A fluorescent protein screening marker staygold-levE r Gene and its construction method and application

Abstract

The present invention relates to a fluorescent protein screening marker staygold-levE r The invention provides a fluorescent protein screening marker staygold-levE for screening L-valine production strains. r The present invention discloses a gene and a method for constructing the gene fragment. Compared with other similar gene screening markers, the fluorescence intensity of the screening marker provided by the present invention is positively correlated with the L-valine concentration, which can significantly improve the screening efficiency of L-valine high-yielding strains. The screening marker can save screening time and cost, and can improve screening efficiency, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to a fluorescent protein screening marker staygold-levE r The present invention relates to genes and their construction methods and applications, belonging to the field of bioengineering technology. Background Art

[0002] Currently, microbial fermentation has become the most widely used production method in the world, and the production scale is huge. Microbial fermentation utilizes the metabolic capacity of microorganisms to absorb energy provided by the outside world and synthesize various amino acid products such as L-valine in the microorganism. At the same time, gene editing or special mutagenesis treatment of the microorganism can be performed to select auxotrophic strains or L-valine structural analogs that can relieve feedback repression and feedback inhibition in metabolic regulation, thereby strengthening the L-valine metabolic pathway in the microbial metabolic pathway and increasing L-valine accumulation. Currently, using L-valine structural analog aminoethylcysteine ​​through physical or chemical mutagenesis, a large-capacity L-valine strain mutation library containing positive and negative mutations can be constructed. 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. Chinese patent document CN116103361A (application number 202111325512.1) uses a highly sensitive codon modification indicator gene to establish an amino acid high-yield strain screening technology based on resistance activity selection and fluorescence intensity screening, providing a new screening system for the screening of amino acid high-yield strains, while providing a convenient and accurate high-throughput screening method for the modification of key enzymes in the amino acid synthesis pathway. Chinese patent document CN116376946A (application number 202111597667.0) provides a method suitable for screening strains with high yields of natural amino acids. The provided screening method is carried out according to the following steps: using the translation element of the protein amino acid as a screening marker, replacing part or all of the codons of a certain amino acid in its nucleic acid sequence with a nucleotide sequence combination that does not encode amino acids, and transforming the screening marker into the mutant library of the microorganism. By detecting the phenotypic differences of the strains caused by the screening marker, strains with high amino acid yields can be selected from the mutant strains. Currently, there are few screening markers developed for L-valine production strains.

[0003] After ARTP mutagenesis combined with high-throughput screening to enhance L-valine carbon flux and output, optimize transcription factor expression, and maintain intracellular cofactor balance, Escherichia coli achieved an L-valine production of 92g / L, a state-of-the-art. Despite significant success in genetic modification of E. coli, effective methods for screening high-yielding strains have yet to be discovered. With the increasing global demand for L-valine, breeding high-yield, low-cost, and genetically stable production strains has become a key goal for the industry. Therefore, the design of specific, high-performance L-valine selection markers remains essential. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides a fluorescent protein screening marker staygold-levE r Genes and their construction methods and applications.

[0005] Terminology Notes:

[0006] Rare codons: The frequencies of use of different codons in different species and organisms are not completely evenly distributed. The most frequently used codons are the best codons, while the codons that are not frequently used are called rare codons or codons with low utilization rates. If all the codons for a certain amino acid in a gene are replaced with the rare codons corresponding to the amino acid, when the concentration of a certain amino acid in the cell drops significantly, tRNA will not be able to load the amino acid. Only when the amino acid concentration increases significantly can the amino acid loading level of tRNA return to normal, thereby accelerating the translation of genes rich in rare codons.

[0007] The technical solutions of the present invention are as follows:

[0008] A fluorescent protein screening marker staygold-levE r The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0009] The above fluorescent protein screening marker staygold-levE r The gene construction method comprises the following steps:

[0010] The codons for L-valine in the nucleotide sequences of the fluorescent protein genes Staygold and levE were replaced with the rare codon GTC to obtain the codon-substituted fluorescent protein gene Staygold and the codon-substituted gene levE, respectively. The two codon-substituted genes were connected with a flexible protein peptide to obtain the fluorescent protein selection marker staygold-levE. r Gene.

[0011] Preferably according to the present invention, the nucleotide sequence of the fluorescent protein gene Staygold after the codon replacement is shown as SEQ ID NO.1.

[0012] Preferably according to the present invention, the nucleotide sequence of the gene levE after the codon replacement is shown as SEQ ID NO.2.

[0013] A recombinant vector comprising the fluorescent protein screening marker staygold-levE r Gene.

[0014] According to the present invention, preferably, the vectors used include pET-22b(+) and pUC-57(+).

[0015] A recombinant bacterium comprising the fluorescent protein screening marker staygold-levE r Gene.

[0016] According to the present invention, preferably, the hosts used include Escherichia coli CGMCC NO.1.366 and Escherichia coli BL21 (DE3).

[0017] The above fluorescent protein screening marker staygold-levE r The application of genes, recombinant vectors or recombinant bacteria in screening high-yielding strains of L-valine.

[0018] Beneficial effects:

[0019] 1. The present invention provides a fluorescent protein screening marker staygold-levE for screening L-valine production strains r The present invention discloses a gene and a method for constructing the gene fragment. Compared with other similar gene screening markers, the fluorescence intensity of the screening marker provided by the present invention is positively correlated with the L-valine concentration, which can significantly improve the screening efficiency of L-valine high-yielding strains. The screening marker can save screening time and cost, and can improve screening efficiency, and has broad application prospects.

[0020] 2. In the present invention, the gene fragment obtained by replacing the L-valine codon in the nucleotide sequence of the gene levE with the rare codon GTC plays a rate-limiting role. The levE gene fragment without rare codon replacement or other gene fragments after rare codon replacement do not play the role of rate-limiting gene. The role of the fluorescent protein staygold gene after rare codon replacement in the present invention is to facilitate subsequent high-throughput sorting. In high-yield strains, L-valine accumulates more, which can meet the requirements of the fluorescent protein screening marker staygold-levE. rThe requirement for efficient gene translation increases the expression of fluorescent proteins, so high-yield strains can be screened by detecting fluorescence intensity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Agarose gel electrophoresis of PCR amplification products;

[0022] Figure 2 The recombinant E. coli BL21(DE3) / pUC-57(+)-staygold-levE at different L-valine concentrations r Fluorescence intensity histogram;

[0023] Figure 3 The figure is a histogram of the codon usage frequency of Escherichia coli;

[0024] Figure 4 Recombinant E. coli BL21(DE3) / pUC-57(+)-staygold-levE for different fermentation times r Fluorescence intensity histogram;

[0025] Figure 5 The figure is a scatter plot of L-valine production of the sorted Escherichia coli mutant strains. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0027] Any unspecified details in the examples were based on conventional techniques in the art; all experimental materials and reagents not described in detail were commercially available. The vectors and host bacteria used in the examples were purchased from Sangon Biotech (Shanghai) Co., Ltd. and are commercially available. Escherichia coli CGMCC No. 1.366 was purchased from the China General Microbiological Culture Collection Center and does not involve proprietary microorganisms.

[0028] Example 1

[0029] staygold-levE r Fragment construction

[0030] The nucleotide sequence of the fluorescent protein gene Staygold was searched through the National Center for Biotechnology Information (NCBI) database of the United States. The NCBI accession number is: LC601652.1. The sequence contains 11 L-valine codons, including 3 codons GTC, 5 codons GTT, 2 codons GTA, and 1 codon GTG. 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 nucleotide sequence after replacement is shown in SEQ ID NO.1. The nucleotide sequence of the gene levE with a high proportion of L-valine codons was selected from the genome of Escherichia coli CGMCC NO.1.366. The sequence contains 22 L-valine codons, including 3 codons GTC, 9 codons GTT, 4 codons GTA, and 1 codon GTG. 6, the L-valine codons GTT, GTA, GTG in the sequence are replaced with the rare codon GTC of L-valine in Escherichia coli, and the nucleotide sequence after replacement is shown in SEQ ID NO.2; the levE after codon replacement is connected to the levE r The fragment was connected with the fluorescent protein gene Staygold after codon replacement and synthesized to obtain staygold-levE r Fragment, resulting staygold-levE 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.

[0031] Example 2

[0032] Recombinant plasmid construction

[0033] The vector plasmid pUC-57(+) was treated with FastDigest EcoRI and FastDigest HindIII double enzyme digestion reaction system, and then cloned with staygold-levE which was also treated with FastDigest EcoRI and FastDigest HindIII double enzyme digestion reaction system through seamless cloning system. r The fragments were connected to obtain the recombinant vector pUC-57(+)-staygold-levE r .

[0034] The double enzyme digestion reaction system is shown in Table 1, with a total system volume of 20 μL; the seamless cloning system is shown in Table 2, with a total system volume of 10 μL.

[0035] Table 1. Double enzyme digestion reaction system

[0036]

[0037] The double enzyme digestion reaction conditions are as follows:

[0038] React at 37°C for 30 min; inactivate at 80°C for 5 min and store at 4°C.

[0039] Table 2. Seamless cloning system

[0040]

[0041] The seamless cloning procedure is as follows:

[0042] The recombination reaction was carried out at 50°C for 15 min, and then the temperature was lowered to 4°C or immediately cooled on ice.

[0043] Example 3

[0044] Competent cell transformation

[0045] Take the recombinant vector pUC-57(+)-staygold-levE 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.

[0046] The formula of the LB liquid culture medium is: 0.5% yeast powder, 1% peptone, 1% sodium chloride, and solvent water.

[0047] The formula of LB solid culture medium is: yeast powder 0.5%, peptone 1%, sodium chloride 1%, agar powder 2%, solvent water.

[0048] Example 4

[0049] Positive colony screening and verification

[0050] The positive recombinant colonies of Example 3 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 primers P1-F and P1-R to obtain an amplified product. The amplified product was verified by agarose gel electrophoresis; the primer sequences are as follows:

[0051] P1-F:5'-CGCCCTTATTCCCTTTTTTGCGG-3'

[0052] P1-R:5'-TCACCGGCTCCAGATTTATCAGC-3'

[0053] The PCR amplification system is shown in Table 3, and the total system volume is 20 μL.

[0054] Table 3. PCR amplification system

[0055]

[0056] The PCR amplification procedure is as follows:

[0057] 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, 30 cycles; extension at 72°C for 10 min, storage at 4°C;

[0058] Agarose gel electrophoresis was used to test the PCR products. The results showed that primers P1-F and P1-R could amplify a specific gene band with a size of about 650 bp. Figure 1 , which is close to the theoretical value of 651 bp, indicating that staygold-levE r The fragment was successfully inserted, and the recombinant E. coli BL21 (DE3) / pUC-57 (+) -staygold-levE was obtained. r .

[0059] Example 5

[0060] L-valine addition concentration and fluorescence intensity detection

[0061] The recombinant E. coli BL21 (DE3) / pUC-57 (+)-staygold-levE 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 ≈1.0, inoculated with a volume ratio of 2% in 50 mL of LB liquid medium containing ampicillin at a final concentration of 100 μg / mL, and cultured at 37 ° C and 200 r / min until OD 600≈1.0, L-valine hydrochloride was added to the culture medium at a final concentration of 0, 1, 2, 3, and 4 g / L, and IPTG was added at a final concentration of 0.5 μM to induce the expression of fluorescent protein. No IPTG was added to the control group. Three parallel experiments were set for each gradient. The induction conditions were 25°C, 200 r / min, and cultured for 14 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.

[0062] Comparative Example 1

[0063] Search and analyze the usage frequency of each codon in E. coli on the NCBI official website. The results are as follows Figure 3 As shown, the codons corresponding to L-valine are GUU, GUC, GUA and GUG, among which GUA is the least frequently used codon. According to the method described in Example 1, the gene staygold-levE in which the codons for L-valine are all GUA was constructed. rGUA , and the fluorescence intensity of gene expression was detected according to Examples 2 to 5. Without the addition of L-valine, the fluorescence intensity results of the system at different fermentation times were as follows: Figure 4 As shown, the results showed that the recombinant E. coli BL21 (DE3) / pUC-57 (+) -staygold-levE r The fluorescence intensity expressed in the system is too high, reaching 8×10 8 Therefore, GUA is not a rare codon for L-valine and cannot be used to construct a fluorescent protein selection marker gene.

[0064] Comparative Example 2

[0065] The gene rplW, which has the second highest proportion of L-valine in its amino acid sequence, was screened from the genome of Escherichia coli CGMCC No. 1.366. The sequence contained 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. The nucleotide sequence after replacement is shown in SEQ ID NO. 5. Staygold-rplW was obtained by gene synthesis according to the methods described in Examples 1 to 4. r fragment and construct the recombinant vector pUC-57(+)-staygold-rplW rThe 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 5. The results showed that the 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.

[0066] Experimental Example 1

[0067] Applications of fluorescent protein selection markers

[0068] (1) Fluorescent protein screening marker transformation

[0069] Take the recombinant vector pUC-57(+)-staygold-levE r 10 μL was transformed into L-valine-producing Escherichia coli CGMCCNO.1.366 competent cells and cultured for 12 to 16 hours. The operation steps were the same as those in Example 3.

[0070] (2) Positive colony screening

[0071] The positive recombinant colonies in step (1) were picked and verified, and the steps were the same as in Example 4 to obtain CGMCCNO.1.366 / pUC-57(+)-staygold-levE r .

[0072] (3) ARTP mutagenesis of recombinant strains

[0073] The single colony in step (2) was inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C and 200 r / min until OD 600 It is 0.6-0.8 and is exposed to ARTP for 120s.

[0074] The ARTP parameters were set as follows: incident power of 120 W, gas volume of 10 SLM, and helium pressure of 120 MPa.

[0075] (4) Inducible expression of fluorescent protein

[0076] 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 The pH value was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and induced at 25°C for 12 h.

[0077] (5) High-throughput screening of mutant strains

[0078] 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 ≈1.0. Flow cytometry was used to analyze strain clustering, with excitation at 488 nm, fluorescence detection at 535 nm, sample pressure at 60 psi, and a nozzle diameter of 70 μm. Data were analyzed using Beckman Summit 5.2 software. A gate was set at 0.01% of the total cells, and cells with high fluorescent protein expression were collected and transferred to a 96-well plate containing 200 μL of LB liquid medium. The plates were cultured at 37°C for 36 h.

[0079] (6) L-valine fermentation test

[0080] The bacterial liquid in the 96-well plate obtained in step (5) 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%. Fermentation was continued in a microplate incubator at 37°C and 600 rpm for 48 hours. After fermentation, 100 μL of the fermentation liquid was centrifuged at 5000 rpm for 5 minutes per well. The supernatant was diluted to an L-valine concentration of 0.1 to 1 g / L and the L-valine concentration was measured using a biosensor analyzer.

[0081] The fermentation medium formula is: glucose 2.0%, corn steep liquor 0.05%, molasses 1.6%, phosphoric acid 0.06%, (NH4)2SO4 1.20%, MgSO4 0.20%, KCl 0.05%, betaine 0.05%, FeSO4 0.03%, MnSO4 0.03%, ZnSO4 0.005%, CuSO4 0.005%, vitamin B1 0.0005%, L-threonine 0.025%, and solvent water.

[0082] Experimental Example 2

[0083] Experimental Example 1: 240 strains of CGMCC NO.1.366 / pUC-57(+)-staygold-levE were obtained by sorting. r Among the mutant strains, 127 strains had fluorescence intensities higher than the average value before sorting. 113 strains with low fluorescence intensities were excluded from the preliminary screening, and strains with relatively high fluorescence intensities were retained for further research. Among the sorted strains, 23 strains with the highest fluorescence intensities were selected for deep-well plate fermentation for 24 hours to measure the production of L-valine. Among them, the highest value of L-valine production in multiple parallel fermentations of the original strain Escherichia coli CGMCCNO.1.366 was 0.21 g / L. Figure 5As shown, among the 23 mutant strains, 18 strains with improved L-valine production were screened, with a screening efficiency of 78.2%, the highest yield of 0.306 g / L, and a yield increase of 45.7%.

[0084] In summary, the fluorescent protein screening marker staygold-levE provided by the present invention r The gene can effectively screen strains with improved L-valine production.

Claims

1. A fluorescent protein screening marker staygold-levE r A gene characterized by The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. The fluorescent protein screening marker according to claim 1 staygold-levE r A method for constructing a gene, characterized in that: The steps include: Fluorescent protein gene Staygold and genes levE The codon for L-valine in the nucleotide sequence of was replaced with the rare codon GTC to obtain the fluorescent protein genes after codon replacement. Staygold and genes after codon substitution levE, The two codon-replaced genes were connected using a flexible protein peptide to obtain a fluorescent protein screening marker. staygold- levE r Gene; The fluorescent protein gene after the codon replacement Staygold The nucleotide sequence is shown in SEQ ID NO.1; The gene after the codon substitution levE The nucleotide sequence is shown in SEQ ID NO.

2.

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

4. The recombinant vector according to claim 3, wherein The vectors used included pET-22b(+) and pUC-57.

5. A recombinant bacterium, characterized in that Comprising the fluorescent protein screening marker according to claim 1 staygold-levE r Gene.

6. The recombinant bacterium according to claim 5, characterized in that The host used was Escherichia coli CGMCC NO.1.366 or Escherichia coli BL21 (DE3).

7. The fluorescent protein screening marker according to claim 1 staygold-levE r Use of the gene, the recombinant vector according to claim 3 or the recombinant bacteria according to claim 5 in screening L-valine high-producing strains.

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