Staygold-ynamr gene for screening fluorescent protein and application thereof

Abstract

The application provides a fluorescent protein screening marker staygold-ynaM r The application belongs to the technical field of bioengineering, and relates to a fluorescent protein gene staygold and a nucleotide sequence of a gene ynaM The codon of L-isoleucine in the nucleotide sequence is replaced by a rare codon ATA to obtain a fluorescent protein gene after codon replacement staygold and a gene after codon replacement ynaM r The two genes after codon replacement are connected by a flexible protein peptide to obtain the fluorescent protein screening marker staygold- ynaM r The gene can obviously improve the screening efficiency of an L-isoleucine high-yield strain.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a fluorescent protein screening marker. staygold-ynaM r Genes and their applications. Background Technology

[0002] L-Isoleucine, as one of the essential amino acids, has a variety of physiological functions, including regulating blood circulation, improving glucose metabolism, maintaining liver function, promoting muscle growth and repair, and enhancing endurance. It has wide applications in the food, feed, and pharmaceutical industries, and shows great market potential.

[0003] Microbial fermentation is a highly efficient and environmentally friendly core technology for the production of L-isoleucine. Microbial fermentation utilizes the powerful metabolic network of microorganisms (such as Corynebacterium glutamicum and Escherichia coli) to efficiently convert carbon sources (such as glucose) into the target amino acid. Although microbial fermentation has made significant progress in the industrial production of L-isoleucine, one of its core bottlenecks lies in the efficiency of selecting high-yield strains.

[0004] Modern breeding techniques, such as gene-editing tools like CRISPR-Cas9 and methods like chemical and physical mutagenesis, can select strains that overcome feedback repression and inhibition in metabolic regulation, or mutant strains resistant to L-isoleucine structural analogs, aiming to enhance the L-isoleucine biosynthetic pathway. However, efficiently and accurately screening truly high-yielding and genetically stable L-isoleucine-producing strains from a vast mutant library remains a key challenge restricting further industry development. Therefore, developing a universal, high-throughput screening marker has become a crucial direction for improving the screening efficiency of L-isoleucine-producing strains. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a fluorescent protein screening marker. staygold-ynaM r Genes and their applications, this fluorescent protein selection marker staygold-ynaM r Genes can significantly improve the screening efficiency of high-yield L-isoleucine strains, enhancing sensitivity and accuracy.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0007] This invention provides a fluorescent protein screening marker. staygold-ynaM r Gene, the fluorescent protein selection marker staygold-ynaM r Genes are formed by flexible protein peptides staygold r Genes and faithr It is formed by the connection of genes.

[0008] The staygold r Gene replaced by ATA staygold Genes following the L-isoleucine codon in a gene.

[0009] The faith r Gene replaced by ATA faith Genes following the L-isoleucine codon in a gene.

[0010] Preferably, the staygold and faith The codons for L-isoleucine are ATA, ATT, and ATC.

[0011] Preferably, the staygold r The nucleotide sequence of the gene is shown in SEQ ID NO.1; staygold 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.

[0012] Preferably, the fluorescent protein screening marker staygold-ynaM r The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0013] This invention provides the fluorescent protein screening marker. staygold-ynaM r The method for constructing genes includes the following steps: staygold and faith The L-isoleucine codon in the sample was replaced with ATA, and the following results were obtained: staygold r Genes and faith r Genes will staygold r Genes and faith r Genes are linked using flexible protein peptides to obtain fluorescent protein selection markers. staygold-ynaM r Gene.

[0014] This invention provides a recombinant vector containing the fluorescent protein selection marker. staygold- faith r Gene.

[0015] Preferably, the starting vector of the recombinant vector is pET-22b(+) or pUC-57(+).

[0016] This invention provides a recombinant bacterium containing the fluorescent protein selection marker. staygold-ynaM r Gene or the recombinant vector.

[0017] Preferably, the starting strain of the recombinant bacteria is Corynebacterium glutamicum ATCC 13032 or Escherichia coli. E. coli BL21.

[0018] This invention provides the fluorescent protein screening marker. staygold-ynaM r The application of the gene, the recombinant vector, or the recombinant bacteria in screening for high-yielding L-isoleucine strains.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a fluorescent protein screening marker for screening L-isoleucine-producing strains. staygold- faith r This selection marker gene, compared to other similar selection markers, exhibits a positive correlation between fluorescence intensity and L-isoleucine concentration, significantly improving the screening efficiency of high-yielding L-isoleucine strains. This selection marker can save screening time and costs.

[0021] Furthermore, the fluorescent protein screening marker of the present invention staygold-ynaM r The gene is prepared by replacing the L-isoleucine codon in the original nucleotide sequence with the rare codon ATA. The screening marker is prepared by fusing two genes, which can enhance sensitivity and improve screening efficiency and accuracy. Attached Figure Description

[0022] Figure 1 This is an agarose gel electrophoresis image of the PCR amplification products in Example 2.

[0023] Lanes 1-3 are staygold-ynaM r .

[0024] Figure 2 The recombinant bacteria at different L-isoleucine concentrations in Example 3 E. coli BL21(DE3) / pUC-57(+)- staygold-ynaM r The fluorescence intensity curve.

[0025] Figure 3 For the recombinant bacteria at different L-isoleucine concentrations in Comparative Example 1 E. coli BL21(DE3) / pUC-57(+)- staygold-ynfTr The fluorescence intensity curve.

[0026] Figure 4 For the recombinant bacteria at different L-isoleucine concentrations in Comparative Example 2 E. coli BL21(DE3) / pUC-57(+)- staygold r The fluorescence intensity curve.

[0027] Figure 5 The scatter plot shows the L-isoleucine production of the Corynebacterium glutamicum ATCC 13032 mutant strain selected in the application example. Detailed Implementation

[0028] This invention provides a fluorescent protein screening marker. staygold-ynaM r Gene, the fluorescent protein selection marker staygold-ynaM r The gene replaces ATA with a flexible protein peptide. staygold and faith The gene is formed by linking the L-isoleucine codon. The ATA described in this invention is a rare codon. staygold and faith The codons for L-isoleucine are ATA, ATT, and ATC.

[0029] In this invention, ATA is replaced. staygold The nucleotide sequence following the L-isoleucine codon is shown in SEQ ID NO. 1; ATA substitution faith The nucleotide sequence following the L-isoleucine codon 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 label of this invention... staygold- faith r The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0030] The present invention also provides the fluorescent protein screening marker. staygold-ynaM r The method for constructing genes includes the following steps: staygold and faith The L-isoleucine codon was replaced with ATA, and the codons after the replacements were obtained. staygold After gene and codon substitution faith r Genes, after replacing two codons, are linked together using flexible protein peptides to obtain fluorescent protein selection markers. staygold-ynaM r Gene. The codon substitution described in this invention staygoldThe nucleotide sequence of the gene is shown in SEQ ID NO.1; after codon substitution... faith 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 of this invention... staygold-ynaM r The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0031] The present invention also provides a recombinant vector containing the fluorescent protein selection marker. staygold-ynaM r Genes. The starting vector of the recombinant vector described in this invention includes pET-22b(+) or pUC-57(+).

[0032] The present invention also provides a recombinant bacterium containing the fluorescent protein selection marker. staygold- faith r The gene or the recombinant vector. The starting strain of the recombinant bacteria of this invention includes Corynebacterium glutamicum ATCC 13032 or Escherichia coli. E. coli BL21.

[0033] The present invention also provides the fluorescent protein screening marker. staygold-ynaM r The application of the gene, the recombinant vector, or the recombinant bacteria in screening for high-yielding L-isoleucine strains.

[0034] In this invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0035] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0036] The vectors and host bacteria involved in the embodiments of this invention were all purchased from Sangon Biotech (Shanghai) Co., Ltd., and are commercially available products. Corynebacterium glutamicum ATCC 13032 was purchased from the China General Microbiological Culture Collection Center.

[0037] Example 1 staygold-ynaM r Fragment construction

[0038] Search for fluorescent protein genes in the National Center for Biotechnology Information (NCBI) database. staygoldThe nucleotide sequence, NCBI accession number LC601652.1, contains 8 L-isoleucine codons, including 1 codon ATA, 3 codons ATC, and 4 codons ATT. The L-isole codons ATC and ATT in the sequence were replaced with the rare L-isole codon ATA. staygold r The nucleotide sequence is shown in SEQ ID NO.1.

[0039] From E. coli ( Escherichia coli Genes with a high proportion of L-isoleucine codons were selected from the K-12 MG1655 genome. faith The nucleotide sequence contains 11 L-isoleucine codons, including 7 codons ATA, 2 codons ATC, and 2 codons ATT. The L-isoleucine codons ATC and ATT are replaced with the rare L-isoleucine codon ATA. faith r The nucleotide sequence is shown in SEQ ID NO.2.

[0040] After codon replacement via flexible linker peptide faith r Fragment and codon substitution fluorescent protein gene staygold r Connect and synthesize to obtain staygold-ynaM r Fragment, obtained staygold-ynaM r The nucleotide sequence of the fragment is shown in SEQ ID NO.3. The nucleotide sequence encoding the flexible linker peptide is shown in SEQ ID NO.4.

[0041] Example 2

[0042] 1. Construction of recombinant plasmids

[0043] The vector plasmid pUC-57(+) was used with FastDigest. EcoRI FastDigest Indian After treatment with the III double enzyme digestion system, it was then processed using a seamless cloning system and combined with the same FastDigest enzyme. EcoRI FastDigest Indian III double enzyme digestion staygold-ynaM r Fragment ligation yielded the recombinant vector pUC-57(+)- staygold-ynaM r .

[0044] The double enzyme digestion reaction system is shown in Table 1, with a total volume of 20 μL; the double enzyme digestion reaction conditions are as follows:

[0045] 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 on ice.

[0046] The seamless cloning system is shown in Table 2, with a total volume of 10 μL.

[0047] Table 1. Double enzyme digestion reaction system

[0048]

[0049] Table 2 Seamless Cloning System

[0050]

[0051] 2. Transformation of competent cells

[0052] Take the recombinant vector pUC-57(+)- staygold-ynaM r Add 10 μL of E. coli. E. coli In BL21(DE3) competent cells, the mixture was gently tapped against the tube wall to mix, and then incubated on ice for 30 min. The cells were then heat-shocked in a 42°C water bath for 90 s, immediately placed on ice for 3 min, and 900 μL of antibiotic-free LB liquid medium was added to the centrifuge tube. The mixture was incubated at 37°C and 200 rpm for 1 h in a shaker. After incubation, the bacterial suspension was centrifuged at 5000 rpm for 2 min, and 900 μL of supernatant was discarded. The remaining bacterial cells were resuspended and evenly spread using a sterile spreader onto LB agar plates containing 100 μg / mL ampicillin. The plates were then incubated upside down in a 37°C incubator for 16 h.

[0053] The above LB liquid medium formula is: 0.5% yeast extract, 1% peptone, 1% sodium chloride, and water as solvent. The LB solid medium formula is: 0.5% yeast extract, 1% peptone, 1% sodium chloride, water as solvent, and 2% agar powder.

[0054] 3. Screening and verification of positive colonies

[0055] Select the positive recombinant colonies from step 2 and inoculate them into LB liquid medium containing 100 μg / mL ampicillin. Incubate overnight at 37°C. After incubation, use the bacterial culture as a template and P1 and P2 as primers for PCR amplification to obtain the amplified gene product. staygold-ynaM rThe primer sequences are P1 (sequence shown in SEQ ID NO. 6) and P2 (sequence shown in SEQ ID NO. 7). The PCR amplification system is shown in Table 3, with a total volume of 20 μL. The PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 57℃ annealing for 1 min, 72℃ extension for 4 min, 30 cycles; 72℃ extension for 10 min, and storage at 4℃.

[0056] Table 3 PCR amplification system

[0057]

[0058] The amplification products were verified using agarose gel electrophoresis. The results showed that primers P1 and P2 could amplify a specific gene band of approximately 700 bp. Figure 1 This is close to the theoretical value of 672bp, indicating that staygold-ynaM r Fragment insertion was successful, and recombinant bacteria were obtained. E. coli BL21 (DE3) / pUC-57(+)- staygold-ynaM r .

[0059] Example 3: Detection of L-Isoleucine Concentration and Fluorescence Intensity

[0060] The recombinant bacteria successfully verified in Example 2 E. coli BL21 (DE3) / pUC-57(+)- staygold-ynaM r Streak the bacteria on LB solid medium, pick a single colony and inoculate it into LB liquid medium, then incubate at 37°C and 200 rpm until OD reaches zero. 600 =1.0, and inoculated at a volume ratio of 2% into 50 mL of LB liquid medium containing a final concentration of 100 μg / mL ampicillin, and cultured at 37 °C and 200 r / min until OD. 600 =1.0, L-isoleucine was added to the culture medium at final concentrations of 0, 0.1, 0.2, 0.3, and 0.4 g / L, followed by the addition of 1 mM IPTG to induce the expression of fluorescent protein. The control group received no IPTG. Each gradient was tested in triplicate. Induction conditions were 28℃, 200 rpm for 18 h. After induction, 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 L-isoleucine concentration and fluorescence intensity is shown in [reference needed]. Figure 2 The results showed that within a certain concentration range (0-0.3 g / L), the fluorescence intensity increased significantly with the increase of L-isoleucine addition; however, when the concentration exceeded 0.3 g / L, the fluorescence intensity tended to stabilize.

[0061] Comparative Example 1

[0062] From E. coli ( Escherichia coli The gene with the second highest proportion of L-isoleucine in the amino acid sequence of the K-12 substr. MG1655 genome was selected. infT The sequence contains nine L-isoleucine codons, including five ATA, two ATC, and two ATT. The L-isoleucine codons ATC and ATT in the sequence were replaced with the rare L-isoleucine codon ATA. The resulting nucleotide sequence is shown in SEQ ID NO. 5. Following the method described in Example 1 or 2, the sequence was obtained through gene synthesis. staygold-ynfT r Fragments were extracted and the recombinant vector pUC-57(+)- was constructed. staygold-ynfT r And recombinant bacteria. Subsequently, following the method in Example 3, the relationship between the fluorescence intensity of gene expression and the concentration of L-isoleucine in the fermentation broth was detected, and the results are as follows: Figure 3 As shown, containing staygold-ynfT r The fluorescence intensity of the fluorescently selected transformant strains was not significantly correlated with the concentration of L-isoleucine in the fermentation broth.

[0063] Comparative Example 2

[0064] Replaced with the rare codon ATA staygold r Genes were used in experiments. Following the methods described in Example 1 or 2, genes were synthesized to obtain... staygold r Fragments were extracted and the recombinant vector pUC-57(+)- was constructed. staygold r And recombinant bacteria. Subsequently, the relationship between the fluorescence intensity of gene expression and the concentration of L-isoleucine in the fermentation broth was detected according to the method in Example 3. Figure 4 The results showed that it contained staygold r The fluorescence intensity of the fluorescently selected transformant strains was not significantly correlated with the concentration of L-isoleucine in the fermentation broth.

[0065] Application example: Application of fluorescent protein for screening marker genes

[0066] (1) Preparation of Corynebacterium glutamicum ATCC 13032 competent cells

[0067] Single colonies of *Corynebacterium glutamicum* ATCC 13032 were picked and cultured in seed culture medium until the bacterial concentration reached OD500. 600The concentration was 0.9, and the cells were placed on ice to cool to 4°C. After cooling, the cells were centrifuged and washed four times with pre-cooled electroporation buffer. The cells were then resuspended in electroporation buffer to obtain competent Corynebacterium glutamicum ATCC 13032 cells.

[0068] The seed culture medium has the following components per liter: 10g peptone, 5g yeast extract, 10g sodium chloride, 5g glucose, and the remainder is water;

[0069] The electroporation buffer solution has the following components per liter: 90g sorbitol, 90g mannitol, 100mL glycerol, and the remainder is water.

[0070] (2) Fluorescent protein screening labeling transformation

[0071] The recombinant vector pUC-57(+)- obtained in Example 2 staygold-ynaM r 10 μL of the solution was electroporated into competent Corynebacterium glutamicum ATCC13032 cells, transferred to liquid resuscitation medium, and cultured at 30°C for 14 h. Transformants were then screened.

[0072] The liquid resuscitation medium has the following components per liter: 10g peptone, 5g yeast extract, 10g sodium chloride, 90g sorbitol, 70g mannitol, and the remainder water;

[0073] The conditions for high-voltage electric shock conversion are: 2200V electric shock for 5ms.

[0074] (3) Screening of positive colonies

[0075] The positive recombinant colonies from step (1) were selected and verified, following the same procedure as the positive colony screening and verification method in Example 2, resulting in *Corynebacterium glutamicum* ATCC 13032 / pUC-57(+)- staygold-ynaM r Recombinant bacteria.

[0076] (4) Recombinant strain ARTP mutagenesis

[0077] Pick a single colony from step (3) and inoculate it into LB liquid medium containing 100 μg / mL ampicillin. Incubate at 30°C and 200 r / min until OD. 600 The concentration was set to 0.8. 10 μL of diluted bacterial solution was spread evenly on the surface of a sterile stainless steel slide and exposed to ARTP for 85 s. The ARTP parameters were set as follows: incident power 120 W, gas flow rate 10 SLM, and helium pressure 120 MPa.

[0078] (5) Induced expression of fluorescent proteins

[0079] After mutagenesis, stainless steel slides containing the mutagenic bacterial solution were placed in 1 mL of LB liquid medium containing 100 μg / mL ampicillin, shaked for 1 min, and cultured at 37 °C and 200 r / min until OD. 600 The concentration was 0.8, and IPTG was added to a final concentration of 1 mM. The mixture was induced at 28°C for 18 h.

[0080] (6) High-throughput screening of mutant strains

[0081] Take 1 mL of the bacterial culture obtained after induction in step (5), wash and resuspend it in 0.1% PBS buffer (pH=7.0) and dilute to OD. 600 =1.0. Flow cytometry was used to analyze bacterial populations. Excitation light was set at 488 nm, fluorescence detection at 535 nm, sample pressure at 60 psi, and nozzle diameter at 70 μm. Beckman Summit 5.2 software was used for data analysis. A gate of 0.01% of total cells was set to collect cells with high fluorescent protein expression levels into 96-well plates containing 200 μL of LB liquid medium, and the plates were incubated at 30°C for 24 h.

[0082] (7) L-Isoleucine fermentation test

[0083] The bacterial culture obtained in step (6) was used as the seed culture and transferred at a volume ratio of 10% to a deep-well plate containing 1 mL of fermentation medium. Fermentation was continued in a microplate incubator at 37°C, 800 r / min, and for 24 h. The fermentation medium was formulated as follows: yeast extract 0.2%, glucose 0.5%, phosphate 0.06%, (NH4)2SO4 1%, MgSO4 0.20%, KCl 0.05%, FeSO4 0.03%, MnSO4 0.03%, vitamin B1 0.0005%, and water as solvent.

[0084] Following the methods described in the examples and application examples, 173 ATCC 13032 / pUC-57(+)- strains were obtained through sorting. staygold- faith r The mutant strains were selected, and the 50 strains with the highest fluorescence intensity were subjected to shake-flask fermentation at 30℃ and 200 rpm for 24 h. The yield of L-isoleucine was then measured. Among them, the original strain ATCC 13032 showed the highest L-isoleucine yield in multiple parallel fermentations at 0.548 g / L. Figure 5 As shown, among the 50 mutant strains, 41 strains with increased L-isoleucine production were screened, with a screening efficiency of 82%.

[0085] In summary, the fluorescent protein screening markers provided by this invention... staygold-ynaMr The gene can effectively screen strains that increase L-isoleucine production.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fluorescent protein selection marker staygold-ynaM r The application of genes in screening high-yielding L-isoleucine strains is characterized by, The fluorescent protein selection marker staygold-ynaM r Genes are formed by flexible protein peptides staygold r Genes and ynaM r Genes linked together; The staygold r Gene replaced by ATA staygold Genes following the L-isoleucine codon in a gene; The ynaM r Gene replaced by ATA ynaM Genes following the L-isoleucine codon in a gene; The staygold and ynaM The codons for L-isoleucine are ATA, ATT, and ATC; The staygold r The nucleotide sequence of the gene is shown in SEQ ID NO.1; The ynaM r The nucleotide sequence of the gene is shown in SEQ ID NO.2; The nucleotide sequence of the flexible protein peptide is shown as SEQ ID NO.4; The fluorescent protein selection marker staygold-ynaM r The nucleotide sequence of the gene is shown in SEQ ID NO.3; The fluorescent protein selection marker staygold-ynaM r The method for constructing genes is as follows: staygold and ynaM The L-isoleucine codon in the sample was replaced with ATA, and the following results were obtained: staygold r Genes and ynaM r Genes will staygold r Genes and ynaM r Genes are linked using flexible protein peptides to obtain fluorescent protein selection markers. staygold-ynaM r Gene; The fluorescent protein selection marker staygold-ynaM r The method for applying genes in screening high-yielding L-isoleucine strains involves constructing a selection marker containing a fluorescent protein. staygold-ynaM r Gene recombinant vectors were used to construct recombinant bacteria containing the above recombinant vectors, and then the above recombinant bacteria were used to screen for high L-isoleucine-producing strains; The starting vector of the recombinant vector is pET-22b(+) or pUC-57(+); The recombinant bacteria originated from Corynebacterium glutamicum. Corynebacterium glutamicum ATCC 13032 or E. coli Escherichia coli BL21(DE3).

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