. This experiment further confirmed the precise regulation of the expression level of the target gene controlled by the ydgI gene promoter by betaine.

[0072]

[0073] Table 1 Effect of betaine on E.coli DH5α-pSTV28-P ydgI Effect of -GFP strain on fluorescence intensity

[0074]

[0075] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for regulating the expression intensity of the ydgI gene promoter, characterized in that: The method comprises adding betaine to an expression system containing a ydgI gene promoter, and regulating the expression intensity of the ydgI gene promoter to decrease in the logarithmic phase of microbial growth and / or increase in the stable phase.

2. The method according to claim 1, characterized in that The expression intensity of the ydgI gene promoter decreased by 60%-70% during the logarithmic phase of microbial growth.

3. The method according to claim 1, characterized in that The expression intensity of the ydgI gene promoter is increased by 110%-160% during the stable growth period of the microorganism.

4. The method according to claim 1, characterized in that The expression system is an Escherichia coli expression system.

5. The method according to claim 4, characterized in that The expression intensity of the ydgI gene promoter decreased by 60%-70% during fermentation for 8-12 hours, and increased by 110%-160% during fermentation for 16-20 hours.

6. The method according to claim 1, characterized in that The amount of betaine added is 1 g / L at a working concentration in the fermentation system.

7. The method according to claim 1, characterized in that The nucleotide sequence of the ydgI gene promoter is shown in SEQ ID No.

1.

8. A method for regulating the expression level of a target gene, the method comprising adding betaine to an expression system containing the target gene and its upstream ydgI gene promoter, so as to regulate the expression level of the target gene to decrease in the logarithmic phase of microbial growth and / or increase in the stable phase.

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