Method for increasing adenosine yield

Abstract

The present disclosure provides modified bacteria that produce adenosine wherein the following modifications are included as compared to the pre-modified bacteria: comprising a modification that has reduced phosphoenolpyruvate carboxykinase (pckA) activity and / or a modification that has reduced pyruvate kinase (pyk) activity, further comprising a modification that has reduced glucose-6-phosphate isomerase (pgii) activity, and comprising a modification that has reduced glucose-6-phosphate isomerase (pgii) activity, or comprising a modification that has reduced glucose-6-phosphate isomerase (pgii) activity, or a modification that has reduced glucose-6-phosphate isomerase (pgii) activity. In addition, methods of increasing adenosine production using the modified bacteria are also provided.

Description

Technical Field

[0001] This disclosure belongs to the field of microbial engineering technology, and in particular relates to a method for producing adenosine by using bacterial fermentation. Background Technology

[0002] Nucleosides are a general term for a class of glycosides. They are components of nucleic acids and nucleotides. Nucleosides are formed by the condensation of D-ribose or D2-deoxyribose with pyrimidine or purine bases. Nucleosides are generally colorless crystals, insoluble in common organic solvents, readily soluble in hot water, and have a melting point of 160–240℃. Nucleosides formed from D-ribose are called ribonucleosides, which participate in the composition of RNA; nucleosides formed from D-α-deoxyribose are called deoxyribonucleosides, which participate in the composition of DNA. D-ribose condenses with adenine, guanine, cytosine, thymine, or uracil to form the corresponding adenine ribonucleosides, guanine ribonucleosides, cytosine ribonucleosides, thymine ribonucleosides, and uracil ribonucleosides, which are abbreviated as adenosine (A), guanine (G), cytosine (C), thymine (T), and uridine (U), respectively. Adenosine is an endogenous nucleoside found throughout human cells. It can directly enter the myocardium, be phosphorylated to produce adenosine monophosphate, participate in myocardial energy metabolism, and also participate in the dilation of coronary arteries, increasing blood flow. Adenosine has physiological effects on the cardiovascular system and many other systems and tissues of the body. Besides being used as a specific drug for treating heart disease, adenosine is also an important intermediate in the synthesis of adenosine triphosphate (ATP), adenine, adenosine monophosphate, and vidarabine, and is widely used in the pharmaceutical industry. Currently, microbial fermentation is the main method for producing nucleosides, and the main microorganisms used include Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus pumilus.

[0003] Microbial engineering aims to modify microorganisms for engineered production through genetic and metabolic engineering techniques. Researchers typically conduct breeding and modification of bacterial strains by understanding the bacterial nucleotide metabolic pathways and regulatory mechanisms, gaining a deep understanding of the strain's genetic background and characteristics. Through metabolic engineering, they purposefully modify the strains to obtain high-yield nucleoside production strains with superior traits. However, the fermentation performance of current nucleoside-producing strains remains poor, and the nucleoside conversion rate is still low, failing to meet the demands of large-scale industrial production. To further improve adenosine conversion rate and yield, this disclosure provides a high-yield adenosine mutant strain, which can increase adenosine yield and conversion rate. Summary of the Invention

[0004] This disclosure provides a modified engineered bacterium for producing adenosine, wherein, compared with the strain before modification, it includes modifications that reduce the activity of phosphoenolpyruvate carboxykinase (pckA) and / or modifications that reduce the activity of pyruvate kinase (pyk).

[0005] In some embodiments, the modified engineered bacteria comprise a modification that reduces phosphoenolpyruvate carboxykinase (pckA) activity. In some embodiments, the modified engineered bacteria comprise a modification that reduces pyruvate kinase (pyk) activity. In some embodiments, the modified engineered bacteria comprise both modifications that reduce phosphoenolpyruvate carboxykinase (pckA) activity and modifications that reduce pyruvate kinase (pyk) activity.

[0006] In some embodiments, the modified engineered bacteria further comprises a modification that reduces the activity of glucose-6-phosphate isomerase (pgi), preferably, the modification that reduces the activity of glucose-6-phosphate isomerase (pgi) is a substitution of the A1T start codon in the ORF region of pgi.

[0007] In one specific embodiment, the modified engineered bacteria comprises a modification that reduces the activity of phosphoenolpyruvate carboxykinase (pckA) and a modification that reduces the activity of glucose-6-phosphate isomerase (pgi). Preferably, the modification that reduces the activity of glucose-6-phosphate isomerase (pgi) is a substitution of the A1T start codon in the ORF region of pgi.

[0008] In one specific embodiment, the modified engineered bacteria comprises modifications that reduce pyruvate kinase (pyk) activity and modifications that reduce glucose-6-phosphate isomerase (pgi) activity. Preferably, the modification that reduces glucose-6-phosphate isomerase (pgi) activity is a substitution of the A1T start codon in the ORF region of pgi.

[0009] In one specific embodiment, the modified engineered bacteria comprises modifications that reduce the activity of phosphoenolpyruvate carboxykinase (pckA), pyruvate kinase (pyk), and glucose-6-phosphate isomerase (pgi). Preferably, the modification that reduces the activity of glucose-6-phosphate isomerase (pgi) is a substitution of the A1T start codon in the ORF region of pgi.

[0010] In some embodiments, the engineered bacteria are preferably bacteria. In some specific embodiments, the bacteria producing adenosine are Bacillus bacteria, preferably Bacillus subtilis.

[0011] In some specific embodiments, the modification that reduces the activity of phosphoenolpyruvate carboxykinase (pckA) is a partial or complete knockout of the pckA gene, preferably the phosphoenolpyruvate carboxykinase (pckA) with reduced activity is shown in SEQ ID NO: 1.

[0012] In some specific embodiments, the modification that reduces pyruvate kinase (pyk) activity is a partial or complete knockout of the pyk gene, preferably the pyruvate kinase (pyk) with reduced activity as shown in SEQ ID NO: 2.

[0013] In some specific embodiments, the modified glucose-6-phosphate isomerase (pgi) comprises the nucleic acid sequence shown in SEQ ID NO: 4; or the nucleic acid sequence of the modified glucose-6-phosphate isomerase (pgi) includes a codon A1T substitution compared to the sequence shown in SEQ ID NO: 3. On the other hand, this disclosure provides the use of the modified bacteria as described above in increasing adenosine production.

[0014] In another aspect, this disclosure provides a method for producing adenosine, comprising culturing the modified engineered bacteria as described above in a culture medium. Detailed Implementation

[0015] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0016] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0017] Table 1. Instrument Information

[0018] Instrument Name Manufacturer model Refrigerated centrifuge Eppendrof Eppendrof 5804R Electroconverter BIO-RAD BIO-RAD MicroPulse

[0019] Table 2. Reagents used in this invention

[0020]

[0021]

[0022] Table 3. Primer sequence information

[0023]

[0024]

[0025] Table 4. Gene Sequence Information

[0026]

[0027]

[0028]

[0029] Reagent preparation method:

[0030] (1) Growth medium: It consists of two solutions: 2×LB medium and 2×sorbitol. The two reagents must be mixed in equal proportions before use. The preparation methods for the two reagents are as follows:

[0031] (2) 2×LB medium: 10g / L peptone, 10g / L sodium chloride, 5g / L yeast powder, 50mg / L guanine, distilled water to make up to volume, dispensed into 250ml Erlenmeyer flasks at 50ml / bottle, sterilized at 121℃ for 20min, and ready for use.

[0032] (3) 2× Sorbitol: Sorbitol 91.1g / L, distilled water to a final volume, dispensed into 150ml Erlenmeyer flasks at 50ml / bottle, sterilized at 121℃ for 20min, and set aside.

[0033] (4) Washing culture medium: Weigh 91.1 g / L of sorbitol, 91.1 g / L of mannitol and 126 g / L of glycerol, add ultrapure water to make up to 1000 ml, sterilize at 121℃ for 20 min, and set aside.

[0034] (5) Resuscitation medium: It consists of two reagents: 2×LB medium and 2×(sorbitol + mannitol). The 2×LB medium is prepared as described in 1), and the 2×(sorbitol + mannitol) is a mixed solution of 91.1 g / L sorbitol and 69.2 g / L mannitol.

[0035] Example

[0036] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0037] Examples 1 and 2 describe the construction process of the starting bacteria B. subtilis AR5 and SMA18b of this invention.

[0038] Example 1: Construction of B. subtilis AR5 starting strain

[0039] 1.1 Construction of B. subtilis168(Δupp) strain

[0040] The markerless gene editing method for strain construction is based on the two-step integration mediated by temperature-sensitive plasmids and the UPP reverse screening principle. The editing process can be found in the reference "A markerless gene replacement method for B. amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production[J].(Applied Microbiology and Biotechnology,2014,98(21):8963-8973.Zhang W,Gao W,Feng J,et al."). The specific construction process is as follows:

[0041] Using *B. subtilis168* strain (available from Beijing BioVideo Biotechnology Co., Ltd.) as the starting strain, primers 1f / 1r and 2f / 2r (Table 3) were used. Using the *B. subtilis168* genome as a template, pfu DNA polymerase was used to amplify the upstream and downstream homologous arms, upp-L and upp-R, respectively. The obtained fragments were then recovered via gel extraction. Using the obtained left and right homologous arms at an equimolar ratio (total amount controlled below 20 ng) as templates, primers 1f / 2r were used for amplification, followed by fusion PCR to obtain fragments upp-L and upp-R. The pKSV7 (tool vector) plasmid (available from the BioVector plasmid vector strain cell protein antibody gene depository) was digested with SalI / PstI. Upp-L, upp-R, and the linearized plasmid were simultaneously gel-extracted. The fragments and linearized vector were then processed using T4 gel extraction. DNA ligase was used to ligate the cells overnight at 22°C, and the resulting cells were transformed into DH5α competent cells. Single clones were obtained and colony PCR was performed for verification. The correct plasmid was named pKSU and transformed into B. subtilis168 using Spizizen. Transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed cells were inoculated into 5 mL LB broth, cultured at 42°C and 200 rpm for 12 h, and passaged for one generation. The transformed cells were then diluted and plated on LB agar plates containing 5 μg / mL chloramphenicol to obtain the first recombinant. The first recombinant was inoculated into 5 mL LB broth, cultured at 42°C and 200 rpm for 12 h, and passaged for one generation. The transformed cells were then diluted and plated on LB agar plates containing 0.8 μM 5-FU to screen for the second recombinant, thus obtaining the B. subtilis168(Δupp) strain.

[0042] 1.2 Construction of B. subtilis AR1 strain

[0043] Using primers 3f / 3r and 4f / 4r, and with the B. subtilis 168 genome as a template, the left and right homologous arm fragments of the purD gene (purD encodes phosphoribosamine-glycine ligase) were amplified. P116L -L and purD P116L -R, and gel collect; using the above homologous arms as templates and primers 3f / 4r, the fusion fragment purD was amplified to obtain P116L -L and purD P116L -R and gel collection: The pKSU plasmid from Example 1.1 was double-digested with SalI / PstI to obtain a linearized plasmid, which was then gel collected. The gel-collected fragment and the linearized plasmid were ligated and transformed to obtain the plasmid pKSU-purD. P116L The plasmid was transformed into B. subtilis168(Δupp) strain, and the strain was screened according to the screening method in Example 1.1. The strain containing the proline mutation to leucine at position 116 of the ORF region of the purD gene was named B. subtilis AR1.

[0044] 1.3 Construction of B. subtilis AR3 strain

[0045] Using primers 5f / 5r and 6f / 6r, respectively, and the genome of B. subtilis 168 as a template, the left and right homologous arms of the purR gene (encoding the Pur operon repressor protein) were amplified. A65D -L and purR A65D -R, and gel-collected; using the above homologous arms as templates and primers 5f / 6r, the fusion fragment purR was amplified to obtain A65D -LR, gel collection; following the plasmid construction method in Example 1.1, the linearized pKSU (SalI / PstI digested) plasmid after gel collection was combined with purR A65D -L and purR A65D The -R fragment was ligated and transformed, and screened to obtain the plasmid pKSU-purR. A65D The plasmid was transformed into B. subtilis AR1 strain, and the strain was screened according to the screening method in Example 1.1. The strain containing the alanine mutation to aspartic acid at position 65 of the purR gene ORF was named B. subtilis AR3.

[0046] 1.4 Construction of B. subtilis AR5 strain

[0047] Using primers 7f / 7r and 8f / 8r, and with the B. subtilis 168 genome as a template, the left and right homologous arm fragments of the guaB gene (encoding inosine dehydrogenase) were amplified. G279R-L and guaB G279R -R, and gel collect; using the above homologous arm as a template and primers 7f / 8r, the fusion fragment guaB was amplified to obtain G279R -L and guaB G279R -R, gel collection; following the plasmid construction method in Example 1.1, the linearized pKSU (SalI / PstI digested) plasmid after gel collection was combined with guaB G279R -L and guaB G279R The -R fragment was ligated and transformed, and the plasmid pKSU-guaB was obtained by screening. G279R The plasmid was transformed into B. subtilis AR3 strain, and the strain was screened according to the screening method in Example 1.1. The strain containing the glycine-arginine point mutation at position 279 of the guaB gene was named B. subtilis AR5. 1.5 Construction of B. subtilis AR7 strain

[0048] Using primers 9f / 9r and 10f / 10r from Table 3, and with the genome of B. subtilis AR5 strain as a template, the left and right homologous arm fragments of the yerA gene (encoding adenine deaminase) were amplified. G209E -L and yerA G209E -R, the fragment was recovered by gel extraction, and using the recovered fragment as a template, fusion PCR was performed with 9f / 10r primers to obtain yerA. G209E -L and yerA G209E The full-length R fragment was ligated to the linearized pKSU plasmid from Example 1.1 and transformed. The resulting plasmid was named pKSU-yerA. G209E The plasmid was transformed into B. subtilis AR5 strain, and the strain was screened according to the screening method in Example 1.1. The strain containing the point mutation of glycine at position 209 of the year gene to glutamic acid was named B. subtilis AR7.

[0049] Construction of 1.6B. subtilis AR9 strain

[0050] Using primers 11f / 11r and 12f / 12r from Table 3, and with the genome of B. subtilis AR5 strain as a template, the left and right homologous arm fragments of the adeC gene (encoding adenine deaminase) were amplified. P135S -L and adeC P135S -R, the fragment was recovered by gel extraction, and using the recovered fragment as a template, fusion PCR was performed with 11f / 12r primers to obtain adeC. P135S -L and adeC P135SThe full-length R fragment was ligated to the linearized pKSU plasmid from Example 1.1 and transformed. The resulting plasmid was named pKSU-adeC. P135S The plasmid was transformed into B. subtilis AR7 strain, and the strain was screened according to the screening method in Example 1.1. The strain containing the proline mutation to serine point mutation at position 135 of the adeC gene was named B. subtilis AR9.

[0051] Example 2: Construction of SMA18b starting strain

[0052] Using primers 13f / 13r and 14f / 14r in Table 3, and with the genome of B. subtilis AR5 strain as a template, the left and right homologous arm fragments ΔpurR-L and ΔpurR-R of the knocked-out purR gene (encoding a purine operon repressor protein) were amplified. The fragments were recovered by gel electrophoresis, and using the recovered fragments as templates, fusion PCR was performed with primers 13f / 14r to obtain the full-length fragments ΔpurR-L and ΔpurR-R. These fragments were then ligated to the linearized pKSU plasmid in Example 1.1 and transformed. The resulting plasmid was named pKSU-ΔpurR. The plasmid was transformed into B. subtilis AR9 strain, and the strain was screened according to the screening method in Example 1.1. The resulting purR knockout strain was named SMA18b.

[0053] Example 3: Construction of pckA (encoding phosphoenolpyruvate carboxykinase) knockout engineered strain SMA354b

[0054] The genetic modification method was performed according to the reference (Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System, Applied and Environmental Microbiology, Josef Altenbuchner et al. 2016), and the specific steps are as follows:

[0055] 3.1. Plasmid Construction

[0056] 3.1.1 Construction of pJOE8999-pckA-N20-LR plasmid

[0057] a) Extract the pJOE8999 plasmid and perform double digestion with BsaI and Xba1. The digestion system is as follows:

[0058] Table 5. BsaI and XbaI enzyme digestion systems

[0059] reagents Volume / μL pJOE8999 plasmid Maximum 2.5μg BsaI 2.5 XbaI 2.5 Buffer 5 water Make up to 50

[0060] The reaction was carried out at 37℃ for 2 hours. The product was then recycled and kept for later use.

[0061] b) Using pJOE8999 plasmid as a template, the N20 fragment was amplified with primers pckA-f1 / pckA-f / T7P-r. Using the SMA18b strain genome as a template, the left and right homologous arm fragments were amplified with primers pckA-1f / pckA-1r and pckA-2f / pckA-2r, respectively. The full-length fragment pckA-LR was obtained by fusion PCR.

[0062] The linearized pJOE8999 vector and the full-length fragment obtained above were ligated according to the ClonExpress II One Step Cloning Kit instructions, transformed into Trans-T1 cells, and cultured overnight at 37°C. Single colonies were identified by PCR using 8999-F1 and 8999-R1 and then sequenced for verification. The final constructed plasmid pJOE8999-pckA-N20-LR was named pMA204.

[0063] 3.2. Strain Construction

[0064] a) Transform at least 1 μg of the above pMA204 plasmid into competent cells of B. subtilis AR5 strain, add 4 ml of recovery buffer, recover for 1 h, centrifuge and discard the supernatant, plate on LB + guanine (50 mg / mL) plates containing 5 μg / mL kanamycin and 0.2% (w / w) mannose for screening, and incubate at 30℃ for about 48 h to grow single colonies; verify the grown transformants by PCR using identification primers pckA-F / pckA-R and by sequencing (sequencing primers pckA-F / pckA-R).

[0065] b) Pick a single colony of the correctly sequenced strain and incubate it overnight at 37°C in LB + guanine liquid medium; transfer 5 μL of the bacterial culture to LB + guanine liquid medium and incubate at 37°C for 8 h, then dilute 10 μL. 5 The plasmid was spread onto antibiotic-free LB agar plates and incubated at 37°C until single colonies grew. Single colonies were then selected and spotted onto both antibiotic-free LB agar plates and LB agar plates containing 5 mg / mL kanamycin. Strains that grew in antibiotic-free medium but not in antibiotic-resistant medium were identified as plasmid-loss strains. These plasmid-loss strains were then selected for further PCR verification (using primers on the outer homologous arms to avoid false positives) and sequencing verification. Strains that were correctly verified were considered successfully modified strains and named SMA354b (B. subtilis AR5△pckA).

[0066] Example 4: Construction of pckA knockout engineered strain SMA356c

[0067] The plasmid pMA204 obtained in Example 3 was transformed into competent cells of SMA18b strain. The strain obtained by screening according to the strain screening method in Example 1.1 was named SMA356c (SMA18b△pckA).

[0068] Example 5: Construction of pyk (encoding pyruvate kinase) knockout engineered strain SMA340b

[0069] 5.1 Plasmid Construction

[0070] 5.1.1 Construction of pJOE8999-pyk-N20 plasmid

[0071] a) Extract pJOE8999 plasmid (HonorGene, product code: HG-VCH1431), and digest it with BsaI. The digestion system is as follows:

[0072] Table 6. BsaI digestion system

[0073] reagents Volume / μL pJOE8999 plasmid Maximum 1μg BsaI(Eco31I) 2 Buffer G 2 water Make up to 20

[0074] The reaction was carried out at 37℃ for 2 hours. The enzyme digestion product was then recovered by gel extraction for later use.

[0075] b) Add 10 μL each of pyk-N20-f1 and pyk-N20-r1 primers (10 μM each) to a PCR tube; amplify the primers at 98℃ for 2 min, then decrease the temperature from 98℃ to 4℃ at a rate of 0.1℃ / s; after amplification, dilute the primer 10-fold, name it gene-N20, and ligate it with the obtained linearized pJOE8999 plasmid at 22℃ overnight. The ligation system is shown in the table below:

[0076] Table 7. Connection System

[0077]

[0078]

[0079] c) The above ligation product was transformed into competent cells of Trans-T1 strain and cultured overnight at 37°C until a single colony grew. PCR verification was performed using 8999-F1 and 8999-R1, and the correct transformants were sequenced for verification.

[0080] 2.2 Construction of pJOE8999-pyk-N20-LR plasmid

[0081] d) Using the SMA18b strain genome as a template, the left and right homologous arm fragments were amplified using primers pyk-1f / pyk-1r and pyk-2f / pyk-2r, respectively, and the full-length fragment pyk-LR was obtained by fusion PCR.

[0082] e) The pJOE8999-pyk-N20 plasmid obtained in 5.1.1 was reacted with SalI and XbaI restriction endonucleases in a metal bath at 37℃ for 2 h. The enzyme digestion system is shown in the table below:

[0083] Table 8. SalI and XbaI digestion systems

[0084] reagents Volume / μL Linearized pJOE8999-N20 plasmid Maximum 0.35μg SalI 2.5 XbaI 2.5 Buffer 5 water Make up to 50

[0085] The linearized pJOE8999-pgi-N20 plasmid obtained above was ligated to the full-length fragment pyk-LR according to the ClonExpressII One Step Cloning Kit instructions, transformed into Trans-T1 cells, and cultured overnight at 37°C. Single colonies were identified by PCR using 8999-F1 and 8999-R1 and then sequenced for verification. The final plasmid pJOE8999-pyk-N20-LR was constructed and named pMA112.

[0086] The pMA112 plasmid was transformed into B. subtilis AR5 strain, and the resulting strain was named SMA340b. The strain screening method is as described in Example 1.1. The identification and sequencing primers were pyk-F / pyk-R.

[0087] Example 6: Construction of pyk knockout strain SMA342c

[0088] The plasmid pMA112 from Example 5 was transformed into competent cells of SMA18b strain. The strain screening method was the same as in Example 1.1. The identification and sequencing primers were pyk-F / pyk-R. The obtained strain was named SMA342c.

[0089] Example 7: Construction of pgi (encoding glucose-6-phosphate isomerase) attenuated engineered strain SMA300a

[0090] The plasmid construction method is as described in Example 5. The primers used for constructing the N20 plasmid were pgi-N20-f1 / pgi-N20-r1. Using the SMA18b strain genome as a template, the left and right homologous arms were amplified using pgi-1f / pgi-1r and pgi-2f / pgi-2r, respectively, and then fused PCR was performed. The resulting plasmid was named pMA197. The pMA197 plasmid was transformed into B. subtilis AR5, and the resulting strain containing a mutant strain with the first nucleotide A of the pgi gene ORF region mutated to T was named SMA340b. The strain construction method is as described in Example 1.1. PCR verification and sequencing verification were performed using the identification primers pgijd-f / pgi-R (sequencing primers pgi-F / pgi-R).

[0091] Example 8: Construction of PGI-weakened engineered strain SMA301c

[0092] The plasmid pMA197 obtained in Example 7 was transformed into the SMA18b strain. The strain construction method was the same as in Example 1.1. PCR verification and sequencing verification were performed using the identification primers pgijd-f / pgi-R (sequencing primers pgi-F / pgi-R). The strain obtained was named SMA301c.

[0093] Example 9: Construction of the pckA and pyk knockout superimposed strain SMA357d

[0094] The pMA204 plasmid obtained in Example 3 was transformed into the SMA340b strain, and the strain obtained by screening according to the strain construction method in Example 2 was named SMA357d.

[0095] Example 10: Construction of the pckA and pyk knockout superimposed strain SMA358a

[0096] The pMA204 plasmid obtained in Example 3 was transformed into the SMA342c strain, and the strain obtained by screening according to the strain construction method in Example 2 was named SMA358a.

[0097] Example 11: Construction of the superimposed pckA knockout and pgi weakening strain SMA350a

[0098] The pMA204 plasmid was transformed into the SMA300a strain, and the strain obtained by screening according to the strain construction method in Example 2 was named SMA350a.

[0099] Example 12: Construction of the superimposed pckA knockout and pgi weakening strain SMA352d

[0100] The pMA204 plasmid was transformed into the SMA301c strain, and the strain obtained by screening according to the strain construction method in Example 2 was named SMA352d.

[0101] Example 13: Construction of the superimposed strain SMA344c of pyk knockout and pgi weakening

[0102] The pMA112 plasmid was transformed into the SMA300a strain, and the resulting strain was named SMA344c. The strain construction method is as described in Example 2. The identification and sequencing primers were pyk-F / pyk-R.

[0103] Example 14: Construction of the superimposed strain SMA343b of pyk knockout and pgi weakening

[0104] The pMA112 plasmid was transformed into the SMA301c strain, and the resulting strain was named SMA343b. The strain construction method is as described in Example 2. The identification and sequencing primers were pyk-F / pyk-R.

[0105] Example 15: Construction of SMA360c, a three-site superposition strain of pckA knockout, pyk knockout, and pgi weakening

[0106] The pMA204 plasmid was transformed into the SMA344c strain, and the resulting strain was named SMA360c. The strain construction method is as described in Example 2. The identification and sequencing primers were pckA-F / pckA-R.

[0107] Example 16: Construction of SMA359b, a three-site superposition strain of pckA knockout, pyk knockout, and pgi weakening

[0108] The pMA204 plasmid was transformed into the SMA343b strain, and the resulting strain was named SMA359b. The strain construction method is the same as in Example 2. The identification and sequencing primers were pckA-F / pckA-R.

[0109] Example 17: Shake-flask verification of adenosine strains

[0110] The recombinant bacteria constructed above were fermented to verify the adenosine production performance. The method for verifying adenosine yield through fermentation is as follows:

[0111] 1. Incubate the bacterial strain preserved in glycerol at 37°C overnight and cut out single clones.

[0112] 2. Inoculate a single colony into 30 mL of seed culture medium and incubate at 110 rpm and 37°C for 7–8 hours. The seed culture medium formula is as follows (g / L): glucose 20 g, yeast extract 5 g, corn steep liquor powder 5 g, potassium dihydrogen phosphate 3 g, magnesium sulfate 0.5 g, ferrous sulfate 0.02 g, manganese sulfate 0.01 g, guanine 0.05 g, pH 7.0–7.2.

[0113] 3. Transfer the inoculum at a rate of 10% (v / v) to 30 ml of fermentation medium, and incubate at 35°C for 48 hours using a shaker at 130 rpm. The fermentation medium is formulated as follows (g / L): 80 g of edible glucose, 3.5 g of yeast extract, 3 g of potassium dihydrogen phosphate, 25 g of ammonium sulfate, 0.01 g of manganese sulfate, 5 g of magnesium sulfate heptahydrate, 10 g of monosodium glutamate, 15 g of corn steep liquor powder, 0.05 g of guanine, 25 g of calcium carbonate, and pH 7.0–7.2.

[0114] 4. The glycosides produced in the fermentation broth were detected using liquid chromatography, and the results are shown in the table.

[0115] Table 9. Shake-flask data of adenosine strains

[0116]

[0117]

[0118] Table 10. Data of Adenosine-containing strain 5L

[0119]

[0120] Table 11. Data of Adenosine-containing strains at 50L

[0121]

[0122] By incorporating via reference

[0123] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0124] Equivalence

[0125] This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. A modified engineered bacterium for producing adenosine, wherein the modified engineered bacterium comprises a modification of reduced phosphoenolpyruvate carboxykinase (pckA) activity and / or a modification of reduced pyruvate kinase (pyk) activity as compared to the strain prior to the modification.

2. The modified engineered bacterium of claim 1, wherein the modified engineered bacterium comprises: 1) a modification of reduced phosphoenolpyruvate carboxykinase (pckA) activity; or 2) a modification of reduced pyruvate kinase (pyk) activity; or 3) a modification of reduced phosphoenolpyruvate carboxykinase (pckA) activity and a modification of reduced pyruvate kinase (pyk) activity.

3. The modified engineered bacterium of claim 1 or 2, wherein the modified engineered bacterium further comprises a modification of reduced glucose-6-phosphate isomerase (pgi) activity, preferably the modification of reduced glucose-6-phosphate isomerase (pgi) activity is an A1T substitution in the start codon of the ORF region of pgi.

4. The modified engineered bacterium of any one of claims 1 to 3, wherein the engineered bacterium is preferably a bacterium.

5. The modified engineered bacterium of claim 4, wherein the bacterium is a Bacillus bacterium, preferably a Bacillus subtilis.

6. The modified engineered bacterium of any one of claims 1 to 5, wherein the modification of reduced phosphoenolpyruvate carboxykinase (pckA) activity is a partial or complete knock-out of the pckA gene, preferably the reduced phosphoenolpyruvate carboxykinase (pckA) is as set forth in SEQ ID NO:

1.

7. The modified engineered bacterium of any one of claims 1 to 5, wherein the modification of reduced pyruvate kinase (pyk) activity is a partial or complete knock-out of the pyk gene, preferably the reduced pyruvate kinase (pyk) is as set forth in SEQ ID NO:

2.

8. The modified engineered bacterium of any one of claims 3 to 5, wherein, 8. The modified glucose-6-phosphate isomerase (pgi) comprises a nucleic acid sequence as set forth in SEQ ID NO: 4; or the nucleic acid sequence of the modified glucose-6-phosphate isomerase (pgi) comprises a codon A1T substitution as compared to the sequence set forth in SEQ ID NO:

3.

9. Use of the modified bacterium of any one of claims 1 to 8 for increasing production of adenosine.

10. A method for producing adenosine, comprising culturing the modified engineered bacterium of any one of claims 1 to 8 and isolating adenosine.

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