Method for constructing nucleoside-producing strain and method for producing nucleoside

Abstract

The present application provides a nucleoside production strain construction method and a nucleoside production method. The glucose / mannose transporter GlcP (encoded by the glcP gene) of Bacillus subtilis or Bacillus amyloliquefaciens is modified, so that the GlcP protein activity is enhanced, and the microorganism can efficiently and quickly produce guanine nucleoside or hypoxanthine nucleoside, thereby providing an effective means for large-scale production of nucleosides, and having a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering technology, specifically, it relates to a method for constructing nucleoside-producing strains and a method for producing nucleosides. 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.

[0003] Guanosine and inosine have wide applications in the food and pharmaceutical industries. In the food sector, guanosine and inosine are important precursors to disodium guanylate and disodium inosinate, respectively. Disodium guanylate and disodium inosinate are used in combination as flavor enhancers, widely applied in condiments such as chicken bouillon and soy sauce. In the pharmaceutical sector, guanosine and inosine serve as intermediates for various antiviral drugs, such as acyclic guanosine, triazole nucleoside, and guanosine triphosphate sodium, all of which require guanosine as a raw material for synthesis. Inosine is an important precursor to inosine monophosphate, which in turn serves as a precursor for the synthesis of adenosine monophosphate (AMP) and guanosine monophosphate (GMP). It is suitable for treating various causes of leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, cirrhosis, and can also be used to treat central retinitis and optic nerve atrophy.

[0004] Currently, microbial fermentation is the main method for producing nucleosides, primarily using microorganisms such as Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus pumilus. In the selection and modification of growth strains, high-yielding nucleosides are selectively bred using ultraviolet mutagenesis and diethyl sulfate mutagenesis; alternatively, based on a thorough understanding of the metabolic pathways and regulatory mechanisms of nucleotides in bacteria, and by using metabolic engineering techniques to purposefully modify the strains, superior traits and high nucleosides can be obtained. However, the fermentation performance of current nucleoside strains remains relatively poor, and the conversion rate of nucleosides is still low, failing to meet the demands of large-scale industrial production. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a nucleoside-producing strain and a method for producing nucleosides.

[0006] To achieve the objectives of this invention, in a first aspect, the present invention provides a modified microorganism, wherein the activity of its glucose transporter and / or mannose transporter GlcP (encoded by the glcP gene) is enhanced compared to the unmodified microorganism, and the microorganism has enhanced nucleoside production capacity compared to the unmodified microorganism. In this invention, the microorganism is a species of Bacillus or Escherichia, preferably Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Escherichia coli, etc., more preferably Bacillus subtilis or Bacillus amyloliquefaciens, such as strain Ba 8333.

[0007] The construction process of strain Ba 8333 is as follows (see CN202111266398.X): using strain DSM7 (ATCC23350, see the literature Genome sequence of B. amyloliquefaciens type strain DSM7) T Using the genome of *B. amyloliquefaciens* FZB42 (which reveals differences to plant-associated B. amyloliquefaciens FZB42) as a template, and guaB-1f / 1r and guaB-2f / 3r as primers, two fragments were amplified using Phusion high-fidelity polymerase (New England BioLabs). The two fragments were then fused using primer guaB-1f / 3r to obtain the recombinant fragment (ORF region nucleotide sequence shown in SEQ ID NO:1). The guaB... L454FThe fragment and pKSU plasmid (pKSU plasmid was kindly provided by Professor Wang Shufang of Nankai University, see 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 DOI:10.1007 / s00253-014-5824-2) were digested with SalI / PstI, assembled, and transformed to obtain the recombinant plasmid pKSU-guaB. L454F Transformed into Ba 836 strain (see CN112574934A), transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, thus obtaining guaB. L454F The point mutation strain was obtained as Ba 837. The pBE43 plasmid (the PBE43 plasmid was synthesized in its entirety; see reference: Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens) was extracted and linearized using KpnI / SalI. The mutated purR sequence (R2, SEQ ID NO:2) and the mutated tal sequence (L5, SEQ ID NO:3) were fused by PCR to obtain the R2+L5 fragment. This fragment was then ligated into the linearized PBE43 plasmid using an assembly kit to construct the plasmid PBE43-R2+L5. This plasmid was transformed into Ba 837 strain to obtain the starting strain Ba 8333.

[0008] The enhancement of glucose transporter and / or mannose transporter activity is achieved by selecting from 1) to 6) below, or an optional combination thereof:

[0009] 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the protein;

[0010] 2) Enhanced by increasing the copy number of the gene encoding the protein on the chromosome;

[0011] 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the protein on the chromosome;

[0012] 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the protein;

[0013] 5) Enhancement is achieved by altering the amino acid sequence of the protein;

[0014] 6) Enhancement is achieved by altering the nucleotide sequence of the protein-coding gene.

[0015] Furthermore, the enhanced activity of glucose transporters and / or mannose transporters is achieved by inserting the P43 promoter upstream of the start codon of the glcP gene.

[0016] Furthermore, the enhanced activity of glucose transporters and / or mannose transporters is achieved by inserting a two-copy glcP gene driven by the P43 promoter at the α-amylase gene.

[0017] Furthermore, the enhanced activity of glucose transporter and / or mannose transporter is achieved by inserting a P43 promoter upstream of the start codon of the glcP gene and inserting two copies of the glcP gene driven by the P43 promoter at the α-amylase gene.

[0018] Furthermore, the enhancement of glucose transporter and / or mannose transporter activity is achieved by inserting a P43 promoter upstream of the start codon of the glcP gene, inserting two copies of the glcP gene driven by the P43 promoter at the α-amylase gene, and mutating amino acid 206 of the glucose transporter and / or mannose transporter from H to R.

[0019] Preferably, the microorganism is Bacillus subtilis or Bacillus amyloliquefaciens.

[0020] In this invention, the glucose / mannose transporter GlcP has a reference sequence number of KS08_00900 on NCBI, or an amino acid sequence with 90% similarity to it. The α-amylase gene has a reference sequence number of KS08_07940 on NCBI, or a nucleotide sequence with 97% similarity to it.

[0021] Secondly, the present invention provides a method for constructing a nucleoside-producing strain, the method comprising: using genetic engineering techniques to enhance the glcP gene in a microorganism with nucleoside-producing capacity to obtain a strain with enhanced glucose transporter and / or mannose transporter activity.

[0022] The enhancement method is selected from the following 1) to 6), or any combination thereof:

[0023] 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the protein;

[0024] 2) Enhanced by increasing the copy number of the gene encoding the protein on the chromosome;

[0025] 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the protein on the chromosome;

[0026] 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the protein;

[0027] 5) Enhancement is achieved by altering the amino acid sequence of the protein;

[0028] 6) Enhancement is achieved by altering the nucleotide sequence of the protein-coding gene.

[0029] Thirdly, the present invention provides a method for producing nucleosides, the method comprising the following steps:

[0030] a) Cultivate the microorganisms to obtain a culture of the microorganisms;

[0031] b) Collect the resulting nucleosides from the culture obtained in step a).

[0032] The nucleosides include inosine, guanosine, and other nucleosides or their corresponding nucleoside derivatives, such as hypoxanthine, inosine, guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc.

[0033] Fourthly, the present invention provides the application of the modified microorganism or the nucleoside-producing strain constructed according to the above method in nucleoside fermentation production or increasing nucleoside fermentation yield.

[0034] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0035] The engineered strain Ba 8334 (with a strong promoter P43 inserted before the start codon ATG of the glcP gene) increased guanosine production from 9.8 g / L to 11.5 g / L and glycoside conversion rate by 1.5% compared to the starting strain Ba 8333. The engineered strain Ba 8335 (with two copies of the glcP gene containing the P43 promoter inserted at the KS08_07940 gene) increased guanosine production from 9.8 g / L to 12.0 g / L and glycoside conversion rate by 2.6% compared to the starting strain B.a8333. Combining these two enhancement methods yielded the mutant strain Ba 8336, which increased guanosine production from 12.0 g / L to 13.0 g / L and glycoside conversion rate by 1%. Detailed Implementation

[0036] The present invention aims to provide a method for producing purine nucleosides using microorganisms, as well as a novel microorganism used in the method that can efficiently produce purine nucleosides and a method for constructing the microorganism.

[0037] Research has found that modifying the glucose / mannose transporter GlcP (encoded by the glcP gene) of Bacillus subtilis or Bacillus amyloliquefaciens enhances the activity of the GlcP protein, enabling the microorganism to produce guanine or hypoxanthine nucleosides efficiently and rapidly. Furthermore, a new microorganism capable of producing nucleosides efficiently has been successfully created, thus completing this invention.

[0038] This invention is the first to obtain mutants of Bacillus amyloliquefaciens that can efficiently produce nucleosides by inserting a strong promoter in front of the original promoter or by increasing the copy number in an ectopic manner, and successfully constructs a microorganism capable of producing nucleosides efficiently.

[0039] The present invention adopts the following technical solution:

[0040] This invention provides a *Bacillus amyloliquefaciens* strain whose intracellular gene encoding the glucose / mannose transporter *glcP* has a strong P43 promoter inserted before the start codon, and / or two copies of the *glcP* gene containing the P43 promoter are inserted at the KS08_07940 gene. By introducing the corresponding promoter sequence into a nucleoside-producing bacterium through genetic engineering, the ability of the strain to produce nucleosides is enhanced compared to the unmodified strain.

[0041] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0042] The primers used in the following examples are shown in Table 1:

[0043] Table 1

[0044]

[0045]

[0046] Example 1: Construction of glcP promoter-enhanced strains

[0047] Using the genome of *Bacillus amyloliquefaciens* ATCC13952 as a template, the upstream and downstream homologous arms of *glcP* were amplified using primer pairs P43-glcP-1f / P43-glcP-1r and P43-glcP-2f / P43-glcP-2r with Pfu high-fidelity DNA polymerase. The P43 fragment was then amplified using primers P43-F1 / P43-R1 with the PBE43 plasmid (the PBE43 plasmid was synthesized in its entirety; see the reference "Effects of overexpression of key enzyme genes on guanosine accumulation in *Bacillus amyloliquefaciens*") as a template. The obtained fragments were then recovered via gel electrophoresis. Finally, the three fragments were fused using primers P43-glcP-1f / P43-glcP-2r to obtain the full-length P43-glcP fragment, which was then recovered via gel electrophoresis. The pKSU plasmid (the pKSU plasmid was kindly provided by Professor Wang Shufang of Nankai University; see 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 DOI:10.1007 / s00253-014-5824-2) was double-digested with XbaI / PstI and then recovered by gel extraction. The linearized plasmid and the P43-glcP fragment were assembled using an assembly kit and transformed into TransT1 competent cells. The recombinant plasmid pKSU-P43-glcP was obtained by identification and screening. Transformed into strain Ba 8333, transformants were screened on LB plates containing 2.5 μg / mL chloramphenicol at 30°C. The obtained transformants were inoculated into 5 mL LB liquid medium, cultured at 42°C and 200 rpm for 12 h and passaged for one generation. The transformed transformants were then diluted and plated onto LB plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. The primary recombinants were inoculated into 5 mL LB liquid medium, cultured at 42°C and 200 rpm for 12 h and passaged for one generation. The primary recombinants were then diluted and plated onto LB plates containing 0.8 μM 5-FU to screen for secondary recombinants. The strain with glcP promoter enhancement was obtained and named Ba 8334.

[0048] Example 2: Construction of a strain with a two-copy glcP gene enhancement

[0049] Using primer pairs glcP2nd-1f / glcP2nd-1r, P43-glcP-2f / glcP2nd-2r, glcP2nd-3f / glcP2nd-3r, and the ATCC13952 genome as templates, the left homologous arm fragments glcP2nd-L, glcP, and glcP2nd-R were amplified and recovered via gel electrophoresis. These four fragments—glcP2nd-L, the P43-glcP fragment obtained in Example 1, glcP, and glcP2nd-R—were fused using primers glcP2nd-1f / glcP2nd-3r to obtain the full-length glcP2nd fragment. The pKSU-glcP2nd plasmid was constructed according to the plasmid construction method in Example 1 and transformed into the Ba 8333 strain. Following the strain selection method in Example 1, a strain containing two copies of glcP was obtained and named B.a8335.

[0050] Example 3: Construction of glcP two-copy gene and its in situ promoter-enhanced strain

[0051] Using Ba 8335 strain as the starting strain, the pKSU-P43-glcP plasmid obtained in Example 1 was transformed into it, and the positive strain obtained by screening according to the method of screening strains in Example 1 was named Ba 8336.

[0052] Example 4 Construction of glcP point mutant strain

[0053] Using primer pairs glcP-1f / 1r and glcP-2f / 2r, and with the Ba 8336 genome as a template, the left homologous arm glcP-L and the right homologous arm glcP-R were amplified and recovered via gel extraction. Using the recovered fragments as templates, the glcP-1f and glcP-2r primers were used for fragment fusion to obtain the full-length fragment. The ORF frame sequence of the gene containing the point mutation is shown in SEQ ID NO:4, and its translated amino acid sequence is shown in SEQ ID NO:5. pKSU-glcP was obtained according to the plasmid construction method in Example 1. H206R The plasmid was transformed into strain Ba 8336, and strains containing glcP were screened according to the strain screening method in Example 1. H206R The point mutation strain is named Ba 8337.

[0054] Example 5: Performance Test of Engineered Bacteria in Nucleoside Production

[0055] 1. Incubate the bacterial strain preserved in glycerol at 37°C overnight and cut out single clones.

[0056] 2. Pick a single colony and inoculate it into 30 mL of seed culture medium (20 g / L glucose, 5 g / L yeast extract, 5 g / L corn steep liquor powder, 3 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.02 g / L ferrous sulfate, 0.01 g / L manganese sulfate, pH 7.0-7.2), and incubate at 37℃ and 110 rpm for 7-8 h.

[0057] 3. Transfer the inoculum at a rate of 10% v / v to 30 mL of fermentation medium (120 g / L glucose, 3.5 g / L yeast extract, 3 g / L potassium dihydrogen phosphate, 25 g / L ammonium sulfate, 0.01 g / L manganese sulfate, 5 g / L magnesium sulfate, 10 g / L monosodium glutamate, 15 g / L corn steep liquor powder, 25 g / L calcium carbonate, pH 7.0–7.2), and incubate at 35°C for 70 h at a shaking speed of 130 rpm.

[0058] 4. The glycosides produced in the fermentation broth were detected using liquid chromatography (Table 2).

[0059] Table 2. Evaluation results of guanosine and inosine production by the mutant strain during shake-flask fermentation (mean of three replicates)

[0060] strain Total glycoside yield (g / L) Guanosine production (g / L) Inosine production (g / L) <![CDATA[OD 562 ]]> Glycoside production enhancement Ba 8333 11.2 9.8 1.0 28.3 - Ba 8334 13.2 11.5 1.3 28.5 17.9％ Ba 8335 13.8 12.0 1.3 27.6 23.2％ Ba 8336 14.5 13.1 1.0 27.6 29.5％ Ba 8337 15.9 15.0 0.5 29.0 42.0％

[0061] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Langfang Meihua Biotechnology Development Co., Ltd. <120> Methods for constructing nucleoside-producing strains and methods for producing nucleosides <130> KHP221110901.7 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1467 <212> DNA <213> Bacillus amyloliquefaciens <400> 1 atgtgggaaa gtaaattttc aaaagaaggc ttaacgttcg atgatgtact gctcgtacca 60 gctcaatcag acgtacttcc gcgtgatgtg gatttgtctg ttgaactgac aaaaacgtta 120 aagcttaata ttcctgtcat cagtgcagga atggatacag taacagaatc agcaatggcg 180 attgcgatgg cccgacaagg cggcttgggc attattcata aaaacatgtc catcgaacag 240 caggctgaac atgttgacaa agtcaaacgt tctgaacggg gcgttattac aaatcccttc 300 ttttaacac ctgatcatca agtattcgat gcggagcatt tgatggggaa atacagaatt 360 tccggtgtc cgatcgtaga tataaagac gatcaaaagc tggtcggtat cattacaaac 420 cgcgatcttc gctttatctc tgattattca atgaaaatca gtgatgttat gacaaaagaa 480 gagctggtta cggctcctgt gggaaccaca ttagacgaag cggaaaaaat cttgcagaag 540 cataaaattg aaaaacttcc attagtggat gaccaaaaca attaaaagg tcttatcacg 600 atcaaagata ttgaaaaggt tatcgaattc ccgaattcat ctaaagatga acacggacgc 660 ctgatcgtcg gcgctgcggt aggcgtgaca ggtgatacaa tgactcgtgt cagcaagctt 720 gttgaagcga atgtcgacgt tatcgtggtt gatacggctc acggacattc cagaggcgta 780 ctgaacacag ttgcgaaaat ccgtgagaca tatcctgaat tgaacattat cgcaggaaat 840 gttgctacgg ctgaagcgac aaaggctttg attgaagccg gagcaaacat tgtaaaagtg 900 ggaatcggac ctggatctat ctgtacgaca cgcgtcgttg caggcgtagg tgtaccgcaa 960 atcactgcga tttatgattg tgccactgaa gcgagaaaac acggcgcaac aattatcgcg 1020 gacggcggta ttaaattctc cggagatatt acgaaagcat tggcatccgg cggacatgct 1080 gtcatgcttg gaagcctgct tgccggtact tcagaaagcc cgggcgaaac tgaaatctat 1140 caaggcagaa gatttaaagt gtatcgcggt atgggttctg tcgctgccat ggaaaaaggc 1200 agtaaagacc gatatttcca agaagaaaat aagaaattcg tccctgaagg tatcgaagga 1260 cggactccgt acaaaggtcc tgtagaagaa acagtgtatc agcttgtcgg cggtcttcgt 1320 tcaggtatgg gatattgcgg ttcaaaagac ttgcgcgctt ttagagaaga agctcaattt 1380 atccgtatga caggagcagg tcttcgcgaa agccatccgc atgatgtcca aatcacgaag 1440 gaatcaccaa actacacaat ctcataa 1467 <210> 2 <211> 931 <212> DNA <213> Artificial Sequence <400> 2 acagaatgct cttgattaaa tccgtatgtt aagttatatt ttttttaatt tttcggattt 60 tgggggtaag ttcatgaagt ttcgtcgcag cggcagattg gtggacttaa caaattattt 120 gttaacccat ccgcatgaat taataccgtt aacgtttttc tcagaacggt atcaatcagc 180 aaagtcatca atcagtgaag atttaacaat tattaaacag accttcgaac agcaaggcat 240 tgggacgctg cttacagtgc cgggagctgc cggaggcgtc aaatatattc cgaaagtaaa 300 acaggctgaa gcagaagcgt ttatacagga gctgggacag tctttagtaa atcctgagcg 360 tatccttccg ggcggttatg tatatttaac ggatatctta ggcaaacctt ctgtcctctc 420 taatgcaggc aggctttttg cttccgtttt tgcggagcgg gagattgatg tggtgatgac 480 cgttgcgaca aaaggaatcc ctcttgctta cgcagcggcc agttatctga acgttccggt 540 tgtcatcgta cgaaaagaca ataaagtgac ggaaggctct acagtgagca tcaattatgt 600 atcagggtcg tctaaccgca ttcaaacaat gtcgcttgcg aaaagaagtt tggccacggg 660 gtcgaacgtt ttgattattg acgactttat gaaagccggc ggcacaatta acggcatgat 720 cagcctgctt gatgagttta atgcgaacgt cgcgggtata ggcgtcttgg ttgaagctga 780 gggagtgaat gaacggcttg tcgatgaata catgtcgctg cttacccttt caaccatcaa 840 catgaaagac aaaacgattg agattcaaaa cggcaatttt ctgcgatttt ttaaagaaca 900 gcatttaaag aatggggaga cagaaaaatg a 931 <210> 3 <211> 839 <212> DNA <213> Artificial Sequence <400> 3 atgttatttt tcattgatac agcaaacatt gacgagatta aagaagctta tgaacttggc 60 gttcttgccg gagttacgac aaaccctagt ttagtggcaa aagaagctga cgtgtctttc 120 catgacagac tgcgtgagat tacggaagtc gtgaaaggat ctgtaagcgc ggaagtcatt 180 tccctgaacg ctgaagaaat gattgaagaa ggtaaagaac ttgcgaaaat cgcgccgaat 240 atcacggtaa aaattccgat gacgtctgaa ggattaaaag ccgtaaaagc gctgagcgac 300 ctgaacatta aaacaaacgt gacgctcatc ttcagcgcca accaggcgct tttggccgcc 360 agagccggag cgacttatgt ttctccgttc ttaggccgtc tggatgatat cggtcataac 420 ggtcttgaac tgatttcaga aataagacag atttttgacc ttcatgacat tgatacacaa 480 atcatcgcag cttccatccg tcatgcgcag cacgtgactg aagccgcttt acgcggtgcc 540 catatcggta cgatgccgct gaaagttatt caccagctga caaagcaccc gttaacggat 600 aaaggcatcg agcaattctt ggcagactgg aataaataag ggcgaaaagg gcggcaaacc 660 ggttgcatcc gtttgccgca cccttatgtt ttcctgtttg acggcgggcc ttcaatatca 720 aagttccccg gaatgtaaac ccggcgggtc tctatgcatc ctatgttaaa aatgccccgc 780 aaagcttcgc tattttcttc ctgttatttt ataatgtcct tgtattcttt ctcttcgaa 839 <210> 4 <211> 1215 <212> DNA <213> Artificial Sequence <400> 4 atgatgaaaa ctcgtctgct gtggataagc tgtttttctt atggatcgat tgcctttaca 60 cttgttattc ttggagccgt actccctgag ttactcacac attattcaca aacctacagc 120 aatggcggag tactagtatt ttctcagttc atgggcttcc tcgtgggtgt catcggaatg 180 ccttacatgg tgaaaaagtt cggggcgcaaa aacgtcgtta tctttggcct ggcactcatc 240 agctgtgagg ttttcatcac cttcctgccg ccctggccgc tgctctttct tctcgtcagt 300 atagcaggac tcggtgctgg attggtgga tcctgcgtag gcacgattat cctcactgct 360 attaagaaa gacaagccgt ggctatgagc aagatggaag tggcttacgg attaggggcg 420 ctgttcatgc ctctgctttc aggctttctt atcaacagcc acatgtggac aattgctttt 480 tttagtattag ggttatccag ttttgcactt ttaatcgcat ggagcaaat gagttttggg agcattgatc agcttctcat acgcaaagat gtctcttccg acggcactaa aaaagaaagc accggctatc ggtcacgtgg attgctgttt attgccctgg cggctgctta cttcttcttt 660 tacggaggca gtgaagtttc aattgtacat tttatcccct ccatcttcgc tgagaaatgg 720 gatatcccca actccttagc aacgataaca gtcaccgtgt attggactgg gatgattatc ggcaggttat taacaggtcc ggtatctgaa aagctgacat atcaccgtta tctccgtatt ataagcgttg gcggcctggc tgcacttgct gtattagcac taagtaaaag cgtatggttc 900 ggttttgccc tttgcttctt tttaggactg ttcatggccg gcatgtttgc aatagcccta 960 atcatcacca atcattttta cccaggaaag acagaaacaa ctaccagtat tctgcttgcc 1020 tcaaacggat tagggggttc actccttccg atcgccgtcg gctggagctt ggatgagtat 1080 cccgcacaaa ccgcgttctg gctgttcact gcactgatgc tcctgatgct gctgattgtg 1140 ttcagtttaa gaatgctcga gacaataaaa tcaaacagtc ttcaaaatca cagcagcaaa 1200 gcaaaatcaa tataa 1215 <210> 5 <211> 404 <212> PRT <213> Artificial Sequence <400> 5 Met Met Lys Thr Arg Leu Leu Trp Ile Ser Cys Phe Ser Tyr Gly Ser 1 5 10 15 Ile Ala Phe Thr Leu Val Ile Leu Gly Ala Val Leu Pro Glu Leu Leu 20 25 30 Thr His Tyr Ser Gln Thr Tyr Ser Asn Gly Gly Val Leu Val Phe Ser 35 40 45 Gln Phe Met Gly Phe Leu Val Gly Val Ile Gly Met Pro Tyr Met Val 50 55 60 Lys Lys Phe Gly Arg Lys Asn Val Val Ile Phe Gly Leu Ala Leu Ile 65 70 75 80 Ser Cys Glu Val Phe Ile Thr Phe Leu Pro Pro Trp Pro Leu Leu Phe 85 90 95 Leu Leu Val Ser Ile Ala Gly Leu Gly Ala Gly Leu Val Glu Ser Cys 100 105 110 Val Gly Thr Ile Ile Leu Thr Ala Ile Lys Glu Arg Gln Ala Val Ala 115 120 125 Met Ser Lys Met Glu Val Ala Tyr Gly Leu Gly Ala Leu Phe Met Pro 130 135 140 Leu Leu Ser Gly Phe Leu Ile Asn Ser His Met Trp Thr Ile Ala Phe 145 150 155 160 Leu Val Leu Gly Leu Ser Ser Phe Ala Leu Leu Ile Ala Trp Lys Gln 165 170 175 Met Ser Phe Gly Ser Ile Asp Gln Leu Leu Ile Arg Lys Asp Val Ser 180 185 190 Ser Asp Gly Thr Lys Lys Glu Ser Thr Gly Tyr Arg Ser Arg Gly Leu 195 200 205 Leu Phe Ile Ala Leu Ala Ala Ala Tyr Phe Phe Phe Tyr Gly Gly Ser 210 215 220 Glu Val Ser Ile Val His Phe Ile Pro Ser Ile Phe Ala Glu Lys Trp 225 230 235 240 Asp Ile Pro Asn Ser Leu Ala Thr Ile Thr Val Thr Val Tyr Trp Thr 245 250 255 Gly Met Ile Ile Gly Arg Leu Leu Thr Gly Pro Val Ser Glu Lys Leu 260 265 270 Thr Tyr His Arg Tyr Leu Arg Ile Ile Ser Val Gly Gly Leu Ala Ala 275 280 285 Leu Ala Val Leu Ala Leu Ser Lys Ser Val Trp Phe Gly Phe Ala Leu 290 295 300 Cys Phe Phe Leu Gly Leu Phe Met Ala Gly Met Phe Ala Ile Ala Leu 305 310 315 320 Ile Ile Thr Asn His Phe Tyr Pro Gly Lys Thr Glu Thr Thr Thr Ser 325 330 335 Ile Leu Leu Ala Ser Asn Gly Leu Gly Gly Ser Leu Leu Pro Ile Ala 340 345 350 Val Gly Trp Ser Leu Asp Glu Tyr Pro Ala Gln Thr Ala Phe Trp Leu 355 360 365 Phe Thr Ala Leu Met Leu Leu Met Leu Leu Ile Val Phe Ser Leu Arg 370 375 380 Met Leu Glu Thr Ile Lys Ser Asn Ser Leu Gln Asn His Ser Ser Lys 385 390 395 400 Ala Lys Ser Ile

Claims

1. A modified microorganism, characterized in that, Compared to unmodified microorganisms, the microorganisms exhibit enhanced activity of glucose transporters and / or mannose transporters, and also have enhanced guanosine production capacity compared to unmodified microorganisms. The enhancement of glucose transporter and / or mannose transporter activity is achieved by a combination of the following: 1) to 3) below. 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the protein; 2) Enhancement is achieved by increasing the copy number of the gene encoding the protein on the chromosome; 3) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the protein; The microorganism is Bacillus amyloliquefaciens strain Ba 8333; The construction method of the Ba 8333 strain includes: using the genome of strain DSM7 with accession number ATCC23350 as a template, and using guaB-1f / 1r and guaB-2f / 3r as primers, two fragments are amplified using Phusion superfidelity polymerase; the two fragments are fused using primer guaB-1f / 3r to obtain the recombinant fragment with the ORF region nucleotide sequence as shown in SEQ ID NO:1; and the ORF region nucleotide sequence as shown in SEQ ID NO:1 is then used to construct the recombinant fragment. L454F The fragment and pKSU plasmid were digested with SalI / PstI, assembled, and transformed to obtain the recombinant plasmid pKSU-guaB. L454F Transformed into Ba 836 strain, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30℃. The obtained transformants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h, and passaged for one generation. The transformed transformants were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. The primary recombinants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h, and passaged for one generation. The primary recombinants were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, thus obtaining guaB. L454F The point mutation strain was obtained as Ba 837. The pBE43 plasmid was extracted and linearized using KpnI / SalI. Fusion PCR was performed on the mutated purR sequence (as shown in SEQ ID NO:2) and the mutated tal sequence (as shown in SEQ ID NO:3) to obtain the R2+L5 fragment. This fragment was then ligated into the linearized pBE43 plasmid using an assembly kit to construct the plasmid PBE43-R2+L5. This plasmid was transformed into the Ba 837 strain to obtain strain Ba 8333. The primer sequences are as follows: guaB-1f: 5′-taaggatcctctagagtcgacaCGCGTCGTTGCAGGCGTA-3′; guaB-1r: 5′-attgagcttcttctctAaaagcgcgcaagtctttt-3′; guaB-2f: 5′-gacttgcgcgctttTagagaagaagctcaattta-3′; guaB-3r: 5′-gccaagcttgcatgcctgcagCATAATCATCAGGAGTATACGTTTGGT-3′; The reference sequence number for the glucose transporter and / or mannose transporter on NCBI is KS08_00900, and its encoding gene is... glcP Gene.

2. The microorganism according to claim 1, characterized in that, The enhancement of glucose transporter and / or mannose transporter activity is achieved through... glcP This is achieved by inserting the P43 promoter upstream of the gene start codon.

3. The microorganism according to claim 1, characterized in that, The enhanced activity of glucose transporters and / or mannose transporters is achieved through the insertion of two copies of the α-amylase gene driven by the p43 promoter. glcP It is achieved through genes.

4. The microorganism according to claim 1, characterized in that, The enhancement of glucose transporter and / or mannose transporter activity is achieved through... glcP The P43 promoter is inserted upstream of the gene start codon, and two copies of the gene driven by the P43 promoter are inserted at the α-amylase gene. glcP It is achieved through genes.

5. A modified microorganism, characterized in that, Compared to unmodified microorganisms, the microorganisms exhibit enhanced activity of glucose transporters and / or mannose transporters, and also have enhanced guanosine production capacity compared to unmodified microorganisms. Among them, the enhancement of glucose transporter and / or mannose transporter activity is achieved through... glcP The P43 promoter is inserted upstream of the gene start codon, and two copies of the gene driven by the P43 promoter are inserted at the α-amylase gene. glcP The gene is obtained by further overexpressing glucose transporter and / or mannose transporter mutants, wherein the glucose transporter and / or mannose transporter mutants are obtained by mutating amino acid 206 of the glucose transporter and / or mannose transporter from H to R. The microorganism is Bacillus amyloliquefaciens strain Ba 8333; The construction method of the Ba 8333 strain includes: using the genome of strain DSM7 with accession number ATCC23350 as a template, and using guaB-1f / 1r and guaB-2f / 3r as primers, two fragments are amplified using Phusion superfidelity polymerase; the two fragments are fused using primer guaB-1f / 3r to obtain the recombinant fragment with the ORF region nucleotide sequence as shown in SEQ ID NO:1; and the ORF region nucleotide sequence as shown in SEQ ID NO:1 is then used to construct the recombinant fragment. L454F The fragment and pKSU plasmid were digested with SalI / PstI, assembled, and transformed to obtain the recombinant plasmid pKSU-guaB. L454F Transformed into Ba 836 strain, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30℃. The obtained transformants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h, and passaged for one generation. The transformed transformants were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. The primary recombinants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h, and passaged for one generation. The primary recombinants were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, thus obtaining guaB. L454F The point mutation strain was obtained as Ba 837. The pBE43 plasmid was extracted and linearized using KpnI / SalI. Fusion PCR was performed on the mutated purR sequence (as shown in SEQ ID NO:2) and the mutated tal sequence (as shown in SEQ ID NO:3) to obtain the R2+L5 fragment. This fragment was then ligated into the linearized pBE43 plasmid using an assembly kit to construct the plasmid PBE43-R2+L5. This plasmid was transformed into the Ba 837 strain to obtain strain Ba 8333. The primer sequences are as follows: guaB-1f: 5′-taaggatcctctagagtcgacaCGCGTCGTTGCAGGCGTA-3′; guaB-1r: 5′-attgagcttcttctctAaaagcgcgcaagtctttt-3′; guaB-2f: 5′-gacttgcgcgctttTagagaagaagctcaattta-3′; guaB-3r: 5′-gccaagcttgcatgcctgcagCATAATCATCAGGAGTATACGTTTGGT-3′; The reference sequence number for the glucose transporter and / or mannose transporter on NCBI is KS08_00900, and its encoding gene is... glcP Gene.

6. A method for constructing a guanosine-producing strain, characterized in that, The method includes: using genetic engineering techniques to enhance genes in microorganisms capable of producing guanosine. glcP Expression was performed to obtain strains with enhanced glucose transporter and / or mannose transporter activity; The enhancement method is selected from the following 1) to 3), or a combination thereof: 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the protein; 2) Enhancement is achieved by increasing the copy number of the gene encoding the protein on the chromosome; 3) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the protein; The microorganism is Bacillus amyloliquefaciens strain Ba 8333; The construction method of the Ba 8333 strain includes: using the genome of strain DSM7 with accession number ATCC23350 as a template, and using guaB-1f / 1r and guaB-2f / 3r as primers, two fragments are amplified using Phusion superfidelity polymerase; the two fragments are fused using primer guaB-1f / 3r to obtain the recombinant fragment with the ORF region nucleotide sequence as shown in SEQ ID NO:1; and the ORF region nucleotide sequence as shown in SEQ ID NO:1 is then used to construct the recombinant fragment. L454F The fragment and pKSU plasmid were digested with SalI / PstI, assembled, and transformed to obtain the recombinant plasmid pKSU-guaB. L454F Transformed into Ba 836 strain, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30℃. The obtained transformants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h, and passaged for one generation. The transformed transformants were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. The primary recombinants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h, and passaged for one generation. The primary recombinants were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, thus obtaining guaB. L454F The point mutation strain was obtained as Ba 837. The pBE43 plasmid was extracted and linearized using KpnI / SalI. Fusion PCR was performed on the mutated purR sequence (as shown in SEQ ID NO:2) and the mutated tal sequence (as shown in SEQ ID NO:3) to obtain the R2+L5 fragment. This fragment was then ligated into the linearized pBE43 plasmid using an assembly kit to construct the plasmid PBE43-R2+L5. This plasmid was transformed into the Ba 837 strain to obtain strain Ba 8333. The primer sequences are as follows: guaB-1f: 5′-taaggatcctctagagtcgacaCGCGTCGTTGCAGGCGTA-3′; guaB-1r: 5′-attgagcttcttctctAaaagcgcgcaagtctttt-3′; guaB-2f: 5′-gacttgcgcgctttTagagaagaagctcaattta-3′; guaB-3r: 5′-gccaagcttgcatgcctgcagCATAATCATCAGGAGTATACGTTTGGT-3′; The reference sequence number for the glucose transporter and / or mannose transporter on NCBI is KS08_00900, and its encoding gene is... glcP Gene.

7. A method for constructing a guanosine-producing strain, characterized in that, The method includes: using genetic engineering techniques to enhance genes in microorganisms capable of producing guanosine. glcP Expression was performed to obtain strains with enhanced glucose transporter and / or mannose transporter activity; Among them, the enhanced activity of glucose transporters and / or mannose transporters is achieved through... glcP The P43 promoter is inserted upstream of the gene start codon, and two copies of the gene driven by the P43 promoter are inserted at the α-amylase gene. glcP The gene is obtained by further overexpressing glucose transporter and / or mannose transporter mutants, wherein the glucose transporter and / or mannose transporter mutants are obtained by mutating amino acid 206 of the glucose transporter and / or mannose transporter from H to R. The microorganism is Bacillus amyloliquefaciens strain Ba 8333; The construction method of the Ba 8333 strain includes: using the genome of strain DSM7 with accession number ATCC23350 as a template, and using guaB-1f / 1r and guaB-2f / 3r as primers, two fragments are amplified using Phusion superfidelity polymerase; the two fragments are fused using primer guaB-1f / 3r to obtain the recombinant fragment with the ORF region nucleotide sequence as shown in SEQ ID NO:1; and the ORF region nucleotide sequence as shown in SEQ ID NO:1 is then used to construct the recombinant fragment. L454F The fragment and pKSU plasmid were digested with SalI / PstI, assembled, and transformed to obtain the recombinant plasmid pKSU-guaB. L454F Transformed into Ba 836 strain, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30℃. The obtained transformants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h, and passaged for one generation. The transformed transformants were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. The primary recombinants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h, and passaged for one generation. The primary recombinants were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, thus obtaining guaB. L454F The point mutation strain was obtained as Ba 837. The pBE43 plasmid was extracted and linearized using KpnI / SalI. Fusion PCR was performed on the mutated purR sequence (as shown in SEQ ID NO:2) and the mutated tal sequence (as shown in SEQ ID NO:3) to obtain the R2+L5 fragment. This fragment was then ligated into the linearized pBE43 plasmid using an assembly kit to construct the plasmid PBE43-R2+L5. This plasmid was transformed into the Ba 837 strain to obtain strain Ba 8333. The primer sequences are as follows: guaB-1f: 5′-taaggatcctctagagtcgacaCGCGTCGTTGCAGGCGTA-3′; guaB-1r: 5′-attgagcttcttctctAaaagcgcgcaagtctttt-3′; guaB-2f: 5′-gacttgcgcgctttTagagaagaagctcaattta-3′; guaB-3r: 5′-gccaagcttgcatgcctgcagCATAATCATCAGGAGTATACGTTTGGT-3′; The reference sequence number for the glucose transporter and / or mannose transporter on NCBI is KS08_00900, and its encoding gene is... glcP Gene.

8. A method for producing guanosine, characterized in that, The method includes the following steps: a) Cultivate the microorganism according to any one of claims 1-5 to obtain a culture of the microorganism; b) Collect the guanosine produced from the culture obtained in step a).

Images