A fluorescent protein screening marker staygold-ygjH r Genes and their applications

Abstract

The present invention provides a fluorescent protein screening marker staygold-ygjH r The invention relates to a gene and its application, which belongs to the field of bioengineering technology. The invention replaces the codon of L-valine in the nucleotide sequence of the fluorescent protein gene Staygold and the gene ygjH with the rare codon GTC, and obtains the fluorescent protein gene Staygold after the codon replacement. r and the gene ygjH after codon substitution r The two codon-replaced genes were connected using a flexible protein peptide to obtain the fluorescent protein screening marker staygold‑ygjH r Gene. Experimental verification has shown that this gene can significantly improve the efficiency of screening for high-L-valine-producing strains.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a fluorescent protein screening marker staygold-ygjH r Genes and their applications. Background Art

[0002] Microbial fermentation is an efficient and environmentally friendly biotechnology for producing amino acids, leveraging the metabolic capacity of microorganisms to synthesize target amino acids. Since its application in L-valine production, this field has undergone continuous technological innovation and optimization. Today, microbial fermentation has become the mainstream method for L-valine production. The core of microbial fermentation is to utilize the metabolic capacity of microorganisms to convert external carbon sources into bioactive molecules such as amino acids. Currently, gene editing technologies such as the CRISPR-Cas9 system, or chemical and physical mutagenesis, have enabled the generation of auxotrophic strains that can overcome feedback repression and feedback inhibition in metabolic regulation or strains resistant to L-valine analogs. These strains can enhance the L-valine biosynthesis pathway in microbial metabolic pathways, thereby increasing L-valine accumulation and production. Despite this, effective methods for screening high-yielding strains remain limited, requiring further research and development.

[0003] At present, a large-capacity L-valine strain mutant library containing positive and negative mutations can be constructed by physical or chemical mutagenesis using aminoethylcysteine, a structural analog of L-valine. However, the limitations of traditional screening methods lead to low efficiency in screening high-yielding 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 number of analogs increases the difficulty of obtaining new mutant strains from the screened strains, making it difficult to meet the needs of industrial production. Therefore, there is a need for a screening marker that can be widely applied to different types of microbial cells for high-throughput screening of L-valine-producing strains. With the increasing demand for L-valine, breeding high-yield, low-cost, and genetically stable production strains has become a key goal of the industry. There are few screening markers developed for L-valine-producing strains, so designing an L-valine screening marker with high specificity and high performance is a technical problem that needs to be solved at this stage. Summary of the Invention

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

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a fluorescent protein screening marker staygold-ygjH r gene, the fluorescent protein screening marker staygold-ygjH r Gene by flexible protein peptide will staygold r Gene and ygjH r Gene connection; the staygold r The gene is the gene after GTC replaces the L-valine codon in the Staygold gene, and the ygjH r The gene is the one obtained by replacing the L-valine codon in the ygjH gene with GTC.

[0007] Preferably, the L-valine codons in the Staygold gene and the ygjH gene are GTT, GTA and GTG.

[0008] Preferably, the staygold r The nucleotide sequence of the gene is shown in SEQ ID NO.1; 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.

[0009] Preferably, the fluorescent protein screening marker staygold-ygjH r The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0010] The present invention provides the fluorescent protein screening marker staygold-ygjH r The gene construction method comprises the following steps: replacing the L-valine codon in Staygold and ygjH with GTC to obtain the codon-substituted Staygold and ygjH respectively. r ygjH after gene and codon substitution r Gene, the gene after the two codons are replaced is connected with a flexible protein peptide to obtain the fluorescent protein screening marker staygold-ygjH r Gene.

[0011] The present invention provides a recombinant vector containing the fluorescent protein screening marker staygold-ygjH r Gene.

[0012] Preferably, the starting vector of the recombinant vector includes pET-22b(+) or pUC-57(+).

[0013] The present invention provides a recombinant bacterium, wherein the recombinant bacterium contains the fluorescent protein screening marker staygold-ygjH r gene or the recombinant vector.

[0014] Preferably, the starting strain of the recombinant bacteria includes Escherichia coli CGMCC NO.1.366 or Escherichia coli BL21.

[0015] The present invention provides the fluorescent protein screening marker staygold-ygjH r Application of the gene, the recombinant vector or the recombinant bacteria in screening L-valine high-yielding strains.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a fluorescent protein screening marker staygold-ygjH for screening L-valine production strains r Compared with other similar gene screening markers, the fluorescence intensity of this screening marker gene is positively correlated with the L-valine concentration, which can significantly improve the screening efficiency of L-valine high-yielding strains. This screening marker gene can save screening time and cost.

[0018] Furthermore, the fluorescent protein screening marker staygold-ygjH of the present invention r The gene replaces the L-valine codon in the original nucleotide sequence with the rare codon GTC, and uses the fusion of two genes to prepare a screening marker, which can enhance sensitivity and improve screening efficiency and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The agarose gel electrophoresis diagram of PCR amplification products. Lanes 1-4 are Staygold-ygjH r .

[0020] Figure 2 The recombinant E. coli BL21(DE3) / pUC-57(+)-staygold-ygjH at different L-valine concentrations r Fluorescence intensity curve graph.

[0021] Figure 3 The recombinant E. coli BL21(DE3) / pUC-57(+)-staygold-rplW was generated at different L-valine concentrations. r Fluorescence intensity curve graph.

[0022] Figure 4The recombinant E. coli BL21(DE3) / pUC-57(+)-staygold was generated at different L-valine concentrations. r Fluorescence intensity curve graph.

[0023] Figure 5 The figure is a scatter plot of L-valine production of the sorted Escherichia coli mutant strains. DETAILED DESCRIPTION

[0024] The present invention provides a fluorescent protein screening marker staygold-ygjH r gene, the fluorescent protein screening marker staygold-ygjH r Gene by flexible protein peptide will staygold r Gene and ygjH r Gene connection; the staygold r The gene is the gene after GTC replaces the L-valine codon in the Staygold gene, and the ygjH r The gene is a gene obtained by replacing the L-valine codon in the ygjH gene with GTC. The GTC described in the present invention is a rare codon. The L-valine codons in the Staygold gene and the ygjH gene described in the present invention are GTT, GTA, and GTG.

[0025] In the present invention, the staygold r The nucleotide sequence of the gene is shown in SEQ ID NO.1; 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.

[0026] The present invention also provides the fluorescent protein screening marker staygold-ygjH r The gene construction method comprises the following steps: replacing the L-valine codon in Staygold and ygjH with GTC to obtain the codon-substituted Staygold and ygjH respectively. r ygjH after gene and codon substitution r Gene, Staygold r Gene and ygjH r The gene is connected using a flexible protein peptide to obtain the fluorescent protein selection marker staygold-ygjH r The Staygold gene of the present invention rThe nucleotide sequence of the gene is shown in SEQ ID NO.1; 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.

[0027] The present invention also provides a recombinant vector containing the fluorescent protein screening marker staygold-ygjH r The starting vector of the recombinant vector of the present invention includes pET-22b(+) or pUC-57(+).

[0028] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium contains the fluorescent protein screening marker staygold-ygjH r The starting strain of the recombinant bacteria of the present invention includes Escherichia coli CGMCC NO.1.366 or Escherichia coli BL21.

[0029] The present invention also provides the fluorescent protein screening marker staygold-ygjH r Application of the gene, the recombinant vector or the recombinant bacteria in screening L-valine high-yielding strains.

[0030] 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.

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] The vectors and host bacteria involved in the examples of the present invention were purchased from Sangon Biotech (Shanghai) Co., Ltd. and are common commercial products. Escherichia coli CGMCC No. 1.366 was purchased from the China General Microbiological Culture Collection Center.

[0033] Example 1 staygold-ygjH r Fragment construction

[0034] The nucleotide sequence of the fluorescent protein gene Staygold was searched through the National Center for Biotechnology Information (NCBI) database. The NCBI accession number is: LC601652.1. The sequence contains 11 L-valine codons, including 3 GTC codons, 5 GTT codons, 2 GTA codons, and 1 GTG codon. The L-valine codons GTT, GTA, and GTG in the sequence were replaced with the rare codon GTC for L-valine in Escherichia coli. r The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0035] 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. The sequence contains 16 L-valine codons, including 3 GTC codons, 1 GTT codon, 3 GTA codons, and 9 GTG codons. The L-valine codons GTT, GTA, and GTG in the sequence were replaced with the rare codon GTC for L-valine in Escherichia coli. The replaced ygjH r The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0036] ygjH was linked to r Gene and fluorescent protein gene Staygold r Connect and synthesize to obtain staygold-ygjH r Fragment, resulting staygold-ygjH r The nucleotide sequence of the fragment is shown in SEQ ID NO. 3. The nucleotide sequence encoding the flexible connecting peptide is shown in SEQ ID NO. 4.

[0037] Example 2

[0038] 1. Construction of recombinant plasmid

[0039] The vector plasmid pUC-57(+) was treated with FastDigest EcoRI and FastDigest HindIII double enzyme digestion reaction system, and then cloned with staygold-ygjH by seamless cloning system. r The fragments were connected to obtain the recombinant vector pUC-57(+)-staygold-ygjH r .

[0040] The double enzyme digestion reaction system is shown in Table 1, with a total system volume of 20 μL. The double enzyme digestion reaction conditions are as follows:

[0041] Incubate at 37°C for 30 minutes, inactivate at 80°C for 5 minutes, and store at 4°C. The seamless cloning procedure is as follows: perform the recombination reaction at 50°C for 15 minutes, then reduce the temperature to 4°C or immediately cool on ice.

[0042] The seamless cloning system is shown in Table 2, and the total system is 10 μL.

[0043] Table 1 Double enzyme digestion reaction system

[0044]

[0045] Table 2 Seamless cloning system

[0046]

[0047] 2. Competent Cell Transformation

[0048] Take the recombinant vector pUC-57(+)-staygold-ygjH r 10 μL of the culture medium was added to 100 μL of E. coli BL21 (DE3) competent cells. The tube was gently tapped to mix. The tube was allowed to stand on ice for 30 minutes. Heat shock was performed in a 42°C water bath for 90 seconds. The tube was immediately placed on ice for 2-3 minutes. 900 μL of LB liquid medium without resistance was added to the centrifuge tube and incubated in a shaker at 37°C and 200 rpm for 1 hour. The incubated bacterial solution was centrifuged at 5000 rpm for 2 minutes. After discarding 900 μL of the supernatant, the remaining bacterial cells were resuspended and evenly spread on LB solid medium plates containing 100 μg / mL ampicillin using a sterile spreader. The plates were incubated upside down in a 37°C constant temperature incubator for 12-16 hours.

[0049] The LB liquid medium formula is: 0.5% yeast powder, 1% peptone, 1% sodium chloride, and solvent water. The LB solid medium formula is: 0.5% yeast powder, 1% peptone, 1% sodium chloride, solvent water, and 2% agar powder.

[0050] 3. Positive colony screening and verification

[0051] The positive recombinant colonies from step 2 were picked and inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C overnight. After the culture was completed, PCR amplification was performed using the bacterial liquid as a template and 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, with a total volume of 20 μL. The PCR amplification procedure is as follows: 95°C pre-denaturation for 5 min; 94°C denaturation for 30 sec, 55°C annealing for 1 min, 72°C extension for 4 min, 30 cycles; 72°C extension for 10 min, and storage at 4°C.

[0052] Table 3 PCR amplification system

[0053]

[0054] The amplified product was verified by agarose gel electrophoresis. The results showed that primers P1 and P2 could amplify a specific gene band with a size of about 1000 bp. Figure 1 , which is close to the theoretical value of 1026 bp, indicating that staygold-ygjH r The fragment was successfully inserted, and the recombinant E. coli BL21 (DE3) / pUC-57(+)-staygold-ygjH was obtained. r .

[0055] Example 3

[0056] The recombinant E. coli BL21 (DE3) / pUC-57(+)-staygold-ygjH was successfully verified in Example 2. r Streak culture on LB solid medium, pick a single colony and inoculate it into LB liquid medium, and culture at 37°C and 200 r / min until OD 600 The inoculum volume ratio was 2% and the culture medium was inoculated into 50 mL of LB liquid medium containing ampicillin at a final concentration of 100 μg / mL. The culture was cultured at 37 ° C and 200 r / min until the OD 600 The concentration of L-valine was about 1.0. After adding L-valine at a final concentration of 0, 0.1, 0.2, 0.3, and 0.4 g / L to the culture medium, IPTG was added at a final concentration of 0.25 μM to induce the expression of fluorescent protein. No IPTG was added to the control group. Three parallel experiments were set up for each gradient. The induction conditions were 28°C and 200 r / min for 18 h. After the induction, the fluorescence intensity was measured using a microplate reader at an excitation wavelength of 488 nm and a detection wavelength of 535 nm. The relationship between the L-valine addition concentration and the fluorescence intensity is shown in Figure 2. Figure 2 The results showed that the fluorescence intensity was positively correlated with the amount of L-valine added.

[0057] Comparative Example 1

[0058] The gene rplW with the second highest proportion of L-valine in the amino acid sequence was screened from the genome of Escherichia coli CGMCC NO.1.366. The sequence contains 14 L-valine codons, including 4 GTC codons, 6 GTT codons, 2 GTA codons, and 2 GTG codons. The L-valine codons GTT, GTA, and GTG in the sequence were replaced with the rare codon GTC for L-valine in Escherichia coli. After the 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, staygold-rplW was obtained by gene synthesis. r fragment and construct the recombinant vector pUC-57(+)-staygold-rplW r The relationship between the fluorescence intensity of gene expression and the concentration of L-valine in the fermentation broth was then detected according to the method of Example 3. Figure 3 The results showed that staygold-rplW r There was no significant correlation between the fluorescence intensity of the transformed strain with the fluorescence selection marker and the concentration of L-valine in the fermentation broth.

[0059] Comparative Example 2

[0060] Staygold after using rare codon GTC replacement r Gene (staygold constructed in Example 1) r According to the methods described in Examples 1 and 2, staygold was obtained by gene synthesis. r fragment and construct the recombinant vector pUC-57(+)-staygold r The relationship between the fluorescence intensity of gene expression and the concentration of L-valine in the fermentation broth was then detected according to the method of Example 3. Figure 4 The results showed that staygold r There was no significant correlation between the fluorescence intensity of the transformed strain with the fluorescence selection marker and the concentration of L-valine in the fermentation broth.

[0061] Experimental Example 1: Fluorescent protein screening marker gene staygold-ygjH r Application

[0062] (1) Fluorescent protein screening marker transformation

[0063] Take the recombinant vector pUC-57(+)-staygold-ygjH constructed above r10 μL was transformed into L-valine-producing Escherichia coli CGMCC NO.1.366 competent cells and cultured for 12-16 hours. The operation steps were the same as those in Example 2.

[0064] (2) Positive colony screening

[0065] The positive recombinant colonies in step (1) were picked and verified, and the steps were the same as in Example 2 to obtain the recombinant bacteria CGMCCNO.1.366 / pUC-57(+)-staygold-ygjH r .

[0066] (3) ARTP mutagenesis of recombinant strains

[0067] The single colony in step (2) was picked and inoculated into LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C and 200 r / min until OD 600 The ARTP parameters were set as follows: incident power 120 W, gas volume 10 SLM, and helium pressure 120 MPa.

[0068] (4) Inducible expression of fluorescent protein

[0069] After the mutagenesis, the stainless steel disc with the mutagenic bacterial solution was placed in 1 mL of LB liquid medium containing 100 μg / mL ampicillin, shaken for 1 min, and cultured at 37°C and 200 rpm until the OD 600 When the pH value was 0.6-0.8, IPTG was added to a final concentration of 0.25 mM and induced at 28°C for 18 h.

[0070] (5) High-throughput screening of mutant strains

[0071] Initial screening: Take 1 mL of the bacterial solution obtained after induction in step (4), wash and resuspend in 0.1% PBS buffer (pH = 7.0) and dilute to OD 600 The fluorescence intensity was about 1.0. Flow cytometry was used to analyze the strain clustering, with excitation light at 488 nm, fluorescence detection at 535 nm, sample pressure at 60 psi, nozzle diameter set to 70 μm, and data analysis using Beckman Summit 5.2 software. A gate of 0.01% of the total cells was set, and cells with high fluorescent protein expression were collected into a 96-well plate containing 200 μL LB liquid medium and cultured at 37 ° C on the 96-well plate for 24 hours. 186 strains of CGMCCNO.1.366 / pUC-57(+)-staygold-ygjH were obtained by the above high-throughput screening. r Mutant strains.

[0072] Rescreening: The bacterial suspension from the 96-well plate of the 186 mutant strains obtained above was used as a seed solution and re-transferred to a deep-well plate containing 1 mL of fermentation medium at a volume ratio of 10%. IPTG was added to a final concentration of 0.5 mM to induce fluorescent protein expression. Fermentation continued in a microplate incubator at 37°C and 800 rpm for 24 hours. Fluorescence intensity was measured using a microplate reader, and the 50 strains with the strongest fluorescence were selected. The fermentation medium formula was as follows: 0.2% yeast extract, 0.5% glucose, 0.06% phosphoric acid, 1% (NH4)2SO4, 0.20% MgSO4, 0.05% KCl, 0.03% FeSO4, 0.03% MnSO4, 0.0005% vitamin B1, and water as solvent.

[0073] (6) L-valine fermentation test

[0074] The 50 strains with the highest fluorescence intensity were fermented in shake flasks at 37°C and 200 rpm for 24 hours, and the L-valine production was determined. The L-valine production of the original strain Escherichia coli CGMCC NO.1.366 was also tested in multiple parallel fermentations, with the highest value being 0.685 g / L. Figure 5 As shown, among the 50 mutant strains, 39 strains with improved L-valine production were screened, and the screening efficiency reached 78%.

[0075] In summary, the fluorescent protein screening marker staygold-ygjH provided by the present invention r The gene can effectively screen strains with improved L-valine production.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fluorescent protein screening marker staygold-ygjH r A gene characterized by The fluorescent protein screening marker staygold-ygjH r Gene by flexible protein peptide will staygold r Gene and ygjH r Genes are connected; The StayGold r The gene is the gene after GTC replaces the L-valine codon in the Staygold gene, and the ygjH r The gene is the one after GTC replaces the L-valine codon in the ygjH gene; The L-valine codons in the Staygold gene and the ygjH gene are GTT, GTA, and GTG; The StayGold r The nucleotide sequence of the gene is shown in SEQ ID NO.1; 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 r The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. Fluorescent protein screening marker staygold-ygjH as claimed in claim 1 r A method for constructing a gene, characterized in that: The method comprises the following steps: replacing the L-valine codon in Staygold and ygjH with GTC to obtain Staygold and ygjH after codon replacement. r ygjH after gene and codon substitution r Gene, Staygold r Gene and ygjH r The gene is connected using a flexible protein peptide to obtain the fluorescent protein selection marker staygold-ygjH r Gene.

3. A recombinant vector, characterized in that The recombinant vector contains the fluorescent protein screening marker staygold-ygjH according to claim 1 r Gene.

4. The recombinant vector according to claim 3, wherein The starting vector of the recombinant vector includes pET-22b(+) or pUC-57(+).

5. A recombinant bacterium, characterized in that The recombinant bacteria contains the fluorescent protein screening marker staygold-ygjH according to claim 1 r The gene or the recombinant vector according to claim 3 or 4; The starting strain of the recombinant bacteria is Escherichia coli CGMCCNO.1.366 or Escherichia coli BL21.

6. The fluorescent protein screening marker staygold-ygjH as claimed in claim 1 r Use of the gene, the recombinant vector according to claim 3 or 4, or the recombinant bacterium according to claim 5 in screening L-valine high-producing strains.

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