5 '-UTR element and application thereof in production of L-arginine

Abstract

The invention provides a 5 'UTR element containing a nucleotide sequence as shown in SEQ ID NO: 11 and application of the 5' UTR element in production of L-arginine. The invention also provides a functional expression element containing the 5 'UTR element and a promoter, a gene expression cassette containing a target gene and the functional expression element, a recombinant expression vector and a corynebacterium glutamicum engineering bacterium containing the gene expression cassette or the recombinant expression vector.

Description

A 5'UTR element and its application in L-arginine production Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a 5'UTR element and application thereof in L-arginine production. Background Art

[0002] L-arginine, also known as 2-amino-5-guanidine valeric acid, has a molecular formula of C6H 14 N4O2 is widely used in medicine, food, and feed additives. It can be used as a balancer for amino acid nutrient infusions and injections; as a health supplement additive to enhance muscle vitality and immunity; and as a feed additive to increase animal fecundity.

[0003] Currently, L-arginine is mainly produced by microbial fermentation, with Escherichia coli and Corynebacterium glutamicum being the most commonly used industrial fermentation methods. During strain metabolic modification, existing techniques typically increase the expression of L-arginine biosynthesis genes by increasing the copy number of key enzyme genes (argCJBDFR) in the arginine biosynthesis pathway via plasmids. However, high-copy plasmids can increase the metabolic burden on bacteria and have poor stability.

[0004] Regulating gene expression at the transcription and translation level is the most direct and convenient way, and is realized by using a functional expression component, i.e., a promoter-5 '-untranslated region (PUTR). Therefore, by replacing PUTRs of different strengths, it is possible to achieve fine-tuning of gene expression levels, thereby alleviating the metabolic burden caused by the imbalance of cell growth and target product synthesis. However, prior art has not yet used PUTR to increase the synthesis of L-arginine in Corynebacterium glutamicum.

[0005] Therefore, there is an urgent need to develop efficient gene expression elements to increase the expression of key enzymes in the L-arginine biosynthesis pathway in Corynebacterium glutamicum and coordinate the balanced expression of multiple genes to optimize the L-arginine biosynthesis pathway.

[0006] Summary of the Invention

[0007] To overcome the above-mentioned defects, the purpose of the present invention is to screen a 5'UTR element that enhances gene expression from the genome of Corynebacterium glutamicum, and use this 5'UTR element in combination with a high-strength promoter to regulate the expression of key enzymes in the L-arginine metabolic pathway in Corynebacterium glutamicum, thereby increasing the L-arginine production of the engineered bacteria.

[0008] To achieve the above object, the present invention first uses flow cytometry sorting technology to screen the 5'UTR sequence that can promote the expression of green fluorescent protein gene from the genome of Corynebacterium glutamicum, and screens the effective optimal 5'UTR sequence by gradually shortening the length of the 5'UTR sequence. Then, the optimal 5'UTR sequence is combined with multiple high-strength promoters P ilvA 、P thrC 、P EF-tu and P dapA The recombinant expression vector was constructed and the compatibility of the 5'UTR sequence with the promoter was verified by fluorescence intensity characterization. EF-tu -The functional expression element composed of the optimal 5'UTR is used to construct a recombinant strain of Corynebacterium glutamicum expressing the L-arginine arg operon gene (argCJBDFR) to produce L-arginine through fermentation.

[0009] In a first aspect, the present invention provides a coryneform bacterium 5'UTR element obtained based on screening of a 5'UTR library, wherein the 5'UTR element comprises a nucleotide sequence as shown in SEQ ID NO: 11.

[0010] Preferably, the nucleotide sequence of the 5'UTR element is shown in any one of SEQ ID NOs: 7, 9-11.

[0011] The second aspect of the present invention provides a functional expression element, the functional expression element comprises a promoter and the 5'UTR element of the first aspect of the present invention, the promoter is selected from P EF-tu 、P ilvA 、P thrC 、P tac and P dapA .

[0012] Preferably, the promoter is P EF-tu .

[0013] A third aspect of the present invention provides a gene expression cassette comprising the functional expression element described in the second aspect, a target gene, and a terminator. The target gene is an arginine operon gene argCJBDFR, whose NCBI GeneIDs are as follows: GeneID 69621907 for argC; GeneID 69621908 for argJ; GeneID 69621909 for argB; GeneID 69621910 for argD; GeneID 69621911 for argF; and GeneID 69621912 for argR.

[0014] The fourth aspect of the present invention provides a recombinant expression vector comprising the 5'UTR element described in the first aspect of the present invention, the functional expression element described in the second aspect of the present invention, or the gene expression cassette described in the third aspect of the present invention. Preferably, the backbone of the recombinant vector is pXMJ19.

[0015] The fifth aspect of the present invention provides an engineered strain of Corynebacterium glutamicum, comprising the gene expression cassette of the third aspect of the present invention or the recombinant vector of the fourth aspect of the present invention. Preferably, the starting strain is Corynebacterium glutamicum H1, deposited with CCTCC NO: M 2020644, or C. glutamicum ATCC 13032.

[0016] The sixth aspect of the present invention provides a method for producing L-arginine by fermenting the engineered Corynebacterium glutamicum bacteria described in the fifth aspect of the present invention in a culture medium.

[0017] Preferably, the culture medium comprises 12.5% ​​glucose, 0.15% dipotassium hydrogen phosphate, 0.1% urea, 4.5% ammonium sulfate, 0.5% yeast extract, 5.5% corn steep liquor, 0.05% magnesium sulfate, 0.05% biotin, 0.1% vitamin B1, 10 drops / L of corn oil, 4.0% molasses, 0.3% silk peptide powder, 0.5% light calcium carbonate, and the balance is deionized water, with a pH of 7; the percentages are weight-volume percentages (g / mL).

[0018] The fermentation conditions are selected from one or more of the following: temperature 30°C; tank pressure: 0.03-0.08 MPa; pH 7.0 preferably controlled by ammonia water; dissolved oxygen 30%; when the residual sugar content is lower than 3.0%, glucose is added to maintain the residual sugar content at 1.5-3.0%; and the fermentation time is more than 72 hours.

[0019] The seventh aspect of the present invention provides the use of the 5'UTR element of the first aspect of the present invention, the functional expression element of the second aspect of the present invention, the gene expression cassette of the third aspect of the present invention, and the recombinant expression vector of the fourth aspect of the present invention in preparing engineered Corynebacterium glutamicum.

[0020] In an eighth aspect, the present invention provides the use of the 5'UTR element described in the first aspect of the present invention, the functional expression element described in the second aspect of the present invention, the gene expression cassette described in the third aspect of the present invention, the recombinant expression vector described in the fourth aspect of the present invention, and the engineered Corynebacterium glutamicum described in the fifth aspect of the present invention in the production of L-arginine.

[0021] The beneficial effects of the present invention are:

[0022] The expression vector constructed by combining the screened 5'UTR element with a high-strength promoter regulated the expression of the arginine operon gene, and the L-arginine production increased from 66.83 g / L in the control group to 103.98 g / L, an increase of 55.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a plasmid P EF-tu -UTR3 14bp Atlas.

[0024] Figure 2 shows the EGFP fluorescence intensity of recombinant bacteria under the regulation of different 5'UTRs.

[0025] Figure 3 shows the changes in fluorescence intensity of recombinant bacteria with different lengths of UTR3.

[0026] Figure 4 shows UTR3 14bp Fluorescent protein expression intensity under different promoters. DETAILED DESCRIPTION

[0027] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0028] Plasmid pXMJ19 was purchased from Prudin Biotechnology (Beijing) Co., Ltd., E. coli DH5α and E. coli BL21 are commonly used in the field; DNA polymerase (Q5 High-Fidelity DNA Polymerase) was purchased from Gene Co., Ltd.; restriction endonucleases (EcoRI, Hind III), DNA marker, plasmid extraction kit, and DNA gel recovery and purification kit were all purchased from Takara Bioengineering (Dalian) Co., Ltd.; bacterial genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., Onestep clonning cloning and recombination kit, and Klenow enzyme were purchased from NEB Beijing Company; chloramphenicol was purchased from Biosharp Company; molasses was purchased from Chifeng Botian Sugar Industry, Inner Mongolia; and all other chemical reagents such as glucose were of analytical grade from national pharmaceutical companies. Plasmid extraction procedures refer to the instructions for the plasmid mini-extraction kit; DNA gel recovery procedures refer to the instructions for the DNA gel recovery kit; Corynebacterium glutamicum genome extraction procedures refer to the instructions for the bacterial genomic DNA extraction kit; DNA fragment recombination and ligation procedures refer to the instructions for the Onestep clonning cloning and recombination kit; Corynebacterium glutamicum competent cell preparation and transformation methods refer to the method of van der Rest et al. (ME van der Rest, C. Lange, D. Molenaar. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl. Microbiol. Biotechnol. 1999, 52: 541-545). Amino acid content in the fermentation broth was determined using an amino acid analyzer (Hitachi 8800).

[0029] Fluorescence intensity was measured at 488 nm (excitation) and 523 nm (emission) wavelengths using a microplate reader (SpectraMax i3x). The background fluorescence value (FPbg) of the strain containing the pXMJ19-egfp plasmid and the background optical density (ODbg) of the culture medium were used for correction, and the relative fluorescence intensity was calculated according to the equation:

[0030]

[0031] Example 1 Construction of pXMJ19-egfp plasmid

[0032] Amplification of the egfp fragment: Using the egfp gene sequence (NCBI Sequence ID: LC336974.1) as a template, primers F-egfp (SEQ ID NO: 1) and R-egfp (SEQ ID NO: 2) were designed for PCR amplification using Q5 High-Fidelity DNA Polymerase. The PCR reaction system (50 μl) consisted of 10 μl of 5× Q5 reaction buffer, 1 μl of 10 mM dNTPs, 2.5 μl of each primer, template depending on sample concentration, 0.5 μl of Q5 enzyme, and water to 50 μl. Reaction conditions were: 98°C for 30 s, 98°C for 10 s, 55°C-72°C for 30 s, 72°C for 45 s, 33 cycles, and 72°C for 2 min. PCR was performed by 1% agarose gel electrophoresis and the PCR product was purified using a DNA gel extraction kit.

[0033] egfp-F: 5'-GCGTCGACATGGTGAGCAAG-3'(Sal I) (SEQ ID NO: 1)

[0034] egfp-R: 5'-CGCGAATTCTTACTTGTACAGCTCGTCC-3'(EcoRI) (SEQ ID NO: 2)

[0035] The pXMJ19 empty vector and the egfp fragment were double-digested with EcoRI and Sal I endonucleases, respectively. The digestion products were recovered using a gel recovery kit and recombined using the Onestep clonning cloning and recombination kit (i.e., the double-digested pXMJ19 and egfp fragments). The double-digested products were transformed into E. coli BL21 and plated on LB plates containing chloramphenicol. After overnight incubation at 37°C, positive transformants were selected and sent to Jinkairui Bioengineering Co., Ltd. for sequencing. The transformants with the correct sequencing results contained the recombinant plasmid pXMJ19-egfp, which was named P tac -egfp.

[0036] Example 2 Construction of 5'UTR Plasmid Library

[0037] Extract the C. glutamicum H1 genome of Corynebacterium glutamicum according to the steps in the DNA extraction kit instructions and save it for later use. Use an ultrasonic cell disruptor to fragment the genome, optimize the disruption time and power to make the genome fragments between 50-300bp. Set the ultrasonic cell disruptor parameters to 10%-20% power, and continuously adjust the disruption power and disruption time to achieve the ideal disruption effect. Concentrate the disrupted cells, add 2 times the volume of anhydrous ethanol to the disrupted genome fragments and precipitate at -20℃. Collect the DNA fragments by centrifugation for 2 hours, dry them, dissolve them in sterile water, and store them at -20℃ until use. tac -egfp was used as a template, and the PstI site between Hind III and Sal I in the plasmid multiple cloning site was used as the starting point. The plasmid was linearized using primers P-UTR-F (SEQ ID NO: 3) / P-UTR-R (SEQ ID NO: 4) to obtain a linearized reporter plasmid P tac -egfp. The cohesive ends of the fragmented DNA fragments were filled with Klenow enzyme. 1X NE Buffer was added with 33 μM dNTPs and 1 unit of Klenow enzyme per μg of DNA. The reaction was incubated at 25°C for 30 minutes. Finally, EDTA was added to a final concentration of 10 mM and the reaction was stopped by heating at 75°C for 20 minutes.

[0038] P-UTR-F: 5'-CTGCAGGTCGACTCTAGAGGATCC-3' (SEQ ID NO: 3)

[0039] P-UTR-R: 5'-GCATGCAAGCTTAATTGCATGCAAG-3' (SEQ ID NO: 4)

[0040] The blunt-end DNA fragment was ligated with the linearized reporter plasmid P using the Onestep clonning kit. tac -egfp recombination, and the recombinant product was transformed into E. coli DH5α to obtain the 5'UTR plasmid library.

[0041] Note: C. glutamicum H1 was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on October 26, 2020. The strain name is Corynebacterium glutamicum H1, the classification number is Corynebacterium glutamicum H1, and the preservation number is CCTCC NO: M 2020644.

[0042] Example 3 Screening and Identification of 5'UTR

[0043] The constructed 5'UTR plasmid library and P tac -egfp plasmids were transformed into C. glutamicum H1 and plated on chloramphenicol-resistant plates. tac -egfp plasmid was used as a control strain and named H1-P tac -egfp. After the plasmid library was transformed, the transformants were washed off the resistance plate and washed three times with PBS. The washed transformants were sorted by flow cytometry and the fluorescence value was higher than that of the control strain H1-P tac -egfp (without 5'UTR) mutants, the obtained target cells were spread on LB agar plates containing antibiotics and screened using a 96-well plate. After culturing for 48 h, transformants with strong fluorescence were selected, and the plasmids were extracted for sequencing.

[0044] The fluorescence intensity of four recombinant bacteria was significantly higher than that of other strains, named H1-UTR1, H1-UTR2, H1-UTR3 and H1-UTR4 (Figure 2). Among them, the recombinant bacteria containing the UTR3 sequence (H1-UTR3) had the highest fluorescence intensity, while the control strain H1-P tac The fluorescence expression level of -egfp was 90,000, and the fluorescence expression level of H1-UTR3 was 1,400,000, which increased the expression level of the reporter gene by 15.5 times compared with the control strain. The plasmids in the four recombinant strains screened were sequenced to confirm the information of the four random sequences inserted upstream of the reporter gene egfp (Table 1).

[0045] Table 1 Screened 5'UTR highly expressed sequences:

[0046]

[0047]

[0048] Example 4 Optimization of 5'UTR

[0049] UTR3 with the highest expression intensity was used as the sequence for subsequent optimization. Due to its short sequence, in order to identify the core sequence of UTR3, it was directly sent to GenScript to synthesize a truncated sequence (Table 2) and add the restriction enzyme sites Hind III and Sal I.

[0050] Table 2 Sequences of different lengths after UTR3 truncations:

[0051]

[0052] P tacThe -egfp vector and the above UTR3 truncated fragment were double-digested with Hind III and Sal I endonucleases, respectively. After gel recovery, the double-digested products were recombined using the Onestep clonning cloning and recombination kit, transformed into E. coli DH5α, and spread on LB plates containing chloramphenicol. After overnight incubation at 37°C, positive transformants were selected and sent to Biotechnology for sequencing. The verified recombinant plasmids were named P tac -UTR3 18bp -egfp,P tac -UTR3 16bp -egfp and P tac -UTR3 14bp -egfp.

[0053] The above truncated sequence recombinant vectors were transformed into C. glutamicum H1 and named H1-P tac -UTR3 18bp -egfp, H1-P tac -UTR3 16bp -egfp, H1-P tac -UTR3 14bp -eGFP and measured the fluorescence intensity. The results showed that the shortened UTR3 18bp 、UTR3 16bp and UTR3 14bp Its fluorescence value was relative to that of the control strain H1-P tac -eGFP still significantly increased, which contained UTR3 14bp Sequence of recombinant bacteria (H1-P tac -UTR3 14bp -egfp) had the highest expression intensity. This sequence increased the expression of the reporter gene by 21.4% compared to the original UTR3 sequence and by 18.9 times compared to the control strain (Figure 3). 14bp The sequence length is 14bp.

[0054] Example 5 Compatibility study between 5'UTR and promoter

[0055] To verify UTR3 14bp The compatibility of the element with different promoters was studied by using different promoters P ilvA 、P thrC 、P EF-tu and P dapA Replace P respectively tac -egfp and P tac -UTR3 14bp -P in the egfp recombinant vector tacThe promoter sequence used was sent to GenScript for synthesis (Table 3) and restriction enzyme sites Nar I and Hind III were added at the beginning and end of the sequence.

[0056] The promoter elements and P were synthesized by Nar I and Hind III endonucleases, respectively. tac -egfp and P tac -UTR3 14bp -egfp was double-digested to remove LacIq and the original P tac The new promoter fragment was recombined using the Onestep clonning kit, and the recombinant product was transformed into E. coli DH5α and spread on LB plates containing chloramphenicol. After overnight culture at 37°C, positive transformants were selected and sent to Biotechnology for sequencing. The recombinant plasmids obtained are shown in Table 3 below.

[0057] Table 3 UTR3 14bp Recombinant plasmids combined with different promoters

[0058]

[0059]

[0060] The above recombinant plasmids were transformed into C. glutamicum H1 and the fluorescence intensity was measured. The fluorescence intensity ratio of the recombinant strains was shown in Table 4. The results showed that after the promoters with different strengths were replaced, the fluorescence expression levels changed (Figure 4). 14bp The elements can enhance gene expression at the translation level, and the fluorescence intensity after enhancement is increased by 15 to 20 times compared with the control, proving that the UTR element is applicable under the regulation of different promoters (Table 4).

[0061] Table 4 UTR3 14bp Comparison of translation enhancement levels under the regulation of different promoters

[0062]

[0063] Example 6 Construction of arg operon gene-enhanced recombinant bacteria

[0064] The arg operon in Corynebacterium glutamicum includes six genes, argC, J, B, and F, with NCBI GeneIDs as follows: argC GeneID 69621907; argJ GeneID 69621908; argB GeneID 69621909; argD GeneID 69621910; argF GeneID 69621911; and argR GeneID 69621912. It involves the arginine metabolic synthesis pathway. Therefore, increasing the expression of arg operon genes is an effective strategy to increase arginine production. At the same time, in order to verify the UTR3 obtained after screening, 14bp glutamicum, we selected the P EF-tu -UTR3 14bp promoter combination to construct arginine fermentation recombinant strains. EF-tu -egfp and P EF-tu -UTR3 14bp -egfp sequence in the control group to construct the recombinant plasmid P EF-tu -arg and overexpression plasmid P EF-tu -UTR3 14bp -arg. Upstream and downstream primers were designed to amplify the arg operon gene and restriction sites Sal I and EcoR I were introduced at the beginning and end. PCR was performed on the Corynebacterium glutamicum genomic template using primers F-arg and R-arg, using high-fidelity Q5 DNA Polymerase. The PCR reaction system (50 μl) consisted of: 10 μl of 5×Q5 reaction buffer, 1 μl of 10 mM dNTPs, 2.5 μl of each primer, template depending on sample concentration, 0.5 μl of Q5 enzyme, and water to 50 μl. Reaction conditions were: 98°C for 30 seconds, 98°C for 10 seconds, 55°C-72°C for 30 seconds, 72°C for 6 minutes, 30 cycles, and 72°C for 2 minutes. Detection was performed by 1% agarose gel electrophoresis, and the PCR product was purified and recovered using a DNA gel recovery kit.

[0065] F-arg 5'-GCGTCGACATGATAATCAAGGTTGCAATCGCAGG-3'(Sal I) (SEQ ID NO: 16)

[0066] R-arg 5'-CGGAATTCTTAAGTGGTGCGCCCGCTGAGTAAT-3'(EcoRI)(SEQ ID NO: 17)

[0067] P EF-tu-egfp and P EF-tu -UTR3 14bp The -egfp vector and arg operon fragment were double-digested with Sal I and EcoRI endonucleases, respectively. After gel recovery, the double-digested products were recombined using the Onestep clonning cloning and recombination kit, transformed into E. coli BL21, and plated on a plate containing chloramphenicol. After overnight incubation at 37°C, positive transformants were selected and double-digested for verification. The recombinant plasmids were named P EF-tu -arg and overexpression plasmid P EF-tu -UTR3 14bp -arg (containing UTR elements) and sent to Bioengineering for sequencing.

[0068] According to the conventional scheme, P EF-tu -arg and P EF-tu -UTR3 14bp -arg were transformed into C. glutamicum H1 and C. glutamicum 13032, respectively. The recombinant C. glutamicum produced by the transformation experiments were named: H1-P EF-tu -arg, H1-P EF-tu -UTR3 14bp -arg, C.glutamicum 13032-P EF-tu -arg and C. glutamicum 13032-P EF-tu -UTR3 14bp -arg.

[0069] Example 7 Effect of using 5'UTR to increase arg operon gene expression on arginine production

[0070] The formula of arginine seed culture medium is: glucose 3.0%, dipotassium hydrogen phosphate 0.2%, urea 0.12%, ammonium sulfate 0.5%, yeast extract 0.5%, corn steep liquor 5.5%, magnesium sulfate 0.04%, biotin 50ug / L, silk peptide powder 1.0%, corn oil 10 drops / L, and light calcium carbonate 1%.

[0071] The formula of the arginine fermentation medium is: 12.5% ​​glucose, 0.15% dipotassium hydrogen phosphate, 0.1% urea, 4.5% ammonium sulfate, 0.5% yeast extract, 5.5% corn steep liquor, 0.05% magnesium sulfate, 0.05% biotin, 0.1% vitamin B1, 10 drops / L of corn oil, 4.0% molasses, 0.3% silk peptide powder, 0.5% light calcium carbonate, and the balance is deionized water. The pH is 7; the percentages are weight-to-volume (g / mL) percentages.

[0072] Taking strains C.glutamicum H1, C.glutamicum 13032, C.glutamicum 13032-P EF- tu -arg and recombinant bacteria H1-P EF-tu -arg was used as the control, and the recombinant bacteria H1-P EF-tu -UTR3 14bp -arg and C. glutamicum 13032-P EF-tu -UTR3 14bp -arg was loaded into a 5L fermentation tank for culture. A single colony was picked from the plate and inoculated into the seed culture medium, and cultured on a shaking table at a temperature of 32°C and a rotation speed of 200r / min for 12 hours to obtain the seed liquid; according to the 5% inoculation amount, the seed culture liquid was merged into the feeding bottle, the feeding pipe was connected, the seed culture liquid was inoculated into the fermentation medium, and the fermentation culture was started. The temperature was controlled at 30°C, the tank pressure was: 0.03~0.08Mpa, the pH was controlled at 7.0 with ammonia water, and the dissolved oxygen was maintained at 30% by adjusting the rotation speed and ventilation volume. When the residual sugar content was lower than 3.0%, 80% glucose was added to maintain the residual sugar content at 1.5~3.0%, and the fermentation time was more than 72 hours. After the fermentation was completed, the fermentation liquid was centrifuged and the supernatant was derivatized, and the main amino acids were analyzed by HPLC. The experimental results are shown in Table 4. Compared with the control strain, the recombinant bacteria H1-P EF-tu -UTR3 14bp -arg had the highest arginine production, and the total arginine production increased from 66.83 g / L to 103.98 g / L, an increase of 55.6%.

[0073] Table 5 Amino acid production

[0074]

[0075] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0076] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A 5'UTR element, characterized in that The 5'UTR element includes the nucleotide sequence shown in SEQ ID NO:

11.

2. The 5'UTR element according to claim 1, wherein The nucleotide sequence of the 5'UTR element is shown in any one of SEQ ID NOs: 7, 9-11.

3. A functional expression element, characterized in that The functional expression element comprises a promoter and the 5'UTR element according to claim 1 or 2.

4. The functional expression element according to claim 3, wherein The promoter is P EF-tu 、P ilvA 、P thrC 、P tac or P dapA ; Preferably, the promoter is P EF-tu .

5. A gene expression cassette, characterized in that It comprises the functional expression element according to claim 3 or 4, a target gene and a terminator.

6. The gene expression cassette according to claim 5, wherein The target gene is the arginine operon gene argCJBDFR; preferably, the NCBI GeneIDs of the argCJBDFR are as follows: GeneID of argC is 69621907; GeneID of argJ is 69621908; GeneID of argB is 69621909; The GeneID of argD is 69621910; The GeneID of argF is 69621911; the GeneID of argR is 69621912.

7. A recombinant expression vector, characterized in that: It comprises the 5'UTR element according to claim 1 or 2, the functional expression element according to claim 3 or 4, or the gene expression cassette according to claim 5 or 6.

8. The recombinant expression vector according to claim 7, wherein The backbone plasmid of the recombinant expression vector is pXMJ19.

9. An engineered bacterium of Corynebacterium glutamicum, characterized in that: The engineered Corynebacterium glutamicum comprises the gene expression cassette according to claim 5 or 6, or the recombinant expression vector according to claim 7 or 8.

10. The engineered Corynebacterium glutamicum according to claim 9, wherein The starting strain of the engineered Corynebacterium glutamicum bacteria is Corynebacterium glutamicum H1, with a preservation number of CCTCC NO: M 2020644 or Corynebacterium glutamicum ATCC 13032.

11. A method for producing L-arginine, characterized in that: The L-arginine is obtained by fermenting the engineered Corynebacterium glutamicum according to claim 9 or 10 in a culture medium.

12. The method according to claim 11, wherein The culture medium comprises: Glucose 12.5%, dipotassium hydrogen phosphate 0.15%, urea 0.1%, ammonium sulfate 4.5%, yeast extract 0.5%, corn steep liquor 5.5%, magnesium sulfate 0.05%, biotin 0.05%, vitamin B1 0.1%, corn oil 10 drops / L, molasses 4.0%, silk peptide powder 0.3%, light calcium carbonate 0.5%, the balance is deionized water, pH 7; the percentages are weight-volume percentages (g / mL).

13. The method according to claim 11, wherein The fermentation conditions are selected from one or more of the following: Temperature 30℃; Tank pressure: 0.03~0.08Mpa; pH 7.0 is preferably controlled by ammonia; Dissolved oxygen 30%; When the residual sugar content is lower than 3.0%, glucose is added to maintain the residual sugar content at 1.5-3.0%; The fermentation time is more than 72 hours.

14. Use of the 5'UTR element according to claim 1 or 2, the functional expression element according to claim 3 or 4, the gene expression cassette according to claim 5 or 6, or the recombinant expression vector according to claim 7 or 8 in preparing engineered Corynebacterium glutamicum.

15. Use of the 5'UTR element according to claim 1 or 2, the functional expression element according to claim 3 or 4, the gene expression cassette according to claim 5 or 6, the recombinant expression vector according to claim 7 or 8, or the engineered Corynebacterium glutamicum according to claim 9 or 10 in the production of L-arginine.

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