WO2024077445A1 - 一种5'utr元件及其在l-精氨酸生产中的应用 - Google Patents

一种5'utr元件及其在l-精氨酸生产中的应用 Download PDF

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WO2024077445A1
WO2024077445A1 PCT/CN2022/124402 CN2022124402W WO2024077445A1 WO 2024077445 A1 WO2024077445 A1 WO 2024077445A1 CN 2022124402 W CN2022124402 W CN 2022124402W WO 2024077445 A1 WO2024077445 A1 WO 2024077445A1
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corynebacterium glutamicum
arginine
geneid
gene
utr
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刘瑾
皮莉
林丽春
孙悦
邢盼盼
苏海霞
查丽燕
李坤
李晓波
陈磊
魏子翔
桂小华
王筱蒙
朱程军
杨璇
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武汉远大弘元股份有限公司
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
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Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a 5'UTR element and application thereof in L-arginine production.
  • L-arginine also known as 2-amino-5-guanidine valeric acid, has a molecular formula of C 6 H 14 N 4 O 2 and 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 product additive to enhance muscle vitality and immunity; as a feed additive to increase the female birth rate of animals, etc.
  • L-arginine is mainly produced by microbial fermentation, and most of the industrial production is by Escherichia coli and Corynebacterium glutamicum.
  • the prior art generally increases the expression of L-arginine synthesis genes by increasing the copy number of key enzyme genes (argCJBDFR) in the arginine synthesis pathway through plasmids.
  • argCJBDFR key enzyme genes
  • high-copy plasmids may increase the metabolic burden of bacteria and have poor stability.
  • Regulating gene expression at the transcription and translation level is the most direct and convenient way, which is achieved 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.
  • PUTR promoter-5'-untranslated region
  • 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 the 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.
  • the present invention uses flow cytometer sorting technology to screen and obtain a 5'UTR sequence that can promote the enhanced expression of green fluorescent protein gene from the Corynebacterium glutamicum genome, and screens an effective optimal 5'UTR sequence by gradually shortening the 5'UTR sequence length. Then, the optimal 5'UTR sequence is combined with multiple high-intensity promoters P ilvA , P thrC , P EF-tu and P dapA to construct a recombinant expression vector, and the adaptability of the 5'UTR sequence and the promoter is verified by fluorescence intensity characterization.
  • the functional expression element composed of the optimal 5'UTR of P EF-tu is selected to construct the recombinant bacteria of Corynebacterium glutamicum of L-arginine arg operon gene (argC J BDFR), and L-arginine is produced by fermentation.
  • the first aspect of the present invention provides a 5’UTR element of Corynebacterium obtained based on 5’UTR library screening, wherein the 5’UTR element comprises a nucleotide sequence as shown in SEQ ID NO: 11.
  • nucleotide sequence of the 5’UTR element is as shown in any one of SEQ ID NO: 7, 9 to 11.
  • the second aspect of the present invention provides a functional expression element, comprising a promoter and the 5'UTR element of the first aspect of the present invention, wherein the promoter is selected from P EF-tu , P ilvA , P thrC , P tac and P dapA .
  • the promoter is P EF-tu .
  • the third aspect of the present invention provides a gene expression cassette, which comprises the functional expression element, target gene and terminator described in the second aspect.
  • the target gene is the arginine operon gene argCJBDFR, and its NCBI GeneID is as follows: GeneID of argC is 69621907; GeneID of argJ is 69621908; GeneID of argB is 69621909; GeneID of argD is 69621910; GeneID of argF is 69621911; GeneID of argR is 69621912.
  • the fourth aspect of the present invention provides a recombinant expression vector, the recombinant expression vector comprises 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.
  • the backbone of the recombinant vector is pXMJ19.
  • the fifth aspect of the present invention provides an engineered bacterium of Corynebacterium glutamicum, wherein the engineered bacterium of Corynebacterium glutamicum comprises the gene expression cassette described in the third aspect of the present invention or the recombinant vector described in the fourth aspect of the present invention.
  • the starting strain is Corynebacterium glutamicum H1, with a deposit number of CCTCC NO: M 2020644, or Corynebacterium glutamicum ATCC 13032.
  • the sixth aspect of the present invention provides a method for producing L-arginine by fermenting the engineered bacteria of Corynebacterium glutamicum described in the fifth aspect of the present invention in a culture medium.
  • the culture medium includes 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 % is weight volume percentage g/mL.
  • the fermentation conditions are selected from one or more of the following: temperature 30°C; tank pressure: 0.03-0.08Mpa; 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.
  • the seventh aspect of 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 and the recombinant expression vector described in the fourth aspect of the present invention in the preparation of Corynebacterium glutamicum engineered bacteria.
  • the eighth aspect of 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 Corynebacterium glutamicum engineered bacteria described in the fifth aspect of the present invention in the production of L-arginine.
  • the expression vector constructed by combining the screened 5'UTR element with a high-strength promoter was used to regulate the expression of the arginine operon gene.
  • the L-arginine production was increased from 66.83 g/L in the control group to 103.98 g/L, an increase of 55.6%.
  • Figure 1 is a map of plasmid PEF -tu -UTR3 14bp .
  • Figure 2 shows the EGFP fluorescence intensity of the recombinant bacteria under the regulation of different 5’UTRs.
  • Figure 3 shows the changes in fluorescence intensity of recombinant bacteria with different lengths of UTR3.
  • FIG4 shows the expression intensity of fluorescent protein of UTR3 14bp under different promoters.
  • Plasmid pXMJ19 was purchased from Protin Biotechnology (Beijing) Co., Ltd., E. coli DH5 ⁇ and E. coli BL21 are commonly used in this field; DNA polymerase (Q5 High-Fidelity DNA Polymerase) was purchased from Gene Co., Ltd.; restriction endonucleases (EcoRI, Hind III), DNA marker, plasmid extraction kit, DNA gel recovery and purification kit were all purchased from Takara Biotechnology (Dalian) Co., Ltd.; bacterial genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., Onestep cloning cloning recombination kit, Klenow enzyme was purchased from NEB Beijing Company; chloramphenicol was purchased from Biosharp Company; molasses was purchased from Chifeng Botian Sugar Industry, Inner Mongolia; glucose and other chemical reagents were of analytical grade national medicine.
  • DNA polymerase Q5 High-Fidelity DNA Polymerase
  • the plasmid extraction operation steps refer to the instructions of the plasmid mini-extraction kit;
  • the DNA gel recovery operation steps refer to the instructions of the DNA gel recovery kit;
  • the glutamicum genome extraction operation steps refer to the instructions of the bacterial genome DNA extraction kit;
  • the DNA fragment recombination and connection operation steps refer to the instructions of the Onestep cloning cloning recombination kit;
  • the preparation and transformation method of Corynebacterium glutamicum competent cells refer to the method of van der Rest et al. (M.E. 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).
  • the amino acid content in the fermentation broth was detected by an amino acid analyzer (Hitachi 8800).
  • Fluorescence intensity determination method The fluorescence value of the target cells was measured using a microplate reader (SpectraMax i3x) at wavelengths of 488 nm (excitation) and 523 nm (emission).
  • 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:
  • Amplification of egfp fragment Using the egfp gene sequence (NCBI sequence ID: LC336974.1) as a template, design primers F-egfp (SEQ ID NO: 1) and R-egfp (SEQ ID NO: 2), use high-fidelity Q5 High-Fidelity DNA Polymerase for PCR amplification, PCR reaction system (50 ⁇ l): 5 ⁇ Q5 reaction buffer 10 ⁇ l, 10mM dNTP 1 ⁇ l, primers 2.5 ⁇ l each, template depending on sample concentration, Q5 enzyme 0.5 ⁇ l, add water to 50 ⁇ l.
  • PCR reaction system 50 ⁇ l: 5 ⁇ Q5 reaction buffer 10 ⁇ l, 10mM dNTP 1 ⁇ l, primers 2.5 ⁇ l each, template depending on sample concentration, Q5 enzyme 0.5 ⁇ l, add water to 50 ⁇ l.
  • Reaction conditions 98°C30s, 98°C10s, 55°C-72°C30s, 72°C45s, 33 cycles; 72°C2min. 1% agarose gel electrophoresis detection and DNA gel recovery kit purification and recovery of PCR products.
  • the pXMJ19 empty vector and the egfp fragment were double-digested with EcoRI and Sal I nucleases, respectively.
  • the digestion products were recovered by a gel recovery kit, and the double-digestion products (i.e., pXMJ19 and egfp fragments after double digestion) were recombined using the Onestep clonning cloning recombination kit, transformed into E. coli BL21, and spread on an LB plate containing chloramphenicol. After overnight culture at 37°C, positive transformants were selected and sent to Jinkairui Bioengineering Co., Ltd. for sequencing.
  • the transformants with correct sequencing contained the recombinant plasmid pXMJ19-egfp, and the plasmid was named Ptac -egfp.
  • the genome of Corynebacterium glutamicum H1 was extracted and stored for later use.
  • the genome was fragmented using an ultrasonic cell disruptor, and the fragmentation time and power were optimized to make the genome fragments between 50-300 bp.
  • the parameters of the ultrasonic cell disruptor were set to a power of 10%-20%, and the fragmentation power and fragmentation time were continuously adjusted to achieve the ideal fragmentation effect.
  • the cells after the fragmentation were concentrated, and the fragmented genome fragments were added with 2 times the volume of anhydrous ethanol to precipitate at -20°C.
  • the DNA fragments were collected by centrifugation for 2 hours, dried, dissolved with sterile water, and stored at -20°C until use.
  • the PstI site between Hind III and Sal I in the plasmid multiple cloning site was used as the starting point, and the primers P-UTR-F (SEQ ID NO: 3)/P-UTR-R (SEQ ID NO: 4) were used to amplify and linearize the plasmid to obtain the linearized reporter plasmid Ptac -egfp.
  • the DNA fragments after the genome of the grain stick were broken were filled with Klenow enzyme to make up the sticky ends of the DNA.
  • 33 ⁇ M dNTP and 1 unit of Klenow enzyme were added to the 1X NE Buffer reaction buffer. The mixture was incubated at 25°C for 30 minutes. Finally, EDTA was added to a final concentration of 10 mM and heated at 75°C for 20 minutes to stop the reaction.
  • P-UTR-F 5’-CTGCAGGTCGACTCTAGAGGATCC-3’ (SEQ ID NO: 3)
  • P-UTR-R 5’-GCATGCAAGCTTAATTGCATGCAAG-3’ (SEQ ID NO: 4)
  • the blunt-end DNA fragments were recombined with the linearized reporter plasmid Ptac -egfp using the Onestep clonning cloning recombination kit, and the recombinant products were transformed into E. coli DH5 ⁇ to obtain the plasmid library of 5'UTR.
  • 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
  • the preservation number is CCTCC NO: M 2020644.
  • the constructed 5'UTR plasmid library and P tac -egfp plasmid were transformed into C. glutamicum H1, respectively, and spread on chloramphenicol resistant plates.
  • the strain transformed with P tac -egfp plasmid was used as the control strain, named H1-P tac -egfp.
  • the transformants were washed off the resistant plate and washed three times with PBS.
  • the washed transformants were sorted by flow cytometry for mutants with higher fluorescence values than the control strain H1-P tac -egfp (without 5'UTR).
  • the obtained target cells were spread on LB agar plates containing antibiotics and screened with 96-well plates. After 48 hours of culture, the transformants with stronger fluorescence were selected, and the plasmids were extracted for sequencing.
  • 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).
  • the recombinant bacteria containing the UTR3 sequence (H1-UTR3) had the highest fluorescence intensity.
  • the fluorescence expression of the control strain H1- Ptac -egfp was 90,000, and the fluorescence expression of H1-UTR3 was 1,400,000, which increased the expression of the reporter gene by 15.5 times relative to the control strain.
  • the plasmids in the four recombinant bacteria screened were sequenced to determine the information of the four random sequences inserted upstream of the reporter gene egfp (Table 1).
  • 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 restriction sites Hind III and Sal I.
  • the Ptac -egfp vector and the above UTR3 truncated fragments were double-digested with Hind III and Sal I nucleases, respectively. After gel recovery, the double-digested products were recombined using the Onestep clonning cloning recombination kit, transformed into E.coli DH5 ⁇ , and spread on LB plates containing chloramphenicol. After overnight culture at 37°C, positive transformants were selected and sent to Biotechnology for sequencing.
  • the verified correct recombinant plasmids were named Ptac -UTR3 18bp -egfp, Ptac -UTR3 16bp -egfp, and Ptac - UTR3 14bp -egfp, respectively.
  • H1-P tac -UTR3 18bp -egfp H1-P tac -UTR3 16bp -egfp
  • H1-P tac -UTR3 14bp -egfp H1-P tac -UTR3 14bp -egfp
  • the Ptac promoter in the Ptac -egfp and Ptac- UTR3 14bp -egfp recombinant vectors was replaced by different promoters PilvA , PthrC , PEF-tu and PdapA , respectively.
  • the promoter sequences used were sent to GenScript for synthesis (Table 3) and restriction sites Nar I and Hind III were added at the beginning and end of the sequence.
  • the synthesized promoter element, Ptac -egfp and Ptac -UTR3 14bp -egfp were double-digested with Nar I and Hind III nuclease, respectively, LacIq and the original Ptac promoter were removed, and the new promoter fragment was recombined using the Onestep clonning cloning recombination kit.
  • the recombinant product was transformed into E.coli DH5 ⁇ and spread on an LB plate containing chloramphenicol. After overnight culture at 37°C, positive transformants were selected and sent to Biotechnology for sequencing.
  • the re-obtained recombinant plasmids are shown in Table 3.
  • the above recombinant plasmids were transformed into C. glutamicum H1 and the fluorescence intensity was measured.
  • the fluorescence intensity ratio of the obtained recombinant strains is shown in Table 4.
  • the results showed that after the replacement of promoters with different strengths, the fluorescence expression levels changed ( Figure 4).
  • the UTR3 14bp element can enhance the expression of the gene at the translation level.
  • the enhanced fluorescence intensity can be increased by 15 to 20 times compared with the control, proving that the UTR element can be applied under the regulation of different promoters (Table 4).
  • the arg operon in Corynebacterium glutamicum includes six genes: argC, J, B, D, and FR, and their NCBI GeneIDs are as follows: GeneID of argC is 69621907; GeneID of argJ is 69621908; GeneID of argB is 69621909; GeneID of argD is 69621910; GeneID of argF is 69621911; and GeneID of argR is 69621912. It involves the arginine metabolic synthesis route. Therefore, improving the expression of arg operon genes is an effective strategy to increase arginine production.
  • Upstream and downstream primers were designed to amplify the arg operon gene and the restriction sites Sal I and EcoR I were introduced at the beginning and end.
  • the PCR was performed on the glutamicum genome template with primers F-arg and R-arg, and the high-fidelity Q5 High-Fidelity DNA Polymerase was used for PCR amplification.
  • the PCR reaction system (50 ⁇ l) was: 5 ⁇ Q5 reaction buffer 10 ⁇ l, 10mM dNTP 1 ⁇ l, primers 2.5 ⁇ l each, template depending on the sample concentration, Q5 enzyme 0.5 ⁇ l, and water was added to 50 ⁇ l.
  • the reaction conditions were: 98°C for 30 s, 98°C for 10 s, 55°C-72°C for 30 s, 72°C for 6 min, 30 cycles; 72°C for 2 min.
  • the PCR product was detected by 1% agarose gel electrophoresis and purified using a DNA gel recovery kit.
  • the P EF -tu -egfp and P EF -tu -UTR3 14 bp -egfp vectors and arg operon fragments were double-digested with Sal I and EcoRI nucleases, respectively. After gel recovery, the double-digestion products were recombined using the Onestep clonning cloning recombination kit, transformed into E. coli BL21, and spread on a plate containing chloramphenicol. After overnight culture at 37°C, positive transformants were selected and double-digestion verification was performed.
  • the recombinant plasmids were named P EF -tu -arg and overexpression plasmid P EF -tu -UTR3 14 bp -arg (containing UTR elements), respectively, and sent to Bioengineering for sequencing.
  • Example 7 Effect of using 5'UTR to increase arg operon gene expression on arginine production
  • arginine seed culture medium 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, light calcium carbonate 1%.
  • 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 syrup, 0.05% magnesium sulfate, 0.05% biotin, 0.1% vitamin B1, 10 drops/L 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 % is the weight volume (g/mL) percentage.
  • strains C.glutamicum H1, C.glutamicum 13032, C.glutamicum 13032-P EF- tu -arg and recombinant bacteria H1-P EF-tu -arg as controls, recombinant bacteria H1-P EF-tu -UTR3 14bp -arg and C.glutamicum 13032-P EF-tu -UTR3 14bp -arg were placed in a 5L fermenter for culture.
  • a single colony was picked from a plate and inoculated into a seed culture medium, and cultured on a shaking table at a temperature of 32°C and a rotation speed of 200r/min for 12h to obtain a seed solution; the seed culture solution was merged into a feed bottle according to a 5% inoculation amount, a feed pipe was connected, and the seed culture solution was inoculated into the fermentation medium to start fermentation culture.
  • the temperature was controlled at 30°C, the tank pressure was 0.03-0.08Mpa, the pH was controlled at 7.0 by ammonia water, the dissolved oxygen was maintained at 30% by adjusting the rotation speed and ventilation volume, and 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 72h.
  • the fermentation liquid was centrifuged and the supernatant was derivatized, and the main amino acids were analyzed by HPLC.
  • Table 4 The experimental results are shown in Table 4.
  • the recombinant strain H1-P EF-tu -UTR3 14bp -arg had the highest arginine production, and the total arginine production increased from 66.83g/L to 103.98g/L, an increase of 55.6%.

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Abstract

提供了一种包含SEQ ID NO:11所示核苷酸序列的5'UTR元件及其在L-精氨酸生产中的应用。还提供了包含该5'UTR元件和启动子的功能性表达元件、包含目的基因和该功能性表达元件的基因表达盒、重组表达载体和包含该基因表达盒或重组表达载体的谷氨酸棒杆菌工程菌。

Description

一种5’UTR元件及其在L-精氨酸生产中的应用 技术领域
本发明属于生物技术领域，具体涉及一种5’UTR元件及其在L-精氨酸生产中的应用。
背景技术
L-精氨酸(L-arginine)，即2-氨基-5-胍基戊酸，分子式C 6H 14N 4O 2，广泛应用于医药、食品和饲料添加剂中。可作为氨基酸营养输液和注射液的平衡剂；作为保健品添加剂增强肌肉活力和增强免疫力；作为饲料添加剂提高动物的产雌率等。
目前L-精氨酸主要通过微生物发酵法生产，工业上采用的多为大肠杆菌和谷氨酸棒杆菌。在菌株代谢改造过程中，现有技术一般通过质粒增加精氨酸合成途径关键酶基因(argCJBDFR)的拷贝数来增加L-精氨酸合成基因的表达量。但高拷贝质粒可能会增加细菌的代谢负担且稳定性较差。
在转录和翻译水平上调节基因表达是最直接、最方便的方式，通过使用功能性表达组件，即启动子-5’-非翻译区(PUTR)来实现。因此，通过替换不同强度的PUTR，可以实现基因表达水平的微调，从而缓解细胞生长和目标产物合成的不平衡造成的代谢负担。然而，现有技术还未有在谷氨酸棒杆菌中使用PUTR来增加L-精氨酸的合成。
因此，亟需开发高效的基因表达元件，增加谷氨酸棒杆菌中L-精氨酸合成途径中关键酶的表达和协调多基因平衡表达来优化L-精氨酸的合成途径。
发明内容
为克服上述缺陷，本发明的目的在于从谷氨酸棒杆菌基因组中筛选一种增强基因表达的5’UTR元件，并利用该5’UTR元件与高强度启动子组合调 控谷氨酸棒杆菌中L-精氨酸代谢途径关键酶的表达，从而提高工程菌的L-精氨酸产量。
为达到上述目的，首先，本发明使用流式细胞仪分选技术从谷氨酸棒杆菌基因组中筛选得到能够促进绿色荧光蛋白基因表达增强的5’UTR序列，通过逐步缩短5’UTR序列长度筛选有效的最优5’UTR序列。然后，利用该最优5’UTR序列与多个高强度启动子P ilvA、P thrC、P EF-tu和P dapA组合构建重组表达载体，通过荧光强度表征验证该5’UTR序列与启动子的适配性。最后，选用P EF-tu-最优5’UTR组成的功能性表达元件，构建L-精氨酸arg操纵子基因(argCJBDFR)的谷氨酸棒杆菌重组菌，发酵生产L-精氨酸。
本发明第一方面提供一种基于5’UTR文库筛选得到的棒状杆菌5’UTR元件，所述5’UTR元件包含如SEQ ID NO：11所示的核苷酸序列。
优选地，所述5’UTR元件的核苷酸序列如SEQ ID NO：7、9～11任一所示。
本发明第二方面提供一种功能性表达元件，所述功能性表达元件包含启动子和本发明第一方面所述的5’UTR元件，所述启动子选自P EF-tu、P ilvA、P thrC、P tac和P dapA。
优选地，所述启动子为P EF-tu。
本发明第三方面提供一种基因表达盒，所述基因表达盒包含第二方面所述的功能性表达原件、目的基因和终止子。所述的目的基因为精氨酸操纵子基因argCJBDFR，其NCBI GeneID分别如下所示：argC的GeneID为69621907；argJ的GeneID为69621908；argB的GeneID为69621909；argD的GeneID为69621910；argF的GeneID为69621911；argR的GeneID为69621912。
本发明第四方面提供一种重组表达载体，所述重组表达载体包含本发明第一方面所述的5’UTR元件、本发明第二方面所述的功能性表达元件或本发明第三方面所述的基因表达盒。优选地，所述重组载体的骨架为pXMJ19。
本发明第五方面提供一种谷氨酸棒杆菌工程菌，所述谷氨酸棒杆菌工程菌包含本发明第三方面所述的基因表达盒或本发明第四方面所述的重组载体。优选地，所述出发菌株为谷氨酸棒状杆菌C.glutamicum H1，保藏编号为CCTCC NO:M 2020644，或谷氨酸板状杆菌C.glutamicum ATCC 13032。
本发明第六方面提供一种在培养基中发酵本发明第五方面所述的谷氨酸棒杆菌工程菌生产L-精氨酸的方法。
优选地，所述培养基包括葡萄糖12.5％，磷酸氢二钾0.15％，尿素0.1％，硫酸铵4.5％，酵母膏0.5％，玉米浆5.5％，硫酸镁0.05％，生物素0.05％，维生素B1 0.1％，玉米油10滴/L，糖蜜4.0％，丝肽粉0.3％，轻质碳酸钙0.5％，余量为去离子水，pH为7；所述％为重量体积百分比g/mL。
所述发酵条件选自以下的一种或多种：温度30℃；罐压：0.03～0.08Mpa；pH7.0优选通过氨水控制；溶氧30％；当残糖含量低于3.0％时，流加葡萄糖，维持残糖含量在1.5～3.0％；发酵时间为72h以上。
本发明第七方面提供本发明第一方面所述的5’UTR元件、本发明第二方面所述的功能性表达元件、本发明第三方面所述的基因表达盒和本发明第四方面所述的重组表达载体在制备谷氨酸棒杆菌工程菌中的应用。
本发明第八方面提供本发明第一方面所述的5’UTR元件、本发明第二方面所述的功能性表达元件、本发明第三方面所述的基因表达盒、本发明第四方面所述的重组表达载体和本发明第五方面所述的谷氨酸棒杆菌工程菌在L-精氨酸生产中的应用。
本发明的有益效果在于：
利用筛选的5’UTR元件与高强启动子组合构建的表达载体调控精氨酸操纵子基因的表达，L-精氨酸产量由对照组的66.83g/L提高到103.98g/L，提高了55.6％。
附图说明
图1为质粒P EF-tu-UTR3 14bp图谱。
图2为不同5’UTR调控下重组菌的egfp荧光强度。
图3为不同长度UTR3重组菌荧光强度变化。
图4为UTR3 14bp在不同启动子下荧光蛋白表达强度。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
质粒pXMJ19购自普如汀生物技术(北京)有限公司、E.coli DH5α、E.coli BL21为本领域常规使用；DNA聚合酶(Q5High-Fidelity DNA Polymerase)购自基因有限公司；限制性内切酶(EcoRI、Hind III)、DNAmarker、质粒提取试剂盒、DNA胶回收纯化试剂盒，均购自Takara宝生物工程(大连)有限公司；细菌基因组DNA提取试剂盒购自天根生化科技(北京)有限公司、Onestep clonning克隆重组试剂盒、Klenow酶购自NEB北京公司；氯霉素购自Biosharp公司；糖蜜购自内蒙古赤峰博天糖业；葡萄糖等其余化学药品试剂均为国药分析纯。质粒提取操作步骤参照质粒小提取试剂盒说明书；DNA胶回收操作步骤参照DNA胶回收试剂盒说明书；谷氨酸棒杆菌基因组提取操作步骤参照细菌基因组DNA提取试剂盒说明书；DNA片段重组连接操作步骤参照Onestep clonning克隆重组试剂盒明书；谷氨酸棒杆菌感受态的制备及转化方法参照vander Rest等的方法(M.E.vander 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)。通过氨基酸分析仪(日立8800) 进行发酵液中的氨基酸含量检测。
荧光强度的测定方法：在488nm(激发)和523nm(发射)波长，使用酶标仪(SpectraMax i3x)测定目标细胞的荧光值。使用含有pXMJ19-egfp质粒的菌株的背景荧光值(FPbg)和培养基的背景光密度(ODbg)进行校正，并根据方程计算相对荧光强度:
Figure PCTCN2022124402-appb-000001
实施例1 pXMJ19-egfp质粒构建
扩增获得egfp片段：以egfp基因序列(NCBI序列ID：LC336974.1)为模板，设计引物F-egfp(SEQ ID NO：1)和R-egfp(SEQ ID NO：2)，使用高保真Q5 High-Fidelity DNA Polymerase聚合酶进行PCR扩增，PCR反应体系(50μl)为：5×Q5 reaction buffer 10μl、10mM dNTP 1μl、引物各2.5μl、模板视样品浓度而定、Q5酶0.5μl、加水至50μl。反应条件为：98℃30s，98℃10s，55℃-72℃30s，72℃45s，33个循环；72℃2min。1％琼脂糖凝胶电泳检测并用DNA胶回收试剂盒纯化回收PCR产物。
egfp-F：5’-GCGTCGACATGGTGAGCAAG-3’(Sal I)(SEQ ID NO：1)
egfp-R：5’-CGCGAATTCTTACTTGTACAGCTCGTCC-3’(EcoRI)(SEQ ID NO：2)
将pXMJ19空载载体和egfp片段分别用EcoRI与Sal I核酸内切酶进行双酶切，通过胶回收试剂盒回收酶切产物，用Onestep clonning克隆重组试剂盒重组上述双酶切产物(即双酶切后的pXMJ19和egfp片段)，转化至E.coli BL21，涂布于含氯霉素的LB平板，经过37℃过夜培养后，挑选阳性转化子送金开瑞生物工程有限公司测序，测序正确的转化子即含有重组质粒pXMJ19-egfp，将该质粒命名为P tac-egfp。
实施例2 5’UTR的质粒文库构建
按照DNA提取试剂盒说明书的步骤提取谷氨酸棒状杆菌基因组C.glutamicum H1并保存备用。使用超声波细胞破碎仪将基因组片段化，优化破碎时间和功率使基因组片段在50-300bp之间。超声波细胞破碎仪参数设置为功率10％-20％，不断调整破碎功率和破碎时间，达到理想的破碎效果。浓缩破碎后的细胞，将破碎后的基因组片段加2倍体积的无水乙醇在-20℃进行沉降，2h离心收集DNA片段，烘干后用无菌水溶解，并保存在-20℃直到使用。以质粒P tac-egfp为模板，以该质粒多克隆位点中Hind III和Sal I中间的PstI位点为起始点，使用引物P-UTR-F(SEQ ID NO：3)/P-UTR-R(SEQ ID NO：4)扩增将质粒线性化获得线性化的报告质粒P tac-egfp。将谷棒基因组破碎后的DNA片段使用Klenow酶补平DNA的粘性末端，在1X NE Buffer反应缓冲液中分别添加：33μM的dNTP，每微克DNA添加1单位Klenow酶。在25℃下孵化30min。最后加入EDTA至10mM的终浓度，在75℃下加热20min，停止反应。
P-UTR-F：5’-CTGCAGGTCGACTCTAGAGGATCC-3’(SEQ ID NO：3)
P-UTR-R：5’-GCATGCAAGCTTAATTGCATGCAAG-3’(SEQ ID NO：4)
用Onestep clonning克隆重组试剂盒将上述平末端DNA片段与线性化的报告质粒P tac-egfp重组，重组产物转化到E.coli DH5α中得到5’UTR的质粒文库。
注：C.glutamicum H1于2020年10月26日保藏在中国典型培养物保藏中心(地址：中国武汉武汉大学)，菌种名称为谷氨酸棒杆菌H1，分类号为Corynebacterium glutamicum H1，保藏编号为CCTCC NO：M 2020644。
实施例3 5’UTR的筛选及鉴定
将构建的5’UTR质粒文库和P tac-egfp质粒分别转化到谷氨酸棒状杆菌C.glutamicum H1中，并涂布与氯霉素抗性平板上，转化有P tac-egfp质粒的菌 种作为对照菌株，命名H1-P tac-egfp。质粒文库转化后，从抗性平板上将转化子冲洗下来并用PBS洗涤3遍，洗涤后的转化子通过流式细胞仪进行分选荧光值高于对照菌株H1-P tac-egfp(不含5’UTR)的突变体，得到的目标细胞涂布在含有抗生素的LB琼脂平板上并用96孔板复筛，培养48h后挑选荧光较强的转化子，并提取质粒进行测序。
有四个重组菌荧光强度明显高于其他菌株，命名为H1-UTR1、H1-UTR2、H1-UTR3和H1-UTR4(图2)，其中含有UTR3序列的重组菌(H1-UTR3)荧光强度最高，对照菌株H1-P tac-egfp荧光表达量在90000，H1-UTR3的荧光表达量在1400000，相对对照菌种使报告基因的表达量提高了15.5倍。对筛选得到的四株重组菌中质粒进行测序确定了报告基因egfp上游插入的这四个随机序列信息(表1)。
表1筛选到的5’UTR高表达序列：
Figure PCTCN2022124402-appb-000002
Figure PCTCN2022124402-appb-000003
实施例4 5’UTR的优化
以表达强度最高的UTR3作为后续优化的序列，由于序列较短，为了鉴定UTR3的核心序列，直接送金斯瑞公司合成截短序列(表2)并添加酶切位点Hind III和Sal I。
表2 UTR3截短后的不同长度序列：
Figure PCTCN2022124402-appb-000004
将P tac-egfp载体和以上UTR3截短片段分别用Hind III与Sal I核酸内切酶进行双酶切，胶回收后用Onestep clonning克隆重组试剂盒重组上述双酶切产物，转化至E.coli DH5α，涂布于含氯霉素的LB平板，经过37℃过夜培养后，挑选阳性转化子，送生工测序，将验证正确的重组质粒分别命名为P tac-UTR3 18bp-egfp、P tac-UTR3 16bp-egfp和P tac-UTR3 14bp-egfp。
将以上截短序列重组载体转化到C.glutamicum H1中分别命名为H1-P tac-UTR3 18bp-egfp、H1-P tac-UTR3 16bp-egfp、H1-P tac-UTR3 14bp-egfp并测定荧光强度。结果显示，缩短后的UTR3 18bp、UTR3 16bp和UTR3 14bp其荧光值相对于对照菌株H1-P tac-egfp仍显著提高，其中含有UTR3 14bp序列的重组菌(H1-P tac- UTR3 14bp-egfp)表达强度最高。该序列相对原始UTR3序列使报告基因的表达量提高了21.4％，相对对照菌种使报告基因的表达量提高了18.9倍(图3)，UTR3 14bp序列长度为14bp。
实施例5 5’UTR与启动子的适配性考察
为验证UTR3 14bp元件与不同启动子的适配性，用不同的启动子P ilvA、P thrC、P EF-tu和P dapA分别替换P tac-egfp和P tac-UTR3 14bp-egfp重组载体中的P tac启动子，所使用的启动子序列送金斯瑞公司合成(表3)并在序列首尾添加酶切位点Nar I和Hind III。
分别用Nar I和Hind III核酸内切酶对上述合成的启动子元件、P tac-egfp和P tac-UTR3 14bp-egfp进行双酶切，去掉LacIq和原来的P tac启动子，将新的启动子片段用Onestep clonning克隆重组试剂盒重组，重组产物转化至E.coli DH5α，涂布于含氯霉素的LB平板，经过37℃过夜培养后，挑选阳性转化子，送生工测序，重新获得的重组质粒如下表3。
表3 UTR3 14bp与不同启动子组合的重组质粒
Figure PCTCN2022124402-appb-000005
Figure PCTCN2022124402-appb-000006
将以上重组质粒转化到C.glutamicum H1中并测定荧光强度，得到的重组菌株荧光强度比见表4。结果显示不同强度的启动子替换后，荧光表达水平各有变化(图4)，UTR3 14bp元件均能增强基因在翻译水平的表达，加强后的荧光强度与对照相比均可提高15至20倍，证明该UTR元件在不同启动子调控下均可适用(表4)。
表4 UTR3 14bp在不同启动子调控下的翻译增强水平比较
Figure PCTCN2022124402-appb-000007
实施例6构建arg操纵子基因增强重组菌
谷氨酸棒状杆菌中arg操纵子包括argCJBDFR六个基因，其NCBI GeneID分别如下所示：argC的GeneID为69621907；argJ的GeneID为69621908；argB的GeneID为69621909；argD的GeneID为69621910；argF的GeneID为69621911；argR的GeneID为69621912。涉及到精氨酸代谢合成路线。因此提高arg操纵子基因的表达是提高精氨酸产量的有效策略。同时，为了验证筛选后得到的UTR3 14bp元件在谷氨酸棒状杆菌中对促进基因表达的作用，我们选用翻译增强水平最高的P EF-tu-UTR3 14bp启动子组合来构建精氨酸发酵重组菌株。用arg操纵子分别替换P EF-tu-egfp和P EF-tu-UTR3 14bp-egfp中的egfp序列，构建对照组重组质粒P EF-tu-arg和过表达质粒P EF-tu-UTR3 14bp-arg。设计上下游引物扩增arg操纵子基因并在首尾引入酶切位点Sal I和EcoR I，用引物F-arg和R-arg对谷氨酸棒状杆菌基因组模板进行PCR，高保真Q5 High-Fidelity DNA Polymerase聚合酶PCR扩增。PCR反应体系(50μl)为：5×Q5 reaction buffer 10μl、10mM dNTP 1μl、引物各2.5μl、模板视样品浓度而定、Q5酶0.5μl、加水至50μl。反应条件为：98℃30s，98℃10s，55℃-72℃30s，72℃6min，30个循环；72℃2min。1％琼脂糖凝胶电泳检测且DNA胶回收试剂盒纯化回收PCR产物。
F-arg 5’-GCGTCGACATGATAATCAAGGTTGCAATCGCAGG-3’(Sal I)(SEQ ID NO：16)
R-arg 5’-CGGAATTCTTAAGTGGTGCGCCCGCTGAGTAAT-3’(EcoRI)(SEQ ID NO：17)
将P EF-tu-egfp和P EF-tu-UTR3 14bp-egfp载体和arg操纵子片段分别用Sal I与EcoRI核酸内切酶进行双酶切，胶回收后用Onestep clonning克隆重组试剂盒重组上述双酶切产物，转化至E.coli BL21，涂布于含氯霉素的平板，经过37℃过夜培养后，挑选阳性转化子，进行双酶切验证，将重组质粒分别 命名为P EF-tu-arg和过表达质粒P EF-tu-UTR3 14bp-arg(含有UTR元件)，并送生工测序。
根据常规方案，将P EF-tu-arg和P EF-tu-UTR3 14bp-arg分别转化到谷氨酸棒状杆菌C.glutamicum H1和C.glutamicum 13032中，由转化实验产生的重组谷氨酸棒状杆菌分别被命名为：H1-P EF-tu-arg、H1-P EF-tu-UTR3 14bp-arg、C.glutamicum 13032-P EF-tu-arg和C.glutamicum 13032-P EF-tu-UTR3 14bp-arg。
实施例7利用5’UTR提高arg操纵子基因表达对精氨酸产量的影响
精氨酸种子培养基配方为：葡萄糖3.0％，磷酸氢二钾0.2％，尿素0.12％，硫酸铵0.5％，酵母膏0.5％，玉米浆5.5％，硫酸镁0.04％，生物素50ug/L，丝肽粉1.0％，玉米油10滴/L，轻质碳酸钙1％。
精氨酸发酵培养基配方为：葡萄糖12.5％，磷酸氢二钾0.15％，尿素0.1％，硫酸铵4.5％，酵母膏0.5％，玉米浆5.5％，硫酸镁0.05％，生物素0.05％，维生素B1 0.1％，玉米油10滴/L，糖蜜4.0％，丝肽粉0.3％，轻质碳酸钙0.5％，余量为去离子水，pH为7；所述％为重量体积(g/mL)百分比。
以菌株C.glutamicum H1、C.glutamicum 13032、C.glutamicum 13032-P EF- tu-arg和重组菌H1-P EF-tu-arg为对照，将重组菌H1-P EF-tu-UTR3 14bp-arg和C.glutamicum 13032-P EF-tu-UTR3 14bp-arg装入5L发酵罐进行培养。从平板上挑取单菌落接种至种子培养基中，于温度为32℃、转速为200r/min的条件下摇床培养12h，得到种子液；按照5％接种量，将种子培养液进行合并至补料瓶中，连接补料管道，将种子培养液接种至发酵培养基中，开始发酵培养。控温30℃，罐压：0.03～0.08Mpa，氨水控制pH在7.0，通过调整转速及通气量维持溶氧在30％，当残糖含量低于3.0％时，流加80％的葡萄糖，维持残糖含量在1.5～3.0％，发酵时间为72h以上。发酵结束后，发酵液离心取上清衍生化后，使用HPLC对各主要氨基酸进行分析，其实验结果统计如表4所示，与对照菌株相比，重组菌H1-P EF-tu-UTR3 14bp-arg的精氨酸产量 最高，精氨酸总产量由之前的66.83g/L提高到103.98g/L，提高了55.6％。
表5氨基酸产量
Figure PCTCN2022124402-appb-000008
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技 术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (15)

  1. 一种5’UTR元件，其特征在于，所述5’UTR元件包括如SEQ ID NO:11所示的核苷酸序列。
  2. 如权利要求1所述的5’UTR元件，其特征在于，所述5’UTR元件的核苷酸序列如SEQ ID NO:7、9～11中任一所示。
  3. 一种功能性表达元件，其特征在于，所述功能性表达元件包含启动子和如权利要求1或2所述的5’UTR元件。
  4. 如权利要求3所述的功能性表达元件，其特征在于，所述启动子为P EF-tu、P ilvA、P thrC、P tac或P dapA；
    优选地，所述启动子为P EF-tu。
  5. 一种基因表达盒，其特征在于，其包括如权利要求3或4所述的功能性表达元件、目的基因和终止子。
  6. 如权利要求5所述的基因表达盒，其特征在于，所述目的基因为精氨酸操纵子基因argCJBDFR；优选地，所述argCJBDFR的NCBI GeneID分别如下所示：argC的GeneID为69621907；argJ的GeneID为69621908；argB的GeneID为69621909；argD的GeneID为69621910；argF的GeneID为69621911；argR的GeneID为69621912。
  7. 一种重组表达载体，其特征在于，其包括如权利要求1或2所述的5’UTR元件、权利要求3或4所述的功能性表达元件或权利要求5或6所述的基因表达盒。
  8. 如权利要求7所述的重组表达载体，其特征在于，所述重组表达载体的骨架质粒为pXMJ19。
  9. 一种谷氨酸棒杆菌工程菌，其特征在于，所述谷氨酸棒杆菌工程菌包含权利要求5或6所述的基因表达盒，或权利要求7或8所述的重组表达载体。
  10. 如权利要求9所述的谷氨酸棒杆菌工程菌，其特征在于，所述的谷氨酸棒杆菌工程菌的出发菌株为谷氨酸棒状杆菌C.glutamicum H1，保藏编号为CCTCC NO:M 2020644或谷氨酸板状杆菌C.glutamicum ATCC 13032。
  11. 一种生产L-精氨酸的方法，其特征在于，其在培养基中发酵如权利要求9或10所述的谷氨酸棒杆菌工程菌，获得所述L-精氨酸。
  12. 如权利要求11所述的方法，其特征在于，所述培养基包括：
    葡萄糖12.5％，磷酸氢二钾0.15％，尿素0.1％，硫酸铵4.5％，酵母膏0.5％，玉米浆5.5％，硫酸镁0.05％，生物素0.05％，维生素B1 0.1％，玉米油10滴/L，糖蜜4.0％，丝肽粉0.3％，轻质碳酸钙0.5％，余量为去离子水，pH为7；所述％为重量体积百分比g/mL。
  13. 如权利要求11所述的方法，其特征在于，所述发酵的条件选自以下一种或多种：
    温度30℃；
    罐压：0.03～0.08Mpa；
    pH7.0优选通过氨水控制；
    溶氧30％；
    当残糖含量低于3.0％时，流加葡萄糖，维持残糖含量在1.5～3.0％；
    发酵时间为72h以上。
  14. 如权利要求1或2所述的5’UTR元件、权利要求3或4所述的功能性表达元件、权利要求5或6所述的基因表达盒、权利要求7或8所述的重组表达载体在制备谷氨酸棒杆菌工程菌中的应用。
  15. 如权利要求1或2所述的5’UTR元件、权利要求3或4所述的功能性表达元件、权利要求5或6所述的基因表达盒、权利要求7或8所述的重组表达载体、权利要求9或10所述的谷氨酸棒杆菌工程菌在L-精氨酸生产中的应用。
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