WO2022082362A1 - 代谢酪氨酸的非病原性细菌基因表现系统及转化株、其用于制备降低尿毒素的组合物的用途以及利用其代谢酪氨酸的方法 - Google Patents

代谢酪氨酸的非病原性细菌基因表现系统及转化株、其用于制备降低尿毒素的组合物的用途以及利用其代谢酪氨酸的方法 Download PDF

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WO2022082362A1
WO2022082362A1 PCT/CN2020/121895 CN2020121895W WO2022082362A1 WO 2022082362 A1 WO2022082362 A1 WO 2022082362A1 CN 2020121895 W CN2020121895 W CN 2020121895W WO 2022082362 A1 WO2022082362 A1 WO 2022082362A1
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nucleic acid
tyrosine
transformant
metabolizing
acid fragment
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PCT/CN2020/121895
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English (en)
French (fr)
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黄一修
陈振暐
桥本昌征
吴意珣
蓝以峻
宋婷涵
谭富文
曾慧燕
杨峻松
陈彦中
杨謦潞
陈湖庭
林孟谦
徐子桓
邢季语
于心婕
谢洁恩
蔡佳桦
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陈振暐
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to a bacterial gene expression system, in particular to a non-pathogenic bacterial gene expression system that metabolizes tyrosine, a transformant containing the same and its application.
  • Chronic kidney disease is a disease in which kidney function is gradually decreased due to irreversible damage to the kidneys. Patients with diabetes, hypertension and/or gout are at high risk of CKD. With the development of CKD, urinary toxins will gradually accumulate in the blood of patients, thereby interfering with the metabolism and function of systemic cells, and finally causing the failure of multiple organs. Among urinary toxins, the accumulation of p-cresol is not only associated with the worsening of CKD, but also is highly correlated with the concurrent cardiovascular disease in CKD patients. Therefore, in addition to controlling the above-mentioned chronic diseases, the method of slowing down or preventing chronic kidney disease also includes reducing the amount of p-cresol in the body.
  • P-cresol is a product of tyrosine metabolism by intestinal bacteria. In healthy humans, there are few gut bacteria that make p-cresol, and p-cresol can be excreted in the urine. Conversely, CKD alters the physiologic environment, thereby altering the gut microbiome, in which p-cresol production increases if specific species (eg, Clostridium difficile) overgrow. Furthermore, due to impaired kidney function, p-cresol cannot be excreted in the urine, causing p-cresol to accumulate in the blood and attack the kidneys, thereby exacerbating the worsening of CKD.
  • specific species eg, Clostridium difficile
  • Methods to reduce p-cresol include dietary control, hemodialysis and/or urea toxin adsorbent (eg: AST-120), however, tyrosine is widely present in food, so the effect of reducing p-cresol by dietary control is limited.
  • p-cresol has a high affinity for proteins, so it is difficult to remove p-cresol by hemodialysis.
  • urine toxin adsorbents can improve the efficacy of CKD.
  • one aspect of the present invention is to provide a non-pathogenic bacterial gene expression system that metabolizes tyrosine, wherein the above gene expression system comprises a special tyrosine-ammonia lyase (TAL) fragment, and the TAL Operatively linked to specific ribose binding sites.
  • TAL tyrosine-ammonia lyase
  • Another aspect of the present invention is to provide a transformant that metabolizes tyrosine, which comprises a host cell and the above-mentioned non-pathogenic bacterial gene expression system is located in the host cell, so that tyrosine can be metabolized to p-coumaric acid.
  • Another aspect of the present invention is to provide the use of a transformant for preparing a composition for reducing urinary toxins, which is to use the transformant to metabolize tyrosine into p-coumaric acid, thereby replacing tyrosine to form p-methyl The metabolic pathway of phenol, thereby reducing the content of p-cresol.
  • Another aspect of the present invention is to provide a method for metabolizing tyrosine by a transformant using the above-mentioned transformant to metabolize tyrosine to p-coumaric acid.
  • a non-pathogenic bacterial gene expression system that metabolizes tyrosine
  • the bacterial gene expression system can comprise a first nucleic acid fragment and a second nucleic acid fragment, and the first nucleic acid fragment and the second nucleic acid fragment
  • the first nucleic acid fragment may be, for example, located on a first expression vector, and the first nucleic acid fragment operably linked to the second nucleic acid fragment.
  • the above-mentioned first nucleic acid fragment may comprise the sequence shown in Sequence Identification Number (SEQ ID NO.): 1.
  • the above-mentioned second nucleic acid fragment can comprise the sequence shown in SEQ ID NO.:2.
  • the above-mentioned non-pathogenic bacterial gene expression system that metabolizes tyrosine can selectively comprise a third nucleic acid sequence, wherein the above-mentioned third nucleic acid sequence can comprise the sequence shown in SEQ ID NO.: 3 , and the third nucleic acid sequence may, for example, be located on the second expression vector.
  • a transformant that metabolizes tyrosine wherein the transformant can comprise a host cell and a non-pathogenic bacterial gene expression system is located in the host cell.
  • the above-mentioned non-pathogenic bacterial gene expression system comprises a first nucleic acid fragment and a second nucleic acid fragment, the first nucleic acid fragment and the second nucleic acid fragment can be, for example, on a first expression vector, and the first nucleic acid fragment is operably linked to the second nucleic acid fragment .
  • the above-mentioned first nucleic acid fragment may comprise the sequence shown in SEQ ID NO.: 1
  • the above-mentioned second nucleic acid fragment may comprise the sequence shown in SEQ ID NO.: 2.
  • the above-mentioned transformant can selectively comprise a third nucleic acid sequence, wherein the above-mentioned third nucleic acid sequence can comprise the sequence shown in SEQ ID NO.: 3, and the third nucleic acid sequence can be, for example, located in the 2. On the performance carrier.
  • the host cell is Escherichia coli Nissle 1917.
  • the host cell can be, for example, a carbonic anhydrase gene-deficient transformant.
  • a transformant for preparing a composition for reducing urinary toxin wherein the transformant is the above transformant, whereby tyrosine is metabolized to p-coumaric acid.
  • the above-mentioned composition is a pharmaceutical composition or a food composition.
  • a method for metabolizing tyrosine using a transformant comprising culturing the above transformant in an environment of not less than 5% CO 2 .
  • the production of p-cresol can be reduced by metabolizing tyrosine into p-coumaric acid, thereby reducing the content of urea toxin.
  • FIGS. 1A and 1B are schematic diagrams illustrating a first plasmid and a second plasmid according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram illustrating a third plasmid according to an embodiment of the present invention.
  • FIG. 3A and 3B respectively show the results of culture under 0.04% ( FIG. 3A ) and 5% ( FIG. 3B ) CO 2 according to an embodiment of the present invention.
  • FIG. 4 is a bar graph showing the content of p-coumaric acid produced by different transformants per unit bacterial mass according to an embodiment of the present invention.
  • FIG. 5 is a bar graph showing the production of p-coumaric acid per unit bacterial mass under different tyrosine concentrations according to an embodiment of the present invention.
  • the present invention provides a non-pathogenic bacterial gene expression system that metabolizes tyrosine.
  • tyrosine can be converted into p-coumaric acid. Since p-cresol is a metabolite of tyrosine in some intestinal bacteria, expressing the above gene expression system can reduce the metabolism of tyrosine by the above-mentioned intestinal bacteria, thereby reducing the production of p-cresol.
  • non-pathogenic bacterial gene expression system means that the gene expression system is expressed in bacteria, and the above-mentioned bacteria are not pathogenic.
  • the “gene expression system” is all nucleic acid fragments required for the expression of a specific gene, including nucleic acid fragments and regulatory region fragments of a specific gene, wherein the regulatory region fragments are required for the initiation of gene transcription and/or translation. Nucleic acid fragments, such as ribosome-binding site (RBS) and promoter (promoter), etc., and regulatory region fragments are operably linked to nucleic acid fragments of specific genes.
  • RBS ribosome-binding site
  • promoter promoter
  • Said "operably linked” means that after the nucleic acid fragment of a specific gene is linked with the regulatory region fragment, the specific gene can be expressed, in other words, it is linked in a cis (cis) manner, so that the number of spaces between the two operatively linked fragments can be separated.
  • base pairs not necessarily adjacent. Several base pairs can be, for example, a fragment that is not removed during genetic engineering but does not affect gene expression, or a fragment that is deliberately reserved for genetic engineering operations (eg, providing restriction sites for restriction enzymes).
  • the above-mentioned non-pathogenic bacterial gene expression system comprises a first nucleic acid fragment and a second nucleic acid fragment, wherein the first nucleic acid fragment and the second nucleic acid fragment are located on the same expression vector, and the first nucleic acid fragment is operably linked to the first nucleic acid fragment.
  • Two nucleic acid fragments The above-mentioned first nucleic acid fragment comprises the gene sequence of tyrosine-ammonia lyase (TAL), wherein TAL can catalyze the non-oxidative deamination of tyrosine to obtain p-coumaric acid.
  • TAL tyrosine-ammonia lyase
  • the TAL gene fragment can be, for example, the RsTAL gene of Rhodobacters phaeroides, the RcTAL gene of Rhodobacter capsulatus, the BagA gene of Streptomycessp, or the Saccharothrixespanaensis (registered). Institution: Leibniz Institute DSMZ of German Microorganism and Cell Culture Co., Ltd.; accession number: DSM 4429) Sam8 gene, among which the specificity and efficiency of Sam8-encoded TAL are better than other TALs .
  • the gene sequence of the TAL can be, for example, the SeSam8 gene, as shown in SEQ ID NO.: 1. The above SeSam8 gene is modified from the Sam8 gene according to the codon usage bias of Escherichia coli, so compared with the Sam8 gene, the SeSam8 gene is easier to express in Escherichia coli.
  • the second fragment described above contains the sequence of RBS.
  • RBS is the junction site of ribosome and RNA, so the sequence of RBS can affect the translation efficiency of ribosome, thereby affecting the expression of downstream genes. Therefore, the expression of SeSam8 gene can be changed by connecting different RBS.
  • the original RBS (normal RBS, NRBS) of Sam8 (as shown in SEQ ID NO.: 4) is replaced with the RBS sequence shown in SEQ ID NO.: 2 to improve the expression of the SeSam8 gene in the transformant. The amount of expression in , thereby increasing the yield of p-coumaric acid.
  • the above-mentioned second fragment is operably linked to the strong promoter J23100 of E. coli (as shown in SEQ ID NO.: 5), so as to efficiently express the expressive amount of TAL.
  • the above-mentioned non-pathogenic bacterial gene expression system may optionally comprise a third nucleic acid sequence, wherein the third nucleic acid sequence may be located, for example, on another expression vector, and the third nucleic acid sequence may comprise a tyrosine transporter (TyrP) gene , in order to improve the efficiency of tyrosine entry into cells, thereby increasing the intracellular tyrosine content, thereby enhancing the effect of TAL.
  • the TyrP gene is the TyrP gene of Escherichia coli MG1655 strain, as shown in SEQ ID NO.:3.
  • the RBS and promoter of the TyrP gene are not limited, and can be adjusted according to the different host cells or the needs of the expression level.
  • the third nucleic acid sequence may, for example, be operably linked to the original RBS and promoter (ie, the RBS and promoter of the TyrP gene of E. coli MG1655 strain).
  • the third nucleic acid sequence is operably linked to J23100 (as shown in SEQ ID NO.: 5), wherein J23100 is a strong promoter of E. coli, to increase the expression level of the TyrP gene.
  • the above expression vectors can be located on chromosomes or plasmids, so the TAL gene and TyrP gene can be located in different operons of the same chromosome or plasmid, or located on different chromosomes and/or plasmids. In one embodiment, the different TAL genes and TyrP genes are located on different plasmids.
  • the above-mentioned plasmid comprises an origin of replication, a promoter, and an antibiotic resistance gene and its promoter, wherein the antibiotic resistance gene can be selected according to the conditions of screening bacteria.
  • the above-mentioned plasmid is pSB1C3.
  • the antibiotic resistance gene is (chloramphenicol resistance, CmR).
  • the above-mentioned non-pathogenic bacterial gene expression system can be expressed by a transformant, wherein the transformant comprises a host cell and the above-mentioned non-pathogenic bacterial gene expression system, and the non-pathogenic bacterial gene expression system is located in the host cell.
  • Transformants can be obtained by genetically engineering host cells.
  • the transformant is obtained by transforming a non-pathogenic bacterial gene expression system into a host cell.
  • the means of transformation may be, for example, electroporation.
  • the host cell is Echerichia coli.
  • the host cell is Escherichia coli Nissle 1917, which is harmless to human body and easy to carry out genetic engineering, and is an ideal host cell for expressing the above gene expression system.
  • the above transformants can selectively introduce specific gene defects as a kill switch, so that the transformants are only active in a specific environment (for example, an environment with not less than 5% CO2 ) (for example: metabolism and growth), but inactive outside a specific environment (eg, an environment with less than 5% CO 2 ), thereby ensuring that the transformant is expressed in the target location (eg, the large intestine).
  • a specific environment for example, an environment with not less than 5% CO2
  • metabolism and growth for example: metabolism and growth
  • a specific environment eg, an environment with less than 5% CO 2
  • the above-mentioned specific gene can be a gene that metabolizes CO2 -related enzymes [such as carbonic anhydrase (hereinafter referred to as can) gene], and the above-mentioned "defect" can be, for example, a partial or complete deletion of a specific gene [such as: knockout (knock) -out) or mutation].
  • the host cell of the transformed strain is a can gene-deficient strain, so the transformed strain cannot synthesize a sufficient amount of bicarbonate ions to maintain its physiological function in an environment with a low CO concentration, wherein the sequence of the can gene is as follows. shown in SEQ ID NO.:6.
  • the means of improving biosafety is not limited to the above-mentioned kill switch, and can be other methods to limit the expression of the above-mentioned gene expression system transformation strains to the large intestine, for example: by changing the control group or using small molecular RNA to make it.
  • Other survival genes eg: cell wall synthesis-related genes
  • the above transformants can form another tyrosine metabolic pathway by expressing TAL and/or TyrP to reduce tyrosine content, thereby reducing the production of p-cresol with tyrosine as a precursor, thereby reducing urinary toxins. accumulation, so the above transformants can be used to prepare compositions for reducing urinary toxins.
  • the above-mentioned composition is a pharmaceutical composition or a food composition.
  • FIG. 1A and FIG. 1B are schematic diagrams illustrating the first plasmid 10 and the second plasmid 20 according to an embodiment of the present invention, wherein the arrow 111 indicates the direction of gene transcription.
  • plasmid fragment 190 represents a fragment of pSB1C3 plasmid DNA, wherein plasmid fragment 190 contains chloramphenicol resistance gene (chloramphenicol resistance, CmR) 191 and its promoter 193, and the origin of replication pMB1 fragment 192 .
  • chloramphenicol resistance gene chloramphenicol resistance, CmR
  • the first cloned fragment 110 of the first plasmid 10 comprises gene fragments 140, 200 and 100 (corresponding to SEQ ID NOs.: 5, 2 and 1) arranged in sequence. Between the gene segments 140 and 200, a first linked DNA segment having a sequence as described in SEQ ID NO:7 is also included. Between the gene fragments 200 and 100, a second linked DNA fragment having a sequence as described in SEQ ID NO:8 is also included.
  • the second cloned fragment 120 of the second plasmid 20 comprises gene fragments 140, 210 and 100 (corresponding to SEQ ID NOs.: 5, 4 and 1) arranged in sequence. Between the gene segments 140 and 210, a first linked DNA segment having a sequence as described in SEQ ID NO:7 is also included.
  • FIG. 2 is a schematic diagram illustrating a third plasmid 30 according to an embodiment of the present invention.
  • the third plasmid 30 comprises a third cloned fragment 130 and a plasmid fragment 190, wherein the third cloned fragment 130 comprises gene fragments 140, 220 and 300 (corresponding to SEQ ID NOs.: 4, 9 and 3) in sequence Arrange in order.
  • the lambda red recombineering experiment was carried out on Escherichia coli Nissle 1917 to replace the can gene with the first substitution fragment or the second substitution fragment respectively, thereby establishing the first can gene with a kill switch (kill switch)
  • the knockout bacteria and the second can gene knockout bacteria wherein the first substitution fragment comprises the CmR fragment of the pKD3 plasmid sandwiched between two flippase recognition target (FRT) sites, and the first substitution fragment comprises the FRT site point.
  • the sequences of the CmR fragment and the FRT site are easily obtained by those with common knowledge in the field of the present invention, and will not be repeated here.
  • Lambda red gene recombination method is referred to Doublet's team published an article in the Journal of Microbiological Methods in 2008, which is hereby incorporated by reference.
  • the kill switch is that the organism survives in a specific environment (in this embodiment, high concentration of carbon dioxide), but loses its activity outside the specific environment.
  • whether the above-mentioned kill switch functions is evaluated by observing the growth status of the first can gene knockout bacteria and the second can gene knockout bacteria under different CO 2 concentrations.
  • Escherichia coli Nissle 1917 strain without can gene deletion
  • pKD3 plasmid expression vector of CmR gene
  • the first can gene knockout bacteria and the second can gene knockout bacteria were smeared in Luria broth (LB) medium, and after culturing overnight at 37°C with 0.04% or 5% CO2 (overnight, about 12 hours to 18 hours, the incubation time in this interval will not affect subsequent evaluations), observe the colony growth
  • LB Luria broth
  • CO2 overnight, about 12 hours to 18 hours, the incubation time in this interval will not affect subsequent evaluations
  • FIG. 3A and 3B respectively show the results of culturing under 0.04% ( FIG. 3A ) and 5% ( FIG. 3B ) CO 2 according to an embodiment of the present invention, wherein blocks 310 , 320 , 330 and 340 respectively represent plasmids containing pKD3 Escherichia coli Nissle 1917, or Escherichia coli Nissle 1917 without pKD3 plasmid, the first can gene knockout bacteria and the second can gene knockout bacteria.
  • the first can gene knockout bacteria and the second can gene knockout bacteria can only grow in an environment of not less than 5% CO 2 , but cannot grow in an environment of 0.04% CO 2 , indicating that the first can
  • the kill switch of the knockout strain and the second can knockout strain has the expected effect (making the strain unable to grow in low CO 2 ), and the above-mentioned lambda red gene recombination method can indeed make the can gene lose its normal function.
  • E. coli Nissle 1917 containing pKD3 plasmid can grow in either 5% or 0.04% CO 2 environment, indicating that the CmR gene does not affect the carbon dioxide requirement of E. coli Nissle 1917.
  • Example 3 Assess the ability of different transformants to convert tyrosine into p-coumaric acid
  • the first plasmid, the second plasmid and the third plasmid were transformed into Escherichia coli Nissle 1917 by electroporation to obtain the transformants of Example 1, Example 2, Comparative Example 1 and Comparative Example 2, wherein The transformant of Example 1 contained the first plasmid, the transformant of Example 2 contained the first plasmid and the third plasmid, the transformant of Comparative Example 1 contained the second plasmid, and the transformant of Comparative Example 2 contained the second plasmid and the third plasmid.
  • the above electroporation is well known to those with common knowledge in the field to which the present invention pertains, and will not be repeated here.
  • the above transformants were cultured anaerobically with 6 mL of LB broth overnight at 37°C to obtain a first culture.
  • 60 ⁇ L of the first culture was added to 6 mL of an LB medium having a tyrosine concentration of 2 mM, and anaerobic culture was performed at 37° C. to obtain a second culture.
  • OD600 optical density at 600 nm
  • PD2 solution lysis buffer
  • PD3 solution neutralization buffer
  • a lysate was obtained by treating 250 ⁇ L of the second culture with 43.5 ⁇ L and 12.5 ⁇ L of glacial acetic acid.
  • the separation treatment was performed by mixing the lysate with 50 ⁇ L of n-octanol and centrifuging to obtain a supernatant containing p-coumaric acid.
  • the measurement step is to measure the absorbance at 310 nm of n-octanol solutions of p-coumaric acid with different concentrations to draw a standard curve, and then measure the absorbance at 310 nm of the above-mentioned supernatant to calculate the content of p-coumaric acid from the standard curve. The results are recorded in FIG. 4 .
  • FIG. 4 is a bar graph showing the content of p-coumaric acid per unit of bacteria produced by different transformants according to an embodiment of the present invention, wherein the horizontal axis represents groups, and from left to right are Comparative Example 1 and Comparative Example 2, respectively , Example 1 and Example 1, the vertical axis represents the yield of p-coumaric acid per unit amount of bacteria, and "**", "***” and "****” represent the single factor analysis of variance [ After one-way analysis of variance (ANOVA)] statistical analysis, there were statistically significant differences (p ⁇ 0.01, p ⁇ 0.001 and p ⁇ 0.0001).
  • ANOVA analysis of variance
  • Example 1 As shown in FIG. 4 , compared with Comparative Example 1, the content of p-coumaric acid per unit bacterial count of Example 1 was significantly higher (1.73 times). Since the RBS of Example 1 is B0034 and the RBS of Comparative Example 1 is the original RBS of Sam8 (NRBS), the results show that the type of RBS can indeed effectively affect the expression of TAL, thereby affecting the metabolism of tyrosine to p-coumaric acid s efficiency.
  • Example 1 Comparative Example 1 and Comparative Example 2
  • Example 2 Comparative Example 1 and Comparative Example 2
  • the transformant of Example 2 had a significantly higher yield of p-coumaric acid per unit bacterial load (Example 2 was 1.31 times that of Example 1, and Comparative Example 2 was 1.44 times that of Comparative Example 1). From the above results, it can be seen that the expression of TyrP can increase the tyrosine content into the transformant, thereby improving the metabolic efficiency of the transformant to tyrosine.
  • Example 2 The transformants of the above Example 2 were cultured at 37°C with LB medium containing 0.5 mM, 1 mM and 2 mM tyrosine for 48 hours to obtain a third culture, and then the method of Example 3 was used to measure the third culture. The bacterial content and p-coumaric acid content in the samples. The results are shown in FIG. 5 .
  • FIG. 5 is a bar graph showing the production of p-coumaric acid per unit bacterial mass under different tyrosine concentrations according to an embodiment of the present invention, wherein the horizontal axis represents the tyrosine concentration, and the vertical axis represents the per unit bacterial mass
  • "ns" and "*” respectively indicate no or statistically significant differences (p ⁇ 0.05) after one-way ANOVA.
  • Figure 5 when the concentration of tyrosine in the LB medium was higher, the yield of p-coumaric acid per unit amount of bacteria was greater, indicating that TAL had a dose-dependent relationship with tyrosine.
  • the application of the non-pathogenic bacterial gene expression system and transformant for metabolizing tyrosine of the present invention, the use thereof for preparing the composition for reducing urinary toxin, and the method for metabolizing tyrosine using the same The advantage is that it has a TAL encoding gene, and the TAL encoding gene uses B0034 as RBS, so it can effectively convert tyrosine into p-coumaric acid to reduce the content of tyrosine, thereby reducing the use of tyrosine as a precursor. Therefore, the present invention has the potential to delay the deterioration of CKD by reducing the production of p-cresol of the compound, thereby reducing the content of urea toxin.
  • the present invention uses a specific preparation method, a specific evaluation method and/or a specific dose to illustrate that the non-pathogenic bacterial gene expression system and transformant for metabolizing tyrosine of the present invention are used for the preparation of reduced urinary toxins.
  • the use of the composition and the method for utilizing the composition of the present invention to metabolize tyrosine are only known to anyone with common knowledge in the technical field to which the present invention belongs, the present invention is not limited to this, and within the spirit and scope of the present invention, the Use of a non-pathogenic bacterial gene expression system and transformants that metabolize tyrosine for the preparation of a composition for reducing urinary toxins and a method for using the same to metabolize tyrosine.
  • Other preparation methods, other evaluation methods, or other dosages may also be used. .

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Abstract

一种代谢酪氨酸的非病原性细菌基因表现系统及转化株、其用于制备降低尿毒素的组合物的用途以及利用其代谢酪氨酸的方法，其中上述基因表现系统包含第一核酸片段及第二核酸片段，且第二核酸片段操作地连接第一核酸片段。上述第一核酸片段及第二核酸片段分别包含如序列辨识编号(SEQ ID NO.):1及2所示的序列。含有上述基因表现系统的转化株可通过将酪氨酸转变成对香豆酸来降低酪氨酸含量，从而减少对甲酚的生成，进而减少尿毒素的含量。

Description

代谢酪氨酸的非病原性细菌基因表现系统及转化株、其用于制备降低尿毒素的组合物的用途以及利用其代谢酪氨酸的方法 技术领域
本发明是有关于一种细菌基因表现系统，特别是关于一种代谢酪氨酸的非病原性细菌基因表现系统、含其的转化株及其应用。
背景技术
慢性肾脏病(chronic kidney disease，CKD)是一种肾脏受到不可逆的伤害而导致肾功能逐渐下降的疾病，其中糖尿病、高血压及/或痛风的患者为CKD的高危险群。随着CKD病程的发展，尿毒素会逐渐累积在患者血液中，从而干扰全身性细胞的代谢及功能，最后造成多重器官的衰竭。尿毒素中，对甲酚(p-cresol)的累积不仅与CKD的恶化有关，还与CKD患者并发的心血管疾病高度相关。因此，减缓或预防慢性肾脏病的方法除了控制上述慢性疾病外，还包含降低体内对甲酚含量。
对甲酚是肠道菌代谢酪氨酸的产物。在健康的人体中，制造对甲酚的肠道菌菌种不多，且对甲酚可通过尿液排除。相反地，CKD会改变生理环境，从而改变肠道菌相，其中如果特殊菌种(如：艰难梭状芽孢杆菌，Clostridium difficile)菌种过度生长，对甲酚的产量就会上升。再者，由于肾功能受损，对甲酚也无法通过尿液排除，导致对甲酚在血液中累积并攻击肾脏，从而加剧CKD的恶化。减少对甲酚的方法包含饮食控制、血液透析及/或尿毒素吸附剂(如：AST-120)，然而酪氨酸广泛存在于食物，因此通过饮食控制来降低对甲酚的效果有限。其次，对甲酚与蛋白质的亲和力高，故血液透析难移除对甲酚。再者，目前尚无有力证据证实尿毒素吸附剂具有改善CKD的功效。
有鉴于此，亟需一种有效减少对甲酚的方法，以解决对甲酚造成CKD恶化的问题。
发明内容
因此，本发明的一样态是提供一种代谢酪氨酸的非病原性细菌基因表现系统，其中上述基因表现系统包含特殊的酪氨酸解氨酶(tyrosine-ammonia lyase， TAL)片段，且TAL操作性地连接特殊的核糖结合位点。表现上述细菌基因表现系统，可将酪氨酸代谢成对香豆酸(p-coumaric acid)。
本发明的另一样态是提供一种代谢酪氨酸的转化株，其包含宿主细胞及上述非病原性细菌基因表现系统位于宿主细胞内，故可将酪氨酸代谢成对香豆酸。
本发明的再一样态是提供一种转化株用于制备降低尿毒素的组合物的用途，其是利用上述转化株来将酪氨酸代谢为对香豆酸，借以取代酪氨酸形成对甲酚的代谢路径，从而降低对甲酚含量。
本发明的又一样态是提供转化株代谢酪氨酸的方法，其是利用上述转化株来将酪氨酸代谢成对香豆酸。
根据本发明的上述态样，提供一种代谢酪氨酸的非病原性细菌基因表现系统，其中细菌基因表现系统可包含第一核酸片段及第二核酸片段，第一核酸片段与第二核酸片段可例如位于第一表现载体上，且第一核酸片段操作地连接第二核酸片段。上述第一核酸片段可包含如序列辨识编号(SEQ ID NO.):1所示的序列。上述第二核酸片段，可包含如SEQ ID NO.:2所示的序列。
在本发明的一实施例中，上述代谢酪氨酸的非病原性细菌基因表现系统可选择性包含第三核酸序列，其中上述第三核酸序列可包含如SEQ ID NO.:3所示的序列，且第三核酸序列可例如位于第二表现载体上。
根据本发明的上述态样，提供一种代谢酪氨酸的转化株，其中上述转化株可包含宿主细胞及非病原性细菌基因表现系统位于宿主细胞内。上述非病原性细菌基因表现系统包含第一核酸片段与第二核酸片段，第一核酸片段与第二核酸片段可例如位于第一表现载体上，且第一核酸片段是操作地连接第二核酸片段。上述第一核酸片段可包含如SEQ ID NO.:1所示的序列，且上述第二核酸片段可包含如SEQ ID NO.:2所示的序列。
在本发明的一实施例中，上述转化株可选择性包含第三核酸序列，其中上述第三核酸序列可包含如SEQ ID NO.:3所示的序列，且第三核酸序列可例如位于第二表现载体上。
在本发明的一实施例中，宿主细胞是大肠杆菌(Escherichia coli)Nissle1917。
在本发明的一实施例中，宿主细胞可例如为碳酸酐酶(carbonic anhydrase) 基因缺陷株转化株。
根据本发明的上述态样，提供一种转化株用于制备降低尿毒素的组合物的用途，其中转化株是上述转化株，借以将酪氨酸代谢为对香豆酸。
在本发明的一实施例中，上述组合物是医药组合物或食品组合物。
根据本发明的上述态样，提供一种利用转化株代谢酪氨酸的方法，包含将上述转化株培养于不小于5％CO 2的环境中。
应用本发明的代谢酪氨酸的非病原性细菌基因表现系统及转化株，可通过将酪氨酸代谢成对香豆酸来降低对甲酚的生成，从而降低尿毒素含量。
附图说明
为让本发明的上述和其他目的、特征、优点与实施例能更明显易懂，所附图式的详细说明如下：
图1A及图1B为绘示根据本发明一实施例的第一质粒及第二质粒的示意图。
图2为绘示根据本发明一实施例的第三质粒的示意图。
图3A及图3B分别为显示根据本发明一实施例的0.04％(图3A)及5％(图3B)CO 2下的培养结果。
图4为绘示根据本发明一实施例的不同转化株每单位菌量生产对香豆酸含量的柱形图。
图5为绘示根据本发明一实施例的在不同酪氨酸浓度下每单位菌量产生对香豆酸的柱形图。
其中，附图标记：
10：第一质粒
20：第二质粒
30：第三质粒
100，140，200，210，220，300：基因片段
110：第一克隆片段
111：方向
120：第二克隆片段
130：第三克隆片段
190：质粒片段
191：氯霉素抗性基因
192：复制起始点pMB1片段
193：启动子
310，320，330，340：区块
具体实施方式
本发明所提到的单数形式“一”、“一个”和“所述”包括复数引用，除非上下文另有明确规定。数值范围(如10％～11％的A)若无特定说明皆包含上、下限值(即10％≤A≤11％)；数值范围若未界定下限值(如低于0.2％的B，或0.2％以下的B)，则皆指其下限值可能为0(即0％≤B≤0.2％)。上述用语是用以说明及理解本发明，而非用以限制本发明。
承上所述，本发明提供一种代谢酪氨酸的非病原性细菌基因表现系统。通过表现上述基因表现系统，可将酪氨酸(tyrosine)转变为对香豆酸(p-coumaric acid)。由于对甲酚是部分肠道菌的酪氨酸的代谢产物，因此表达上述基因表现系统可降低上述肠道菌对酪氨酸的代谢，从而降低对甲酚的生成。
所述“非病原性细菌基因表现系统”表示基因表现系统是表现于细菌中，且上述细菌不致病。所述“基因表现系统”是特定基因进行表达所需的所有核酸片段，包含特定基因的核酸片段及调节区(regulatory region)片段，其中调节区片段是基因转录及/或翻译开始进行所需的核酸片段，可例如核糖结合位点(ribosome-binding site，RBS)及启动子(promoter)等，且调节区片段操作性连接特定基因的核酸片段。
所述“操作地连接”表示特定基因的核酸片段与调节区片段连接后，可表达特定基因，换言之，以顺向(cis)的方式连接，因此，操作地连接的两个片段间可间隔数个碱基对，而不一定要相邻连接。数个碱基对可例如进行基因工程时未去除但不影响基因表现的片段，或是为了基因工程的操作(例如：提供限制酶的限制位点)而刻意保留的片段。
详细而言，上述非病原性细菌基因表现系统包含第一核酸片段及第二核酸片段，其中第一核酸片段及第二核酸片段是位于同一表现载体上，且第一核酸片段是操作地连接第二核酸片段。上述第一核酸片段包含酪氨酸解氨酶 (tyrosine-ammonia lyase，TAL)的基因序列，其中TAL可催化酪氨酸的非氧化型脱氨反应，从而获得对香豆酸。在一实施例中，TAL基因片段可例如为球形红杆菌(Rhodobactersphaeroides)的RsTAL基因、荚膜红杆菌(Rhodobactercapsulatus)的RcTAL基因、链霉菌(Streptomycessp)的BagA基因，或西班牙糖丝菌Saccharothrixespanaensis(寄存机构：德国微生物和细胞培养有限公司的莱布尼茨研究所DSMZ；寄存编号：DSM 4429)的Sam8基因，其中相较于其他TAL，Sam8编码(encoded)的TAL的专一性及效率较佳。在一实施例中，TAL的基因序列可例如SeSam8基因，如序列辨识编号(SEQ ID NO.):1所示。上述SeSam8基因是由Sam8基因依据大肠杆菌的密码子偏好性(codon usage bias)改良而成，故相较于Sam8基因，SeSam8基因较容易在大肠杆菌中表现。
上述第二片段包含RBS的序列。在翻译时，RBS是核糖体与RNA的连接位点，因此RBS的序列可影响核糖体的翻译效率，从而影响下游基因的表现量，故可通过连接不同的RBS来改变SeSam8基因的表现量。在一实施例中，将Sam8的原始RBS(normal RBS，NRBS)(如SEQ ID NO.:4所示)取代成如SEQ ID NO.:2所示的RBS序列，以提高SeSam8基因在转化株中的表现量，从而提高对香豆酸的产率。
在一实施例中，上述第二片段操作性地连接大肠杆菌的强启动子J23100(如SEQ ID NO.:5所示)，以有效表达TAL的表现量。
上述非病原性细菌基因表现系统可选择性包含第三核酸序列，其中第三核酸序列可例如位于另一表现载体上，且第三核酸序列可包含酪氨酸运输蛋白(tyrosine transporter，TyrP)基因的序列，以提升酪氨酸进入细胞的效率，从而增加胞内酪氨酸含量，进而提高TAL的作用。在一实施例中，TyrP基因是大肠杆菌MG1655菌株的TyrP基因，如SEQ ID NO.:3所示。
TyrP基因的RBS及启动子不限，可视宿主细胞的不同或是表现量的需求进行调整。在一实施例中，第三核酸序列可例如操作性地连接原本的RBS及启动子(即大肠杆菌MG1655菌株的TyrP基因的RBS及启动子)。在一实施例中，第三核酸序列操作性地连接J23100(如SEQ ID NO.:5所示)，其中J23100是大肠杆菌的强启动子，以提高TyrP基因的表现量。
上述表现载体可位于染色体或质粒，故TAL基因及TyrP基因可位于相同 染色体或质粒的不同操作子(operon)中，或是位于不同的染色体及/或质粒上。在一实施例中，不同TAL基因及TyrP基因是位于不同质粒上。在一实施例中，上述质粒包含复制起始点、启动子，以及抗生素抗性基因及其启动子，其中抗生素抗性基因可依据筛菌的条件进行选择。在一实施例中，上述质粒为pSB1C3。在一实施例中，抗生素抗性基因为(chloramphenicol resistance，CmR)。
上述非病原性细菌基因表现系统可通过转化株表达，其中转化株包含宿主细胞及上述非病原性细菌基因表现系统，且非病原性细菌基因表现系统位于该宿主细胞内。转化株可通过对宿主细胞进行基因工程获得。在一实施例中，转化株是通过将非病原性细菌基因表现系统转化至宿主细胞中获得。在一实施例中，转化的手段可例如电穿孔。在一实施例中，宿主细胞是大肠杆菌(Echerichia coli)。在一实施例中，宿主细胞是大肠杆菌Nissle 1917，其对人体无害，且容易进行基因工程，是表现上述基因表现系统的理想宿主细胞。
为了提升生物安全性，上述转化株可选择性导入特定基因的缺陷作为杀伤开关(kill switch)，以使转化株只在特定环境(例如：CO 2不小于5％的环境)具有活性(例如：代谢及生长)，但在特定环境外(例如：CO 2小于5％的环境)失去活性，从而确保转化株表现于目标位置(如：大肠)。
上述特定基因可以是代谢CO 2相关酵素的基因[例如碳酸酐酶(carbonic anhydrase，以下简称为can)基因]，且上述“缺陷”可例如特定基因的部分或全部缺失[例如：基因剔除(knock-out)或突变]。在一实施例中，转型株的宿主细胞是can基因缺陷株，故转型株无法在低CO 2浓度的环境中合成足量的碳酸氢根离子来维持其生理机能，其中can基因的序列是如SEQ ID NO.:6所示。
补充说明的是，提升生物安全性的手段不限于上述杀伤开关，可为其他限制上述基因表现系统转型株只表现于大肠中的方法，例如：通过改变操控组或利用小分子核糖核酸的方式使其他生存基因(如：细胞壁合成相关基因)只在CO 2浓度高的环境下表现。
上述转化株可通过表现TAL及/或TyrP来形成另一条酪氨酸的代谢路径，以减少酪氨酸含量，从而减少以酪氨酸为前驱物的对甲酚的产生，进而减少尿毒素的累积，故上述转化株可用来制备降低尿毒素的组合物。在一实施例中，上述组合物是医药组合物或食品组合物。
以下利用数个实施例以说明本发明的应用，然其并非用以限定本发明，本 发明技术领域中具有公知常识者，在不脱离本发明的精神和范围内，当可作各种的更动与润饰。
实施例一、制备非病原性细菌基因表现系统
首先，利用分子生物技术将第一质粒、第二质粒及第三质粒的不同组合分别克隆(clone)到pSB1C3质粒DNA中，以形成如图1A、图1B及图2所述的第一质粒10、第二质粒20及第三质粒30。分子生物技术为本发明所属领域具有公知常识者所熟知，不在此赘述。
图1A及图1B为绘示根据本发明一实施例的第一质粒10及第二质粒20的示意图，其中箭头111表示基因转录的方向。如图1A及图1B所示，质粒片段190表示pSB1C3质粒DNA的片段，其中质粒片段190包含氯霉素抗性基因(chloramphenicol resistance，CmR)191及其启动子193，以及复制起始点pMB1片段192。本发明所属领域具有公知常识者易于获得pSB1C3质粒及其序列，不在此赘述。
如图1A所示，第一质粒10的第一克隆片段110包含基因片段140、200及100(对应SEQ ID NOs.:5、2及1)依序顺向排列。在基因片段140及200之间，还包含序列如SEQ ID NO:7所述的第一连结DNA片段。在基因片段200及100之间，还包含序列如SEQ ID NO:8所述的第二连结DNA片段。
如图1B所示，第二质粒20的第二克隆片段120包含基因片段140、210及100(对应SEQ ID NOs.:5、4及1)依序顺向排列。在基因片段140及210之间，还包含序列如SEQ ID NO:7所述的第一连结DNA片段。
图2为绘示根据本发明一实施例的第三质粒30的示意图。如图2所示，第三质粒30包含第三克隆片段130及质粒片段190，其中第三克隆片段130包含基因片段140、220及300(对应SEQ ID NOs.:4、9及3)依序顺向排列。
实施例二、建立具有杀伤开关的转化株
对大肠杆菌Nissle 1917进行lambda红基因重组法(lambda red recombineering experiment)，以分别利用第一取代片段或是第二取代片段来置换can基因，从而建立具有杀伤开关(kill switch)的第一can基因剔除菌及第二can基因剔除菌，其中第一取代片段包含pKD3质粒的CmR片段夹于两个翻转酶识别标靶(flippase recognition target，FRT)位点之间，且第一取代片段包含FRT位点。CmR片段及FRT位点的序列容易为本发明所属领域具有公知常识 者获得，不在此赘述。Lambda红基因重组法是参阅
Figure PCTCN2020121895-appb-000001
Doublet的团队在2008年发表于《微生物学方法期刊》(Journal of Microbiological Methods)的文章，此处一并列为参考文献。
接着，评估第一can基因剔除菌及第二can基因剔除菌是否具有杀伤开关。所述杀伤开关是生物在特定环境(在本实施例中，是指高浓度二氧化碳)下存活，但在特定环境外会失去活性。在本实施例中，通过观察在不同CO 2浓度的环境下，第一can基因剔除菌及第二can基因剔除菌的生长状况来评估上述杀伤开关是否作用。详细而言，将含或不含pKD3质粒(CmR基因的表现载体)的大肠杆菌Nissle 1917(can基因没有被剔除的菌株)、第一can基因剔除菌及第二can基因剔除菌涂抹在Luria broth(LB)培养基上，并于37℃下以0.04％或5％的CO 2培养过夜(overnight，约12小时至18小时，培养时间在此区间中不会影响后续评估)后，观察菌落生长状况，再将结果显示于图3A及图3B。
图3A及图3B分别为显示根据本发明一实施例的0.04％(图3A)及5％(图3B)CO 2下的培养结果，其中区块310、320、330及340分别表示含pKD3质粒的大肠杆菌Nissle 1917、或不含pKD3质粒的大肠杆菌Nissle 1917、第一can基因剔除菌及第二can基因剔除菌。如图3A及图3B所示，第一can基因剔除菌及第二can基因剔除菌只能在不小于5％CO 2环境下生长，而无法在0.04％CO 2环境下生长，显示第一can基因剔除菌及第二can基因剔除菌的杀伤开关具有预期效果(使菌株在低CO 2处无法生长)，且上述lambda红基因重组法可使can基因确实失去正常功能。补充说明的是，含pKD3质粒的大肠杆菌Nissle 1917不论在5％或0.04％CO 2环境中皆可生长，表示CmR基因不影响大肠杆菌Nissle 1917对二氧化碳的需求量。
实施例三、评估不同转化株将酪氨酸转变成对香豆酸的能力
将第一质粒、第二质粒、第三质粒以电穿孔(electroporation)的方式转化到大肠杆菌Nissle 1917中，以获得实施例1、实施例2、比较例1及比较例2的转化株，其中实施例1的转化株包含第一质粒，实施例2的转化株包含第一质粒及第三质粒，比较例1的转化株包含第二质粒，且比较例2包含第二质粒及第三质粒。上述电穿孔为本发明所属领域具有公知常识者所熟知，在此不再赘述。
于37℃下，以6mL的LB培养液对上述转化株进行无氧培养隔夜，以获 得第一培养物。接着，将60μL第一培养物加入酪氨酸浓度为2mM的LB培养液6mL，并于37℃下进行无氧培养，从而获得第二培养物。
接续地，在48小时取样，以测量第二培养物中的含菌量及对香豆酸含量。首先，测量600nm下的光密度(optical density，OD)(表示为OD600)，其中OD600可代表菌量。接着，对第二培养物进行裂解处理、分离处理及测量步骤，以推算对香豆酸含量。详细而言，裂解处理是依序利用美国赛默飞世尔科技股份有限公司生产的小量制备套组(MiniPrep kit)中的PD2溶液(裂解缓冲液)25μL、PD3溶液(中和缓冲液)43.5μL及冰醋酸12.5μL处理250μL的第二培养物，借以获得裂解物(lysate)。分离处理是利用50μL的正辛醇混合裂解物并进行离心，以获得上清液，且上清液中包含对香豆酸。测量步骤是先测量不同浓度的对香豆酸的正辛醇溶液的310nm吸光值，以绘制标准曲线，再测量上述上清液的310nm吸光值，以由标准曲线推算对香豆酸含量。将结果记录于图4中。
图4为绘示根据本发明一实施例的不同转化株每单位菌量生产对香豆酸含量的柱形图，其中横轴表示组别，由左至右分别为比较例1、比较例2、实施例1及实施例1，纵轴表示每单位菌量的对香豆酸产量，且“**”、“***”及“****”分别表示经单因子变异数分析[one-way analysis of variance(ANOVA)]统计分析后，具有统计上显著差异性(p<0.01、p<0.001及p<0.0001)。
如图4所示，相较于比较例1，实施例1的每单位菌量的对香豆酸含量显著较高(1.73倍)。由于实施例1的RBS为B0034，而比较例1的RBS为Sam8的原始RBS(NRBS)，此结果显示RBS的种类确实可有效影响TAL的表现量，从而影响酪氨酸代谢为对香豆酸的效率。
此外，实施例1及实施例2(比较例1及比较例2)的差异在于是否表现TyrP，而如图4的结果显示，相较于实施例1(比较例1)，实施例2(比较例2)的转化株的每单位菌量的对香豆酸产量显著地较高(实施例2为实施例1的1.31倍，且比较例2为比较例1的1.44倍)。由上述结果可知，TyrP的表现可增加进入转化株的酪氨酸含量，从而提高转化株对酪氨酸的代谢效率。
实施例四、评估TAL对酪氨酸的专一性
将上述实施例2的转化株于37℃下分别利用含有0.5mM、1mM及2mM酪氨酸的LB培养液培养48小时，以获得第三培养物，再利用实施例三的方法测量第三培养物中的含菌量及对香豆酸含量。将结果显示于图5。
图5为绘示根据本发明一实施例的在不同酪氨酸浓度下每单位菌量产生对香豆酸的柱形图，其中横轴表示酪氨酸浓度，纵轴表示每单位菌量的对香豆酸产量，且“ns”及“*”分别表示经单因子变异数分析后，没有或具有统计上显著差异性(p<0.05)。如图5所示，当LB培养液的酪氨酸浓度越高，每单位菌量的对香豆酸产量越大，显示TAL对酪氨酸具有剂量依存关系。
由上述实施例可知，应用本发明的代谢酪氨酸的非病原性细菌基因表现系统及转化株、其用于制备降低尿毒素的组合物的用途，以及利用其代谢酪氨酸的方法，其优点在于具有TAL编码(encoding)基因，且TAL编码基因是以B0034作为RBS，故可有效地将酪氨酸转变为对香豆酸，以减少酪氨酸含量，从而减少以酪氨酸为前驱物的对甲酚的生成，进而降低尿毒素含量，故本发明可具有延缓CKD恶化的潜力。
需补充的是，本发明虽以特定的制备方法、特定的评估方法及/或特定的剂量来说明本发明的代谢酪氨酸的非病原性细菌基因表现系统及转化株用于制备降低尿毒素的组合物的用途及利用其代谢酪氨酸的方法，只是本发明所属技术领域中任何具有公知常识者可知，本发明并不限于此，在不脱离本发明的精神和范围内，本发明的代谢酪氨酸的非病原性细菌基因表现系统及转化株用于制备降低尿毒素的组合物的用途及利用其代谢酪氨酸的方法亦可使用其他制备方法、其他评估方法或其他的剂量进行。
虽然本发明已以数个实施例揭露如上，然其并非用以限定本发明，在本发明所属技术领域中任何具有公知常识者，在不脱离本发明的精神和范围内，当可作各种的更动与润饰，因此本发明的保护范围当视所附的权利要求所界定的范围为准。

Claims (9)

  1. 一种代谢酪氨酸的非病原性细菌基因表现系统，其特征在于，包含：
    一第一核酸片段，包含如序列辨识编号(SEQ ID NO.):1所示的序列；以及
    一第二核酸片段，包含如SEQ ID NO.:2所示的序列，且
    其中，该第一核酸片段与该第二核酸片段是位于一第一表现载体上，且该第一核酸片段是操作地连接该第二核酸片段。
  2. 根据权利要求1所述的代谢酪氨酸的非病原性细菌基因表现系统，其特征在于，还包含一第三核酸序列，该第三核酸序列包含如SEQ ID NO.:3所示的序列，且该第三核酸序列位于一第二表现载体上。
  3. 一种代谢酪氨酸的转化株，其特征在于，包含：
    一宿主细胞；以及
    一非病原性细菌基因表现系统位于该宿主细胞内，其中该非病原性细菌基因表现系统包含：
    一第一核酸片段，包含如SEQ ID NO.:1所示的序列；以及
    一第二核酸片段，包含如SEQ ID NO.:2所示的序列，且
    其中，该第一核酸片段与该第二核酸片段是位于一第一表现载体上，且该第一核酸片段是操作地连接该第二核酸片段。
  4. 根据权利要求3所述的代谢酪氨酸的转化株，其特征在于，还包含一第三核酸序列，该第三核酸序列包含如SEQ ID NO.:3所示的序列，且该第三核酸序列位于一第二表现载体上。
  5. 根据权利要求3所述的代谢酪氨酸的转化株，其特征在于，该宿主细胞是大肠杆菌(Escherichia coli)Nissle 1917。
  6. 根据权利要求5所述的代谢酪氨酸的转化株，其特征在于，该宿主细胞是碳酸酐酶(carbonic anhydrase)基因缺陷株。
  7. 一种转化株用于制备降低尿毒素的组合物的用途，其特征在于，该转化株是根据权利要求3至权利要求6任一项所述，借以将酪氨酸代谢为对香豆酸(p-coumaric acid)。
  8. 根据权利要求7所述的转化株用于制备降低尿毒素的组合物的用途，其特征在于，该组合物是一医药组合物或一食品组合物。
  9. 一种利用转化株代谢酪氨酸的方法，其特征在于，包含将根据权利要 求3至权利要求6任一项所述的该转化株培养于不小于5％CO 2的一环境中。
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