WO2022022197A1

WO2022022197A1 - N-terminus coding sequence-based method for modifying regulatory protein expression

Info

Publication number: WO2022022197A1
Application number: PCT/CN2021/102986
Authority: WO
Inventors: 刘松; 徐奎栋; 李江华; 陈坚; 周景文
Original assignee: 江南大学
Priority date: 2020-07-29
Filing date: 2021-06-29
Publication date: 2022-02-03
Also published as: CN111850096A; CN111850096B

Abstract

The present invention relates to the field of genetic engineering. Disclosed is an N-terminus coding sequence-based method for modifying a regulatory protein expression. For the present invention, Bacillus subtilis serves as an expression host, by means of a predictive model, the nucleotide sequence most favorable for promoting gene expression is assessed in an N-terminus coding region synonymous mutation. With the combination of synonymous mutation libraries of the top ten amino acids of the NCS of a superfolder green fluorescent protein (sfGFP), the fluorescent intensity of proteins in the libraries is measured, 172 representative samples are selected, sequenced, and identified, and a statistical method is used to establish a predictive model. Pullulanase of a BlgS signal peptide is optimally integrated via the model, the extracellular enzyme activity of pullulanase can be increased to 2.67 times of that prior to transformation and be reduced by 48%, thus providing a direction for a rational transformation to design an N-terminus gene from scratch, and favoring an easily regulated expression of the gene.

Description

A method for regulating protein expression based on N-terminal coding sequence modification

technical field

The invention relates to a method for regulating protein expression based on the modification of an N-terminal coding sequence, belonging to the technical field of genetic engineering and enzyme engineering

Background technique

Mutation of genes is of great significance for changing the properties of proteins. Usually, mutation sequences with better properties can be found through mutation, thereby improving the application value of proteins. Synonymous mutation of genes is a commonly used mutation method, and the expression levels of synonymous mutations of genes can vary greatly.

The current commonly used method is to construct a synonymous mutation library and combine it with a high-throughput screening strategy to find the best mutant. However, this method is time-consuming, labor-intensive, and specific, and cannot be used to guide the design of other genes. Although some studies have found that synthesizing a series of short peptides is beneficial to widely improve gene expression, this method will have an impact on enzyme activity, because these expression-promoting short peptides occupy the position of the signal peptide, so that the Suitable for extracellular proteins that require the addition of a signal peptide.

Existing methods for improving gene expression are often through the optimization of the untranslated region (5'UTR). However, when the 5'UTR module is strong enough, it is difficult to continue to optimize and significantly increase the expression. However, there are few studies on the N-terminal coding region (NCS). Therefore, it is very important to establish an NCS engineering strategy applicable to a wide range of gene designs.

SUMMARY OF THE INVENTION

The method of the present invention is established based on the bioinformatics analysis of representative samples, and by this method, the nucleotide sequence of the first 30 bases of the N-terminal of any gene can be de novo designed, and synonymous mutation can be performed on it. In the embodiment of this model, the NCS nucleotide sequence of any gene is changed to the target nucleotide sequence by mutating the primers. By optimizing the nucleotide sequence of NCS, the present invention can be used to guide the design of any gene without adding additional amino acid sequence, and the properties of the protein are minimized. It can greatly improve the expression level of the target gene.

The present invention provides a method for screening nucleotide sequences encoding proteins with different expression levels, measuring the values of GC3 and ΔG, and then calculating the relative expression level of the protein by using the following equation, that is, the _PsfGFP value, and screening according to the _PsfGFP value The corresponding nucleotide sequence is obtained; the P _sfGFP value is positively correlated with the actual expression of the protein:

_PsfGFP =274497.657-108717.401×GC3+4886.529×ΔG.

In one embodiment, the GC3 is the content of GC in the first 9n-10n nucleotides of the synonymous codon of the gene encoding the target protein near the N-terminal coding region of ATG, n=3; The ΔG is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region.

In one embodiment, the ΔG is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 96 bp region of the N-terminal coding region.

In one embodiment, the protein is any protein that can be expressed in Bacillus subtilis.

In one embodiment, the protein includes, but is not limited to, pullulanase.

In one embodiment, the amino acid sequence of the pullulanase is shown in SEQ ID NO.19.

In one embodiment, the _PsfGFP value is positively correlated with the actual expression level of the protein.

In one embodiment, the corresponding nucleotide sequences are screened according to the _PsfGFP value.

In one embodiment, the higher the _PsfGFP value, the higher the corresponding protein expression level.

The invention provides a method for regulating the protein expression of genetically engineered bacteria. The length of the N-terminal coding region of the target protein is selected to be 9n-10n nucleotides, and n=3, and a synonymous mutation library is established; GC3 and ΔG parameters of the gene, calculate the relative expression level of each nucleotide sequence according to the equation, select the nucleotide sequence with the required expression level, mutate the N-terminal coding region of the target protein accordingly, and transform it into in host cells;

The equation is: P _sfGFP =274497.657−108717.401×GC3+4886.529×ΔG.

In one embodiment, the GC3 is the content of GC in the first 9n-10n nucleotides of the synonymous codon of the gene encoding the target protein near the N-terminal coding region of ATG, n=3.

In one embodiment, the ΔG is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region.

In an embodiment of the present invention, when the protein expression needs to be up-regulated, the nucleotide sequences whose P _sfGFP value in the mutation library is in the top 10% are selected; when the protein expression needs to be down-regulated, the P sfGFP value in the mutation library is selected. _sfGFP values are in the bottom 10% of nucleotide sequences.

In one embodiment, the first 9n-10n nucleotides of the gene encoding the protein of interest near the N-terminal coding region of ATG are mutated to the corresponding nucleotide sequence, n=3.

In one embodiment, the genetically engineered bacteria use Bacillus subtilis as a host.

In one embodiment, the protein includes, but is not limited to, pullulanase.

The invention provides a method for regulating the expression level of pullulanase. The first 9n to 10n nucleotides in the N-terminal coding region of pullulanase are selected, n=3, and synonymous mutation is performed to construct a mutant library, and Calculate the P _sfGFP value, select the corresponding synonymous mutation sequence according to the P _sfGFP value; mutate the N-terminal coding region of the target protein correspondingly, connect it to the expression vector, and construct a recombinant plasmid;

The P _sfGFP value is calculated as follows: P _sfGFP =274497.657-108717.401×GC3+4886.529×ΔG;

The GC3 is the first 9n～10n nucleotides of the gene encoding the target protein near the N-terminal coding region of the ATG synonymous codon, and the third base is the content of GC, n=3;

The ΔG is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region.

In one embodiment, when the expression level of pullulanase needs to be up-regulated, the nucleotide sequence with the _PsfGFP value in the top 10% of the mutation library is selected, and the gene encoding the target protein is placed before the N-terminal coding region of ATG. 9n-10n nucleotides were mutated to the corresponding nucleotide sequence, n=3.

In one embodiment, when the expression level of pullulanase needs to be down-regulated, the nucleotide sequence whose _PsfGFP value in the mutation library is in the lower 10% is selected, and the gene encoding the target protein is placed before the N-terminal coding region of ATG. 9n-10n nucleotides were mutated to the corresponding nucleotide sequence, n=3.

In one embodiment, the recombinant plasmid is introduced into Bacillus subtilis, and the Bacillus subtilis is used to produce the protein.

The present invention also protects the application of the method for screening nucleotide sequences encoding high-expression proteins, or the method for regulating the protein expression of genetically engineered bacteria in regulating the expression of a target protein.

The present invention also protects the application of the method for regulating the expression of pullulanase in regulating pullulanase.

Beneficial effects of the present invention:

By combining sfGFP and modifying the N-terminal coding region of the target gene (synonymous mutation), the present invention explores a formula for instructing the protein to make directional modification, thereby increasing or reducing the expression of the target protein PsfGFP=274497.657-108717.401 ×GC3+4886.529×ΔG. The calculated PsfGFP value is positively correlated with the actual expression level of the protein. According to this formula, to calculate the PsfGFP value is to select the corresponding synonymous mutation sequence as needed. It was applied to transform the N-terminus of pullulanase fused to the nucleotide sequence of the Bgls signal peptide, and the selected synonymous mutation sequence could increase the extracellular enzyme activity by 2.67 times and decrease it by 48%.

Description of drawings

Figure 1 is the map of the sfGFP expression plasmid P43-NMK-sfGFP.

Figure 2 is a graph showing the relative fluorescence intensity of the NCS library of sfGFP.

Figure 3 shows the nucleotide sequence indices and fluorescence values of the 1st to 60th samples among the 172 samples.

Figure 4 shows the nucleotide sequence indices and fluorescence values of the 61st to 120th samples among the 172 samples.

Figure 5 shows the nucleotide sequence indexes and fluorescence values of the 121st to 172nd samples in the 172 samples.

Figure 6 is the distribution of relative fluorescence values before and after transformation.

Figure 7 is the map of pullulanase expression plasmid P43-NMK-Bgls fused with BglS signal peptide.

Figure 8 is a protein gel image of the 5 NCS variants of the BglS signal peptide.

Figure 9 is a graph showing the correlation between the predicted expression value of pullulanase with the addition of five Bgls signal peptide sequences and the measured value of enzyme activity.

detailed description

1. Culture medium composition:

Seed medium (g/L): peptone 10, yeast extract 5, sodium chloride 5;

Fermentation medium (g/L): The following components were dissolved in 0.9L water: peptone 12g, yeast extract 24g, glycerol 4mL.

After each component is dissolved, autoclave; cool to 60°C, add 100mL of sterilized 0.17mol/L KH2PO4, 0.72mol/L K2HPO4 solution (2.31g KH2PO4 and 12.54g K2HPO4 are dissolved in enough water , make the final volume 100mL; filter sterilization with 0.22μm filter);

2. Cultivation method:

Seed culture: Pick a single colony of engineering bacteria and insert it into the seed medium, the culture temperature is 37°C, the shaking speed is 200r/min, and the culture is 24h;

Fermentation culture: The seed culture liquid is inserted into the fermentation medium according to the inoculum amount of 4%, the culture temperature is 37 °C, and the fermentation is carried out for 24 hours.

3. Green fluorescent protein expression and biomass determination

Add the fermentation broth diluted with PBS buffer (100mM and pH 7.2) to the appropriate concentration in the 96-well plate, use Cytation3 cell imaging microplate detector (Berton Instrument Co., Ltd., USA), green fluorescence excitation wavelength: 480nm, green Fluorescence emission wavelength: 520nm, cell growth OD absorption wavelength: 600nm.

One-step cloning kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.

4. SDS-PAGE electrophoresis detection

Glue concentration of 10%

SDS-PAGE gel was used to analyze the protein expression level. MES or MOPS buffer was used as the running buffer, and the loading volume was 10 μL. The electrophoresis voltage was 150V. Specific sample preparation and electrophoresis operations were performed according to the kit instructions. When electrophoresed in MES buffer, the molecular weights (kDa) of the standard protein were: 188, 98, 62, 49, 38, 28, 17, 14, 6 and 3; and when electrophoresed in MOPS buffer, the molecular weight of the standard protein was Molecular weights (kDa) are: 191, 97, 64, 51, 39, 28, 19, 14

5. Assay method of pullulanase activity

Mix 1mL 1g/100mL pullulan polysaccharide substrate and 0.9mL 100mM pH 4.5 acetic acid-sodium acetate buffer evenly, place it in a 60°C water bath to preheat for 10min, add 0.1mL pullulan enzyme solution, react for 10min, Add 3 mL of DNS color developing solution, then boil in a boiling water bath for 7 min, place in ice water to stop the color developing reaction, add 10 mL of deionized water, mix well, and measure the absorbance at 540 nm. The amount of enzyme that generates 1 μmol of reducing sugar per unit time is defined as one unit of enzyme activity.

Example 1: Construction of NCS synonymous mutation library

The PLytr promoter (nucleotide sequence shown in SEQ ID NO.1) was used with primers Lytr-F/Lytr-R (nucleotide sequence shown in SEQ ID NO.2 and 3) and Lytr-F-plasmid/ Lytr-R-plasmid (nucleotide sequence shown in SEQ ID NO. 4 and 5) was connected to the P43NMK plasmid by a one-step cloning kit to construct the plasmid P43NMK-Lytr;

Using the same method, the sfGFP fluorescent protein reporter gene (nucleotide sequence shown in SEQ ID NO.6) was used primers sfGFP-F/sfGFP-R (nucleotide sequence shown in SEQ ID NO.7 and 8) and sfGFP-F-plasmid/sfGFP-R-plasmid (nucleotide sequences shown in SEQ ID NO. 9 and 10) were fused to the downstream of PLytr by a one-step cloning kit to obtain the construction of P43NMK-Lytr_sfGFP, as shown in Figure 1. Show;

Using P43NMK-Lytr_sfGFP as a template and using degenerate primers sfGFP-F-NCS/sfGFP-R-NCS (nucleotide sequences shown in SEQ ID NO. 11 and 12), the first 30 bases of the N-terminal of sfGFP were obtained. Synonymous mutant recombinant plasmids, these recombinant plasmids constitute a synonymous mutant library, which changes the first 30 bases of sfGFP, but the encoded amino acid sequence remains unchanged.

Example 2: Characterization of NCS Synonymous Mutation Libraries

The recombinant plasmids with synonymous mutations constructed in Example 1 were transformed into the expression host Bacillus subtilis WB600, respectively, and the transformed single clones were inoculated into 96 shallow-well plates containing 200 μL of LB seed medium, and cultured for 8 hours;

Then, according to the inoculation amount of 4mL/100mL, it was inoculated into a 96 deep-well plate containing 800μL of TB medium, and cultured for 24 hours to obtain a fermentation broth;

Then the fermentation broth was quickly frozen on ice, and after centrifugation, the supernatant was removed, diluted to an appropriate multiple with PBS buffer (100 mM, pH 7.2), and passed through a Cytation3 cell imaging microplate detector (Borton Instrument Co., Ltd., USA). ) to measure the fluorescence value (excitation light 480, absorption light 520) and OD ₆₀₀ . A total of 8598 single colonies were characterized as shown in Figure 2.

Example 3: Sequence identification and fermentation of representative samples

A total of 8598 monoclonal host cells were characterized in Example 2, and the fluorescence value/OD was defined as the relative fluorescence intensity RFI. According to the level of the RFI value, the monoclonal cells were sorted from high to low, and every 50 cells were selected for sequencing identification (that is, the first One of the 1 to 50 strains was selected, one of the 51 to 100 strains was selected, and so on), and a total of 172 single clones were identified by sequencing.

172 single clones identified by sequencing were inoculated into 250 mL shake flasks containing 20 mL of seed medium, fermented at 37°C and 220 rpm for 8 hours until OD ₆₀₀ was greater than 4, and inoculated into 25 mL of fermentation culture at a ratio of 4 mL/100 mL. The fluorescence value and OD ₆₀₀ of sfGFP were measured after 24 hours of fermentation in a 250 mL shake flask based on the sfGFP. Each group of experiments was set up in 3 parallels. The results are shown in Figures 3 to 5 below.

Example 4: Sequence analysis of nucleotides of samples using bioinformatics tools

11 different nucleotide sequence metrics were created for sequence analysis using CodonW, Nupack, RBS calculator.

(1) Use CodonW to calculate GC, GC3, T3s, C3s, A3s, G3s, CAI, CBI, Fop

GC: G+C content of the target gene;

GC3: The third base of the synonymous codon is the content of GC;

T3s, C3s, A3s, G3s: After a synonymous mutation occurs at the first 30 bases of the N-terminal of the gene, the third synonymous codon is the frequency of T, C, A, and G, respectively;

CAI: codon preference;

CBI: codon preference index;

Fop: frequency of optimal codons (both above calculated ranges are 30 nucleotide sequences for NCS mutations).

(2) Calculate ΔG using Nupack

ΔG: the minimum free energy, the calculated range includes the region from the transcription start site to the downstream of NCS, in this example, 25 bases upstream of ATG (the transcription start site of the PLytr promoter) to 96 bases downstream of ATG were selected base;

(3) Calculate TIR using RBS calculator

TIR: translation initiation rate, the range is the same as the calculation ΔG.

By analyzing the 172 samples with RFI as the dependent variable and 11 nucleotide sequence indicators as the dependent variable, the multiple regression analysis was performed by SPSS, and the method was stepwise regression.

Finally, a regression prediction equation PsfGFP=274497.657-108717.401×GC3+4886.529×ΔG was obtained, see Table 1. And it is used to guide the NCS transformation of the gene. When the NCS is transformed, the corresponding parameters are calculated and brought into the formula, that is, the synonymous mutation sequence with high protein expression can be selected according to the calculated value.

Table 1 Multiple regression analysis

Substitute the sequence of 172 samples in Example 3 into the regression prediction equation, calculate the predicted value, and compare it with the actual fluorescence value measured in Example 3, and perform correlation analysis. As shown in Figure 6, the sequence of The Pearson coefficient between the predicted value and the measured fluorescence value can reach 0.675, and the correlation is very strong, indicating that the regression prediction equation can be used to predict the protein fluorescence value.

Example 5: Using a prediction equation to guide NCS engineering of the signal peptide BglS gene

(1) Construction of P43NMK-Lytr_BglS wild type

The BglS signal peptide (nucleotide sequence shown in SEQ ID NO. 13) was fused to the N-terminal of the pullulanase encoding gene (nucleotide sequence shown in SEQ ID NO. 14) to achieve pullulanase of extracellular expression. The specific method is to clone the BglS signal peptide into the downstream of PLytr in P43NMK-Lytr by using the same one-step cloning method in the example to construct P43NMK-Lytr-BglS, as shown in FIG. 7 .

(2) Construction of P43NMK-Lytr_BglS synonymous mutant plasmid

In order to further improve the extracellular activity of pullulanase, the NCS region of BglS close to ATG was optimized: all the synonymous mutation combinations of the first ten amino acids of BglS were exhausted, and there were 131,072 possibilities; according to the examples 4 equations were used to calculate the GC3 and ΔG of each of the 131072 sequences and the theoretical value of PsfGFP, and according to the predicted value, 5 Bgls variants including wild type were selected: NCS+, NCS+', NCS-wt, NCS -', NCS-.

NCS+ represents the P _sfGFP maximum variant; NCS+' represents the intermediate variant between the maximum and wild type of P _sfGFP ; NCS-wt represents the wild type; NCS- represents the P _sfGFP minimum variant, and NCS-' represents the intermediate variant Between the minimum of _PsfGFP and the intermediate value variant of wild type, it has continuously decreasing predicted expression intensity.

Using the same method as step (1), signal peptide Bgls variants NCS+ (nucleotide sequence shown in SEQ ID NO. 15), NCS+' (nucleotide sequence shown in SEQ ID NO. 16), NCS- ' (the nucleotide sequence is shown in SEQ ID NO. 17), NCS- (the nucleotide sequence is shown in SEQ ID NO. 18), respectively connected to the downstream of the PLytr cloned into P43NMK-Lytr, respectively to obtain The plasmid containing the synonymous mutant sequence of BglS signal peptide; then transform the obtained plasmid into the expression host Bacillus subtilis WB600, inoculate the transformed single clone into a 250 mL shake flask containing 20 mL of LB medium, and ferment at 37°C and 220 rpm for 8 hours Then, make the OD ₆₀₀ reach 4 or more, inoculate it into a 250 mL shake flask containing 25 mL of TB medium at a ratio of 4 mL/100 mL, and ferment at 37 °C and 250 rpm for 30 hours to measure the extracellular enzyme activity of pullulanase. As shown in Figure 9, it was found that its pullulanase extracellular enzyme activity achieved the predicted change in the high, medium and low 5 levels, and had an R ² level of 0.89 with the predicted value.

Table 2 Prediction and actual detection results of NCS mutants of signal peptide BglS

	预测荧光值(×10 ³) Predicted fluorescence value (×10 ³ )	发酵酶活Fermentation enzyme activity
NCS+NCS+	174.32174.32	41.34U/ml41.34U/ml
NCS+’NCS+’	145.86145.86	28.18U/ml28.18U/ml
NCS-wtNCS-wt	117.64117.64	18.26U/ml18.26U/ml
NCS-’NCS-'	63.5363.53	13.44U/ml13.44U/ml
NCS-NCS-	9.419.41	5.83U/ml5.83U/ml

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims

A method for regulating the expression level of pullulanase, characterized in that, selecting the first 9n to 10n nucleotides in the N-terminal coding region of pullulanase, n=3, performing synonymous mutation, constructing a mutant library, and calculating P sfGFP value, select the corresponding synonymous mutation sequence according to the P sfGFP value; carry out the corresponding mutation in the N-terminal coding region of the target protein, connect it to the expression vector, and construct a recombinant plasmid;

The P sfGFP value is calculated as follows: P sfGFP =274497.657-108717.401×GC3+4886.529×ΔG;

The GC3 is a synonymous codon of the first 9n-10n nucleotides of the gene encoding the target protein close to the N-terminal coding region of ATG, and the third base is the content of GC, n=3;

The ΔG is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region;

When the expression level of pullulanase needs to be up-regulated, select the nucleotide sequence with the PsfGFP value in the top 10% in the mutation library;

When the expression level of pullulanase needs to be down-regulated, select the nucleotide sequence whose PsfGFP value in the mutation library is in the lower 10%;

The recombinant plasmid was introduced into Bacillus subtilis, and the Bacillus subtilis was used to produce the protein; the amino acid sequence of the pullulanase was shown in SEQ ID NO.19.
A method for screening nucleotide sequences encoding proteins with different expression levels, characterized in that the values of GC3 and ΔG are measured, and the relative expression level of the protein, that is, the PsfGFP value, is calculated by applying the following equation, and screening is performed according to the PsfGFP value. The corresponding nucleotide sequence is obtained; the P sfGFP value is positively correlated with the actual expression of the protein:

P sfGFP = 274497.657－108717.401×GC3+4886.529×ΔG;

The GC3 is the synonymous codon of the first 9n-10n nucleotides of the gene encoding the target protein close to the N-terminal coding region of ATG. The third base is the content of GC, n=3; the ΔG is the amount of the target gene. The minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter and the 90-99 bp region of the N-terminal coding region.
The method according to claim 2, wherein the ΔG is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the target gene and the 96 bp region of the N-terminal coding region.
The method of claim 3, wherein the protein is any protein that can be expressed in Bacillus subtilis.
The method of claim 4, wherein the protein includes but is not limited to pullulanase.
The method according to claim 5, wherein the amino acid sequence of the pullulanase is as shown in SEQ ID NO.19.
The method according to claim 5, wherein the corresponding nucleotide sequence is screened according to the PsfGFP value.
A method for regulating the protein expression of genetically engineered bacteria, characterized in that, taking the first 9n to 10n nucleotides in the N-terminal coding region of a target protein, n=3, to establish a synonymous mutation library; and calculating the genes in the synonymous mutation library The parameters GC3 and ΔG, calculate the relative expression level of each nucleotide sequence according to the equation, select the nucleotide sequence with the required expression level, mutate the N-terminal coding region of the target protein accordingly, and transform it into host cells middle;

The equation is: P sfGFP =274497.657－108717.401×GC3+4886.529×ΔG;

The GC3 is the synonymous codon of the first 9n-10n nucleotides of the gene encoding the target protein close to the N-terminal coding region of ATG. The third base is the content of GC, n=3; the ΔG is the amount of the target gene. The minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter and the 90-99 bp region of the N-terminal coding region;

When the protein expression needs to be up-regulated, select the nucleotide sequence whose PsfGFP value in the mutation library is in the top 10%;

When the protein expression needs to be down-regulated, select the nucleotide sequence whose PsfGFP value in the mutation library is in the lower 10%.

The ΔG is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the target gene and the 96 bp region of the N-terminal coding region.
The method according to claim 8, wherein the genetically engineered bacteria use Bacillus subtilis as a host.
The method of claim 9, wherein the protein is any protein that can be expressed in Bacillus subtilis.
The method of claim 10, wherein the protein includes but is not limited to pullulanase.
The method according to claim 11, wherein the amino acid sequence of the pullulanase is shown in SEQ ID NO.19.
A method for regulating the expression level of pullulanase, characterized in that, selecting the first 9n to 10n nucleotides in the N-terminal coding region of pullulanase, n=3, performing synonymous mutation, constructing a mutant library, and calculating P sfGFP value, select the corresponding synonymous mutation sequence according to the P sfGFP value; carry out the corresponding mutation in the N-terminal coding region of the target protein, connect it to the expression vector, and construct a recombinant plasmid;

When the expression level of pullulanase needs to be up-regulated, select the nucleotide sequence with the PsfGFP value in the top 10% in the mutation library;

When the expression level of pullulanase needs to be down-regulated, select the nucleotide sequence whose PsfGFP value in the mutant library is in the lower 10%.
The method according to claim 13, wherein the recombinant plasmid is introduced into Bacillus subtilis, and the Bacillus subtilis is used to produce the protein.
The method according to claim 14, wherein the amino acid sequence of the pullulanase is shown in SEQ ID NO.19.
Application of any one of the methods of claims 1 to 12 in regulating the expression level of a target protein.