Method for preparing pseudouridine through whole-cell catalysis of recombinant escherichia coli, recombinant escherichia coli and use

Abstract

A method for preparing pseudouridine through whole-cell catalysis of recombinant Escherichia coli. The recombinant Escherichia coli is obtained by overexpressing pseudouridine-5-phosphoglycosidase, ribokinase, ribonucleoside hydrolase, cytosine deaminase and nucleoside permease, and not expressing pseudouracil transport protein in Escherichia coli E.coHMG1655; and whole-cell catalytic preparation of pseudouridine taking uridine or cytidine as a substrate is realized by using the recombinant Escherichia coli. A novel pseudouridine enzyme catalytic synthesis route is designed, and in optimal reaction conditions, the maximum yield of pseudouridine reaches 27.24 g / L, the conversion rate reaches 90.8%, the production efficiency is 1.135 g·h-1·L-1, and the pseudouridine yield and the production efficiency are high.

Description

A method for preparing pseudouridine using recombinant Escherichia coli whole cells, recombinant Escherichia coli and its application Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular to a method for preparing pseudouridine by utilizing recombinant Escherichia coli whole cells for catalysis, the recombinant Escherichia coli and its application. Background Art

[0002] To date, over 100 different types of RNA modifications have been discovered, most of which are found in rRNA, tRNA, and snRNA. One of the most common and earliest discovered modifications is the isomerization from uridine to pseudouridine. In recent years, with the increasing interest and research in RNA vaccines, the addition of pseudouridine and its analogs to mRNA in place of uridine can improve mRNA stability and enhance its translation capacity, potentially addressing the problem of mRNA drugs being easily recognized and cleared by the immune system, leading to immune side effects. As an RNA metabolite, pseudouridine is excreted exclusively through the kidneys and can be used clinically to monitor tumor development and progression, as well as in the diagnosis and treatment of kidney disease. Pseudouridine has significant application value, and with the growing market demand for pseudouridine, research on its synthesis has intensified.

[0003] In 1961, the method of chemical synthesis of pseudouridine was first reported, but the total yield was only 2%, and the product was a mixture of α and β isomers. At present, the production methods of pseudouridine mainly include chemical synthesis, chemical-enzymatic method and enzyme catalysis. Li Jingjian et al. (202210798651.4) used 2,3,5-tribenzyloxy-D-ribonic acid-1,4-lactone and 2,4-dialkoxy-5-bromopyrimidine as raw materials to obtain pseudouridine through addition, reduction, deprotection and other reactions; Jin Feng et al. (202210473532.1) used D-ribose and uracil as starting reactants and obtained pseudouridine through addition, condensation, lipase splitting, deprotection and other reactions in organic solvents. However, both chemical synthesis methods have the problems of cumbersome steps and low yields. Xiao Cong et al. (2021111581013.9) used a chemical-enzymatic method to synthesize pseudouridine. They first chemically synthesized ribose 5-phosphate, then used pseudouridine-5-phosphatase to catalyze the synthesis of pseudouridine monophosphate from ribose 5-phosphate and uracil. Finally, pseudouridine was dephosphorylated to obtain pseudouridine, with a yield of 7.9 g / L and a conversion rate of 51%. Chen Wei et al. (202111581013.9) cloned the pseudouridine biosynthesis genes pumH, pumJ, and pumD from Streptomyces sp. ID38640 into Escherichia coli, and were able to convert 10 g / L of inosine to 7.2 g / L of pseudouridine in 96 hours. Zhu Wenjuan et al. (202310168205X) used recombinant Escherichia coli to express uridine phosphorylase UdP, purine nucleoside phosphorylase DeoD, pentose phosphate mutase DeoB, and pseudouridine-5-phosphoglycosidase YeiN, respectively. The enzyme solutions were then mixed and uridine was converted to produce 1.16 g / L pseudouridine in 24 hours, with a conversion rate of 95%. However, the above methods generally have the disadvantages of complicated steps, high production costs, low production efficiency, and difficulty in product recovery and purification, resulting in high product prices, which limits their large-scale application in the biological and pharmaceutical fields. Therefore, there is an urgent need to develop a cheap pseudouridine production process suitable for industrial scale-up.

[0004] Whole-cell biocatalysis primarily utilizes intracellular enzymes for catalysis, making it a biotechnology intermediate between fermentation and pure enzyme catalysis. This method not only enables enzyme cascade reactions and improves enzyme catalytic efficiency, but also significantly reduces the enzyme purification process, effectively controlling production costs. Thus, under the protection of the cell membrane or cell wall, intracellular enzymes can possess greater stability and a longer half-life, making it easier to achieve high catalytic efficiency. The production of nucleoside products using whole-cell catalysis has been a hot topic of research in recent years. The present invention constructs a novel enzyme-catalyzed reaction route in Escherichia coli MG1655, achieving a new process for preparing pseudouridine using whole-cell catalysis using uridine or cytidine as substrates.

[0005] Through searching, no patent publication documents related to the patent application of the present invention have been found.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a method for preparing pseudouridine using recombinant Escherichia coli whole cells, recombinant Escherichia coli and its application.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] A recombinant Escherichia coli capable of whole-cell catalytic production of pseudouridine, characterized in that the recombinant Escherichia coli is obtained by overexpressing pseudouridine-5-phosphatase, overexpressing ribokinase, overexpressing ribonucleoside hydrolase, overexpressing cytosine deaminase, overexpressing nucleoside permease, and not expressing pseudouridine transporter in Escherichia coli E. coli MG1655.

[0010] Furthermore, the pseudouridine-5-phosphoglycosidase is a protein encoded by the Escherichia coli yeiN gene, and the nucleotide sequence of the Escherichia coli yeiN gene is shown in SEQ ID NO.1;

[0011] The ribokinase is a protein encoded by the Escherichia coli rbsK gene, and the nucleotide sequence of the Escherichia coli rbsK gene is shown in SEQ ID NO.2;

[0012] The ribonucleoside hydrolase is a protein encoded by the Escherichia coli rihA gene, the Escherichia coli rihB gene, or the Escherichia coli rihC gene. The nucleotide sequence of the Escherichia coli rihA gene is shown in SEQ ID NO.3, the nucleotide sequence of the Escherichia coli rihB gene is shown in SEQ ID NO.4, and the nucleotide sequence of the Escherichia coli rihC gene is shown in SEQ ID NO.5; preferably, the protein encoded by the Escherichia coli rihA gene;

[0013] The cytosine deaminase is a protein encoded by the Escherichia coli codA gene, and the nucleotide sequence of the Escherichia coli codA gene is shown in SEQ ID NO.6;

[0014] The nucleoside permease is a protein encoded by the Escherichia coli nupC gene, and the nucleotide sequence of the Escherichia coli nupC gene is shown in SEQ ID NO.7;

[0015] The pseudouridine transporter is a protein encoded by the Escherichia coli psuT gene, and the nucleotide sequence of the Escherichia coli psuT gene is shown in SEQ ID NO.8.

[0016] Furthermore, the method for constructing the recombinant Escherichia coli strain comprises the following steps:

[0017] (1) The pseudouridine 5-phosphatase gene yeiN with the nucleotide sequence shown in SEQ ID NO.1 was linked to the promoter P trc The fusion fragment P trc -yeiN was ligated into plasmid pTrc99aΔlacI to obtain plasmid P1;

[0018] (2) The ribokinase gene rbsK with the nucleotide sequence shown in SEQ ID NO.2 was linked to the promoter P trc The fusion fragment P trc -rbsK was ligated into plasmid P1 to obtain plasmid P2;

[0019] (3) The ribonucleoside hydrolase gene rihA with the nucleotide sequence shown in SEQ ID NO.3 was linked to the promoter P trc The fusion fragment P trc -rihA, ribonucleoside hydrolase gene rihB and promoter P as shown in SEQ ID NO.4 trc The fusion fragment P trc -rihB, ribonucleoside hydrolase gene rihC and promoter P as shown in SEQ ID NO.5 trc The fusion fragment P trc -rihC were ligated into plasmid P2 to obtain plasmids P3-P5;

[0020] (4) The cytosine deaminase gene codA with the nucleotide sequence shown in SEQ ID NO.6 was linked to the promoter P trc The fusion fragment P trc -codA was ligated into plasmid P3 to obtain plasmid P6;

[0021] (5) The nucleoside permease gene nupC with the nucleotide sequence shown in SEQ ID NO.7 was linked to the promoter P trc The fusion fragment P trc -nupC was integrated into the yjgX pseudogene site of the starting strain to obtain the recombinant strain Ψ1;

[0022] (6) knocking out or inactivating the pseudouridine transporter gene psuT having a nucleotide sequence as shown in SEQ ID NO. 8 from the recombinant strain Ψ1 to obtain the recombinant strain Ψ2;

[0023] (7) The plasmids P3-P6 constructed in (3) and (4) were electroporated into Ψ2 to obtain recombinant strains Ψ3-Ψ6.

[0024] The application of the above method in the preparation of pseudouridine,

[0025] A method for preparing pseudouridine by utilizing whole-cell catalysis of recombinant Escherichia coli. The method comprises overexpressing pseudouridine-5-phosphatase, overexpressing ribokinase, overexpressing ribonucleoside hydrolase, overexpressing cytosine deaminase, and overexpressing nucleoside permease in Escherichia coli MG1655, and not expressing pseudouridine transporter, to obtain recombinant Escherichia coli. The recombinant Escherichia coli is then used to achieve whole-cell catalytic preparation of pseudouridine using uridine or cytidine as a substrate.

[0026] Furthermore, the method specifically includes the following steps:

[0027] (1) Inoculate the activated recombinant E. coli strain into the culture medium and culture at 37°C for 14-16 hours until the bacterial OD 600 The pH value was maintained at 15-20, and ammonia was added to maintain pH 7.0 during the culture. The rotation speed of the shake flask culture was set at 200 r / min, and the dissolved oxygen of the batch culture was set at 30%-40%;

[0028] The culture medium composition is: glucose 20-30g / L, yeast powder 3-8g / L, KH2PO4 1-3g / L, MgSO4·7H2O 0.1-1g / L, FeSO4·7H2O1-5mg / L, VB1, VB3, VB5, VB 12 、V H 1-3 mg / L each, 2 drops of defoamer, the rest is water, pH 7.0-7.5;

[0029] (2) Collect the cultured cells by centrifugation and wash them once with PBS buffer; resuspend the washed cells in the reaction solution and calculate the cell concentration OD 600 The reaction temperature was about 40°C; 5 g / L of uridine or cytidine was added at once during shake flask catalysis, and 5-40 g / L of uridine or cytidine was added during batch feed catalysis; the reaction temperature was 37°C, the reaction time was 24 h, and ammonia was added to maintain the pH at 7.0;

[0030] The reaction solution consists of: glucose 15-25 g / L, Na2HPO4·7H2O 5-8 g / L, KH2PO4 2-4 g / L, NH4Cl 2-4 g / L, NaCl 0.1-1 g / L, MgSO4·7H2O 0.1-1 g / L, and FeSO4 10-100 mg / L.

[0031] The advantages and positive effects achieved by the present invention are:

[0032] 1. The present invention uses ribonucleoside hydrolase to hydrolyze uridine or cytidine into uracil, cytosine and D-ribose; uses ribokinase to phosphorylate D-ribose to 5-phosphate ribose; uses cytosine deaminase to convert cytosine into uracil; uses pseudouridine-5-phosphoglycosidase to catalyze the synthesis of pseudouridine monophosphate from uracil and 5-phosphate ribose; the ATP required for the enzyme cascade reaction and the dephosphorylation process of pseudouridine monophosphate are completed by the cell's own metabolism. The pseudouridine enzyme catalytic reaction route designed by the present invention is different from the existing enzyme catalysis method (202310168205X, 202111581013.9) (Figure 1), and has strong creativity.

[0033] 2. In order to improve the efficiency of whole-cell catalytic preparation of pseudouridine, the present invention integrates the nucleoside permease gene nupC into the chassis cell genome, thereby enhancing the intracellular transport of substrate uridine or cytidine; and knocks out the pseudouridine transporter gene psuT in the chassis cells, thereby reducing the intracellular transport of the product pseudouridine. In addition, the whole-cell catalytic substrate addition method is optimized, and the one-time feeding method is replaced by the feeding method, thereby reducing the additional consumption of substrate by the cell. The present invention designs a novel pseudouridine enzyme catalytic synthesis route. Under the optimal reaction conditions, the maximum yield of pseudouridine reaches 27.24g / L, the conversion rate reaches 90.8%, and the production efficiency is 1.135g·h -1 ·L -1 The pseudouridine yield and production efficiency are the highest values ​​reported by existing enzymatic methods.

[0034] 3. The production of nucleoside products using whole-cell catalysis has been a research hotspot in recent years. This invention constructs a novel enzymatic reaction route. By overexpressing pseudouridine-5-phosphatase, ribokinase, ribonucleoside hydrolase, cytosine deaminase, and nucleoside permease in Escherichia coli MG1655, while de-expressing the pseudouridine transporter, a recombinant E. coli strain with efficient and stable production performance for pseudouridine production is obtained. A whole-cell catalytic method for this is also provided, realizing a new process for the whole-cell catalytic preparation of pseudouridine using uridine or cytidine as substrates. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG1 is a schematic diagram of the pseudouridase catalytic reaction route of the present invention;

[0036] FIG2 is a schematic diagram of the construction of the constitutive plasmid pTrc99aΔlacI of the present invention;

[0037] FIG3 is a schematic diagram of the construction of the recombinant plasmid P1 of the present invention;

[0038] FIG4 is a schematic diagram of the construction of the recombinant plasmid P2 of the present invention;

[0039] FIG5 is a schematic diagram of the construction of the recombinant plasmid P3 of the present invention;

[0040] FIG6 is a schematic diagram of the construction of the recombinant plasmid P4 of the present invention;

[0041] FIG7 is a schematic diagram of the construction of the recombinant plasmid P5 of the present invention;

[0042] FIG8 is a schematic diagram of the construction of the recombinant plasmid P6 of the present invention;

[0043] Figure 9 is an electrophoretic diagram of the construction and verification of the nudG integration fragment of the present invention; wherein: M: 1 kb DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: target gene; 4: overlapping fragment; 5: original bacterial PCR fragment; 6: positive single colony PCR identification fragment;

[0044] Figure 10 is an electrophoretic diagram of the construction and verification of the psuT gene knockout fragment in the present invention; wherein: M: 1 kb DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: overlapping fragment; 4: original bacterial PCR fragment; 5: positive single colony PCR identification fragment;

[0045] FIG11 is a diagram showing the shake flask catalysis results of Example 3 of the present invention;

[0046] Figure 12 is a graph showing the results of fed-batch catalysis in Example 5 of the present invention;

[0047] FIG13 is a diagram showing the catalytic results of Examples 4 and 6 of the present invention. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0049] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0050] A recombinant Escherichia coli capable of whole-cell catalytic production of pseudouridine, characterized in that the recombinant Escherichia coli is obtained by overexpressing pseudouridine-5-phosphatase, overexpressing ribokinase, overexpressing ribonucleoside hydrolase, overexpressing cytosine deaminase, overexpressing nucleoside permease, and not expressing pseudouridine transporter in Escherichia coli E. coli MG1655.

[0051] Preferably, the pseudouridine-5-phosphoglycosidase is a protein encoded by the yeiN gene of Escherichia coli, and the nucleotide sequence of the yeiN gene of Escherichia coli is shown in SEQ ID NO.1;

[0052] The ribokinase is a protein encoded by the Escherichia coli rbsK gene, and the nucleotide sequence of the Escherichia coli rbsK gene is shown in SEQ ID NO.2;

[0053] The ribonucleoside hydrolase is a protein encoded by the Escherichia coli rihA gene, the Escherichia coli rihB gene, or the Escherichia coli rihC gene. The nucleotide sequence of the Escherichia coli rihA gene is shown in SEQ ID NO.3, the nucleotide sequence of the Escherichia coli rihB gene is shown in SEQ ID NO.4, and the nucleotide sequence of the Escherichia coli rihC gene is shown in SEQ ID NO.5; preferably, the protein encoded by the Escherichia coli rihA gene;

[0054] The cytosine deaminase is a protein encoded by the Escherichia coli codA gene, and the nucleotide sequence of the Escherichia coli codA gene is shown in SEQ ID NO.6;

[0055] The nucleoside permease is a protein encoded by the Escherichia coli nupC gene, and the nucleotide sequence of the Escherichia coli nupC gene is shown in SEQ ID NO.7;

[0056] The pseudouridine transporter is a protein encoded by the Escherichia coli psuT gene, and the nucleotide sequence of the Escherichia coli psuT gene is shown in SEQ ID NO.8.

[0057] Preferably, the method for constructing the recombinant Escherichia coli strain comprises the following steps:

[0058] (1) The pseudouridine 5-phosphatase gene yeiN with the nucleotide sequence shown in SEQ ID NO.1 was linked to the promoter P trc The fusion fragment P trc -yeiN was ligated into plasmid pTrc99aΔlacI to obtain plasmid P1;

[0059] (2) The ribokinase gene rbsK with the nucleotide sequence shown in SEQ ID NO.2 was linked to the promoter P trc The fusion fragment P trc -rbsK was ligated into plasmid P1 to obtain plasmid P2;

[0060] (3) The ribonucleoside hydrolase gene rihA with the nucleotide sequence shown in SEQ ID NO.3 was linked to the promoter P trc The fusion fragment P trc -rihA, ribonucleoside hydrolase gene rihB and promoter P as shown in SEQ ID NO.4 trc The fusion fragment P trc-rihB, ribonucleoside hydrolase gene rihC and promoter P as shown in SEQ ID NO.5 trc The fusion fragment P trc -rihC were ligated into plasmid P2 to obtain plasmids P3-P5;

[0061] (4) The cytosine deaminase gene codA with the nucleotide sequence shown in SEQ ID NO.6 was linked to the promoter P trc The fusion fragment P trc -codA was ligated into plasmid P3 to obtain plasmid P6;

[0062] (5) The nucleoside permease gene nupC with the nucleotide sequence shown in SEQ ID NO.7 was linked to the promoter P trc The fusion fragment P trc -nupC was integrated into the yjgX pseudogene site of the starting strain to obtain the recombinant strain Ψ1;

[0063] (6) knocking out or inactivating the pseudouridine transporter gene psuT having a nucleotide sequence as shown in SEQ ID NO. 8 from the recombinant strain Ψ1 to obtain the recombinant strain Ψ2;

[0064] (7) The plasmids P3-P6 constructed in (3) and (4) were electroporated into Ψ2 to obtain recombinant strains Ψ3-Ψ6.

[0065] (8) the promoter P trc The nucleotide sequence is shown in SEQ ID NO.9.

[0066] The application of the above method in the preparation of pseudouridine,

[0067] A method for preparing pseudouridine by utilizing whole-cell catalysis of recombinant Escherichia coli. The method comprises overexpressing pseudouridine-5-phosphatase, overexpressing ribokinase, overexpressing ribonucleoside hydrolase, overexpressing cytosine deaminase, and overexpressing nucleoside permease in Escherichia coli MG1655, and not expressing pseudouridine transporter, to obtain recombinant Escherichia coli. The recombinant Escherichia coli is then used to achieve whole-cell catalytic preparation of pseudouridine using uridine or cytidine as a substrate.

[0068] Preferably, the method specifically includes the following steps:

[0069] (1) Inoculate the activated recombinant E. coli strain into the culture medium and culture at 37°C for 14-16 hours until the bacterial OD 600 The pH value was maintained at 15-20, and ammonia was added to maintain pH 7.0 during the culture. The rotation speed of the shake flask culture was set at 200 r / min, and the dissolved oxygen of the batch culture was set at 30%-40%;

[0070] The culture medium composition is: glucose 20-30g / L, yeast powder 3-8g / L, KH2PO4 1-3g / L, MgSO4·7H2O 0.1-1g / L, FeSO4·7H2O1-5mg / L, VB1, VB3, VB5, VB 12 、V H 1-3 mg / L each, 2 drops of defoamer, the rest is water, pH 7.0-7.5;

[0071] (2) Collect the cultured cells by centrifugation and wash them once with PBS buffer; resuspend the washed cells in the reaction solution and calculate the cell concentration OD 600 The reaction temperature was about 40°C; 5 g / L of uridine or cytidine was added at once during shake flask catalysis, and 5-40 g / L of uridine or cytidine was added during batch feed catalysis; the reaction temperature was 37°C, the reaction time was 24 h, and ammonia was added to maintain the pH at 7.0;

[0072] The reaction solution consists of: glucose 15-25 g / L, Na2HPO4·7H2O 5-8 g / L, KH2PO4 2-4 g / L, NH4Cl 2-4 g / L, NaCl 0.1-1 g / L, MgSO4·7H2O 0.1-1 g / L, and FeSO4 10-100 mg / L.

[0073] Specifically, the relevant preparation and detection are as follows:

[0074] Example 1: Construction of recombinant plasmid

[0075] 1. Construction of constitutive plasmid

[0076] The pTrc99a plasmid used in the present invention contains the lacI gene that expresses the lactose operon repressor. When expressing the plasmid, isopropylβ-D-thiogalactoside (IPTG) needs to be added for induction. However, IPTG can easily affect bacterial growth. Therefore, a constitutive plasmid pTrc99aΔlacI was constructed. By designing primers 1-S and 1-A with restriction enzyme sites Apa I at both ends, a linearized pTrc99a vector without lacI was amplified by PCR, and then the recombinase II caused it to self-circularize, constructing the constitutive plasmid pTrc99aΔlacI. The schematic diagram of the plasmid construction is shown in Figure 2.

[0077] 2. Construction of recombinant plasmid P1

[0078] According to the desired overexpressed pseudouridine-5-phosphatase gene yeiN, appropriate restriction enzyme cutting sites Sal I and Hind III were selected, and the plasmid pTrc99aΔlacI was double-digested; using the E. coli MG1655 genome as a template, PCR amplification was performed to obtain the plasmid containing the Sal I and Hind III restriction enzyme cutting sites and P trc The target gene fragment of the promoter (SEQ ID NO.9 is its sequence); the PCR product was recovered and purified using a direct recovery kit, and the enzyme cleavage product was recovered and purified using a gel recovery kit and then recombinase The recombinant plasmid P1 containing the pseudouridine-5-phosphatase gene yeiN was obtained after homologous recombination under the action of II and then transformed into Escherichia coli competent cells DH5α. After screening and colony PCR identification, the recombinant plasmid P1 containing the pseudouridine-5-phosphatase gene yeiN was obtained. The schematic diagram of the plasmid construction is shown in Figure 3.

[0079] 3. Construction of recombinant plasmid P2

[0080] According to the desired overexpressed ribokinase gene rbsK, appropriate restriction sites BamH Ⅰ and Xba Ⅰ were selected and double-digested on plasmid P1. Using the E. coli MG1655 genome as a template, PCR amplification was performed to obtain a plasmid containing BamH Ⅰ and Xba Ⅰ restriction sites and P trc The target gene fragment of the promoter; the PCR product was recovered and purified with a direct recovery kit, and the enzyme digestion product was recovered and purified with a gel recovery kit and then The recombinant plasmid P2 containing the ribokinase gene rbsK was obtained after homologous recombination under the action of II and then transformed into Escherichia coli competent cells DH5α. After screening and colony PCR identification, the recombinant plasmid P2 containing the ribokinase gene rbsK was obtained. The schematic diagram of the plasmid construction is shown in Figure 4.

[0081] 4. Construction of recombinant plasmid P3

[0082] According to the desired overexpressed ribonucleoside hydrolase gene rihA, suitable restriction sites SacⅠ and KpnⅠ were selected and double-digested on plasmid P2. Using the E. coli MG1655 genome as a template, PCR amplification was performed to obtain the plasmid containing SacⅠ and KpnⅠ restriction sites and P trc The target gene fragment of the promoter; the PCR product was recovered and purified with a direct recovery kit, and the enzyme digestion product was recovered and purified with a gel recovery kit and then The recombinant plasmid P3 containing the ribokinase gene rihA was obtained after homologous recombination under the action of II and then transformed into Escherichia coli competent cells DH5α. After screening and colony PCR identification, the recombinant plasmid P3 containing the ribokinase gene rihA was obtained. The schematic diagram of the plasmid construction is shown in Figure 5.

[0083] 5. Construction of recombinant plasmid P4

[0084] According to the desired overexpressed ribonucleoside hydrolase gene rihB, suitable restriction sites SacⅠ and KpnⅠ were selected to perform double restriction digestion on plasmid P2. Using the E. coli MG1655 genome as a template, PCR amplification was performed to obtain a plasmid containing the SacⅠ and KpnⅠ restriction sites and P trc The target gene fragment of the promoter; the PCR product was recovered and purified with a direct recovery kit, and the enzyme digestion product was recovered and purified with a gel recovery kit and then The recombinant plasmid P4 containing the ribokinase gene rihB was obtained after homologous recombination under the action of II and then transformed into Escherichia coli competent cells DH5α. After screening and colony PCR identification, the recombinant plasmid P4 containing the ribokinase gene rihB was obtained. The schematic diagram of the plasmid construction is shown in Figure 6.

[0085] 6. Construction of recombinant plasmid P5

[0086] According to the desired overexpressed ribonucleoside hydrolase gene rihC, suitable restriction sites SacⅠ and KpnⅠ were selected and double-digested on plasmid P2. Using the E. coli MG1655 genome as a template, PCR amplification was performed to obtain the plasmid containing SacⅠ and KpnⅠ restriction sites and P trc The target gene fragment of the promoter; the PCR product was recovered and purified with a direct recovery kit, and the enzyme digestion product was recovered and purified with a gel recovery kit and then The recombinant plasmid P5 containing the ribokinase gene rihC was obtained after homologous recombination under the action of II and then transformed into Escherichia coli competent cells DH5α. After screening and colony PCR identification, the recombinant plasmid P5 containing the ribokinase gene rihC was obtained. The schematic diagram of the plasmid construction is shown in Figure 7.

[0087] 7. Construction of recombinant plasmid P6

[0088] According to the desired overexpressed ribonucleoside hydrolase gene codA, appropriate restriction sites Nco I and EcoRI were selected to perform double restriction digestion on plasmid P3. Using the E. coli MG1655 genome as a template, PCR amplification was performed to obtain the plasmid containing Nco I, EcoRI restriction sites and P trc The target gene fragment of the promoter; the PCR product was recovered and purified with a direct recovery kit, and the enzyme digestion product was recovered and purified with a gel recovery kit and then The recombinant plasmid P6 containing the ribokinase gene codA was obtained after homologous recombination under the action of II and then transformed into Escherichia coli competent cells DH5α. The recombinant plasmid P6 containing the ribokinase gene codA was obtained after screening and colony PCR identification. The schematic diagram of the plasmid construction is shown in Figure 8.

[0089] 8. Primers used in the construction of recombinant plasmids

[0090] The primers involved in the above construction process are shown in the table below:

[0091] Example 2: Construction of recombinant strains

[0092] 1. Gene Editing Methods

[0093] The present invention employs a CRISPR / Cas9-mediated gene editing method, which can be performed with reference to the literature (Metabolic Engineering, 2015, 31:13-21). CRISPR / Cas9 is a precise and efficient new gene-targeted modification technology. The two plasmids used in this method are pGRB and pREDCas9. The pREDCas9 plasmid is a temperature-sensitive plasmid that carries a gRNA plasmid elimination system, the lambda phage Red recombination system, and the Cas9 protein expression system. It confers spectinomycin resistance (working concentration: 100 mg / L) and an optimal incubation temperature of 32°C. The pGRB plasmid, based on the pUC18 backbone, contains the promoter J23100, a gRNA-Cas9 binding region sequence, and a terminator sequence. It confers ampicillin resistance (working concentration: 100 mg / L) and an optimal incubation temperature of 37°C.

[0094] 2. Construction of recombinant strain Ψ1

[0095] Using the E. coli MG1655 genome as a template, upstream homology arm primers (UP-yjgX-S, UP-yjgX-A) and downstream homology arm primers (DN-yjgX-S, DN-yjgX-A) were designed according to the upstream and downstream sequences of its yjgX gene, and its upstream and downstream homology arm fragments were amplified by PCR; primers (P trc -nupC-S, nupC-P trc -A), amplify the nupC gene fragment, promoter P trc The terminator T is designed in the downstream primer of the upstream homology arm and the upstream primer of the nupC gene. trc The upstream primer of the downstream homology arm and the downstream primer of the nupC gene are designed. The above fragments are fused by overlapping PCR to obtain the integrated fragment of the nupC gene (upstream homology arm-P trc -nupC-T trc-downstream homology arm), a DNA fragment containing the target sequence of the target gene was prepared by annealing primers gRNA-yjgX-S and gRNA-yjgX-A, and recombined with the linearized pGRB vector to obtain the recombinant pGRB-yjgX. The recombinant fragment and plasmid pGRB-yjgX were electroporated into competent cells of E.coli MG1655 containing the pREDCas9 plasmid, and the positive strains were screened and then the plasmid was eliminated to obtain strain Ψ1. The electrophoresis diagram of the construction of the nupC integration fragment and the PCR verification of the positive strain is shown in Figure 9. Among them, the length of the upstream homology arm is 561bp, the length of the nupC gene fragment is 1326bp, the length of the downstream homology arm is 505bp, and the total length of the integration fragment is 2335bp. During PCR verification, the length of the PCR amplification fragment of the positive bacteria should be 2335bp, and the length of the PCR amplification fragment of the original bacteria should be 1286bp.

[0096] 3. Construction of recombinant strain Ψ2

[0097] Using the E. coli MG1655 genome as a template, upstream homology arm primers (UP-psuT-S, UP-psuT-A) and downstream homology arm primers (DN-psuT-S, DN-psuT-A) were designed based on the upstream and downstream sequences of its psuT gene (as shown in SEQ ID NO.8), and PCR amplification of its upstream and downstream homology arm fragments was performed. The above fragments were fused by overlapping PCR to obtain a knockout fragment of the psuT gene (upstream homology arm-downstream homology arm). A DNA fragment containing the target sequence of the target gene was prepared by annealing primers gRNA-psuT-S and gRNA-psuT-A, and recombined with the linearized pGRB vector to obtain recombinant pGRB-psuT. The recombinant fragment and plasmid pGRB-psuT were electroporated into competent cells of Ψ1 containing the pREDCas9 plasmid, and after screening for positive strains, the plasmid was eliminated to obtain strain Ψ2. The electrophoresis diagram of the construction of the psuT knockout fragment and PCR verification of the positive strains is shown in Figure 10. The upstream homology arm is 283 bp long, the downstream homology arm is 492 bp long, and the total length of the overlapping fragment is 775 bp. During PCR verification, the PCR amplification fragment for positive bacteria should be 775 bp long, and the PCR amplification fragment for the original bacteria should be 1970 bp long.

[0098] 4. Construction of recombinant strains Ψ3-Ψ6

[0099] The recombinant plasmids P3-P6 constructed in Example 1 were extracted from DH5α and then electroporated into competent cells of the recombinant strain Ψ2. After screening and colony PCR identification, recombinant strains Ψ3-Ψ6 containing the recombinant plasmids P3-P6 were obtained.

[0100] 5. Primers used in strain construction

[0101] The primers involved in the above construction process are shown in the table below:

[0102] Example 3: Shake flask experiment of whole cell catalysis of uridine to produce pseudouridine

[0103] The shake flask culture method is as follows:

[0104] Inoculate an appropriate amount of activated strains into the culture medium and culture at 37°C for 14-16 hours until the bacterial OD 600 The pH value reached 15-20, and ammonia was added to maintain pH 7.0 during the culture. The rotation speed of the shake flask culture was set at 200 r / min. The culture medium composition was: glucose 25 g / L, yeast powder 5 g / L, KH2PO4 1.2 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 2.8 mg / L, VB1, VB3, VB5, VB 12 、V H 1.3 mg / L of each, 2 drops of defoamer, the rest is water, pH 7.0-7.5.

[0105] The cultured cells were collected by centrifugation and washed once with PBS buffer. The washed cells were resuspended in the reaction solution and the cell concentration was OD 600 The catalytic reaction temperature was 37°C for 24 hours, and ammonia was added to maintain the pH at 7.0. The reaction solution composition was: glucose 20 g / L, Na2HPO4·7H2O 6.8 g / L, KH2PO4 3 g / L, NH4Cl 3 g / L, NaCl 0.5 g / L, MgSO4·7H2O 0.5 g / L, and FeSO4 20 mg / L.

[0106] Shake flask catalysis was performed using strains Ψ3-Ψ5, with 5 g / L of the substrate uridine added at once. The results are shown in Figure 11. The three strains showed varying pseudouridine production, with strain Ψ3, overexpressing rihA, accumulating the highest amount of pseudouridine at 3.55 g / L, 22.4% and 105.6% higher than strains Ψ4, overexpressing rihB, and Ψ5, overexpressing rihC, respectively. Furthermore, strains Ψ3 and Ψ4 showed no excess uridine, while strain Ψ5 did. These results demonstrate that RihA is significantly more potent at hydrolyzing uridine than RihB and RihC, making it a more suitable enzyme for whole-cell catalysis of pseudouridine.

[0107] Example 4: Shake flask experiment of whole cell catalyzed cytidine production of pseudouridine

[0108] The shake flask culture method is as follows:

[0109] Inoculate an appropriate amount of activated strains into the culture medium and culture at 37°C for 14-16 hours until the bacterial OD 600 The pH value reached 15-20, and ammonia was added to maintain pH 7.0 during the culture. The rotation speed of the shake flask culture was set at 200 r / min. The culture medium composition was: glucose 25 g / L, yeast powder 5 g / L, KH2PO4 1.2 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 2.8 mg / L, VB1, VB3, VB5, VB 12 、V H 1.3 mg / L of each, 2 drops of defoamer, the rest is water, pH 7.0-7.5.

[0110] The cultured cells were collected by centrifugation and washed once with PBS buffer. The washed cells were resuspended in the reaction solution and the cell concentration was OD 600 The catalytic reaction temperature was 37°C for 24 hours, and ammonia was added to maintain the pH at 7.0. The reaction solution composition was: glucose 20 g / L, Na2HPO4·7H2O 6.8 g / L, KH2PO4 3 g / L, NH4Cl 3 g / L, NaCl 0.5 g / L, MgSO4·7H2O 0.5 g / L, and FeSO4 20 mg / L.

[0111] Strain Ψ6 was used for shake flask catalysis. 5 g / L of substrate cytidine was added at one time during the catalytic process. After 24 hours of reaction, the pseudouridine yield reached 3.03 g / L. The results are shown in FIG13 .

[0112] Example 5: Fed-batch experiment of whole-cell catalytic uridine production of pseudouridine

[0113] Inoculate an appropriate amount of activated strains into the culture medium and culture at 37°C for 14-16 hours until the bacterial OD 600 The pH value was maintained at 15-20. During the culture, ammonia was added to maintain pH 7.0. The dissolved oxygen in batch culture was set at 30%-40%. The culture medium composition was: glucose 25g / L, yeast powder 5g / L, KH2PO41.2g / L, MgSO4·7H2O 0.5g / L, FeSO4·7H2O 2.8mg / L, VB1, VB3, VB5, VB 12 、V H 1.3 mg / L of each, 2 drops of defoamer, the rest is water, pH 7.0-7.5.

[0114] The cultured cells were collected by centrifugation and washed once with PBS buffer. The washed cells were resuspended in the reaction solution and the cell concentration was OD 600The catalytic reaction temperature was 37°C for 24 hours, and ammonia was added to maintain the pH at 7.0. The reaction solution composition was: glucose 20 g / L, Na2HPO4·7H2O 6.8 g / L, KH2PO4 3 g / L, NH4Cl 3 g / L, NaCl 0.5 g / L, MgSO4·7H2O 0.5 g / L, and FeSO4 20 mg / L.

[0115] Strain Ψ5 was used for fed-batch catalysis, with uridine added at final concentrations of 5 g / L, 10 g / L, 20 g / L, 30 g / L, and 40 g / L, respectively. The results are shown in Figure 12. When the final concentration of uridine was 5 g / L, the accumulation of pseudouridine in strain Ψ5 reached 3.89 g / L, a 9.6% increase compared to a single-batch feed, demonstrating that the fed-batch method helps reduce the excess consumption of uridine. When the final concentration of uridine was increased from 5 g / L to 30 g / L, the accumulation of pseudouridine increased from 3.89 g / L to 27.24 g / L, and the conversion rate increased from 77.8% to 90.8%. When the final concentration of uridine was 40 g / L, the uridine was not completely consumed, with 4.23 g / L remaining. The accumulation of pseudouridine reached 30.86 g / L, with a conversion rate of 77.2%.

[0116] Example 6: Fed-batch experiment of whole-cell catalyzed cytidine production of pseudouridine

[0117] Inoculate an appropriate amount of activated strains into the culture medium and culture at 37°C for 14-16 hours until the bacterial OD 600 The pH value was maintained at 15-20. During the culture, ammonia was added to maintain pH 7.0. The rotation speed of the shake flask culture was set at 200 r / min. The dissolved oxygen of the batch culture was set at 30%-40%. The culture medium composition was: glucose 25g / L, yeast powder 5g / L, KH2PO4 1.2g / L, MgSO4·7H2O 0.5g / L, FeSO4·7H2O 2.8mg / L, VB1, VB3, VB5, VB 12 、V H 1.3 mg / L of each, 2 drops of defoamer, the rest is water, pH 7.0-7.5.

[0118] The cultured cells were collected by centrifugation and washed once with PBS buffer. The washed cells were resuspended in the reaction solution and the cell concentration was OD 600The catalytic reaction temperature was 37°C for 24 hours, and ammonia was added to maintain the pH at 7.0. The reaction solution composition was: glucose 20 g / L, Na2HPO4·7H2O 6.8 g / L, KH2PO4 3 g / L, NH4Cl 3 g / L, NaCl 0.5 g / L, MgSO4·7H2O 0.5 g / L, and FeSO4 20 mg / L.

[0119] Batch fed-batch catalysis was performed using strain Ψ6. During the catalytic process, substrate cytidine was added at a final concentration of 30 g / L. The results are shown in FIG13 . After 24 hours of reaction, the pseudouridine yield was 23.76 g / L, and the conversion rate was 85.9%.

[0120] The relevant sequences in the present invention are as follows:

[0121] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A recombinant Escherichia coli capable of whole-cell catalytic production of pseudouridine, characterized in that: The recombinant Escherichia coli is obtained by overexpressing pseudouridine-5-phosphatase, overexpressing ribokinase, overexpressing ribonucleoside hydrolase, overexpressing cytosine deaminase, overexpressing nucleoside permease, and not expressing pseudouridine transporter in Escherichia coli E. coli MG1655.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The pseudouridine-5-phosphatase is a protein encoded by the yeiN gene of Escherichia coli, and the nucleotide sequence of the yeiN gene of Escherichia coli is shown in SEQ ID NO.1; The ribokinase is a protein encoded by the Escherichia coli rbsK gene, and the nucleotide sequence of the Escherichia coli rbsK gene is shown in SEQ ID NO.2; The ribonucleoside hydrolase is a protein encoded by the Escherichia coli rihA gene, the Escherichia coli rihB gene, or the Escherichia coli rihC gene, the nucleotide sequence of the Escherichia coli rihA gene is shown in SEQ ID NO.3, the nucleotide sequence of the Escherichia coli rihB gene is shown in SEQ ID NO.4, and the nucleotide sequence of the Escherichia coli rihC gene is shown in SEQ ID NO.5; The cytosine deaminase is a protein encoded by the Escherichia coli codA gene, and the nucleotide sequence of the Escherichia coli codA gene is shown in SEQ ID NO.6; The nucleoside permease is a protein encoded by the Escherichia coli nupC gene, and the nucleotide sequence of the Escherichia coli nupC gene is shown in SEQ ID NO.7; The pseudouridine transporter is a protein encoded by the Escherichia coli psuT gene, and the nucleotide sequence of the Escherichia coli psuT gene is shown in SEQ ID NO.

8.

3. The recombinant Escherichia coli according to claim 2, characterized in that: The method for constructing the recombinant Escherichia coli strain comprises the following steps: (1) The pseudouridine-5-phosphatase gene yeiN with the nucleotide sequence shown in SEQ ID NO.1 was linked to the promoter P trc The fusion fragment P trc -yeiN was ligated into plasmid pTrc99aΔlacI to obtain plasmid P1; (2) The ribokinase gene rbsK with the nucleotide sequence shown in SEQ ID NO.2 was linked to the promoter P trc The fusion fragment P trc -rbsK was connected to plasmid P1 to obtain plasmid P2; (3) The ribonucleoside hydrolase gene rihA with the nucleotide sequence shown in SEQ ID NO.3 was linked to the promoter P trc The fusion fragment P trc -rihA, ribonucleoside hydrolase gene rihB and promoter P as shown in SEQ ID NO.4 trc The fusion fragment P trc -rihB, ribonucleoside hydrolase gene rihC and promoter P as shown in SEQ ID NO.5 trc The fusion fragment P trc -rihC were ligated into plasmid P2 to obtain plasmids P3-P5; (4) The cytosine deaminase gene codA with the nucleotide sequence shown in SEQ ID NO.6 was linked to the promoter P trc Fusion Fragment P trc -codA was ligated to plasmid P3 to obtain plasmid P6; (5) The nucleoside permease gene nupC with the nucleotide sequence shown in SEQ ID NO.7 and the promoter P trc The fusion fragment P trc -nupC was integrated into the yjgX pseudogene site of the starting strain to obtain the recombinant strain Ψ1; (6) knocking out or inactivating the pseudouridine transporter gene psuT having a nucleotide sequence as shown in SEQ ID NO.8 from the recombinant strain Ψ1 to obtain the recombinant strain Ψ2; (7) The plasmids P3-P6 constructed in (3) and (4) were respectively electroporated into Ψ2 to obtain recombinant strains Ψ3-Ψ6.

4. Use of the method according to any one of claims 1 to 3 in the preparation of pseudouridine.

5. A method for preparing pseudouridine using recombinant Escherichia coli whole cells, characterized in that: The method overexpresses pseudouridine-5-phosphatase, ribokinase, ribonucleoside hydrolase, cytosine deaminase and nucleoside permease in Escherichia coli MG1655, and does not express pseudouridine transporter, so as to obtain recombinant Escherichia coli, and utilizes the recombinant Escherichia coli to prepare pseudouridine by whole-cell catalysis using uridine or cytidine as substrate.

6. The method according to claim 5, characterized in that: The specific steps include: (1) Inoculate the activated recombinant E. coli strain into the culture medium and culture at 37°C for 14-16 hours until the bacterial OD 600 Reach 15-20, during the culture period, pH 7.0 was maintained by feeding ammonia water, the rotation speed of the shake flask culture was set to 200r / min, and the dissolved oxygen of the batch culture was set to 30%-40%; The culture medium composition is: glucose 20-30g / L, yeast powder 3-8g / L, KH2PO41-3g / L, MgSO4·7H2O 0.1-1g / L, FeSO4·7H2O 1-5mg / L, VB1, VB3, VB5, VB 12 、V H 1-3 mg / L each, 2 drops of defoamer, the rest is water, pH 7.0-7.5; (2) Collect the cultured cells by centrifugation and wash them once with PBS buffer; resuspend the washed cells in the reaction solution and calculate the cell concentration OD 600 40; 5 g / L of substrate uridine or cytidine was added at one time during shake flask catalysis, and 5-40 g / L of substrate uridine or cytidine was added during batch feed catalysis; the catalytic reaction temperature was 37°C, the reaction time was 24 h, and ammonia water was added to maintain pH 7.0; The reaction solution consists of: glucose 15-25 g / L, Na2HPO4·7H2O 5-8 g / L, KH2PO4 2-4 g / L, NH4Cl 2-4 g / L, NaCl 0.1-1 g / L, MgSO4·7H2O 0.1-1 g / L, FeSO4 10-100 mg / L.

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