Heterologous transport protein YdhP and application of heterologous transport protein YdhP in production of beta-thymidine from escherichia coli

Abstract

The invention discloses a heterologous transport protein YdhP and application thereof in production of beta-thymidine from escherichia coli, and belongs to the technical field of microbial engineering. According to the present invention, by introducing the heterologous transport protein ydhP derived from Ralstania mannitolyticus, the extracellular secretion of beta-thymidine can be effectively promoted, and the intracellular feedback regulation limitation can be reduced; during shake flask fermentation, the yield of beta-thymidine is increased to 1.6 g / L (increased by about 45%), and the by-product deoxyuridine is reduced by about 50%; the yield in a 5 L fermentation tank reaches 20 g / L, which is obviously improved compared with that of a control strain (12 g / L), and an efficient and stable engineering strain and a fermentation process are provided for industrial production of beta-thymidine.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, and in particular to the heterologous transporter protein YdhP and its application in the production of β-thymidine in Escherichia coli. Background Technology

[0002] β-Thymidine (TdR) is a natural nucleoside composed of 2-deoxyribose and thymine bases. Its chemical name is 1-(2-deoxy-β-D-ribofuranosyl)-5-methyluracil, and its molecular formula is C2. 10 H 14 N₂O₅ has a relative molecular mass of 242.23. TdR is a white or off-white crystalline powder at room temperature with a melting point of 185℃. Its solubility in water at room temperature is approximately 40 g / L, increasing in hot water. It is readily soluble in alkaline aqueous solutions, pyridine, and glacial acetic acid. Its solubility in most common organic solvents (such as ethanol, methanol, and chloroform) is generally low or poor. Dimethyl sulfoxide (DMSO) is commonly used to prepare high-concentration thymidine stock solutions. TdR has significant applications primarily in the pharmaceutical field. For example, TdR is a key precursor to the anti-AIDS drugs zidovudine (AZT) and stavudine (D4T), with zidovudine being widely used in AIDS treatment research.

[0003] Currently, there are two main strategies for TdR production: chemical synthesis and microbial fermentation. Chemical synthesis suffers from complex procedures, severe environmental pollution, and high costs. Developing inexpensive and efficient production methods is crucial to meeting the market demand for TdR. With the rapid development of systems metabolic engineering and synthetic biology, TdR production processes based on microbial fermentation have become a research hotspot in recent years.

[0004] Constructing efficient and stable TdR production cell factories is the core of developing green and economical TdR production processes. In microorganisms, there are two main synthetic pathways for pyrimidine nucleosides: one is the de novo synthesis of pyrimidine nucleosides using matrices such as glucose and nitrogen sources; the other is the salvage pathway, where thymine bases, deoxyribose, and phosphate produced by nucleotide metabolism are re-ribosylated and phosphorylated to generate pyrimidine nucleosides.

[0005] In *E. coli*, the de novo TdR synthesis pathway is lengthy, and its metabolic flux is regulated by negative feedback from multiple pathways. Furthermore, the de novo TdR synthesis pathway is related to DNA synthesis, exhibits poor substrate selectivity for several key enzymes, and involves numerous competing pathways in TdR synthesis. *E. coli* itself possesses pathways for the synthesis and degradation of pyrimidine nucleosides; by regulating these pathways through genetic engineering, TdR production can be increased. Core strategies include: blocking TdR degradation pathways, enhancing precursor supply, optimizing TdR synthesis pathways, and balancing intracellular redox balance to modify the metabolic flux of the TdR synthesis pathway in chassis microorganisms. In a recent study, systems metabolic engineering was used to modify *E. coli*, introducing the TdR synthesis pathway into wild-type *E. coli* MG1655. Through multiple rounds of genetic optimization, the engineered strain achieved a TdR yield of 11.10 g / L in 5 L fermentation, with a yield of 0.23 g / (L·h) (Yao et al., 2024). In Escherichia coli, its own nucleoside transport system mainly relies on transport proteins such as NupC and NupG, but experimental results show that the transport efficiency for TdR is low and lacks specificity (Yang et al., 2017). Summary of the Invention

[0006] The purpose of this invention is to provide a heterotransporter protein YdhP and its application in the production of β-thymidine in Escherichia coli, in order to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is the application of the ydhP gene or its encoded protein in promoting the production of β-thymidine by microorganisms, wherein the nucleotide sequence of the ydhP gene is shown in SEQ ID NO.6 and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.7.

[0008] The second technical solution of the present invention is a recombinant expression vector that promotes the production of β-thymidine by microorganisms, which is constructed with pEZ07 as the backbone and contains the ydhP gene.

[0009] The third technical solution of the present invention is an engineered bacterium that produces high levels of β-thymidine. It uses Escherichia coli W3110 as the starting strain, knocks out the TdR degradation genes deoA, tdk, and udp, and integrates the TdR operon NrdA into W3110, and includes the recombinant expression vector.

[0010] The fourth technical solution of the present invention is the application of the recombinant expression vector or the engineered bacteria in the preparation of β-thymidine.

[0011] The fifth technical solution of the present invention is a method for preparing β-thymidine, which utilizes the engineered bacteria to produce β-thymidine through fermentation under IPTG induction.

[0012] Based on the above technical solution, the present invention has the following technical effects: This invention introduces Ralstonia mannitolilytica The heterotransporter ydhP, derived from this strain, effectively promotes the extracellular secretion of β-thymidine and reduces intracellular feedback regulation. In shake-flask fermentation, it increased β-thymidine yield to 1.6 g / L (an increase of approximately 45%), while reducing the byproduct deoxyuridine by approximately 50%. In a 5 L fermenter, the yield reached 20 g / L, a significant increase compared to the control strain (12 g / L), providing an efficient and stable engineered strain and fermentation process for the industrial production of β-thymidine. Attached Figure Description

[0013] Figure 1 This is a chromatogram of HPLC peaks.

[0014] Figure 2 This is the result of fermentation of transport proteins. Here, ydhP represents a transport protein derived from the large intestine, and RaydhP represents... Ralstonia mannitolilytica, Ra The source is the transport protein ydhP. Detailed Implementation

[0015] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0016] This invention provides the application of the ydhP gene or its encoded protein in promoting the production of β-thymidine by microorganisms. The nucleotide sequence of the ydhP gene is shown in SEQ ID NO.6, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.7.

[0017] This invention also provides a recombinant expression vector that promotes the production of β-thymidine by microorganisms, which is constructed with pEZ07 as the backbone and contains the ydhP gene.

[0018] This invention also provides an engineered bacterium that produces high levels of β-thymidine. The bacterium is Escherichia coli W3110 as the starting strain. The degradation genes deoA, tdk, and udp of TdR are knocked out. At the same time, the TdR operon NrdA is integrated into W3110, and the recombinant expression vector is also included.

[0019] The present invention also provides the application of the recombinant expression vector or the engineered bacteria in the preparation of β-thymidine.

[0020] This invention also provides a method for preparing β-thymidine, which utilizes the engineered bacteria to produce β-thymidine through fermentation under IPTG induction.

[0021] In some specific implementations, the concentration of IPTG in the culture medium is 0.5-1 mM.

[0022] The ydhP transporter (protein_id=YBO73487.1) is a transporter not currently reported to be associated with β-thymidine transport. It belongs to the MFS (Major Facilitator Superfamily). Ralstonia mannitollytica The source of ydhP can further increase the fermentation yield of β-thymidine.

[0023] Experimental Method 1 of this invention: Method for verifying the production of L-tryptophan by recombinant strains through shake-flask fermentation 1. Reagents (1) LB medium: Each liter of medium contains 5g of yeast extract, 10g of sodium chloride, 10g of peptone, and deionized water to a final volume of 1L (by J. Sambrook, translated by Huang Peitang, Molecular Cloning Guide, 2002, 1595).

[0024] The above solution was sterilized by high-pressure steam at a temperature of 121°C for 20-30 minutes.

[0025] (2) Fermentation medium (per liter): 30g glucose, 200mL 5N-5 times salt solution, 1mL TM3 trace element solution (formula: 2.0g zinc chloride tetrahydrate, 2.0g calcium chloride hexahydrate, 2.0g sodium molybdate dihydrate, 1.9g copper sulfate pentahydrate, 0.5g boric acid, 100mL 1M hydrochloric acid, and deionized water to 1L), 10mg ferric citrate, 246mg magnesium sulfate heptahydrate, 111mg calcium chloride, 1μg thiamine, and sterile deionized water to 1L.

[0026] The 5N-5 times salt solution consists of 75.6g of disodium hydrogen phosphate dodecahydrate, 15g of potassium dihydrogen phosphate per liter, 2.5g of sodium chloride, and 25g of ammonium chloride, diluted to 1L with deionized water; the TM3 trace element solution consists of 2.0g of zinc chloride tetrahydrate, 2.0g of calcium chloride hexahydrate, 2.0g of sodium molybdate dihydrate, 1.9g of copper sulfate pentahydrate, 0.5g of boric acid, 100mL of 1M hydrochloric acid, diluted to 1L with deionized water.

[0027] The above solution was autoclaved at 121°C for 20-30 minutes. An empty shake flask was prepared simultaneously.

[0028] 2. Instruments: constant temperature shaker incubator.

[0029] 3. Method: Shake flask fermentation process: (1) Inoculate the recombinant strain into 3 mL of LB medium containing antibiotics and culture it in a shaker at 37°C and 250 rpm; (2) Take 500 μL of the seed after 16 h of culture and transfer it to 2 mL of LB liquid medium containing antibiotics and culture it in a shaker at 37°C and 250 rpm for 4 h; (3) Transfer all 2.5 mL of secondary seed into a shake flask containing 17.5 mL of fermentation medium and culture it in a shaker at 37°C and 250 rpm for 4 h; (4) Add IPTG to make the final concentration 1 mM, adjust the shaker temperature to 34°C, and continue to culture for about 20 h. Take 1 mL of fermentation liquid or dilute it with 1 mL to a suitable multiple and centrifuge it (12000 rpm, 1 min). Take the supernatant for detection. For the detection method, please refer to Experimental Method 3.

[0030] Experimental Method 2 of this Invention: Validation of the Production of β-thymidine by Recombinant Strains in a 5L Fermenter 1. Reagents Fermentation medium (per liter): 50g glucose, 10g ammonium sulfate, 10g sodium chloride, 5g potassium dihydrogen phosphate, 1g magnesium sulfate heptahydrate, 105mg calcium chloride, 10mg zinc chloride, 1mL TM3 trace element solution, 94mg ferric citrate, and diluted with deionized water.

[0031] The TM3 trace element solution consists of 1.31 g zinc chloride, 1.01 g calcium chloride, 1.18 g ammonium molybdate tetrahydrate, 3.9 g copper sulfate, 7.5 g manganese sulfate monohydrate, 10 mL 1M hydrochloric acid, and diluted to 1 L with deionized water.

[0032] Prepare 2L of fermentation medium for a 5L fermenter, with a feed medium containing 500g of glucose per liter.

[0033] 2. Fermentation process First, prepare the seed culture. Pick single clones from LB agar plates and incubate them overnight at 37°C in LB tubes containing antibiotics. Inoculate 2% (v:v) of the culture into a 500 mL shake flask containing 100 mL of LB medium and incubate at 37°C for 4 hours until the OD reaches 1.5–2. Then, inoculate 5% (v:v) of the culture into a 5 L fermenter containing 2 L of fermentation medium and incubate at 37°C. Control the pH to 6.9 with ammonia and maintain dissolved oxygen at 30% by coupling the feed speed with dissolved oxygen. Once dissolved oxygen rebounds, fix the feed speed and maintain dissolved oxygen levels between 30% and 45%. After 6 hours of fermentation, when the OD reaches 15–20 from 600, add IPTG to a final concentration of 0.5 mM for induction. Lower the temperature to 34°C and begin testing after 24 hours of fermentation. The detection method is described in Experimental Method 3.

[0034] Experimental Method 3: HPLC determination of β-thymidine in fermentation broth The fermentation broth was diluted with methanol to a suitable ratio, centrifuged and filtered through a 0.22 μm filter membrane, and detected by high performance liquid chromatography (HPLC).

[0035] The HPLC parameters were as follows: SB-AQ column (4.6*150*5µm) was used. The mobile phase consisted of: A: 2.73g potassium dihydrogen phosphate diluted to 1L with water, pH adjusted to 3.3 with phosphoric acid; B: methanol, initial ratio A:B = 100:0; column flow rate: 1mL / min; column temperature: 30℃; wavelength: 209nm; injection volume: 2µl (after 4-fold dilution); detection time: 15min. The elution gradient was: mobile phase ratio: methanol 0% for 0.1-2 minutes; methanol ratio increased from 0% to 35% for 2-9 minutes, maintained for 11 minutes, then decreased from 35% to 0% for 11-11.5 minutes. Detection was performed using a UV detector at 209nm. The initial mobile phase flow rate was 1.0mL / min; the loading volume of the fermentation broth was 2µL; column temperature: 30℃. The peak time for β-thymidine was 8.0min, and the peak time for deoxyuridine was 2.7min. The HPLC chromatograms are shown below. Figure 1 As shown.

[0036] Example 1 Construction and host detection of transporter expression libraries Our laboratory selected one through searching, querying, and comparison. Ralstonia mannitolilytica The transporter protein ydhP (protein_id=ANA34848.1) was used as the source. Primers were designed and the protein was seamlessly cloned into the low-copy vector pEZ07 (vector pEZ07 is the same as in Chinese patent application number: 201510093004.3) to obtain a transporter protein expression plasmid pHI468. Taking the construction of pHI468 (pEZ07-cmk-RaydhP-folA-gcvTHP) as an example: In the previous control phase, pHE291 (pEZ07-cmk-ydhP-folA-gcvTHP) was constructed. Using the low-copy expression plasmid pEZ07 as a template, the plasmid was digested with restriction endonucleases NcoI / NcoII. Using the W3110 genome as a template, the gene fragment shown in SEQ ID NO.1 was amplified using primer pairs YHI-980 / YHE-853 (primer pairs are shown in Table 1).

[0037] Using the control plasmid pHE291 (pEZ07-cmk-ydhP-folA-gcvTHP) as a template, gene fragment 1 (SEQ ID NO.2) and gene fragment 3 (SEQ ID NO.4) were amplified using primer pairs YHI-980 / YHI-981 and YHI-1000 / YHI-985, respectively. Using the synthesized gene as a template, gene fragment 2 (SEQ ID NO.3) was amplified using primer pairs YHI-998 / YHI-999 (primer pairs are shown in Table 1).

[0038] Gene fragment 1 sequence (SEQ ID NO.2):

[0039]

[0040]

[0041] A fragment of 1059 bp was obtained and no impurities were observed on electrophoresis. The fragment was then directly purified by column chromatography (Jerui Gel Purification Kit, Shanghai Jerui Biotechnology Co., Ltd.).

[0042]

[0043] The obtained fragment was used to construct EZ clones by digesting and recovering the pEZ07 vector fragment with NcoI / NcoII at a nanomolar ratio of 1:2 (GBclonart Seamless Cloning Kit, Suzhou Shenzhou Gene Co., Ltd.). The recombinant cloning reaction solution was incubated in a 45℃ water bath for 30 min, then transferred to ice and placed for 5 min. TG1-transfected competent cells were added, mixed, and placed for 5 min. After heat shock at 42℃ for 2 min, the cells were placed on ice for 2 min, and then 800 μL of LB recovery medium was added. After recovery and culture for 1 h, the cells were centrifuged and plated on LB plates containing 100 mg / L spectinomycin. The next day, clones were picked and cultured overnight. The plasmid was extracted and verified by enzyme digestion. The final plasmid pEZ07-cmk-RaydhP-folA-gcvTHP was constructed and numbered pHI468.

[0044] The gene sequence of RaydhP is SEQ ID NO.6, and it was then transformed into host SHI53 for shake-flask fermentation.

[0045] Table 1 Primers for construction

[0046]

[0047] SEQ ID NO.7: MFGIGTTEFSPMGLLPVIAEGVHVSIPQAGMLISAYAIGVMLGAPVMTLLLARWPRRSALIALMSIFTLGNLLSAIAPNYTTLLLARLVTSLNHGAFFGLGSVVAASLVPREKQASAVATMFMGLTIANVGGVPAATWLGQMIGWRMSFMATASLGLIAIAGLFAALPKGDAGK MPNLRAELRVLTRPVVVGALLTTVLGAGAMFTLYTYVAPTLAQLTGATPAFVTAMLVLIGVGFSIGNMAGGRLADRSLDGSLIGFLVLLIATMLAFPVLAKTHAGAAAALLVWGIATFAVVPPLQMRVMRAAAEAPGLASSINVGAFNLGNALGAAAGGAVISAGFGYAAVPVAGALI*.

[0048] This embodiment uses *Escherichia coli* W3110 (ATCC27325) (genotype: F-mcrAmcrB IN(rrnD-rrnE)1lambda-) as the base strain. The TdR degradation genes—thymidine phosphorylase deoA (Protein_id=XUW83941.1), thymidine kinase tdk (Protein_id=XUW82731.1), and uridine phosphorylase udp (Protein_id=UKW15609.1)—were knocked out. Simultaneously, the TdR operon (NrdA, GeneID:1258795, NrdB, Protein_id=QPI17454.1, and NrdC, Protein_id=CAM1377120.1)—was integrated into W3110 to obtain the TdR genetically engineered strain SHI53, which is classified and named *Escherichia coli*. Escherichia coli ).

[0049] The transporter-associated plasmid pHI468 constructed above was transformed into host SHI53, resulting in one recombinant strain of each transporter protein. These strains, along with the control strain SHI53 / pHE291, were subjected to shake-flask screening to identify transporters that could help increase TdR production.

[0050] Example 2 The transporter protein ydhP from different sources was initially screened using the shake-flask fermentation comparison method in Experiment 1: Recombinant strains containing transporters from different sources and the control strain SHI53 / pHE291 were inoculated into LB tubes containing 100 mg / L spectinomycin. 500 μL of the overnight cultured seed culture was transferred to 2 mL of LB liquid medium containing antibiotics and cultured at 37°C and 250 rpm for 4 h. The culture was then transferred to shake flasks containing 17.5 mL of fermentation medium and cultured at 37°C and 250 rpm for 4 h. IPTG was added to induce incubation at 34°C overnight, followed by further incubation for approximately 20 h. 0.4 mL of fermentation broth was diluted 3-fold with 0.8 mL of methanol, centrifuged at 12000 rpm for 1 min, and the supernatant was collected for analysis. The analysis method is detailed in Experiment 3. Four clones from each strain were selected for parallel fermentation, and the average value was taken.

[0051] The initial screening results showed: Ralstonia mannitolilytica The transporter protein of ydhP source is higher than Escherichia coli The yield of TdR from ydhP was significantly increased, reaching 1.6 g / L in shake flasks, an increase of approximately 45%, while the yield of the byproduct deoxyuridine was reduced by approximately 50%, demonstrating a clear advantage. Results are attached. Figure 2 .

[0052] Example 3 Validation of superior transport proteins in fermentation tanks A recombinant strain with a dominant transporter protein, screened according to the 5L fermenter fermentation process of Experimental Method 2, was used for fermentation verification to confirm the effect of transporter protein overexpression on TdR yield. The results showed that after 72 hours of fermentation, the TdR yield of the control strain SHI53 / pHE291 was around 12 g / L, while deoxyuridine remained fluctuating at 1 g / L. Ralstonia mannitolilytica The recombinant strain SHI53 / pHI468, which overexpresses the transporter protein of ydhP, has a stable fermentation yield. The yield of deoxyuridine during fermentation is less than 1 g / L, and the final accumulation of TdR after 72 h of fermentation is about 20 g / L.

[0053] Results from the 5L fermenter showed that the TdR yield of the transporter protein ydhP from E. coli was 12 g / L. ， and Ralstonia mannitolilytica The ydhP transporter protein from E. coli was replaced with ydhP from the source, and the yield in the fermenter was increased to 20 g / L. The increased yield may effectively transport TdR to the extracellular space, reducing various in vivo regulation, thereby significantly increasing the yield of TdR.

[0054] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of the ydhP gene or its encoded protein in promoting the production of β-thymidine in microorganisms, characterized in that, The nucleotide sequence of the ydhP gene is shown in SEQ ID NO.6, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

7.

2. A recombinant expression vector that promotes the production of β-thymidine by microorganisms, characterized in that, It is constructed using pEZ07 as a backbone and contains the ydhP gene as described in claim 1.

3. An engineered bacterium that produces high levels of β-thymidine, characterized in that, Using Escherichia coli W3110 as the starting strain, the TdR degradation genes deoA, tdk, and udp were knocked out. At the same time, the TdR operon NrdA was integrated into W3110, and the recombinant expression vector described in claim 2 was included.

4. The application of the recombinant expression vector as described in claim 2 or the engineered bacteria as described in claim 3 in the preparation of β-thymidine.

5. A method for preparing β-thymidine, characterized in that, Using the engineered bacteria described in claim 3, β-thymidine is produced by fermentation under IPTG induction.

6. The preparation method according to claim 5, characterized in that, The concentration of IPTG in the culture medium is 0.5-1 mM.

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