Fluorescent protein selection marker staygold-ygjHr gene and application thereof

Abstract

The invention provides a fluorescent protein selection marker staygold-ygjHr gene and application thereof, and belongs to the technical field of biological engineering. The preparation method comprises the following steps: replacing codons of L-valine in nucleotide sequences of a fluorescent protein gene Staygold and a gene ygjH by using a rare codon GTC to respectively obtain a fluorescent protein gene Staygoldr after codon replacement and a gene ygjHr after codon replacement, and connecting the two segments of genes after codon replacement by using a flexible protein peptide to obtain the fluorescent protein selection marker Staygold-ygjHr gene. Experiments prove that the gene can obviously improve the screening efficiency of L-valine high-yield strains.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a fluorescent protein screening marker staygold-ygjH r gene and its application. Background Art

[0002] The production of amino acids by microbial fermentation is an efficient and environmentally friendly biotechnology that utilizes the metabolic capabilities of microorganisms to synthesize target amino acids. Since microbial fermentation has been applied to the production of L-valine, continuous technological innovation and optimization have been carried out in this field. Nowadays, microbial fermentation has become the mainstream method for L-valine production. The core of microbial fermentation lies in utilizing the metabolic capabilities of microorganisms to convert externally provided carbon sources into bioactive molecules such as amino acids. Currently, through gene editing techniques such as the CRISPR-Cas9 system, or through chemical and physical mutagenesis treatments, auxotrophic strains that can relieve feedback repression and feedback inhibition in metabolic regulation or strains resistant to L-valine structural analogs can be selected. These strains can strengthen the L-valine synthesis pathway in the microbial metabolic pathway, thereby increasing the accumulation and yield of L-valine. Nevertheless, the methods for effectively screening high-yield strains are still limited and further research and development are needed.

[0003] Currently, a large-capacity L-valine strain mutant library containing positive and negative mutations can be constructed by physical or chemical mutagenesis using the L-valine structural analog aminoethylcysteine. However, the limitations of traditional screening methods result in low screening efficiency for high-yield strains of certain amino acids. For example, high concentrations of amino acid analogs can interfere with cell metabolism and even affect cell structure. The long-term use of a limited variety of analogs makes it difficult to obtain new mutant strains from the screened strains, and it is difficult to meet the requirements of industrial production. Therefore, a screening marker that can be widely applied to different types of microbial cells is needed for high-throughput screening of L-valine production strains. With the continuous increase in the demand for L-valine, breeding high-yield, low-cost, and genetically stable production strains has become a key goal in the industry. There are few screening markers developed for L-valine production strains, so designing L-valine screening markers with high specificity and high performance is a technical problem to be solved at the present stage. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a fluorescent protein screening marker staygold-ygjH r gene and its application. This fluorescent protein screening marker staygold-ygjH r gene can significantly improve the screening efficiency of L-valine high-yield strains and enhance the sensitivity and accuracy.

[0005] To solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a fluorescent protein screening marker staygold-ygjH r gene, and the fluorescent protein screening marker staygold-ygjH r gene is formed by connecting the staygold r gene and the ygjH r gene with a flexible protein peptide; the staygold r gene is the gene after replacing the L-valine codon in the Staygold gene with GTC, and the ygjH r gene is the gene after replacing the L-valine codon in the ygjH gene with GTC.

[0006] Preferably, the L-valine codons in the Staygold gene and the ygjH gene are GTT, GTA, and GTG.

[0007] Preferably, the nucleotide sequence of the staygold r gene is as shown in SEQ ID NO.1; the nucleotide sequence of the ygjH r gene is as shown in SEQ ID NO.2; the nucleotide sequence of the flexible protein peptide is as shown in SEQ ID NO.4.

[0008] Preferably, the nucleotide sequence of the fluorescent protein screening marker staygold-ygjH r gene is as shown in SEQ IDNO.3.

[0009] The present invention provides a construction method for the fluorescent protein screening marker staygold-ygjH r gene, which includes the following steps: replacing the L-valine codons in Staygold and ygjH with GTC to obtain the Staygold r gene after codon replacement and the ygjH r gene after codon replacement, and connecting the two genes after codon replacement with a flexible protein peptide to obtain the fluorescent protein screening marker staygold-ygjH r gene.

[0010] The present invention provides a recombinant vector, and the recombinant vector contains the fluorescent protein screening marker staygold-ygjH r gene.

[0011] Preferably, the starting vectors of the recombinant vector include pET-22b(+) or pUC-57(+).

[0012] The present invention provides a recombinant bacterium, which contains the fluorescent protein screening marker staygold-ygjH r gene or the recombinant vector.

[0013] Preferably, the starting strain of the recombinant bacterium includes Escherichia coli CGMCC NO.1.366 or Escherichia coli E.coli BL21.

[0014] The present invention provides the application of the fluorescent protein screening marker staygold-ygjH r gene, the recombinant vector or the recombinant bacterium in screening high-yield L-valine strains.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a fluorescent protein screening marker staygold-ygjH r gene for screening L-valine-producing strains. Compared with other similar gene screening markers, the fluorescence intensity of this screening marker gene shows a positive correlation with the L-valine concentration, which can significantly improve the screening efficiency of high-yield L-valine strains, and this screening marker gene can save screening time and cost.

[0016] Furthermore, the fluorescent protein screening marker staygold-ygjH r gene of the present invention replaces the codon of L-valine in the original nucleotide sequence with the rare codon GTC, and prepares the screening marker by fusing two genes, which can enhance the sensitivity, improve the screening efficiency and accuracy. Description of the Drawings

[0017] Figure 1 It is an agarose gel electrophoresis diagram of PCR amplification products. Among them, lanes 1-4 are all Staygold-ygjH r .

[0018] Figure 2 It is a fluorescence intensity curve diagram of the recombinant bacterium E.coli BL21(DE3) / pUC-57(+)-staygold-ygjH r under different L-valine concentrations.

[0019] Figure 3 It is a fluorescence intensity curve diagram of the recombinant bacterium E.coli BL21(DE3) / pUC-57(+)-staygold-rplW r under different L-valine concentrations.

[0020] Figure 4Fluorescence intensity curves of recombinant E. coli BL21(DE3) / pUC-57(+)-staygold at different L-valine concentrations r .

[0021] Figure 5 Scatter plot of L-valine production of sorted Escherichia coli mutant strains Specific implementation mode

[0022] The present invention provides a fluorescent protein screening marker staygold-ygjH r gene, and the fluorescent protein screening marker staygold-ygjH r gene is formed by connecting the staygold r gene and the ygjH r gene with a flexible protein peptide; the staygold r gene is the gene after replacing the L-valine codon in the Staygold gene with GTC, and the ygjH r gene is the gene after replacing the L-valine codon in the ygjH gene with GTC. The GTC in the present invention is a rare codon. The L-valine codons in the Staygold gene and the ygjH gene in the present invention are GTT, GTA, and GTG.

[0023] In the present invention, the nucleotide sequence of the staygold r gene is shown in SEQ ID NO.1; the nucleotide sequence of the ygjH r gene is shown in SEQ ID NO.2; the nucleotide sequence of the flexible protein peptide is shown in SEQ ID NO.4. The nucleotide sequence of the fluorescent protein screening marker staygold-ygjH r gene in the present invention is shown in SEQ ID NO.3.

[0024] The present invention also provides a construction method of the fluorescent protein screening marker staygold-ygjH r gene, which includes the following steps: replacing the L-valine codons in Staygold and ygjH with GTC to obtain the Staygold r gene after codon replacement and the ygjH r gene after codon replacement, and connecting the Staygold r gene and the ygjH r gene with a flexible protein peptide to obtain the fluorescent protein screening marker staygold-ygjH r gene. The Staygold rThe nucleotide sequence of the gene is shown in SEQ ID NO.1; the ygjH r The nucleotide sequence of the gene is shown in SEQ ID NO.2; the nucleotide sequence of the flexible protein peptide is shown in SEQ ID NO.4. The fluorescent protein screening marker staygold-ygjH of the present invention r The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0025] The present invention also provides a recombinant vector, which contains the fluorescent protein screening marker staygold-ygjH r gene. The starting vectors of the recombinant vectors of the present invention include pET-22b(+) or pUC-57(+).

[0026] The present invention also provides a recombinant bacterium, which contains the fluorescent protein screening marker staygold-ygjH r gene or the recombinant vector. The starting strains of the recombinant bacteria of the present invention include Escherichia coli CGMCC NO.1.366 or Escherichia coli E.coli BL21.

[0027] The present invention also provides the application of the fluorescent protein screening marker staygold-ygjH r gene or the recombinant vector or the recombinant bacterium in screening high-yield L-valine strains.

[0028] In the present invention, unless otherwise specified, all components, reagents or culture media are commercially available products well-known to those skilled in the art.

[0029] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the examples in the present invention. Obviously, the described examples are only a part of the examples of the present invention, rather than all the examples. All other examples obtained by those of ordinary skill in the art based on the examples in the present invention without creative work shall fall within the protection scope of the present invention.

[0030] The vectors and host bacteria involved in the examples of the present invention are all purchased from Sangon Biotech (Shanghai) Co., Ltd. and are ordinary commercially available products. Escherichia coli CGMCC NO.1.366 is purchased from the China General Microbiological Culture Collection Center.

[0031] Example 1 Construction of the staygold-ygjH r fragment The nucleotide sequence of the fluorescent protein gene Staygold was retrieved from the National Center for Biotechnology Information (NCBI) database with the NCBI accession number: LC601652.1. This sequence contains 11 L-valine codons, including 3 codons of GTC, 5 codons of GTT, 2 codons of GTA, and 1 codon of GTG. The L-valine codons GTT, GTA, and GTG in the sequence were replaced with the rare codon GTC of E. coli L-valine, and the resulting staygold r The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0032] The nucleotide sequence of the gene ygjH with a high proportion of L-valine codons was selected from the genome of Escherichia coli CGMCC NO.1.366. This sequence contains 16 L-valine codons, including 3 codons of GTC, 1 codon of GTT, 3 codons of GTA, and 9 codons of GTG. The L-valine codons GTT, GTA, and GTG in the sequence were replaced with the rare codon GTC of E. coli L-valine, and the resulting ygjH r The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0033] The ygjH r gene was linked to the fluorescent protein gene Staygold r through a flexible linker peptide and synthesized to obtain the staygold-ygjH r fragment. The nucleotide sequence of the obtained staygold-ygjH r fragment is shown in SEQ ID NO.3. Among them, the nucleotide sequence encoding the flexible linker peptide is shown in SEQ ID NO.4.

[0034] Example 2 1. Construction of recombinant plasmid The vector plasmid pUC-57(+) was treated with the double digestion reaction system of FastDigest EcoRI and FastDigest HindIII, and then ligated with the staygold-ygjH r fragment that was also digested with FastDigest EcoRI and FastDigest HindIII through a seamless cloning system to obtain the recombinant vector pUC-57(+)-staygold-ygjH r .

[0035] Among them, the double digestion reaction system is shown in Table 1, and the total system is 20 μL; the double digestion reaction conditions are as follows: React at 37°C for 30 min; inactivate at 80°C for 5 min, and store at 4°C. The seamless cloning procedure is as follows: perform the recombination reaction at 50°C for 15 min, then cool to 4°C or immediately place on ice.

[0036] The seamless cloning system is shown in Table 2, and the total volume of the system is 10 μL.

[0037] Table 1 Double digestion reaction system

[0038] Table 2 Seamless cloning system

[0039] 2. Transformation of competent cells Take 10 μL of the recombinant vector pUC-57(+)-staygold-ygjH r and add it to 100 μL of E. coli BL21(DE3) competent cells. Gently flick the tube wall to mix, let it stand on ice for 30 min, heat shock in a 42°C water bath for 90 s, immediately place on ice and let it stand for 2 - 3 min. Add 900 μL of LB liquid medium without antibiotics to the centrifuge tube, and incubate in a shaker at 37°C and 200 r / min for 1 h. Centrifuge the incubated bacterial solution at 5000 r / min for 2 min. After discarding 900 μL of the supernatant, resuspend the remaining bacteria and evenly spread them on an LB solid medium plate containing 100 μg / mL ampicillin using a sterile spreading rod. Incubate the plate upside down in a 37°C constant temperature incubator for 12 - 16 h.

[0040] The formula for the above LB liquid medium is: yeast extract 0.5%, peptone 1%, sodium chloride 1%, solvent water. The formula for the LB solid medium is: yeast extract 0.5%, peptone 1%, sodium chloride 1%, solvent water, agar powder 2%.

[0041] 3. Screening and verification of positive colonies Pick the positive recombinant colonies from step 2, inoculate them into an LB liquid medium containing 100 μg / mL ampicillin, and culture overnight at 37°C. After the culture is completed, use the bacterial solution as a template and perform PCR amplification with P1 and P2 as primers to obtain the amplified product gene Staygold-ygjH rThe primer sequences are: P1: 5’-ATGGAAACCGTCGCTTACGC-3’ (SEQ ID NO.6) and P2: 5’-TTATAAGTGTGCTTCGAGAGTCTC-3’ (SEQ ID NO.7). The PCR amplification system is shown in Table 3, and the total volume of the system is 20 μL. The PCR amplification procedure is as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 1 min, extension at 72°C for 4 min, for 30 cycles; extension at 72°C for 10 min, and preservation at 4°C.

[0042] Table 3 PCR Amplification System

[0043] The amplified product was verified by agarose gel electrophoresis. The results showed that a specific gene band could be amplified using primers P1 and P2, with a size of approximately 1000 bp, as shown in Figure 1 , which was close to the theoretical value of 1026 bp, indicating that the staygold-ygjH r fragment was successfully inserted, and the recombinant bacterium E. coli BL21 (DE3) / pUC-57(+)-staygold-ygjH r was obtained.

[0044] Example 3 The recombinant bacterium E. coli BL21 (DE3) / pUC-57(+)-staygold-ygjH r successfully verified in Example 2 was streaked on an LB solid medium, and a single colony was picked and inoculated into an LB liquid medium. It was cultured at 37°C and 200 r / min until the OD 600 was approximately 1.0. It was then inoculated into an LB liquid medium containing 50 mL with a final concentration of 100 μg / mL ampicillin at an inoculation amount of 2% by volume and cultured at 37°C and 200 r / min until the OD 600 was approximately 1.0. After adding L-valine with final concentrations of 0, 0.1, 0.2, 0.3, and 0.4 g / L to the medium, IPTG with a final concentration of 0.25 μM was added to induce the expression of the fluorescent protein. The control group did not add IPTG. Three parallel experiments were set for each gradient. The induction conditions were culturing at 28°C and 200 r / min for 18 h. After the induction ended, a microplate reader was used to measure the fluorescence intensity at an excitation wavelength of 488 nm and a detection wavelength of 535 nm. The relationship between the added concentration of L-valine and the fluorescence intensity is shown in Figure 2 , and the results showed a positive correlation between the fluorescence intensity and the added amount of L-valine.

[0045] Comparative Example 1 Screen the gene rplW with the second highest proportion of L-valine in the amino acid sequence from the genome of Escherichia coli CGMCC NO.1.366. This sequence contains 14 L-valine codons, including 4 codons of GTC, 6 codons of GTT, 2 codons of GTA, and 2 codons of GTG. Replace the L-valine codons GTT, GTA, and GTG in the sequence with the rare codon GTC of Escherichia coli L-valine. After replacement, rplW r The nucleotide sequence of the gene is shown in SEQ ID NO.5. According to the methods described in Examples 1 and 2, obtain staygold-rplW r fragment by gene synthesis, and construct the recombinant vector pUC-57(+)-staygold-rplW r and the recombinant bacterium. Then, according to the method of Example 3, detect the correlation between the fluorescence intensity of gene expression and the concentration of L-valine in the fermentation broth as shown in Figure 3 , and the results show that there is no obvious correlation between the fluorescence intensity of the transformed strain containing staygold-rplW r fluorescent screening marker and the concentration of L-valine in the fermentation broth.

[0046] Comparative Example 2 The staygold r gene after replacement with the rare codon GTC (the staygold r gene constructed in Example 1). According to the methods described in Examples 1 and 2, obtain staygold r fragments by gene synthesis respectively, and construct the recombinant vector pUC-57(+)-staygold r and the recombinant bacterium. Then, according to the method of Example 3, detect the correlation between the fluorescence intensity of gene expression and the concentration of L-valine in the fermentation broth as shown in Figure 4 , and the results show that there is no obvious correlation between the fluorescence intensity of the transformed strain containing staygold r fluorescent screening marker and the concentration of L-valine in the fermentation broth.

[0047] Experimental Example 1 Application of the fluorescent protein screening marker gene staygold-ygjH r (1) Transformation with the fluorescent protein screening marker Take 10 μL of the recombinant vector pUC-57(+)-staygold-ygjH r constructed above, transform it into the competent cells of L-valine-producing Escherichia coli CGMCC NO.1.366, and culture for 12 - 16 h. The operation steps are the same as those in Example 2.

[0048] ​(2) Screening of positive colonies Pick out the positive recombinant colonies in step (1) and verify them. The steps are the same as those in Example 2 to obtain the recombinant strain CGMCC NO.1.366 / pUC-57(+)-staygold-ygjH r .

[0049] (3) ARTP mutagenesis of recombinant strains Pick out the single colonies in step (2) and inoculate them into LB liquid medium containing 100 μg / mL ampicillin. Culture at 37 °C and 200 r / min until OD 600 is 0.6 - 0.8, and expose to ARTP for 80 s. The ARTP parameters are set as follows: the incident power is 120 W, the gas volume is 10 SLM, and the helium gas pressure is 120 Mpa.

[0050] (4) Induced expression of fluorescent protein After mutagenesis, put the stainless steel small round piece with the mutagenized bacterial liquid into 1 mL of LB liquid medium containing 100 μg / mL ampicillin, shake and elute for 1 min, and culture at 37 °C and 200 r / min until OD 600 is 0.6 - 0.8, add IPTG with a final concentration of 0.25 mM, and induce at 28 °C for 18 h.

[0051] (5) High-throughput screening of mutant strains Primary screening: Take 1 mL of the bacterial liquid obtained after induction in step (4), wash and resuspend it in 0.1% PBS buffer (pH = 7.0) and dilute to OD 600 about 1.0. Analyze the bacterial population by flow cytometry. The excitation light is at 488 nm, the fluorescence detection is at 535 nm, the sample pressure is 60 psi, the nozzle diameter is set to 70 μm, and Beckman Summit 5.2 software is used for data analysis. Set a gate accounting for 0.01% of the total cells, collect the cells with higher fluorescent protein expression into a 96-well plate containing 200 μL of LB liquid medium, and culture at 37 °C on the 96-well plate for 24 h. 186 strains of CGMCC NO.1.366 / pUC-57(+)-staygold-ygjH r mutant strains are obtained by the above high-throughput screening.

[0052] Second screening: The bacterial liquid in the 96-well plate of the 186 mutant strains obtained above was used as the seed liquid, and was re-inoculated into a deep-well plate containing 1 mL of fermentation medium at a volume ratio of 10%. IPTG with a final concentration of 0.5 mM was added to induce the expression of fluorescent protein, and fermentation was continued in a microplate incubator. The fermentation conditions were 37 °C and 800 r / min, and the fermentation time was 24 h. The fluorescence intensity was detected using a microplate reader, and 50 strains with the strongest fluorescence were selected. The formula of the above fermentation medium was: yeast powder 0.2%, glucose 0.5%, phosphoric acid 0.06%, (NH4)2SO4 1%, MgSO4 0.20%, KCl 0.05%, FeSO4 0.03%, MnSO4 0.03%, vitamin B1 0.0005%, and the solvent was water.

[0053] (6)L-valine fermentation test The 50 strains with the highest fluorescence intensity selected above were subjected to shake flask fermentation and cultured in a shaker at 37 °C and 200 r / min for 24 h, and the L-valine yield was measured. At the same time, the parallel fermentation yields of multiple groups of L-valine of the original strain Escherichia coli CGMCC NO. 1.366 were detected, and the highest value was 0.685 g / L. Data statistics were carried out as Figure 5 shown. Among the 50 mutant strains, 39 strains with increased L-valine yield were screened, and the screening efficiency reached 78%.

[0054] In summary, the fluorescent protein screening marker staygold-ygjH r gene provided by the present invention can effectively screen strains with increased L-valine yield.

[0055] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A fluorescent protein screening marker staygold-ygjH r gene, characterized in that The fluorescent protein screening marker staygold-ygjH r gene is formed by connecting the staygold r gene and the ygjH r gene with a flexible protein peptide; the staygold r gene is the gene after replacing the L-valine codon in the Staygold gene with GTC, and the ygjH r gene is the gene after replacing the L-valine codon in the ygjH gene with GTC.

2. The fluorescent protein screening marker staygold-ygjH r gene according to claim 1, characterized in that The L-valine codons in the Staygold gene and the ygjH gene are GTT, GTA, and GTG.

3. The fluorescent protein screening marker staygold-ygjH r gene according to claim 1, characterized in that The said staygold r The nucleotide sequence of the gene is shown in SEQ ID NO.1; the said ygjH r The nucleotide sequence of the gene is shown in SEQ ID NO.2; the nucleotide sequence of the flexible protein peptide is shown in SEQ ID NO.

4.

4. The fluorescent protein screening marker staygold-ygjH r gene according to claim 1, characterized in that The fluorescent protein screening marker staygold-ygjH r The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

5. The construction method of the fluorescent protein screening marker staygold-ygjH r gene according to any one of claims 1-4, characterized in that It includes the following steps: Replace the L-valine codons in Staygold and ygjH with GTC to obtain the codon-replaced Staygold r gene and the codon-replaced ygjH r gene, connect the Staygold r gene and ygjH r gene with a flexible protein peptide to obtain the fluorescent protein screening marker staygold-ygjH r gene.

6. A recombinant vector, characterized in that The recombinant vector contains the fluorescent protein screening marker staygold-ygjH described in any one of claims 1-4 r gene.

7. The recombinant vector according to claim 6, characterized in that The starting vector of the recombinant vector includes pET-22b(+) or pUC-57(+).

8. A recombinant bacterium, characterized in that The recombinant bacterium contains the fluorescent protein screening marker staygold-ygjH described in any one of claims 1-4 r gene or the recombinant vector described in claim 6 or 7.

9. The recombinant bacterium according to claim 8, characterized in that The starting strain of the recombinant bacterium includes Escherichia coli CGMCC NO.1.366 or Escherichia coli E.coli BL21.

10. The application of the fluorescent protein screening marker staygold-ygjH r gene according to any one of claims 1-4, or the recombinant vector according to claims 6 or 7, or the recombinant bacterium according to claims 8 or 9 in screening high L-valine-producing strains.

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