The invention relates to a method for producing 2apos; engineering strain of-deoxyadenosine as well as preparation method and application of engineering strain

Abstract

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to an engineering strain for producing 2 '-deoxyadenosine as well as a preparation method and application thereof, and the engineering strain expresses ketodeoxyadenosine reductase gene cns1 and adenylate dephosphatase gene cns2. The engineering strain prepared by the preparation method disclosed by the invention has obvious advantages in the aspect of producing the 2 '-deoxyadenosine, the yield and the efficiency of synthesizing the 2'-deoxyadenosine by the corynebacterium glutamicum are improved to different degrees by optimizing conditions of a fermentation culture medium, and the yield of the 2 '-deoxyadenosine on a 6L bioreactor reaches 702.3 mg / L.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial genetic engineering, and particularly relates to an engineered strain for producing 2'-deoxyadenosine, a preparation method thereof, and an application thereof. Background Art

[0002] 2'-Deoxyadenosine (dAR) is one of the basic structural units of deoxyribonucleic acid (DNA) and has important biological functions and medicinal development values. In recent years, studies have shown that 2'-deoxyadenosine can not only inhibit glucose-induced insulin secretion, but also promote insulin release by regulating specific phosphodiesterase inhibitors or adenylate cyclase activators. In addition, as an important intermediate for antiviral, anti-tumor, and anti-AIDS drugs, it also shows broad application prospects. At present, the preparation methods of 2'-deoxyadenosine mainly include DNA degradation method, chemical synthesis method, and biocatalytic method. However, the traditional DNA degradation method has poor stability and low yield; the chemical synthesis method has complex steps, high raw material costs, environmental pollution, etc., which cannot meet market requirements; although the biocatalytic method is green and environmentally friendly, there are still problems such as low substrate concentration and high production costs, which limit its large-scale application. Therefore, there is an urgent need to develop a green, economical, and efficient microbial fermentation production process.

[0003] Corynebacterium glutamicum ( Corynebacterium glutamicum ) is an industrial microorganism widely recognized as a "Generally Recognized As Safe (GRAS)" strain, which has the advantages of strong robustness, no endotoxin, and mature genetic manipulation, and is widely used in the industrial production of important metabolites such as amino acids and organic acids. However, there is no report on the synthesis of nucleoside analogs such as cordycepin and 2'-deoxyadenosine using Corynebacterium glutamicum as a chassis cell. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide an engineered strain for producing 2'-deoxyadenosine, a preparation method thereof, and an application thereof.

[0005] To achieve the above technical purpose, the technical solution adopted by the present invention is as follows: The present invention provides an engineered strain for producing 2'-deoxyadenosine, and the engineered strain expresses a ketodeoxyadenosine reductase gene cns1 and an adenosine dephosphatase gene cns2 .

[0006] Further, cns1 and cns2 the genes both originate from Cordyceps militaris (Latin name: Cordyceps militaris ), cns1The nucleotide sequence of the gene is shown as SEQ ID NO.1; cns2 The nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0007] The present invention also provides a method for preparing the above-mentioned engineered strain for producing 2'-deoxyadenosine, including: connecting the ketodeoxyadenosine reductase gene cns1 and the adenylate dephosphatase gene cns2 to an expression plasmid, and then transforming it into the host Corynebacterium glutamicum.

[0008] Preferably, the expression plasmid includes but is not limited to pEC-XK99E, pXMJ19, etc. As long as the plasmids in the prior art can satisfy the expression of the ketodeoxyadenosine reductase gene cns1 and the adenylate dephosphatase gene cns2 in Corynebacterium glutamicum are applicable to the present invention.

[0009] In some embodiments of the present invention, the expression plasmid is pXMJ19.

[0010] In some embodiments of the present invention, the above preparation method includes: first finding the gene sequences of the ketodeoxyadenosine reductase gene cns1 and the adenylate dephosphatase gene cns2 from Cordyceps militaris on NCBI, and their sequences are shown as SEQ ID NO:1 and SEQ ID NO:2. According to the codon usage preference of Corynebacterium glutamicum, after codon optimization of cns1 and cns2 sequences, the gene sequences are artificially synthesized into the pUC57 plasmid to obtain the recombinant plasmid pUC57-cns1-cns2. Using the pUC57-cns1-cns2 plasmid as a template, cns1 and cns2 genes are amplified, and the obtained gene fragments are ligated to the pXMJ19 plasmid digested by Eco R I / Hin d III, and the correct recombinant plasmid pXMJ19-cns1-cns2 is screened out. The correct recombinant plasmid is then transformed into the host Corynebacterium glutamicum to obtain the recombinant strain Cg-dAR.

[0011] The present invention further provides the application of the above-mentioned engineered strain for producing 2'-deoxyadenosine in microbial fermentation. The main purpose of this application is to produce 2'-deoxyadenosine using the engineered strain.

[0012] Preferably, the fermentation process includes: first activating the engineered strain on a plate until monoclonal colonies grow, picking the monoclonal colonies and culturing them first in the LBG medium, and then transferring them to the seed medium for culturing until the optical density (OD562 When the value reaches between 2 and 10, a seed solution is obtained. The seed solution is inoculated into a fermentation medium at an inoculum size of 2% - 20% v / v and fermented to obtain a fermentation broth containing a fermentation product.

[0013] More preferably, the formula of the seed medium is: 15 - 35 g / L sucrose, 5 - 15 g / L peptone, 1 - 10 g / L yeast powder, 1 - 10 g / L ammonium sulfate, 0.1 - 2 g / L magnesium sulfate heptahydrate, 1 - 10 g / L potassium dihydrogen phosphate, 5 - 15 g / L dipotassium hydrogen phosphate, 1 - 10 g / L urea, and the solvent is water; the culture conditions in the seed medium are: culture at 28 - 34 °C and 200 - 250 rpm for 4 - 8 h.

[0014] More preferably, the components of the fermentation medium include a carbon source, a nitrogen source, inorganic salts, and cofactors, among which: The carbon source includes any one or a combination of two of glucose and molasses; preferably a combination of 20 - 50 g / L glucose and 10 - 30 g / L molasses, more preferably a combination of 30 g / L glucose and 20 g / L molasses; The nitrogen source includes any one or a combination of several of yeast extract, ammonium sulfate, urea, corn steep liquor, peptone, and soybean meal powder; preferably a combination of 1 - 5 g / L corn steep liquor and 10 - 20 g / L ammonium sulfate, more preferably a combination of 3 g / L corn steep liquor and 12 g / L ammonium sulfate; The inorganic salts include H2PO4 - 、K + 、Mg 2+ and Mn 2+ and any one or a combination of several of them; preferably a combination of 0.1 - 2 g / L phosphoric acid, 0.5 - 1.5 g / L magnesium sulfate heptahydrate, 0.1 - 5 g / L potassium chloride, and 50 - 200 mg / L manganese sulfate, more preferably a combination of 0.4 g / L phosphoric acid, 0.87 g / L magnesium sulfate heptahydrate, 0.53 g / L potassium chloride, and 100 mg / L manganese sulfate; The cofactors include any one or a combination of several of glycine, adenine, adenosine, CuSO4, ZnSO4, and biotin; preferably a combination of 1 - 10 g / L glycine, 1 - 5 g / L adenine, and 0.1 - 2 mg / L biotin, more preferably a combination of 5 g / L glycine, 2.5 g / L adenine, and 1 mg / L biotin; The fermentation culture conditions in the fermentation medium are: temperature 28 - 34 °C, pH 6 - 7, aeration rate 2 - 8 vvm, and initial stirring speed 400 - 700 rpm.

[0015] More preferably, when the glucose concentration is lower than 1 g / L during the fermentation process, the dissolved oxygen value is controlled at about 30% through the linkage control of dissolved oxygen and rotation speed, and a fed-batch sugar medium is added to maintain the glucose concentration at 1-2 g / L until the end of fermentation; when the ammonium sulfate concentration is lower than 1 g / L during the fermentation process, a fed-batch nitrogen medium is added to maintain the ammonium sulfate concentration at 1-1.5 g / L until the end of fermentation.

[0016] More preferably, the above application further includes: adding isopropyl-β-D-thiogalactoside (IPTG) during the fermentation process; preferably adding 0.01-1 mM IPTG during the fermentation process; more preferably adding 1 mM IPTG.

[0017] More preferably, the fermentation process includes: streaking the constructed engineering strain on a plate and culturing it in an incubator at 30 °C for 24 h until monoclonal colonies grow, picking the monoclonal colonies for activation and inoculating them into LBG medium, culturing them in a shaker at 30 °C and 220 rpm for 12 h. After activation, inoculate them into a baffled shake flask containing seed medium at an inoculation amount of 1% v / v, and culture them at 30 °C and 220 rpm until the OD 562 is 2-10 to obtain a seed solution. Inoculate the seed solution into a bioreactor containing fermentation medium. The formula of the used fermentation medium is: 30 g / L glucose, 20 g / L molasses, 3 g / L corn steep liquor, 12 g / L ammonium sulfate, 0.4 g / L phosphoric acid, 0.87 g / L magnesium sulfate heptahydrate, 0.53 g / L potassium chloride, 100 mg / L manganese sulfate, 1 mg / L biotin, 5 g / L glycine, and 2.5 g / L adenine. The culture conditions are: the temperature is 30 °C, the pH is maintained at 6.5 using ammonia water, the initial aeration rate is 6 vvm, and the initial stirring speed is 550 rpm. When the glucose concentration is lower than 1 g / L during the fermentation process, the dissolved oxygen value is controlled at 30% through the linkage control of dissolved oxygen and rotation speed, and a fed-batch sugar medium is added to maintain the glucose concentration at 1-2 g / L until the end of fermentation; when the ammonium sulfate concentration is lower than 1 g / L during the fermentation process, a fed-batch nitrogen medium is added to maintain the ammonium sulfate concentration at 1-1.5 g / L until the end of fermentation. Among them, the formula of the fed-batch sugar medium is 600 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 1 g / L choline chloride, 0.1 g / L vitamin B1, 1 g / L potassium dihydrogen phosphate, and 1 mg / L biotin; the fed-batch nitrogen medium has a formula of 450 g / L ammonium sulfate.

[0018] Beneficial effects:

[0019] (1) In the present invention, the ketodeoxyadenosine reductase gene is expressed through a plasmid in Corynebacterium glutamicum cns1 and the adenylate dephosphatase gene cns2, the yield of 2'-deoxyadenosine reached 187.6 mg / L.

[0020] (2) By optimizing the fermentation medium conditions, the present invention improved the yield and efficiency of Corynebacterium glutamicum in synthesizing 2'-deoxyadenosine to varying degrees. Finally, when the initial pH of the fermentation medium was 6.5 and 5 g / L of glycine and 2.5 g / L of adenine were added simultaneously, the yield of 2'-deoxyadenosine was increased. The yield in shake flask fermentation reached 239.3 mg / L, and the yield of 2'-deoxyadenosine in a 6 L bioreactor reached 702.3 mg / L. Description of the Drawings

[0021] The attached drawings in the specification, which form a part of this application, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0022] Figure 1 It is the map of the expression plasmid pXMJ19-cns1-cns2, with a size of 10017 bp.

[0023] Figure 2 It is a graph showing the change of 2'-deoxyadenosine production by the engineered strain Cg-dAR over time.

[0024] Figure 3 It is the HPLC detection spectrum of 2'-deoxyadenosine in the fermentation broth of the engineered strain Cg-dAR.

[0025] Figure 4 It is the mass spectrometry identification graph of 2'-deoxyadenosine in the fermentation broth of the engineered strain Cg-dAR.

[0026] Figure 5 It is a schematic diagram for optimizing the concentration of adenine added to the fermentation medium for producing 2'-deoxyadenosine.

[0027] Figure 6 It is a schematic diagram for optimizing the concentration of adenosine added to the fermentation medium for producing 2'-deoxyadenosine.

[0028] Figure 7 It is a schematic diagram for optimizing the initial pH of the fermentation medium for producing 2'-deoxyadenosine.

[0029] Figure 8 It is a schematic diagram for optimizing the concentration of glycine added to the fermentation medium for producing 2'-deoxyadenosine.

[0030] Figure 9 It is a schematic diagram for optimizing the concentration of ZnSO4 added to the fermentation medium for producing 2'-deoxyadenosine.

[0031] Figure 10 It is a schematic diagram for optimizing the concentration of CuSO4 added to the fermentation medium for producing 2'-deoxyadenosine.

[0032] Figure 11 It is a graph showing the change of the optimal combination fermentation production of 2'-deoxyadenosine over time by the engineered strain Cg-dAR using orthogonal experiments.

[0033] Figure 12 It is a graph showing the change of the fermentation production of 2'-deoxyadenosine over time by the engineered strain Cg-dAR in a 6L bioreactor. Detailed implementation manners

[0034] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited do not limit the present invention.

[0035] In the following embodiments, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0036] In the following embodiments, the plasmid pXMJ19 was purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0037] In the following embodiments, the host Corynebacterium glutamicum and the original strain of Corynebacterium glutamicum are both Corynebacterium glutamicum ATCC 13032.

[0038] In the following embodiments, the LBG plate (lysogeny broth medium supplemented with glucose) has the following formula: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 10 g / L glucose. 1.8% (v / v) agar powder was added to the solid medium.

[0039] The seed medium has the following formula: 25 g / L sucrose, 10 g / L peptone, 5 g / L yeast extract, 5 g / L ammonium sulfate, 1 g / L magnesium sulfate heptahydrate, 5 g / L potassium dihydrogen phosphate, 12 g / L dipotassium hydrogen phosphate, 5 g / L urea.

[0040] The fermentation medium includes carbon source, nitrogen source, inorganic salts and cofactors: the carbon source is a combination of 30 g / L glucose and 20 g / L molasses; the nitrogen source is a combination of 3 g / L corn steep liquor and 12 g / L ammonium sulfate; the inorganic salts are a combination of 0.4 g / L phosphoric acid, 0.87 g / L magnesium sulfate heptahydrate, 0.53 g / L potassium chloride, 100 mg / L manganese sulfate; the cofactors are a combination of 5 g / L glycine, 2.5 g / L adenine and 1 mg / L biotin.

[0041] First, according to the codon usage preference of Corynebacterium glutamicum, for the cns1 gene andcns2 After codon optimization of the gene sequences (the sequences are shown in SEQ ID NO:1 and SEQ ID NO:2), the synthetic gene was inserted into the pUC57 plasmid to obtain the recombinant plasmid pUC57-cns1-cns2 for standby.

[0042] Example 1: Construction of the pXMJ19-cns1-cns2 expression plasmid Using the pUC57-cns1-cns2 plasmid as a template and primer 1 / primer 2 (Table 1) as the upstream and downstream primers, the gene was amplified by PCR using 2×Phanta Max Master Mix high-fidelity polymerase. cns1 Using the pUC57-cns1-cns2 plasmid as a template and primer 3 / primer 4 as the upstream and downstream primers, the gene was amplified by PCR using 2×Phanta Max Master Mix high-fidelity polymerase. cns2 The PCR program was: 95°C for 30 s, 55°C for 15 s, 72°C for 30 s / kb, for 30 cycles. The PCR products were purified and verified by 1.5% agarose gel electrophoresis.

[0043] Table 1 Sequences of primers 1-4

[0044] The two PCR products obtained above were subjected to a one-step cloning ligation reaction with the pXMJ19 plasmid fragment treated with restriction enzymes Hin d III / Eco R I. After screening, the recombinant plasmid pXMJ19-cns1-cns2 for expressing the ketodeoxyadenosine reductase gene cns1 and the adenylate dephosphatase gene cns2 was obtained. The plasmid map is shown in Figure 1 .

[0045] Example 2: Construction of engineering strains The expression plasmid pXMJ19-cns1-cns2 constructed in Example 1 was electrotransformed (1800 V, 25 μF, 200Ω, 2 mm) into Corynebacterium glutamicum ATCC 13032. After culturing on an LBG plate containing 6.5 μg / mL chloramphenicol for 2-3 days, the transformants were screened, and then colony PCR verification was carried out to obtain the expression of the ketodeoxyadenosine reductase gene cns1 and the adenylate dephosphatase gene cns2The engineered strain was named Cg-dAR. Primers 5 and 6 were used for PCR verification to confirm that the genes on the expression plasmid pXMJ19-cns1-cns2 had entered the engineered strain Cg-dAR. The sequences of primers 5 and 6 are shown in Table 2.

[0046] Table 2 Sequences of Primers 5 and 6

[0047] Example 3: Identification of Fermentation Products of Engineered Strains (1) The engineered strain Cg-dAR constructed in Example 2 was streaked on a plate and cultured in an incubator at 30 °C for 24 h until monoclonal colonies grew. A monoclonal colony was picked and inoculated into 5 mL of LBG medium and cultured in a shaker at 30 °C and 220 rpm for 12 h. After activation, it was inoculated into a 500 mL baffled shake flask containing 50 mL of seed medium at an inoculation amount of 1% v / v and cultured at 30 °C and 220 rpm until the OD 562 was about 8 to obtain a seed solution.

[0048] (2) The seed solution obtained in step (1) was inoculated into a 500 mL baffled shake flask containing 50 mL of fermentation medium at 10% v / v and cultured at 30 °C and 220 rpm for 72 h to obtain a fermentation broth. Subsequently, the fermentation broth was centrifuged at 12,000 rpm for 5 min to collect the supernatant, and the impurities were removed by filtration through a 0.22 μm filter membrane for subsequent detection and analysis. During the fermentation of the recombinant strain Cg-dAR, 6.5 μg / mL of chloramphenicol needed to be added to the medium; in addition, IPTG with a final concentration of 1 mM was added during the logarithmic growth phase to induce the expression of the target gene.

[0049] (3) Detection of fermentation products: HPLC detection was performed using an Agilent 1260 Infinity II high-performance liquid chromatograph equipped with a variable wavelength detector (VWD) from Agilent Technologies. The chromatographic column was Poroshell 120 EC-C18, the mobile phase was acetonitrile and 0.3% trifluoroacetic acid (2:98, v / v) for isocratic elution, the flow rate was 0.6 mL / min, the column oven was set at 40 °C, and the signals of 2'-deoxyadenosine and other substances were detected at an absorbance of 260 nm. LC-MS / MS analysis was sent to Nanjing Jiangbei New Area Biomedical Public Service Platform Co., Ltd. (Nanjing, China) for analysis.

[0050] From Figure 3 and Figure 4 it can be seen that the expressed genes cns1 and cns2The peak positions of the engineered strain and the 2'-deoxyadenosine standard were consistent, and there was no peak at the corresponding time for the control strain containing the empty vector. The LC-MS / MS results confirmed that the molecular weight of this peak was the same as that of the 2'-deoxyadenosine standard sample, and this peak was the peak of 2'-deoxyadenosine. The results showed ( Figure 2 ), and the 2'-deoxyadenosine production of the engineered strain was 187.6 mg / L. Subsequently, the fermentation conditions of the engineered strain Cg-dAR were optimized.

[0051] Example 4: Optimization of fermentation medium The medium is the necessary nutrient substrate for the growth, reproduction of microorganisms and the synthesis of various metabolites. The components of the medium can significantly regulate the growth rate of the bacterial cells, the selectivity of the metabolic pathway and the yield of the target metabolite. Therefore, the components and culture conditions of the fermentation medium were systematically optimized in this application. The single-factor control variable method was adopted for optimization. The culture was carried out at 30 °C and 220 rpm for 72 h, and the 2'-deoxyadenosine production was measured.

[0052] (1) Different concentrations of the precursor adenine (0.1 g / L, 1 g / L, 2.5 g / L, and 5 g / L) were added to the fermentation medium, and the remaining culture conditions remained unchanged. Each combination was set with three replicates.

[0053] The results were as Figure 5 shown. When the concentration of the precursor adenine was 2.5 g / L, the 2'-deoxyadenosine production was the highest.

[0054] (2) Different concentrations of the precursor adenosine (0.1 g / L, 1 g / L, 2.5 g / L, and 5 g / L) were added to the fermentation medium, and the remaining culture conditions remained unchanged. Each combination was set with three replicates.

[0055] The results were as Figure 6 shown. When the concentration of the precursor adenosine was 2.5 g / L, the 2'-deoxyadenosine production was the highest.

[0056] (3) Different initial fermentation medium pH values (4.0, 5.0, 6.0, 7.0) were set, and the remaining culture conditions remained unchanged. Each combination was set with three replicates.

[0057] The results were as Figure 7 shown. When pH = 6.0, the 2'-deoxyadenosine production was the highest. Under the condition of pH = 7.0, the production was not much different from that at pH = 6.0, indicating that both of these pH conditions were suitable for the synthesis of the product 2'-deoxyadenosine.

[0058] (4) Different concentrations of glycine (0 g / L, 5 g / L, 10 g / L, and 15 g / L) were added to the fermentation medium, and the remaining culture conditions remained unchanged. Each combination was set with three replicates.

[0059] The results are as Figure 8 shown. When the concentration of glycine added was 5 g / L, the yield of 2'-deoxyadenosine was the highest.

[0060] (5) Different concentrations of ZnSO4 (0.1 mM, 0.5 mM, 1 mM, and 2 mM) were added to the fermentation medium, and the remaining culture conditions remained unchanged. Each combination was set with three replicates.

[0061] The results are as Figure 9 shown. The addition of ZnSO4 did not significantly increase the yield of 2'-deoxyadenosine. When the concentration of ZnSO4 was 0.5 mM, the yield of 2'-deoxyadenosine was the highest.

[0062] (6) Different concentrations of CuSO4 (0.1 mM, 0.5 mM, 1 mM, and 2 mM) were added to the fermentation medium, and the remaining culture conditions remained unchanged. Each combination was set with three replicates.

[0063] The results are as Figure 10 shown. When the concentration of CuSO4 added was 1 mM, the yield of 2'-deoxyadenosine was the highest.

[0064] Example 5: Optimization of the Optimal Culture Conditions for 2'-Deoxyadenosine Fermentation by Orthogonal Experiment After optimizing the key parameters of 2'-deoxyadenosine fermentation through the single-factor experiments in Example 4 above, an orthogonal experiment with three factors and three levels was further designed to systematically evaluate the effects of various factors on the yield of 2'-deoxyadenosine. A total of 9 groups of orthogonal experiments were conducted, and each group had 3 parallel experiments. Fermentation experiments were carried out according to the parameters provided in Table 3. After 72 h of fermentation, HPLC was used to quantitatively analyze the content of 2'-deoxyadenosine in the supernatant of the fermentation broth.

[0065] Table 3 Orthogonal Experiment Factor-Level Table

[0066] Table 4 Orthogonal Experiment Scheme and Results

[0067] The fermentation results are shown in Table 4. The range R of adenine is the largest at 28.80, indicating that it has the most significant effect on the dAR yield. Followed by glycine content, the initial pH has a relatively small impact. The optimal combination is 2.5 g / L adenine, initial pH = 6.5, and 5 g / L glycine. The optimal culture conditions obtained from the experiment were verified, and the results are as Figure 11 shown. The optimized culture conditions increased the 2'-deoxyadenosine yield to 239.3 mg / L, a 27.6% increase compared to the non-optimized system.

[0068] Example 6: Production of 2'-deoxyadenosine by the engineered strain in a 6 L bioreactor The constructed engineered strain Cg-dAR was streaked on a plate and cultured in an incubator at 30 °C for 24 h until monoclonal colonies grew. A monoclonal colony was picked and inoculated into 5 mL of LBG medium and cultured in a shaker at 30 °C and 220 rpm for 12 h. After activation, it was inoculated into a 500 mL baffled shake flask containing 50 mL of seed medium at an inoculation amount of 1% v / v and cultured at 30 °C and 220 rpm until the OD 562 was about 8 to obtain the seed solution. 300 mL of the seed solution was inoculated into a 6 L bioreactor containing 1.8 L of fermentation medium. The formula of the fermentation medium used was: 30 g / L glucose, 20 g / L molasses, 3 g / L corn steep liquor, 12 g / L ammonium sulfate, 0.4 g / L phosphoric acid, 0.87 g / L magnesium sulfate heptahydrate, 0.53 g / L potassium chloride, 100 mg / L manganese sulfate, 1 mg / L biotin, 5 g / L glycine, and 2.5 g / L adenine. Culture conditions: The temperature was 30 °C, the pH was maintained at 6.5 using ammonia water, the initial aeration rate was 6 vvm, and the initial stirring speed was 550 rpm. When the glucose concentration was lower than 1 g / L during fermentation, the dissolved oxygen value was controlled at about 30% through the linkage control of dissolved oxygen and rotation speed, and a feeding sugar medium was added to maintain the glucose concentration at 1 - 2 g / L until the end of fermentation; when the ammonium sulfate concentration was lower than 1 g / L during fermentation, a feeding nitrogen medium was added to maintain the ammonium sulfate concentration at 1 - 1.5 g / L until the end of fermentation. Among them, the formula of the feeding sugar medium was: 600 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 1 g / L choline chloride, 0.1 g / L vitamin B1, 1 g / L potassium dihydrogen phosphate, and 1 mg / L biotin; the formula of the feeding nitrogen medium was: 450 g / L ammonium sulfate.

[0069] The results are as Figure 12 shown. The yield of 2'-deoxyadenosine in the 6 L bioreactor reached 702.3 mg / L.

[0070] The present invention provides a method for synthesizing 2'-deoxyadenosine using Corynebacterium glutamicum. There are many methods and approaches to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented using existing technologies.

Claims

1. An engineered strain for producing 2'-deoxyadenosine, characterized in that, The engineered strain expresses the ketodeoxyadenosine reductase gene in the host Corynebacterium glutamicum cns1 and the adenylate dephosphatase gene cns2 , cns1 and cns2 both genes are derived from Cordyceps militaris, cns1 the nucleotide sequence of the gene is as shown in SEQ ID NO.1; cns2 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

2. The preparation method of an engineered strain for producing 2'-deoxyadenosine according to claim 1, characterized in that, including: Connect the ketodeoxyadenosine reductase gene cns1 and the adenylic acid dephosphatase gene cns2 into an expression plasmid and then transform it into the host Corynebacterium glutamicum.

3. Use of an engineered strain for producing 2'-deoxyadenosine as claimed in claim 1 in microbial fermentation.

4. The application according to claim 3, characterized in that The fermentation process includes: First, activate the engineered strain on a plate until monoclonal colonies grow, pick the monoclonal colonies and culture them first in LBG medium, and then transfer them to a seed medium for culture until the optical density OD 562 value reaches between 2 and 10 to obtain a seed solution. Then, inoculate the seed solution into a fermentation medium at an inoculation amount of 2% - 20% v / v for fermentation to obtain a fermentation broth containing the fermentation product.

5. The application according to claim 4, wherein The formula of the seed medium is: 15 - 35 g / L sucrose, 5 - 15 g / L peptone, 1 - 10 g / L yeast powder, 1 - 10 g / L ammonium sulfate, 0.1 - 2 g / L magnesium sulfate heptahydrate, 1 - 10 g / L potassium dihydrogen phosphate, 5 - 15 g / L dipotassium hydrogen phosphate, 1 - 10 g / L urea, and the solvent is water; the culture conditions in the seed medium are: culture at 28 - 34 °C and 200 - 250 rpm for 4 - 8 h.

6. The application according to claim 4, wherein The components of the fermentation medium include a carbon source, a nitrogen source, inorganic salts and cofactors, where: The carbon source includes any one or a combination of two of glucose and molasses; The nitrogen source includes any one or a combination of several of yeast extract, ammonium sulfate, urea, corn steep liquor, peptone and soybean meal powder; The inorganic salts described include H2PO4 - , K + , Mg 2+ and Mn 2+ or a combination of any one or more of them; The cofactors include any one or a combination of several of glycine, adenine, adenosine, CuSO4, ZnSO4 and biotin; The fermentation culture conditions in the fermentation medium are: temperature 28 - 34 °C, pH 6 - 7, aeration rate 2 - 8 vvm, initial stirring speed 400 - 700 rpm.

7. The application according to claim 6, wherein When the glucose concentration is lower than 1 g / L during fermentation, the dissolved oxygen value is controlled at 30% by the linkage of dissolved oxygen and rotation speed, and a fed-batch sugar medium is added to maintain the glucose concentration at 1 - 2 g / L until the end of fermentation; when the ammonium sulfate concentration is lower than 1 g / L during fermentation, a fed-batch nitrogen medium is added to maintain the ammonium sulfate concentration at 1 - 1.5 g / L until the end of fermentation.

8. The application according to claim 4, characterized in that It also includes: Isopropyl-β-D-thiogalactoside (IPTG) is added during fermentation.

9. The application according to claim 4, wherein The fermentation process includes: streaking the constructed engineering strain on a plate and culturing it in an incubator at 30 °C for 24 h until monoclonal colonies grow. Pick a monoclonal colony for activation and inoculate it into LBG medium, and culture it in a shaker at 30 °C and 220 rpm for 12 h. After activation, inoculate it into a baffled shake flask containing seed medium at an inoculation amount of 1% v / v, and culture it at 30 °C and 220 rpm until OD 562 reaches 2 - 10 to obtain a seed solution. Inoculate the seed solution into a bioreactor containing fermentation medium. The formula of the fermentation medium used is: 30 g / L glucose, 20 g / L molasses, 3 g / L corn steep liquor, 12 g / L ammonium sulfate, 0.4 g / L phosphoric acid, 0.87 g / L magnesium sulfate heptahydrate, 0.53 g / L potassium chloride, 100 mg / L manganese sulfate, 1 mg / L biotin, 5 g / L glycine, and 2.5 g / L adenine. The culture conditions are: the temperature is 30 °C, and ammonia water is used to maintain the pH at 6.

5. The initial aeration rate is 6 vvm, and the initial stirring speed is 550 rpm. When the glucose concentration is lower than 1 g / L during fermentation, the dissolved oxygen value is controlled at 30% through the linkage control of dissolved oxygen and rotation speed, and a feeding sugar medium is added to maintain the glucose concentration at 1 - 2 g / L until the end of fermentation; when the ammonium sulfate concentration is lower than 1 g / L during fermentation, a feeding nitrogen medium is added to maintain the ammonium sulfate concentration at 1 - 1.5 g / L until the end of fermentation. Among them, the formula of the feeding sugar medium is 600 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 1 g / L choline chloride, 0.1 g / L vitamin B1, 1 g / L potassium dihydrogen phosphate, and 1 mg / L biotin; the formula of the feeding nitrogen medium is 450 g / L ammonium sulfate.

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