WO2022143225A1 - 一种产假尿苷工程菌及其应用 - Google Patents

一种产假尿苷工程菌及其应用 Download PDF

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WO2022143225A1
WO2022143225A1 PCT/CN2021/139177 CN2021139177W WO2022143225A1 WO 2022143225 A1 WO2022143225 A1 WO 2022143225A1 CN 2021139177 W CN2021139177 W CN 2021139177W WO 2022143225 A1 WO2022143225 A1 WO 2022143225A1
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pseudouridine
fermentation
seq
medium
gene
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French (fr)
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陈伟
郑玲辉
廖云娥
周敏
王雪峰
朱进伟
高祥
陈世敏
彭湘屏
石磊
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浙江珲达生物科技有限公司
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/385—Pyrimidine nucleosides
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
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    • C12Y308/00—Hydrolases acting on halide bonds (3.8)
    • C12Y308/01—Hydrolases acting on halide bonds (3.8) in C-halide substances (3.8.1)
    • C12Y308/01002—(S)-2-Haloacid dehalogenase (3.8.1.2)
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    • C12Y308/01—Hydrolases acting on halide bonds (3.8) in C-halide substances (3.8.1)
    • C12Y308/01009—(R)-2-Haloacid dehalogenase (3.8.1.9)

Definitions

  • the invention relates to the field of biotechnology, in particular to a maternity pseudouridine engineered bacterium and application thereof.
  • ribosomal RNA Ribonucleic acids from yeast which contain a fifth nucleotide[J].
  • Uridine is removed from ribosine by pseudouridine synthase
  • the base is cleaved and reconnected to form a C-C glycosidic bond, which is isomerized to pseudouridine.
  • the chemical structural formulas of uridine and pseudouridine are shown in formula 1.
  • pseudouridine is present in many classes of RNAs in addition to messenger RNAs, including tRNAs and rRNAs.
  • Pseudouridine is the most abundant modified nucleoside in non-coding RNA, and enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure.
  • Pseudouridine is catalyzed by pseudouridine synthase, which isomerizes specific uridines in RNA after transcription, a process known as pseudouridylation.
  • pseudouridine As a nucleotide metabolite, pseudouridine has been paid more and more attention by scholars.
  • Pseudouridine can be used as a potential biomarker for the diagnosis of nephropathy and tumor, monitoring of curative effect, and also has certain reference significance for the diagnosis of other diseases.
  • pseudouridine reduces radiation-induced chromosomal aberrations in human lymphocytes.
  • pseudouridines play a role in RNA stability and/or helping aminoacyltransferases interact with tRNAs.
  • genomic medicines discovered by means of nucleic acid aptamers, RNA interference, etc. have attracted people's interest.
  • pseudouridine is used as a pharmaceutical intermediate, pseudouridine
  • the phosphorus amidine derivatives were used as one of the starting materials for the synthesis of such oligomers.
  • pseudouridine In terms of the preparation of pseudouridine, at present, only the traditional chemical synthesis method can catalyze the synthesis of pseudouridine. However, for the chemical synthesis of pseudouridine, there are long chemical synthesis steps, low yield, and flammable reagents used. Explosive, less safe and a series of problems. In terms of biosynthesis, no microbial strains and technical reports have been found that can be specifically used for the preparation of pseudouridine in large quantities. Therefore, the low-cost and convenient acquisition of pseudouridine has certain drawbacks.
  • pseudouridine can only be prepared by chemical synthesis, and the chemical synthesis process has long synthesis steps, low yield, and the reagents used are inflammable and explosive, and relatively unsafe, and a series of problems
  • An engineered bacterium capable of fermenting pseudouridine can be constructed by genetic engineering, and the production of pseudouridine can be realized through simple fermentation and low cost.
  • the present invention provides a maternity pseudouridine engineering bacterium and application thereof, comprising the following steps: expressing a gene related to pseudouridine synthesis in Escherichia coli by using genetic engineering technology, and obtaining Genetically engineered strains capable of producing pseudouridine;
  • the protein sequences of the pseudouridine synthesis related genes are: adenylate kinase coding gene pumH protein sequence, tRNA pseudouridine synthase coding gene pumJ protein sequence and HAD (halogen acid dehalogenase) protein family hydrolase coding gene pumD protein sequence;
  • the present invention adopts the following technical solutions:
  • a pseudouridine-producing engineering bacterium which utilizes genetic engineering technology to express a gene related to pseudouridine synthesis in a host bacterium to obtain a pseudouridine-producing genetically engineered bacterium.
  • the host bacteria described in the present invention include but are not limited to bacteria, algae and fungi, wherein the bacteria are preferably selected from Escherichia coli, and further, preferably Escherichia coli DH5 ⁇ .
  • the pseudouridine synthesis-related genes are: adenylate kinase encoding gene pumH, tRNA pseudouridine synthase encoding gene pumJ and HAD (halogen acid dehalogenase) protein family hydrolase encoding gene pumD.
  • the described coding gene pumH protein sequence, coding gene pumJ protein sequence and coding gene pumD protein sequence are all derived from Streptomyces sp.ID38640, and are respectively the gene pumH protein sequence shown in SEQ ID No.1 , the gene pumJ protein sequence shown in SEQ ID No.2 and the gene pumD protein sequence shown in SEQ ID No.3.
  • gene pumH, pumJ and pumD its dna sequence is all derived from Streptomyces sp.ID38640, the sequence is respectively nucleotide sequence shown in SEQ ID No.4, SEQ ID No.5 The nucleotide sequence shown and the nucleotide sequence shown in SEQ ID No. 6.
  • the DNA sequences of the genes pumH, pumJ and pumD are optimized according to the codon preference of Escherichia coli, and the optimized sequences are respectively the nucleotide sequence shown in SEQ ID No.7, SEQ ID The nucleotide sequence shown in No. 8 and the nucleotide sequence shown in SEQ ID No. 9.
  • the pumH codon-optimized nucleic acid sequence is connected to the promoter Ptrp sequence before the start codon, pumJ
  • the nucleic acid sequence after codon optimization with pumD is connected to the RBS sequence aaaggaggatatacat before the initiation codon, and the modified sequences are respectively the nucleotide sequence shown in SEQ ID No.10 and the nucleotide sequence shown in SEQ ID No.11. and the nucleotide sequence shown in SEQ ID No. 12.
  • the construction method of the maternity uridine engineering bacteria of the present invention specifically comprises the following steps:
  • pumH+Ptrp sequence and vector pUC18 were digested with HindIII+SphI respectively, then connected with T4 ligase (Thermfisher), and the ligation product was transformed into E. coli competent cells (preferably DH5 ⁇ ) to obtain clone pZH423;
  • pumJ+RBS sequence and vector pZH423 were digested with XbaI+SphI respectively, then connected with T4 ligase (Thermfisher), and the ligation product was transformed into E. coli competent cells (preferably DH5 ⁇ ) to obtain clone pZH424;
  • pumD+RBS sequence and vector pZH424 were digested with XbaI+KpnI respectively, then ligated with T4 ligase (Thermfisher), and the ligation product was transformed into E. coli competent cells (preferably DH5 ⁇ ) to obtain clone pZH425, that is, pseudouridine Engineering bacteria.
  • the method for producing pseudouridine includes the following steps: fermenting and culturing the genetically engineered bacteria described above to obtain pseudouridine.
  • inosine is added to the fermentation medium in the form of an aqueous solution dissolved in sodium hydroxide, and the initial concentration of inosine in the fermentation medium is 1.0-10 g/L.
  • the pH of the fermentation process is maintained between 6.5-7.6, and it is preferably adjusted by a combination of citric acid aqueous solution or acetic acid aqueous solution and ammonia water.
  • the concentration of the citric acid or acetic acid is 50-100 g/L; the preferred concentration of the ammonia water is 13 mol/L.
  • the medium formula (g/L) of the fermentation culture contains the following components: glucose 10-20, KH 2 PO 4 2-6, yeast powder 4-8, ammonium sulfate 3-10, corn Slurry dry powder 10-20, MgSO 4 7H 2 O 2-6; other material components in medium (mg/L): FeSO 4 7H 2 O 100-200, MnSO 4 7H2O 10-30, VB 1 1.3 -3.0, VH 0.6-2.0, cobalt chloride 2-5, zinc sulfate 2-5, calcium chloride 5-10, spectinomycin 50, the balance is water, before sterilization, the pH of the fermentation medium is 2mol/L The hydrochloric acid and 2mol/L sodium hydroxide were adjusted to 7.0.
  • the temperature is controlled in stages, and the fermentation temperature is maintained at 28-30°C for the 10th-20th hour of the fermentation, and then the fermentation temperature is increased to 35-37°C; the fermentation culture period is 72-96h.
  • the present invention provides a kind of maternity pseudouridine engineering bacteria and its application, and the engineering bacteria is derived from the pseudouridine synthesis genes pumH, pumJ and pumD genes in Streptomyces sp. ID38640 cloned. In the body of Escherichia coli, the engineered bacteria that can produce pseudouridine are obtained.
  • the present invention realizes the heterologous expression of pseudouridine in Escherichia coli by genetically modifying Escherichia coli, and further by adding inosine to the culture medium, adopting stage temperature control in the fermentation process, and using citric acid and ammonia water to The regulation of pH greatly promotes the accumulation of pseudouridine, and finally the genetically engineered Escherichia coli can produce more than 7g/L of pseudouridine.
  • the method for constructing the Escherichia coli gene of the invention is simple, the pseudouridine with higher yield can be obtained without adding the inducer isopropyl- ⁇ -D-thiogalactopyranoside in the fermentation process, and the application prospect is broad.
  • Fig. 1 is the mass spectrum of pseudouridine standard substance
  • Fig. 2 is the HPLC detection spectrum of pseudouridine standard substance
  • Fig. 3 is the HPLC spectrum of embodiment 4 pZH425 fermentation broth detection.
  • the strain used in the present invention is Escherichia coli DH5 ⁇ , and the strain preservation number is CCTCC AB 2013329, which was purchased from China Center for Type Culture Collection (CCTCC for short).
  • liquid phase detection method conditions used for pseudouridine titer detection of the present invention are as follows:
  • Chromatographic column Waters XBridge Amide (250mm ⁇ 460mm, 3.5 ⁇ m), mobile phase A: 0.2% (mass volume ratio, unit is g/L) ammonium acetate aqueous solution, mobile phase B: acetonitrile, retention time: 20min, flow rate: 1mL /min, injection volume: 10 ⁇ L, detection wavelength: 255 nm, column temperature: (25 ⁇ 1) °C.
  • the solid medium (g/L) used in the embodiment of the present invention is: yeast extract 5, tryptone 10, NaCl 10, spectinomycin 0.05, the rest are water, before sterilization, the pH of the fermentation medium is 2mol/L The hydrochloric acid and 2mol/L sodium hydroxide were adjusted to 7.0.
  • the seed medium (g/L) used in the embodiment of the present invention is: yeast extract 5, tryptone 10, NaCl 10, glucose 20, spectinomycin 0.05, and the rest are water. 2mol/L hydrochloric acid and 2mol/L sodium hydroxide were adjusted to 7.0.
  • the fermentation medium formula (g/L) used in the embodiment of the present invention is: glucose 10-20, KH 2 PO 4 2-6, yeast powder 4-8, ammonium sulfate 3-10, corn steep liquor dry powder 10-20, MgSO 4 ⁇ 7H 2 O 2-6; other material components in the medium (mg/L): FeSO 4 ⁇ 7H 2 O 100-200, MnSO 4 ⁇ 7H 2 O 10-30, VB 1 1.3-3.0, VH 0.6-2.0 , cobalt chloride 2-5, zinc sulfate 2-5, calcium chloride 5-10, in addition, add spectinomycin 50mg/L to the medium, the balance is water, before sterilization, the pH of the fermentation medium is 2mol/L The hydrochloric acid and 2mol/L sodium hydroxide were adjusted to 7.0.
  • Example 1 Construction of pZH423 HindIII and SphI double digested the DNA fragment of SEQ ID No. 10 synthesized by Shanghai Shenggong to obtain exogenous fragment 1; HindIII and SphI double digested the vector pUC18 to obtain vector fragment 1; 1 was connected with vector fragment 1 to transform E. coli competent cell DH5 ⁇ to obtain recombinant colony pZH423.
  • the pZH425 successfully constructed in Example 3 was inserted into the solid medium on the plate, and after culturing at 36°C for 20 hours, a single colony was picked from the solid medium and inserted into the seed medium, and the seed solution was obtained by culturing at 36°C and 250 rpm for 15 hours. Then according to 20% of the inoculum, draw 4ml of seed liquid into 20ml of fermentation medium, and the medium formula (g/L) of the fermentation culture is as follows: glucose 10, KH 2 PO 4 2, yeast powder 4, ammonium sulfate 3.
  • Corn steep liquor dry powder 10, MgSO 4 7H 2 O 2; other material components in the medium (mg/L): FeSO 4 7H 2 O 100, MnSO 4 7H2O 10, VB 1 1.3, VH 0.6, chlorine Cobalt 2, zinc sulfate 2, calcium chloride 5, and spectinomycin 50 were added to the medium, and the balance was water.
  • the pH of the fermentation medium was 2 mol/L hydrochloric acid and 2 mol/L sodium hydroxide. Adjust to 7.0.
  • the fermentation temperature was maintained at 37° C., and the cultured at 250 rpm for 30 h to obtain a fermentation broth.
  • the fermentation broth was centrifuged at 14,000 ⁇ g for 10 min, and the centrifuged supernatant was taken and passed through a membrane (0.22um water filter), and the supernatant after passing through the membrane was tested by HPLC.
  • a membrane (0.22um water filter)
  • HPLC HPLC
  • the pZH425 successfully constructed in Example 3 was inserted into the solid medium on the plate, and after culturing at 36°C for 20 hours, a single colony was picked from the solid medium and inserted into the seed medium, and the seed liquid was obtained by culturing at 36°C and 250rpm for 15h. Then, according to 20% of the inoculation amount, 2L of seed liquid was sucked into the fermentation medium, and the total volume of the medium after inoculation was 10L.
  • the medium formula (g/L) of the fermentation culture is as follows: glucose 15, KH 2 PO 4 4, yeast powder 6, ammonium sulfate 8, corn steep liquor dry powder 15, MgSO 4 ⁇ 7H 2 O 4; other materials in the medium Components (mg/L): FeSO 4 ⁇ 7H 2 O 150, MnSO 4 ⁇ 7H2O 20, VB 1 2, VH 1, Cobalt Chloride 3.5, Zinc Sulfate 3.5, Calcium Chloride 8,
  • the culture medium was added with Spectacularia
  • dissolve 10g of inosine in 100ml of an aqueous solution containing 1mol/L sodium hydroxide dissolve 10g of inosine in 100ml of an aqueous solution containing 1mol/L sodium hydroxide, and then add the sodium hydroxide aqueous solution containing 10g of inosine into the fermentation medium, the balance is water, before sterilization , the pH of the fermentation medium was adjusted to 7.0 with 2 mol/L hydrochloric acid
  • the pH of the medium was adjusted to 6.5 with citric acid aqueous solution containing 50g/L and ammonia water containing 13mol/L ammonia, and the fermentation temperature was kept at 28°C for 0-10h, and the fermentation temperature was kept at 35° C., aeration during the fermentation process is set to 1.0vvm, and the dissolved oxygen is controlled at 30%-40% through the linkage between the stirring speed and the dissolved oxygen, and the fermentation period is 72 hours to obtain a fermentation broth.
  • the fermentation broth was centrifuged at 14,000 ⁇ g for 10 min, and the centrifuged supernatant was taken and passed through a membrane (0.22um water filter), and the supernatant after passing through the membrane was tested by HPLC.
  • a membrane (0.22um water filter)
  • HPLC HPLC
  • HPLC chromatogram of pZH425 fermentation broth was compared with the standard product, and the fermentation unit of pseudouridine was about 5.4g/L.
  • the pZH425 successfully constructed in Example 3 was inserted into the solid medium on the plate, and after culturing at 36°C for 20 hours, a single colony was picked from the solid medium and inserted into the seed medium, and the seed solution was obtained by culturing at 36°C and 250 rpm for 15 hours. Then, according to 20% of the inoculation amount, 2L of seed liquid was sucked into the fermentation medium, and the total volume of the medium after inoculation was 10L.
  • the pH of the medium was adjusted to 6.8 with acetic acid aqueous solution containing 60g/L and ammonia water containing 13mol/L ammonia, and the fermentation temperature was kept at 28°C during 0-10h, and the fermentation temperature was kept at 35°C from the 11th hour to the end of the fermentation.
  • the aeration during the fermentation process is set to 1.0vvm, and the dissolved oxygen is controlled at 30%-40% through the linkage between the stirring speed and the dissolved oxygen, and the fermentation period is 72h to obtain a fermentation broth.
  • the fermentation broth was centrifuged at 14,000 ⁇ g for 10 min, and the centrifuged supernatant was taken and passed through a membrane (0.22um water filter), and the supernatant after passing through the membrane was tested by HPLC.
  • a membrane (0.22um water filter)
  • HPLC HPLC
  • HPLC chromatogram of pZH425 fermentation broth was compared with the standard product, and the fermentation unit of pseudouridine was about 2.9g/L.
  • the pZH425 successfully constructed in Example 3 was inserted into the solid medium on the plate, and after culturing at 36°C for 20 hours, a single colony was picked from the solid medium and inserted into the seed medium, and the seed solution was obtained by culturing at 36°C and 250 rpm for 15 hours. Then, according to 20% of the inoculation amount, 2L of seed liquid was sucked into the fermentation medium, and the total volume of the medium after inoculation was 10L.
  • the medium formula (g/L) of the fermentation culture is as follows: glucose 20, KH 2 PO 4 6, yeast powder 8, ammonium sulfate 10, corn steep liquor dry powder 20, MgSO 4 7H 2 O 6; other materials in the medium Component (mg/L): FeSO 4 ⁇ 7H 2 O 100, MnSO 4 ⁇ 7H 2 O 10, VB 1 1.3, VH 0.6, cobalt chloride 2, zinc sulfate 2, calcium chloride 5, additionally added to the medium Spectinomycin 50, in addition, dissolve 60g of inosine in 200ml of an aqueous solution containing 1 mol/L sodium hydroxide, and then add the sodium hydroxide aqueous solution containing 60g of inosine into the fermentation medium, the balance is water, before sterilization , the pH of the fermentation medium was adjusted to 7.0 with 2 mol/L hydrochloric acid and 2 mol/L sodium hydroxide.
  • the pH of the medium was adjusted to 7.0 with citric acid aqueous solution containing 80g/L and ammonia water containing 13mol/L ammonia, the fermentation temperature was kept at 30°C for the first 15h, and the fermentation temperature was kept at 37°C from the 16th hour to the end of the fermentation.
  • the ventilation during the fermentation process is set to 1.0vvm, and the dissolved oxygen is controlled at 30%-40% through the linkage between the stirring speed and the dissolved oxygen, and the fermentation period is 84h to obtain a fermentation broth.
  • the fermentation broth was centrifuged at 14,000 ⁇ g for 10 min, and the centrifuged supernatant was taken and passed through a membrane (0.22um water filter), and the supernatant after passing through the membrane was tested by HPLC.
  • a membrane (0.22um water filter)
  • HPLC HPLC
  • HPLC chromatogram of pZH425 fermentation broth was compared with the standard product, and the fermentation unit of pseudouridine was about 6.1g/L.
  • Example 8 Fermentation of pZH425 to produce pseudouridine
  • the pZH425 successfully constructed in Example 3 was inserted into the solid medium on the plate, and after culturing at 36°C for 20 hours, a single colony was picked from the solid medium and inserted into the seed medium, and the seed liquid was obtained by culturing at 36°C and 250rpm for 15h. Then, according to 20% of the inoculation amount, 2L of seed liquid was sucked into the fermentation medium, and the total volume of the medium after inoculation was 10L.
  • the medium formula (g/L) of the fermentation culture is as follows: glucose 20, KH 2 PO 4 6, yeast powder 8, ammonium sulfate 10, corn steep liquor dry powder 20, MgSO 4 7H 2 O 6; other materials in the medium Component (mg/L): FeSO 4 ⁇ 7H 2 O 100, MnSO 4 ⁇ 7H2O 10, VB 1 1.3, VH 0.6, cobalt chloride 2, zinc sulfate 2, calcium chloride 5, in addition to the medium, add Spectacularia
  • 60g of inosine was dissolved in 200ml of an aqueous solution containing 1mol/L sodium hydroxide, and then the sodium hydroxide aqueous solution containing 60g of inosine was added to the fermentation medium, and the balance was water.
  • the pH of the fermentation medium was adjusted to 7.0 with 2 mol/L hydrochloric acid and 2 mol/L sodium hydroxide.
  • the pH of the medium was adjusted to 7.0 with acetic acid aqueous solution containing 80g/L and ammonia water containing 13mol/L ammonia, the fermentation temperature was kept at 30°C for the first 15h, and the fermentation temperature was kept at 37°C from the 16th hour to the end of the fermentation.
  • the ventilation is set to 1.0vvm, and the dissolved oxygen is controlled at 30%-40% through the linkage between the stirring speed and the dissolved oxygen, and the fermentation period is 84h to obtain the fermentation broth.
  • the fermentation broth was centrifuged at 14,000 ⁇ g for 10 min, and the centrifuged supernatant was taken and passed through a membrane (0.22um water filter), and the supernatant after passing through the membrane was tested by HPLC.
  • a membrane (0.22um water filter)
  • HPLC HPLC
  • HPLC chromatogram of pZH425 fermentation broth was compared with the standard product, and the fermentation unit of pseudouridine was about 3.3g/L.
  • the pZH425 successfully constructed in Example 3 was inserted into the solid medium on the plate, and after culturing at 36°C for 20 hours, a single colony was picked from the solid medium and inserted into the seed medium, and the seed liquid was obtained by culturing at 36°C and 250rpm for 15h. Then, according to 20% of the inoculation amount, 2L of seed liquid was sucked into the fermentation medium, and the total volume of the medium after inoculation was 10L.
  • the medium formula (g/L) of the fermentation culture is as follows: glucose 20, KH 2 PO 4 6, yeast powder 7, ammonium sulfate 10, corn steep liquor dry powder 18, MgSO 4 7H 2 O 6; other materials in the medium Component (mg/L): FeSO 4 ⁇ 7H 2 O 200, MnSO 4 ⁇ 7H2O 20, VB 1 2, VH 1, cobalt chloride 4, zinc sulfate 4, calcium chloride 8, in addition, the culture medium was added with Spectacularia In addition, dissolve 100g of inosine in 300ml of aqueous solution containing 1mol/L sodium hydroxide, and then add the sodium hydroxide aqueous solution containing 100g of inosine into the fermentation medium, the balance is water, before sterilization , the pH of the fermentation medium was adjusted to 7.0 with 2 mol/L hydrochloric acid and 2 mol/L sodium hydroxide.
  • the fermentation broth was centrifuged at 14,000 ⁇ g for 10 min, and the centrifuged supernatant was taken and passed through a membrane (0.22um water filter), and the supernatant after passing through the membrane was tested by HPLC.
  • a membrane (0.22um water filter)
  • HPLC HPLC
  • HPLC chromatogram of pZH425 fermentation broth was compared with the standard product, and the fermentation unit of pseudouridine was about 7.2g/L.
  • the fermentation temperature was maintained at 37° C., and the cultured at 250 rpm for 30 h to obtain a fermentation broth.
  • the fermentation broth was centrifuged at 14,000 ⁇ g for 10 min, and the centrifuged supernatant was taken and passed through a membrane (0.22um water filter), and the supernatant after passing through the membrane was tested by HPLC.
  • a membrane (0.22um water filter)
  • HPLC HPLC
  • the pZH425 successfully constructed in Example 3 was inserted into the solid medium on the plate, and after culturing at 36°C for 20 hours, a single colony was picked from the solid medium and inserted into the seed medium, and the seed liquid was obtained by culturing at 36°C and 250 rpm for 15 hours. Then, according to 20% of the inoculation amount, 2L of seed liquid was sucked into the fermentation medium, and the total volume of the medium after inoculation was 10L.
  • the medium formula (g/L) of the fermentation culture is as follows: glucose 20, KH 2 PO 4 6, yeast powder 8, ammonium sulfate 10, corn steep liquor dry powder 20, MgSO 4 7H 2 O 6; other materials in the medium Component (mg/L): FeSO 4 ⁇ 7H 2 O 100, MnSO 4 ⁇ 7H2O 10, VB 1 1.3, VH 0.6, cobalt chloride 2, zinc sulfate 2, calcium chloride 5, in addition to the medium, add Spectacularia
  • 80g of inosine was dissolved in 200ml of an aqueous solution containing 1mol/L sodium hydroxide, and then the sodium hydroxide aqueous solution containing 80g of inosine was added to the fermentation medium, and the balance was water.
  • the pH of the fermentation medium was adjusted to 7.0 with 2 mol/L hydrochloric acid and 2 mol/L sodium hydroxide.
  • the pH of the medium was adjusted to 8.0 with acetic acid aqueous solution containing 100g/L and ammonia water containing 13mol/L ammonia, the fermentation temperature was kept at 30°C for the first 20h, and the fermentation temperature was kept at 37°C from the 20th hour to the end of the fermentation.
  • the ventilation is set to 1.0vvm, and the dissolved oxygen is controlled at 30%-40% through the linkage between the stirring speed and the dissolved oxygen, and the fermentation period is 96h to obtain the fermentation broth.
  • the fermentation broth was centrifuged at 14,000 ⁇ g for 10 min, and the centrifuged supernatant was taken and passed through a membrane (0.22um water filter), and the supernatant after passing through the membrane was tested by HPLC.
  • a membrane (0.22um water filter)
  • HPLC HPLC
  • HPLC chromatogram detected by the fermentation broth of pZH425 was compared with the standard product, and the pseudouridine fermentation unit was basically undetectable.
  • the pZH425 successfully constructed in Example 3 was inserted into the solid medium on the plate, and after culturing at 36°C for 20 hours, a single colony was picked from the solid medium and inserted into the seed medium, and the seed liquid was obtained by culturing at 36°C and 250rpm for 15h. Then, according to 20% of the inoculation amount, 2L of seed liquid was sucked into the fermentation medium, and the total volume of the medium after inoculation was 10L.
  • the medium formula (g/L) of the fermentation culture is as follows: glucose 20, KH 2 PO 4 6, yeast powder 7, ammonium sulfate 10, corn steep liquor dry powder 18, MgSO 4 7H 2 O 6; other materials in the medium Component (mg/L): FeSO 4 ⁇ 7H 2 O 100, MnSO 4 ⁇ 7H2O 10, VB 1 1.3, VH 0.6, cobalt chloride 2, zinc sulfate 2, calcium chloride 5, in addition to the medium, add Spectacularia
  • 50g of inosine was dissolved in 200ml of an aqueous solution containing 1mol/L sodium hydroxide, and then the sodium hydroxide aqueous solution containing 50g of inosine was added to the fermentation medium, and the balance was water.
  • the pH of the fermentation medium was adjusted to 7.0 with 2 mol/L hydrochloric acid and 2 mol/L sodium hydroxide.
  • the pH of the medium was adjusted to 7.2 with 2 mol/L hydrochloric acid and 2 mol/L sodium hydroxide, the fermentation temperature was maintained at 37 °C, and the aeration during the fermentation process was set to 1.0 vvm.
  • the dissolved oxygen is controlled at 30%-40%, the fermentation period is 96h, and the fermentation broth is obtained.
  • the fermentation broth was centrifuged at 14,000 ⁇ g for 10 min, and the centrifuged supernatant was taken and passed through a membrane (0.22um water filter), and the supernatant after passing through the membrane was tested by HPLC.
  • a membrane (0.22um water filter)
  • HPLC HPLC
  • HPLC chromatogram detected by the fermentation broth of pZH425 was compared with the standard product, and the pseudouridine fermentation unit was basically undetectable.
  • HAD haloacid dehalogenase

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Abstract

提供了一种产假尿苷工程菌及其应用，该工程菌是将来源于链霉菌Streptomyces sp.ID38640中的假尿苷合成基因pumH、pumJ和pumD基因克隆至大肠杆菌体内，转化得到可产假尿苷的工程菌。还提供了一种产假尿苷的方法，是将该工程菌进行发酵培养得到假尿苷。

Description

一种产假尿苷工程菌及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种产假尿苷工程菌及其应用。
背景技术
1957年，DAV I S等在核糖体RNA(ribosomal RNA，rRNA)和转运RNA(transferRNA，tRNA)中首次发现一种与尿嘧啶核苷天然结构类似的异构体，其核糖不与尿嘧啶(Uridine，U)N1相连，而与C5相连，形成假尿嘧啶核苷(Pseudouridine，PU)。(DAVIS F F，ALLEN F W.Ribonucleic acids from yeast which contain a fifth nucleotide[J].J Biol Chem，1957，227(2)：907-915.)尿苷通过假尿苷合酶从核糖糖碱基裂解并重新连接生成C-C糖苷键，异构化成假尿苷。尿嘧啶核苷与假尿嘧啶核苷化学结构式如式1所示。
Figure PCTCN2021139177-appb-000001
作为核苷尿苷的一种异构体，假尿苷存在于除信使RNA外的许多类RNA中，包括tRNA和rRNA。假尿苷是非编码RNA中含量最 多的修饰核苷，通过稳定RNA结构来增强转移RNA和核糖体RNA的功能。假尿苷是由假尿苷合酶催化得来的，这种酶在转录后将RNA中特定的尿苷异构化，这个过程被称为假尿苷化。假尿苷作为核苷酸代谢物，对其的研究越来越受到学者们的重视。假尿苷可作为一个潜在的生物标志物，用于肾病与肿瘤的诊断、疗效监测，而且对于其他疾病的诊断也有一定的参考意义。研究表明，假尿苷可以减少辐射引起的人类淋巴细胞染色体畸变。近年来，科学家推测假尿苷在RNA稳定性和/或帮助氨基酰基转移酶与tRNAs相互作用方面发挥作用。另外，随着生物技术的飞速发展，利用核酸适配体、RNA干扰等手段发现的基因组药物引起了人们的兴趣，根据其特殊的生理功能，假尿苷被用作医药中间体，假尿苷的磷脒衍生物被用作合成此类低聚物的起始材料之一。
在假尿苷制备方面，现目前，只有传统的化学合成法可催化合成假尿苷，但是，针对假尿苷的化学合成，存在化学合成步骤长、收率低，且用到的试剂易燃易爆，较不安全等一系列的问题。在生物合成方面，尚未发现可专一大量用于假尿苷制备的微生物菌种和技术报道。因此，假尿苷的低成本便捷式获取存在一定的缺陷。
针对现有技术中假尿苷只能通过化学合成法制备，且化学合成过程中存在的合成步骤长、收率低，且用到的试剂易燃易爆，较不安全等一系列的问题，寻求一种通过基因工程构建可发酵产假尿苷的工程菌，并可通过简单的发酵和较低的成本实现假尿苷的生产。
发明内容
为了解决假尿苷现有制备方法不足的问题，本发明提供了一种产假尿苷工程菌及其应用，包括以下步骤：利用基因工程技术在大肠杆菌中表达假尿苷合成相关的基因，得到可产假尿苷的基因工程菌株；
所述假尿苷合成相关基因的蛋白序列为：腺苷酸激酶编码基因pumH蛋白序列、tRNA假尿苷合酶编码基因pumJ蛋白序列和HAD(卤代酸脱卤酶)蛋白家族水解酶编码基因pumD蛋白序列；
为了解决以上技术问题，本发明采用如下技术方案：
本发明的技术方案之一为：一种产假尿苷工程菌，利用基因工程技术在宿主菌中表达假尿苷合成相关的基因，得到可产假尿苷的基因工程菌。
本发明所述的宿主菌包括不限于细菌、藻和真菌，其中，细菌优选自大肠杆菌，进一步，优选为大肠杆菌DH5α。
进一步，作为优选的，所述假尿苷合成相关的基因为：腺苷酸激酶编码基因pumH、tRNA假尿苷合酶编码基因pumJ和HAD(卤代酸脱卤酶)蛋白家族水解酶编码基因pumD。
进一步，作为优选的，所述的编码基因pumH蛋白序列、编码基因pumJ蛋白序列和编码基因pumD蛋白序列均来源于链霉菌Streptomyces sp.ID38640，分别为SEQ ID No.1所示的基因pumH蛋白序列、SEQ ID No.2所示的基因pumJ蛋白序列和SEQ ID No.3所示的基因pumD蛋白序列。
进一步，作为优选的，所述的基因pumH、pumJ和pumD，其DNA序列均来源于链霉菌Streptomyces sp.ID38640，序列分别如SEQ  ID No.4所示的核苷酸序列、SEQ ID No.5所示的核苷酸序列和SEQ ID No.6所示的核苷酸序列。
进一步，作为优选的，所述的基因pumH、pumJ和pumD，其DNA序列按照大肠杆菌密码子偏好性进行优化，优化后的序列分别如SEQ ID No.7所示的核苷酸序列、SEQ ID No.8所示的核苷酸序列和SEQ ID No.9所示的核苷酸序列。
进一步，作为优选的，包括对所述的密码子偏好性优化后的基因pumH、pumJ和pumD进行修饰，具体为pumH密码子优化后的核酸序列在起始密码子前连接启动子Ptrp序列，pumJ和pumD密码子优化后的核酸序列在起始密码子前连接RBS序列aaaggaggatatacat，修饰后序列分别如SEQ ID No.10所示的核苷酸序列、SEQ ID No.11所示的核苷酸序列和SEQ ID No.12所示的核苷酸序列。
本发明所述的产假尿苷工程菌的构建方法具体包括如下步骤：
(1)pumH+Ptrp序列和载体pUC18分别用HindIII+SphI酶切，然后用T4连接酶(Thermfisher)连接，连接产物转化大肠杆菌感受态细胞(优选为DH5α)，得到克隆pZH423；
(2)pumJ+RBS序列和载体pZH423分别用XbaI+SphI酶切，然后用T4连接酶(Thermfisher)连接，连接产物转化大肠杆菌感受态细胞(优选为DH5α)，得到克隆pZH424；
(3)pumD+RBS序列和载体pZH424分别用XbaI+KpnI酶切，然后用T4连接酶(Thermfisher)连接，连接产物转化大肠杆菌感受态细胞(优选为DH5α)，得到克隆pZH425，即产假尿苷工程菌。
本发明所述的产假尿苷工程菌在产假尿苷中的应用。
进一步，作为优选的，所述的产假尿苷的方法，包括如下步骤：将上述所述的基因工程菌进行发酵培养得到假尿苷。
进一步，作为优选的，向所述的发酵培养基中添加肌苷，以溶于氢氧化钠的水溶液的形式加入，所述的发酵培养基中肌苷的初始终浓度为1.0-10g/L。
进一步，作为优选的，所述的发酵过程pH维持在6.5-7.6之间，优选为采用柠檬酸水溶液或乙酸水溶液和氨水进行组合调节。
进一步，作为优选的，所述的柠檬酸或乙酸的浓度为50-100g/L；所述的氨水优选浓度为13mol/L。
进一步，作为具体的，所述发酵培养的培养基配方(g/L)含有如下组分：葡萄糖10-20，KH 2PO 4 2-6，酵母粉4-8，硫酸铵3-10，玉米浆干粉10-20，MgSO 4·7H 2O 2-6；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 100-200，MnSO 4·7H2O 10-30，VB 1 1.3-3.0，VH 0.6-2.0，氯化钴2-5，硫酸锌2-5，氯化钙5-10，壮观霉素50，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。
进一步，作为优选的，发酵过程中，阶段控温，发酵第10-20h，发酵温度保持在28-30℃，随后，发酵温度升高至35-37℃；发酵培养周期为72-96h。
与现有技术相比，本发明提供了一种产假尿苷工程菌及其应用，所述工程菌是将来源于链霉菌Streptomyces sp.ID38640中的假尿苷 合成基因pumH、pumJ和pumD基因克隆至大肠杆菌体内，转化得到可产假尿苷的工程菌。本发明通过对大肠杆菌进行基因改造，实现了假尿苷在大肠杆菌体内的异源表达，进一步通过向培养基中添加肌苷，在发酵过程中采用阶段控温，并使用柠檬酸和氨水对pH进行调控，极大促进了假尿苷的积累，最终基因工程改造的大肠杆菌可产假尿苷达7g/L以上。本发明大肠杆菌基因构建方法简单，发酵过程中不需要添加诱导剂异丙基-β-D-硫代吡喃半乳糖苷即可获得较高产量的假尿苷，应用前景广阔。
附图说明
图1为假尿苷标准品的质谱图；
图2为假尿苷标准品的HPLC检测图谱；
图3为实施例4 pZH425发酵液检测的HPLC图谱。
具体实施方式
以下实施例对本发明作进一步说明，但有必要指出以下实施例只用于对发明内容的描述，并不构成对本发明保护范围的限制，本发明保护范围以权利要求为准。
本发明所用菌种为大肠杆菌DH5α，菌种保藏编号为CCTCC AB 2013329，购自中国典型培养物保藏中心(China Center for Type Culture Collection，简称CCTCC)。
除特别说明外，本发明实施例中所用的各种材料和试剂都是本领域种常用的材料和试剂，均可以通过常规的商业途径获得。
本发明假尿苷效价检测所用的液相检测方法条件如下：
色谱柱：Waters XBridge Amide(250mm×460mm,3.5μm)，流动相A：0.2％(质量体积比，单位为g/L)乙酸铵水溶液，流动相B：乙腈，保留时间：20min，流速：1mL/min，进样量：10μL，检测波长：255nm，柱温：(25±1)℃。
表1流动相A和B的洗脱程序体积比
Min A B
0 100 0
3 100 900
6 98 2
12 60 40
17 100 0
20 100 0
本发明实施例中所用的固体培养基(g/L)为：酵母提取物5，胰蛋白胨10，NaCl 10，壮观霉素0.05，其余为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。
本发明实施例中所用的种子培养基(g/L)为：酵母提取物5，胰蛋白胨10，NaCl 10，葡萄糖20，壮观霉素0.05，其余为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。
本发明实施例中所用发酵培养基配方(g/L)为：葡萄糖10-20，KH 2PO 4 2-6，酵母粉4-8，硫酸铵3-10，玉米浆干粉10-20，MgSO 4·7H 2O 2-6；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O  100-200，MnSO 4·7H2O 10-30，VB 1 1.3-3.0，VH 0.6-2.0，氯化钴2-5，硫酸锌2-5，氯化钙5-10，另外培养基中加入壮观霉素50mg/L，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。
实施例1：pZH423的构建HindIII和SphI双酶切由上海生工合成的SEQ ID No.10DNA片段，得到外源片段1；HindIII和SphI双酶切载体pUC18，得到载体片段1；将外源片段1与载体片段1连接转化大肠杆菌感受态细胞DH5α，得到重组菌落pZH423。
实施例2：pZH424的构建
XbaI和SphI双酶切上海生工合成的SEQ ID No.11DNA片段，得到外源片段2；XbaI和SphI双酶切载体pZH423，得到载体片段2；将外源片段2与载体片段2连接转化大肠杆菌感受态细胞DH5α，得到重组菌落pZH424。
实施例3：pZH425的构建
XbaI和KpnI双酶切上海生工合成的SEQ ID No.12DNA片段，得到外源片段3；XbaI和KpnI双酶切载体pZH424，得到载体片段3；将外源片段3与载体片段3连接转化大肠杆菌感受态细胞DH5α，得到重组菌落pZH425。
实施例4：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃， 250rpm培养15h得到种子液。再按照20％的接种量，吸取4ml种子液至20ml的发酵培养基中，所述发酵培养的培养基配方(g/L)如下：葡萄糖10，KH 2PO 4 2，酵母粉4，硫酸铵3，玉米浆干粉10，MgSO 4·7H 2O 2；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 100，MnSO 4·7H2O 10，VB 1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，发酵温度保持在37℃，250rpm培养30h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
假尿苷标准品的质谱图(图1)，假尿苷标准品的HPLC检测图谱(图2，保留时间15min)。
pZH425发酵液检测的HPLC图谱(图3)，经与标准品对比测算，假尿苷发酵单位约为0.6g/L。
实施例5：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培 养基配方(g/L)如下：葡萄糖15，KH 2PO 4 4，酵母粉6，硫酸铵8，玉米浆干粉15，MgSO 4·7H 2O 4；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 150，MnSO 4·7H2O 20，VB 1 2，VH 1，氯化钴3.5，硫酸锌3.5，氯化钙8，另外培养基中加入壮观霉素50mg/L，另，将10g肌苷溶解于100ml含有氢氧化钠1mol/L的水溶液中，再将含有10g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有50g/L的柠檬酸水溶液和含氨13mol/L的氨水调控在6.5，发酵温度在0-10h保持在28℃，发酵第11h至发酵结束，发酵温度保持在35℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期72h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为5.4g/L。
实施例6：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃， 250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖15，KH 2PO 4 4，酵母粉6，硫酸铵8，玉米浆干粉15，MgSO 4·7H 2O 4；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 200，MnSO 4·7H2O 30，VB 1 3，VH 2，氯化钴5，硫酸锌5，氯化钙10，另外培养基中加入壮观霉素50mg/L，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有60g/L的乙酸水溶液和含氨13mol/L的氨水调控在6.8，发酵温度在0-10h保持在28℃，发酵第11h至发酵结束，发酵温度保持在35℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期72h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为2.9g/L。
实施例7：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃， 250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH 2PO 4 6，酵母粉8，硫酸铵10，玉米浆干粉20，MgSO 4·7H 2O 6；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 100，MnSO 4·7H 2O 10，VB 1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50，另，将60g肌苷溶解于200ml含有氢氧化钠1mol/L的水溶液中，再将含有60g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有80g/L的柠檬酸水溶液和含氨13mol/L的氨水调控在7.0，发酵温度在前15h保持在30℃，发酵第16h至发酵结束，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期84h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为6.1g/L。
实施例8：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH 2PO 4 6，酵母粉8，硫酸铵10，玉米浆干粉20，MgSO 4·7H 2O 6；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 100，MnSO 4·7H2O 10，VB 1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50mg/L，另，将60g肌苷溶解于200ml含有氢氧化钠1mol/L的水溶液中，再将含有60g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有80g/L的乙酸水溶液和含氨13mol/L的氨水调控在7.0，发酵温度在前15h保持在30℃，发酵第16h至发酵结束，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期84h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为3.3g/L。
实施例9：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH 2PO 4 6，酵母粉7，硫酸铵10，玉米浆干粉18，MgSO 4·7H 2O 6；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 200，MnSO 4·7H2O 20，VB 1 2，VH 1，氯化钴4，硫酸锌4，氯化钙8，另外培养基中加入壮观霉素50mg/L，另，将100g肌苷溶解于300ml含有氢氧化钠1mol/L的水溶液中，再将含有100g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有100g/L的柠檬酸水溶液和含氨13mol/L的氨水调控在7.6，发酵温度在前20h保持在30℃，发酵第20h至发酵结束，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期96h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为7.2g/L。
对比实施例1：改造前大肠杆菌发酵产假尿苷检测
(1)改造前大肠杆菌的发酵培养
取改造前大肠杆菌菌落接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取4ml种子液至20ml的发酵培养基中，所述发酵培养的培养基配方(g/L)如下：葡萄糖10，KH 2PO 4 2，酵母粉4，硫酸铵3，玉米浆干粉10，MgSO 4·7H 2O 2；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 100，MnSO 4·7H2O 10，VB 1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50μg/ml，余量为水，pH7.0。发酵过程中，发酵温度保持在37℃，250rpm培养30h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
经过对HPLC检测图谱的分析，改造前大肠杆菌发酵液中未检测有假尿苷，说明改造前，大肠杆菌不产假尿苷。
对比实施例2：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃ 培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH 2PO 4 6，酵母粉8，硫酸铵10，玉米浆干粉20，MgSO 4·7H 2O 6；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 100，MnSO 4·7H2O 10，VB 1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50mg/L，另，将80g肌苷溶解于200ml含有氢氧化钠1mol/L的水溶液中，再将含有80g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有100g/L的乙酸水溶液和含氨13mol/L的氨水调控在8.0，发酵温度在前20h保持在30℃，发酵第20h至发酵结束，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期96h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位基本检测不到。
对比实施例3：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH 2PO 4 6，酵母粉7，硫酸铵10，玉米浆干粉18，MgSO 4·7H 2O 6；培养基中其他物料组分(mg/L)：FeSO 4·7H 2O 100，MnSO 4·7H2O 10，VB 1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50mg/L，另，将50g肌苷溶解于200ml含有氢氧化钠1mol/L的水溶液中，再将含有50g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调控在7.2，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期96h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位基本检测不到。
SEQIDNo.1：
腺苷酸激酶编码基因pumH蛋白序列
Figure PCTCN2021139177-appb-000002
SEQIDNo.2：
tRNA假尿苷合酶编码基因pumJ蛋白序列
Figure PCTCN2021139177-appb-000003
SEQIDNo.3：
HAD(卤代酸脱卤酶)蛋白家族水解酶编码基因pumD蛋白序列
Figure PCTCN2021139177-appb-000004
SEQIDNo.4：
基因pumH核苷酸序列
Figure PCTCN2021139177-appb-000005
SEQIDNo.5：
基因pumJ核苷酸序列
Figure PCTCN2021139177-appb-000006
Figure PCTCN2021139177-appb-000007
SEQIDNo.6：
基因pumD核苷酸序列
Figure PCTCN2021139177-appb-000008
SEQIDNo.7：
pumH密码子优化后的核酸序列
Figure PCTCN2021139177-appb-000009
Figure PCTCN2021139177-appb-000010
SEQIDNo.8：
pumJ密码子优化后的核酸序列
Figure PCTCN2021139177-appb-000011
Figure PCTCN2021139177-appb-000012
SEQIDNo.9：
pumD密码子优化后的核酸序列
Figure PCTCN2021139177-appb-000013
SEQIDNo.10：
人工合成的pumH和启动子Ptrp序列
Figure PCTCN2021139177-appb-000014
Figure PCTCN2021139177-appb-000015
SEQIDNo.11：
人工合成的pumJ和RBS序列
Figure PCTCN2021139177-appb-000016
Figure PCTCN2021139177-appb-000017
SEQIDNo.12：
人工合成的pumD和RBS序列
Figure PCTCN2021139177-appb-000018

Claims (15)

  1. 一种产假尿苷工程菌，其特征在于，利用基因工程技术在宿主菌中表达假尿苷合成相关的基因，得到可产假尿苷的基因工程菌。
  2. 根据权利要求1所述的方法，其特征在于，所述的宿主菌选自细菌、藻和真菌，优选为大肠杆菌，进一步，优选为大肠杆菌DH5α。
  3. 根据权利要求1所述的方法，其特征在于，所述假尿苷合成相关的基因为：腺苷酸激酶编码基因pumH、tRNA假尿苷合酶编码基因pumJ和HAD(卤代酸脱卤酶)蛋白家族水解酶编码基因pumD。
  4. 根据权利要求1所述的方法，其特征在于，所述的编码基因pumH蛋白序列、编码基因pumJ蛋白序列和编码基因pumD蛋白序列均来源于链霉菌Streptomyces sp.ID38640，分别为SEQ ID No.1所示的基因pumH蛋白序列、SEQ ID No.2所示的基因pumJ蛋白序列和SEQ ID No.3所示的基因pumD蛋白序列。
  5. 根据权利要求2所述的方法，其特征在于，所述的基因pumH、pumJ和pumD，其DNA序列均来源于链霉菌Streptomyces sp.ID38640，序列分别如SEQ ID No.4所示的核苷酸序列、SEQ ID No.5所示的核苷酸序列和SEQ ID No.6所示的核苷酸序列。
  6. 根据权利要求1所述的方法，其特征在于，所述的基因pumH、pumJ和pumD，其DNA序列按照大肠杆菌密码子偏好性进行优化，优化后的序列分别如SEQ ID No.7所示的核苷酸序列、SEQ ID No.8所示的核苷酸序列和SEQ ID No.9所示的核苷酸序列。
  7. 根据权利要求6所述的方法，其特征在于，包括对所述的密码子偏好性优化后的基因pumH、pumJ和pumD进行修饰，具体为 pumH密码子优化后的核酸序列在起始密码子前连接启动子Ptrp序列，pumJ和pumD密码子优化后的核酸序列在起始密码子前连接RBS序列aaaggaggatatacat，修饰后序列分别如SEQ ID No.10所示的核苷酸序列、SEQ ID No.11所示的核苷酸序列和SEQ ID No.12所示的核苷酸序列。
  8. 根据权利要求1～7之一所述的产假尿苷工程菌的构建方法，其特征在于，所述方法包括如下步骤：
    (1)pumH+Ptrp序列和载体pUC18分别用HindIII+SphI酶切，然后用T4连接酶(Thermfisher)连接，连接产物转化大肠杆菌感受态细胞，得到重组菌落pZH423；
    (2)pumJ+RBS序列和载体pZH423分别用XbaI+SphI酶切，然后用T4连接酶(Thermfisher)连接，连接产物转化大肠杆菌感受态细胞，得到重组菌落pZH424；
    (3)pumD+RBS序列和载体pZH424分别用XbaI+KpnI酶切，然后用T4连接酶(Thermfisher)连接，连接产物转化大肠杆菌感受态细胞，得到重组菌落pZH425，即产假尿苷工程菌。
  9. 一种如权利要求1所述的产假尿苷工程菌在产假尿苷中的应用。
  10. 一种产假尿苷的方法，其特征在于：将权利要求1～7之一所述的基因工程菌进行发酵培养得到假尿苷。
  11. 根据权利要求10所述的方法，其特征在于：所述的发酵培养基中添加肌苷，进一步，优选的，所述的肌苷以溶于氢氧化钠的水 溶液的形式加入，所述的发酵培养基中肌苷的初始终浓度为1.0-10g/L。
  12. 根据权利要求10所述的方法，其特征在于：所述的发酵过程pH维持在6.5-7.6之间，进一步，优选为采用柠檬酸水溶液或乙酸水溶液与氨水组合进行调节。
  13. 根据权利要求权利要求12所述的方法，其特征在于：所述的柠檬酸或乙酸的浓度为50-100g/L。
  14. 根据权利要求权利要求10所述的方法，其特征在于：所述发酵培养的培养基配方(g/L)含有如下组分：葡萄糖10-20，KH 2PO 42-6，酵母粉4-8，硫酸铵3-10，玉米浆干粉10-20，MgSO 4·7H 2O 2-6；所述发酵培养基中还包括其他物料组分(mg/L)：FeSO 4·7H 2O 100-200，MnSO 4·7H 2O 10-30，VB 1 1.3-3.0，VH 0.6-2.0，氯化钴2-5，硫酸锌2-5，氯化钙5-10，壮观霉素50，余量为水，pH7.0。
  15. 根据权利要求权利要求12所述的方法，其特征在于：发酵过程中，阶段控温，发酵第10-20h，发酵温度保持在28-30℃，随后，发酵温度升高至35-37℃；发酵培养周期为72-96h。
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