Fluorescent protein selection marker staygold-levEr gene as well as construction method and application thereof

Abstract

The invention relates to a fluorescent protein selection marker staygold-levEr gene as well as a construction method and application thereof, and belongs to the technical field of biological engineering. The invention provides a fluorescent protein selection marker staygold-levEr gene for screening an L-valine production strain and a construction method of a gene fragment, compared with other similar gene selection markers, the selection marker provided by the invention has the advantages that the fluorescence intensity is positively correlated with the concentration of L-valine, and the screening marker has the advantages that the screening marker can be used for screening the L-valine production strain; according to the screening marker, the screening efficiency of the L-valine high-yield strain can be obviously improved, the screening time and cost can be saved, the screening efficiency can be improved, and the screening marker has a wide application prospect.

Description

Technical Field

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

[0002] At present, the production of L-valine by microbial fermentation has become the most widely used production method in the world, and the production scale is huge. The microbial fermentation method utilizes the metabolic capacity of the microorganism itself, absorbs the energy provided by the outside world, synthesizes various amino acid products such as L-valine in the microorganism, and simultaneously performs gene editing or special mutagenesis treatment on the microorganism, which can select and breed resistant strains with nutritional deficiency or L-valine structural analogs that can relieve feedback repression and feedback inhibition in metabolic regulation, thereby strengthening the metabolic pathway of L-valine in the microbial metabolic pathway and increasing the accumulation of L-valine. At present, a large-capacity L-valine strain mutation library containing positive and negative mutations can be constructed by physical or chemical mutagenesis using aminoethylcysteine, an L-valine structural analog. Therefore, there is a need for a screening marker that can be widely used in different types of microbial cells for high-throughput screening of L-valine production 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 vitality selection and fluorescence intensity screening, providing a new screening system for the screening of amino acid high-yield strains, and at the same time 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 screening method provided is carried out according to the following steps: using the translation element of protein amino acids 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 mutation library of the microorganism. By detecting the phenotypic differences of the strains caused by the screening marker, strains with high yields of amino acids can be selected from the mutant strains. At present, there are few screening markers developed for L-valine production strains.

[0003] After ARTP mutagenesis combined with high-throughput screening, enhanced carbon flux and output of L-valine, optimized expression of transcription factors, and balance of intracellular cofactors, the L-valine production of Escherichia coli reached 92g / L, which is the best standard available. Although great success has been achieved in genetic modification of Escherichia coli, no effective method for screening high-yield bacteria has been found. With the increasing global demand for L-valine, breeding high-yield, low-cost, and genetically stable production strains has gradually become a key goal of the industry. Therefore, it is still necessary to design and screen specific, high-performance L-valine screening markers. 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 Note:

[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, and 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 is greatly reduced, tRNA will not be able to load the amino acid. Only when the amino acid concentration is greatly increased can the amino acid loading level of tRNA return to normal, thereby accelerating the translation of genes rich in rare codons.

[0007] The technical solution of the present invention is as follows:

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

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

[0010] The codons of L-valine in the nucleotide sequences of the fluorescent protein gene Staygold and the gene levE were replaced with the rare codon GTC to obtain the fluorescent protein gene Staygold after codon replacement and the gene levE after codon replacement, respectively. The two codon-replaced 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 as shown in SEQ ID NO.1.

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

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

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

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

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

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

[0018] Beneficial effects:

[0019] 1. The present invention provides a fluorescent protein screening marker staygold-levE for screening L-valine production strains r Gene, and a method for constructing the gene fragment. Compared with other similar gene screening markers, the screening marker provided by the invention has a fluorescence intensity that is positively correlated with the L-valine concentration, and 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 the high-yield strain, L-valine accumulates more, which can meet the fluorescent protein screening marker staygold-levE rThe requirement for efficient gene translation increases the expression of fluorescent protein, so high-yield strains can be screened out 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 The fluorescence intensity histogram of

[0023] Figure 3 is a histogram of the codon usage frequency of E. coli;

[0024] Figure 4 The recombinant E. coli BL21(DE3) / pUC-57(+)-staygold-levE was generated at different fermentation times. r The fluorescence intensity histogram of

[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] The contents not described in detail in the examples are all based on the conventional techniques in the art; the experimental materials and reagents not described in detail are all common commercial products. Among them, the vectors and host bacteria involved in the examples 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 Management Center and does not involve patented microbial collection.

[0028] Example 1

[0029] staygold-levE r Construction of fragments

[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 of Escherichia coli L-valine. 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 (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, and GTG in the sequence are replaced with the rare codon GTC of L-valine in Escherichia coli, and the nucleotide sequence after the replacement is shown in SEQ ID NO.2; the levE after the codon replacement is connected by a flexible connecting peptide 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. Among them, 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 restriction enzyme digestion reaction system, and then cloned with staygold-levE after being double digested with FastDigest EcoRI and FastDigest HindIII 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, and the total system is 20 μL; the seamless cloning system is shown in Table 2, and the total system is 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℃ for 30min; inactivate at 80℃ for 5min and store at 4℃.

[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, 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 10μL, add 100μL E. coli BL21 (DE3) competent cells, flick the tube wall to mix, let stand on ice for 30min, heat shock in a 42℃ water bath for 90s, immediately let stand on ice for 2-3min, add 900μL LB liquid medium without resistance to the centrifuge tube, and incubate in a shaker at 37℃, 200r / min for 1h. Centrifuge the incubated bacterial solution at 5000r / min for 2min, discard 900μL of supernatant, resuspend the remaining bacterial cells and evenly spread them on LB solid medium plates containing 100μg / mL ampicillin using a sterile coating rod, and incubate the plates inverted in a 37℃ constant temperature incubator for 12-16h.

[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: 0.5% yeast powder, 1% peptone, 1% sodium chloride, 2% agar powder, and 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 culture 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-F and P1-R as primers to obtain an amplified product, which 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 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 bacteria E.coli BL21(DE3) / pUC-57(+)-staygold-levE was obtained. r .

[0059] Example 5

[0060] L-valine 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 200r / min until OD 600 ≈1.0, inoculated with 2% by volume into 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, after adding L-valine hydrochloride at a final concentration of 0, 1, 2, 3, and 4 g / L to the culture medium, IPTG at a final concentration of 0.5 μM was added 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 and 200 r / min for 14 h. After the induction, the fluorescence intensity was measured using an ELISA reader at an excitation wavelength of 488 nm and a detection wavelength of 535 nm. The relationship between the addition concentration of L-valine and the fluorescence intensity is shown in 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 the codon with the lowest frequency is GUA. According to the method described in Example 1, a gene staygold-levE in which the codons for L-valine are all GUA is constructed. rGUA , and the fluorescence intensity of gene expression was detected according to Examples 2 to 5. Without adding L-valine, the fluorescence intensity results of the system at different fermentation times are 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 screening marker gene.

[0064] Comparative Example 2

[0065] 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 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 of L-valine in Escherichia coli. The nucleotide sequence after replacement is shown in SEQ ID NO.5. According to the method described in Examples 1 to 4, staygold-rplW were obtained by gene synthesis. r fragment and construct the recombinant vector pUC-57(+)-staygold-rplW rThen, the relationship between the fluorescence intensity of gene expression and the concentration of L-valine in the fermentation broth was detected according to the method of Example 5. The results showed that the staygold-rplW r There was no obvious correlation between the fluorescence intensity of the transformed strain with fluorescence screening marker and the concentration of L-valine in the fermentation broth.

[0066] Experimental Example 1

[0067] Application of fluorescent protein selection markers

[0068] (1) Transformation with fluorescent protein screening marker

[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 picked and 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 exposed to ARTP for 120s.

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

[0075] (4) Inducible expression of fluorescent proteins

[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 and eluted for 1 min, and cultured at 37°C and 200 r / min until the OD 600 The pH value was 0.6-0.8, IPTG was added at 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 the strain clustering, with excitation light at 488nm, fluorescence detection at 535nm, sample pressure at 60psi, nozzle diameter set to 70μm, and Beckman Summit 5.2 software for data analysis. A gate of 0.01% of the total cells was set, and cells with higher fluorescent protein expression were collected into a 96-well plate containing 200μL LB liquid medium and cultured on the 96-well plate at 37℃ for 36h.

[0079] (6) L-valine fermentation test

[0080] The bacterial liquid in the 96-well plate obtained in step (5) was used as a seed liquid, and was re-transferred to a deep-well plate containing 1 mL of fermentation medium at a volume ratio of 10%, and continued to ferment in a microplate incubator at 37° C. and 600 r / min for 48 h. After the fermentation was completed, 100 μL of the fermentation liquid was taken from each well and centrifuged at 5000 r / min for 5 min, and the supernatant was diluted to an L-valine concentration of 0.1 to 1 g / L, and the concentration of L-valine was determined using a biosensor analyzer.

[0081] The fermentation medium formula is: glucose 2.0%, corn syrup 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%, 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 before sorting. The initial screening excluded 113 strains with low fluorescence intensity, and retained strains with relatively high fluorescence intensity for further research. Among the sorted strains, the 23 strains with the highest fluorescence intensity 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 0.21g / L. Figure 5As shown, among the 23 mutant strains, 18 strains with improved L-valine production were screened, the screening efficiency reached 78.2%, the highest yield was 0.306 g / L, and the yield was increased by 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 staygold-levE according to claim 1 r A method for constructing a gene, characterized in that: The steps include: The codons of L-valine in the nucleotide sequences of the fluorescent protein gene Staygold and the gene levE were replaced with the rare codon GTC to obtain the fluorescent protein gene Staygold after codon replacement and the gene levE after codon replacement, respectively. The two codon-replaced genes were connected with a flexible protein peptide to obtain the fluorescent protein selection marker staygold-levE r Gene.

3. The construction method according to claim 2, characterized in that: The nucleotide sequence of the fluorescent protein gene Staygold after the codon replacement is shown in SEQ ID NO.

1.

4. The construction method according to claim 2, characterized in that: The nucleotide sequence of the gene levE after the codon replacement is shown in SEQ ID NO.

2.

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

6. The recombinant vector according to claim 5, characterized in that The vectors used included pET-22b(+) and pUC-57(+).

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

8. The recombinant bacterium according to claim 7, characterized in that The hosts used include Escherichia coli CGMCC NO.1.366 and Escherichia coli BL21 (DE3).

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

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