A fluorescent protein selection marker, staygold-ybdJ r Genes and their applications

Abstract

The application provides a fluorescent protein screening marker staygold-ybdJ 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 ybdJ The codon of L-leucine in the nucleotide sequence is replaced by a rare codon TTA to obtain a fluorescent protein gene after codon replacement Staygold and a gene after codon replacement ybdJ r The two genes after codon replacement are connected by a flexible protein peptide to obtain the fluorescent protein screening marker staygold- ybdJ r The gene can obviously improve the screening efficiency of an L-leucine high-yield strain.

Description

Technical Field

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

[0002] Microbial fermentation technology has become a major method for the industrial production of L-leucine due to its high efficiency and environmental friendliness. This technology modifies the metabolic pathways of microorganisms, enabling them to efficiently convert carbon sources into target amino acids. In recent years, with the combined application of gene editing technologies such as CRISPR-Cas9 and traditional mutagenesis methods, researchers have been able to construct engineered strains with metabolic regulatory advantages, such as auxotrophic strains resistant to feedback inhibition or L-leucine analogue-resistant strains, thereby significantly increasing yields.

[0003] Currently, researchers typically utilize structural analogs of L-leucine (such as aminoethylcysteine) combined with physical or chemical mutagenesis techniques to construct large-scale mutant strain libraries, containing both positive and negative mutations. However, traditional screening methods based on amino acid analogs have significant limitations: on the one hand, high concentrations of analogs may adversely affect cell metabolism and structure, reducing screening efficiency; on the other hand, long-term reliance on a few analogs can lead to saturation of the screening system, making it difficult to obtain novel, high-yielding mutant strains with industrial application potential. Therefore, developing a versatile, high-throughput screening marker applicable to multiple microorganisms is crucial for improving the breeding efficiency of L-leucine-producing strains.

[0004] Faced with the continued growth in global demand for L-leucine, developing strains with high yield, good genetic stability, and low cost has become a key focus for the industry. Currently, a critical factor hindering industry development is the lack of efficient and specific screening markers. Therefore, designing and developing novel L-leucine-specific screening systems has become a pressing technical challenge. This breakthrough will significantly improve the screening efficiency of high-yield strains and drive the upgrading and development of the entire industry. Summary of the Invention

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

[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-ybdJ r Gene, the fluorescent protein selection marker staygold-ybdJ r The gene is replaced by a flexible protein peptide that replaces TTA. Staygold and ybdJ Genes linked after the L-leucine codon are formed.

[0008] Preferably, the Staygold and ybdJ The codons for L-leucine are TTA, TTG, CTG, CTC, CTT, and CTA.

[0009] Preferred, TTA replacement Staygold The nucleotide sequence following the L-leucine codon is shown in SEQ ID NO.1; TTA substitution ybdJ The nucleotide sequence following the L-leucine codon is shown in SEQ ID NO.2; the nucleotide sequence of the flexible protein peptide is shown in SEQ ID NO.4.

[0010] Preferably, the fluorescent protein screening marker staygold-ybdJ r The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0011] This invention provides the fluorescent protein screening marker. staygold-ybdJ r A method for constructing genes, characterized by comprising the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Staygold and ybdJ The L-leucine codon in [the sample] was replaced with TTA, and the codons after the replacements were obtained respectively. Staygold After gene and codon substitution ybdJ r Genes, after replacing two codons, are linked together using flexible protein peptides to obtain fluorescent protein selection markers. staygold-ybdJ r Gene.

[0012] This invention provides a recombinant vector containing the fluorescent protein selection marker. staygold- ybdJ r Gene.

[0013] Preferably, the starting vector of the recombinant vector is pJYS2_crtYf.

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

[0015] Preferably, the starting strain of the recombinant bacteria includes Corynebacterium glutamicum SCgG2 or Escherichia coli. E. coli BL21.

[0016] This invention provides the fluorescent protein screening marker. staygold-ybdJ r The application of the gene, the recombinant vector, or the recombinant bacteria in screening for high-yielding L-leucine strains.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention provides a fluorescent protein screening marker for screening L-leucine-producing strains. staygold-ybdJ r This selection marker gene, compared to other similar selection markers, exhibits a positive correlation between fluorescence intensity and L-leucine concentration, significantly improving the screening efficiency of high-yield L-leucine strains. This selection marker can save screening time and costs.

[0019] (2) Fluorescent protein screening marker of the present invention staygold-ybdJ r The gene is prepared by replacing the L-leucine codon in the original nucleotide sequence with the rare codon TTA. The screening marker is prepared by fusing two genes, which can enhance sensitivity and improve screening efficiency and accuracy. Attached Figure Description

[0020] Figure 1 This is an agarose gel electrophoresis image of the PCR amplification products in Example 2;

[0021] Lanes 1-4 are Staygold-ybdJ r .

[0022] Figure 2 The recombinant bacteria at different leucine concentrations in Example 3 E. coli BL21(DE3) / pJYS2_crtYf- staygold-ybdJ r The fluorescence intensity curve.

[0023] Figure 3 For the recombinant bacteria at different leucine concentrations in Comparative Example 1 E. coli BL21(DE3) / pJYS2_crtYf- staygold-yjjZ r The fluorescence intensity curve.

[0024] Figure 4 For the recombinant bacteria at different leucine concentrations in Comparative Example 2 E. coli BL21(DE3) / pJYS2_crtYf- staygoldr The fluorescence intensity curve.

[0025] Figure 5 This is a scatter plot of L-leucine production from the Corynebacterium glutamicum mutant strains selected in the application example. Detailed Implementation

[0026] This invention provides a fluorescent protein screening marker. staygold-ybdJ r Gene, the fluorescent protein selection marker staygold-ybdJ r The gene is replaced by a flexible protein peptide that replaces TTA. Staygold and ybdJ The gene is formed by linking the L-leucine codon. The TTA described in this invention is a rare codon. Staygold and ybdJ The codons for L-leucine are TTA, TTG, CTG, CTC, CTT, and CTA.

[0027] In this invention, TTA is replaced. Staygold The nucleotide sequence following the L-leucine codon is shown in SEQ ID NO.1; TTA substitution ybdJ The nucleotide sequence following the L-leucine 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-ybdJ r The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0028] The present invention also provides the fluorescent protein screening marker. staygold-ybdJ r The method for constructing genes includes the following steps: Staygold and ybdJ The L-leucine codon in [the sample] was replaced with TTA, and the codons after the replacements were obtained respectively. Staygold After gene and codon substitution ybdJ r Genes, after replacing two codons, are linked together using flexible protein peptides to obtain fluorescent protein selection markers. staygold-ybdJ r Gene.

[0029] The present invention also provides a recombinant vector containing the fluorescent protein selection marker. staygold-ybdJ r Gene. The starting vector of the recombinant vector described in this invention is pJYS2_crtYf.

[0030] The present invention also provides a recombinant bacterium containing the fluorescent protein selection marker. staygold- ybdJ r The gene or the recombinant vector. The starting strain of the recombinant bacteria of this invention includes Corynebacterium glutamicum SCgG2 or Escherichia coli. E. coli BL21.

[0031] The present invention also provides the fluorescent protein screening marker. staygold-ybdJ r The application of the gene, the recombinant vector, or the recombinant bacteria in screening for high-yielding L-leucine strains.

[0032] In this invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0033] 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.

[0034] 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 (… Corynebacterium glutamicum SCgG2 was purchased from the China General Microbiological Culture Collection Center.

[0035] Example 1 staygold-ybdJ r Fragment construction

[0036] Search for fluorescent protein genes in the National Center for Biotechnology Information (NCBI) database. Staygold The nucleotide sequence, NCBI accession number LC601652.1, contains 13 L-leucine codons, including 5 TTA, 1 TTG, 1 CTG, 4 CTT, 1 CTA, and 1 CTC. The L-leucine codons TTG, CTG, CTT, CTA, and CTC in the sequence were replaced with the rare L-leucine codon TTA. staygold r The nucleotide sequence is shown in SEQ ID NO.1.

[0037] From E. coli ( Escherichia coli Genes with a high proportion of L-leucine codons were selected from the K-12 substr. MG1655 genome. ybdJThe nucleotide sequence contains 22 L-leucine codons, including 3 TTA, 3 TTG, 13 CTG, 2 CTT, and 1 CTC. The L-leucine codons TTG, CTG, CTT, and CTC in the sequence were replaced with the rare L-leucine codon TTA. The resulting nucleotide sequence is shown in SEQ ID NO.2.

[0038] After codon replacement via flexible linker peptide ybdJ r Fragment and codon substitution fluorescent protein gene Staygold r Connect and synthesize to obtain staygold-ybdJ r Fragment, obtained staygold-ybdJ 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.

[0039] Example 2

[0040] 1. Construction of recombinant plasmids

[0041] The vector plasmid pJYS2_crtYf was processed using 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-ybdJ Fragment ligation yields the recombinant vector pJYS2_crtYf- staygold-ybdJ .

[0042] 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:

[0043] React at 37℃ for 30 min; inactivate at 80℃ for 5 min, and store at 4℃. The seamless cloning procedure is as follows: carry out the recombination reaction at 50℃ for 15 min, and then immediately cool on ice.

[0044] The seamless cloning system is shown in Table 2, with a total volume of 10 μL.

[0045] Table 1. Double enzyme digestion reaction system

[0046]

[0047] Table 2 Seamless Cloning System

[0048]

[0049] 2. Transformation of competent cells

[0050] Take the recombinant vector pJYS2_crtYf- staygold-ybdJ 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 50 μg / mL spectinomycin. The plates were then incubated upside down in a 37°C incubator for 12 h.

[0051] 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.

[0052] 3. Screening and verification of positive colonies

[0053] Select the positive recombinant colonies from step 2 and inoculate them into LB liquid medium containing 50 μg / mL spectinomycin. 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-ybdJ r The primer sequences are: P1: 5'-CACGCTGTCGGCAGAGAAC-3' (SEQ ID NO. 6) and P2: 5'-TGATCTACAACAGTAGAAATTCGGA-3' (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 2 min; 95℃ denaturation for 20 sec, 57℃ annealing for 20 sec, 72℃ extension for 2 min, 35 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0054] Table 3 PCR amplification system

[0055]

[0056] 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 1000 bp. The agarose gel electrophoresis image of the PCR amplification products is shown below. Figure 1 This is close to the theoretical value of 1065bp, indicating that staygold-ybdJ r Fragment insertion was successful, and recombinant bacteria were obtained. E. coli BL21 (DE3) / pJYS2_crtYf- staygold-ybdJ r .

[0057] Example 3: Detection of Leucine Addition Concentration and Fluorescence Intensity

[0058] The recombinant bacteria successfully verified in Example 2 E. coli BL21 (DE3) / pJYS2_crtYf- staygold-ybdJ 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, inoculated at a volume ratio of 2% into 50 mL of LB liquid medium containing a final concentration of 50 μg / mL spectinomycin, and incubated at 37 °C and 200 rpm until OD. 600 =1.0, L-leucine was added to the culture medium at final concentrations of 0, 0.1, 0.3, 0.5, and 0.7 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 525 nm. The relationship between L-leucine concentration and fluorescence intensity is shown in [reference needed]. Figure 2 The results showed that the fluorescence intensity was positively correlated with the amount of L-leucine added.

[0059] Comparative Example 1

[0060] From E. coli ( Escherichia coli The gene with the second highest proportion of L-leucine in the amino acid sequence of the K-12 substr. MG1655 genome was screened. yjjZ The sequence contains 14 L-leucine codons, including 2 TTA, 7 TTG, 8 CTG, 1 CTT, and 1 CTA. The L-leucine codons TTG, CTG, CTT, and CTA in the sequence were replaced with the rare E. coli L-leucine codon TTA. 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-yjjZ r Fragments were extracted and the recombinant vector pJYS2_crtYf- was constructed. staygold-yjjZr And recombinant bacteria. Subsequently, the relationship between the fluorescence intensity of gene expression and the concentration of L-leucine in the fermentation broth was detected according to the method in Example 3, and the results are as follows: Figure 3 As shown, containing staygold-yjjZ r The fluorescence intensity of the fluorescently selected transformant strains was not significantly correlated with the concentration of L-leucine in the fermentation broth.

[0061] Comparative Example 2

[0062] Replaced with the rare codon TTA staygold r Genes were used to conduct the above experiments.

[0063] According to the method described in Example 1 or 2, respectively, through gene synthesis, [the following were obtained] staygold r Fragments were extracted and the recombinant vector pJYS2_crtYf- was constructed. staygold r And recombinant bacteria. Subsequently, the relationship between the fluorescence intensity of gene expression and the concentration of L-leucine in the fermentation broth was detected according to the method in Example 3, and the results are as follows: Figure 4 As shown, containing staygold r The fluorescence intensity of the fluorescently selected transformant strains was not significantly correlated with the concentration of L-leucine in the fermentation broth.

[0064] Application example: Application of fluorescent protein for screening marker genes

[0065] (1) Fluorescent protein screening labeling transformation

[0066] Take the recombinant vector pJYS2_crtYf- obtained in Example 2 staygold-ybdJ r 10 μL was transformed into L-leucine-producing Corynebacterium SCgG2 competent cells and cultured for 12 h, following the same procedure as in Example 2.

[0067] (2) Screening of positive colonies

[0068] Select and verify the positive recombinant colonies from step (1), following the same steps as in Example 2, to obtain the recombinant bacteria Corynebacterium glutamicum SCgG2 / pJYS2_crtYf- staygold-ybdJ r .

[0069] (3) Recombinant strain ARTP mutagenesis

[0070] Pick a single colony from step (2) and inoculate it into LBG liquid medium containing 50 μg / mL spectinomycin, and incubate at 30°C and 200 r / min until OD. 600The value was 0.7, and the exposure time was 60 seconds at ARTP. The ARTP parameters were set as follows: incident power of 120W, gas flow rate of 10 SLM, and helium pressure of 120MPa.

[0071] The above LBG liquid culture medium formula is: 0.5% yeast extract, 1% peptone, 1% sodium chloride, 0.5% glucose, and water as solvent.

[0072] (4) Induced expression of fluorescent proteins

[0073] After mutagenesis, stainless steel discs containing the mutagenic bacterial solution were placed in 1 mL of LBG liquid medium containing 50 μg / mL spectinomycin, shaked for 1 min, and cultured at 30℃ and 200 rpm until OD500 was reached. 600 The concentration was 0.6, and IPTG was added to a final concentration of 1 mM. The mixture was induced at 30°C for 18 h.

[0074] (5) High-throughput screening of mutant strains

[0075] Take 1 mL of the bacterial culture obtained after induction in step (4), 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 525 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 LBG liquid medium, and the plates were incubated at 30°C for 24 h.

[0076] (6) L-Leucine fermentation test

[0077] The bacterial culture obtained in step (5) 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 30℃ and 800 r / min for 24 h. The fermentation medium was formulated as follows: yeast extract 0.5%, glucose 1%, phosphate 0.05%, (NH4)2SO4 1%, MgSO4 0.3%, KCl 0.1%, FeSO4 0.05%, MnSO4 0.05%, vitamin B1 0.0005%, and water as solvent.

[0078] In this application example, flow cytometry was used to screen and obtain 167 strains of Corynebacterium SCgG2 / pJYS2_crtYf- staygold-ybdJ rFifty mutant strains with the highest fluorescence intensity were selected for shake-flask fermentation at 30℃ and 200 rpm for 24 h, and the L-leucine yield was measured. Among them, the original strain *Corynebacterium glutamicum* SCgG2 showed the highest L-leucine yield in multiple parallel fermentations at 0.336 g / L. A scatter plot of L-leucine yield from the isolated *Corynebacterium glutamicum* mutant strains is shown below. Figure 5 As shown, among the 50 mutant strains, 41 strains with increased L-leucine production were screened, with a screening efficiency of 82%.

[0079] In summary, the fluorescent protein screening markers provided by this invention... staygold-ybdJ r The gene can effectively screen strains that increase L-leucine production.

[0080] 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-ybdJ r Genes, characterized by, The fluorescent protein selection marker staygold-ybdJ r The gene was constructed using TTA substitution. Staygold After the L-leucine codon is replaced, the codon is obtained. Staygold Genes, replaced with TTA ybdJ After the L-leucine codon is replaced, the codon is obtained. ybdJ r Genes, after codons are replaced using flexible protein peptides ybdJ r After gene and codon substitution Staygold Gene connection; TTA replacement Staygold The nucleotide sequence following the L-leucine codon is shown in SEQ ID NO.1; TTA substitution ybdJ The nucleotide sequence following the L-leucine 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 selection marker staygold-ybdJ r The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. The fluorescent protein screening marker as described in claim 1 staygold-ybdJ r Genes, characterized by, The Staygold and ybdJ The codons for L-leucine are TTA, TTG, CTG, CTC, CTT, and CTA.

3. A recombinant vector, characterized in that, The recombinant vector contains the fluorescent protein screening marker according to any one of claims 1 or 2. staygold-ybdJ r Gene.

4. The recombinant vector as described in claim 3, characterized in that, The starting vector of the recombinant vector is pJYS2_crtYf.

5. A recombinant bacterium, characterized in that, The recombinant bacteria contains the fluorescent protein screening marker according to any one of claims 1 or 2. staygold-ybdJ r Gene, or the recombinant vector as described in claim 3 or claim 4.

6. The recombinant bacteria as described in claim 5, characterized in that, The starting strains of the recombinant bacteria include Corynebacterium glutamicum SCgG2 or Escherichia coli. E. coli BL21.

7. The fluorescent protein screening marker as described in claim 1 or claim 2 staygold-ybdJ r The use of the gene, or the recombinant vector of claim 3 or claim 4, or the recombinant bacteria of claim 5 or claim 6 in screening for high-yielding L-leucine strains.

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