CN116804178A - Construction method of nucleoside producing engineering bacteria and method for producing nucleosides - Google Patents

Abstract

The invention provides a construction method of nucleoside producing engineering bacteria and a method for producing nucleosides. Modifying the glucokinase (encoded by the glcK gene) of bacillus subtilis or bacillus amyloliquefaciens such that glcK protein activity is enhanced; further, by modifying the glucose/mannose transporter GlcP (encoded by the glcP gene), the activity of the GlcP protein is enhanced, so that the microorganism can efficiently and rapidly generate guanosine or inosine, thereby providing an effective means for mass production of nucleosides and having a wide application prospect.

Description

Construction method of nucleoside producing engineering bacteria and method for producing nucleosides

Technical Field

The invention belongs to the technical field of microbial engineering, and particularly relates to a construction method of nucleoside-producing engineering bacteria and a method for producing nucleosides.

Background

Nucleosides are a generic term for a class of glycosides. Nucleosides are constituents of nucleic acids and nucleotides. The nucleoside is formed by condensing D-ribose or D-Z-deoxyribose with pyrimidine base or purine base. The nucleoside is generally colorless crystals, insoluble in common organic solvents, soluble in hot water and having a melting point of 160-240 ℃. Nucleosides produced from D-ribose are called ribonucleosides, participate in RNA composition, nucleosides produced from D- α -deoxyribose are called deoxyribonucleosides, participate in DNA composition. D-ribose condenses with adenine, guanine, cytosine, thymine, or uracil to form the corresponding adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, thymine ribonucleoside, and uracil ribonucleoside, which are abbreviated as adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U), respectively.

Guanosine (guanosine) and inosine (inosine) have a wide variety of roles in the food and pharmaceutical industries. In the food field, guanosine and inosine are important precursors of disodium guanylate and disodium inosinate, respectively, and the combination of disodium guanylate and disodium inosinate is used as a food flavor enhancer, and is widely applied to condiments such as chicken essence, soy sauce and the like. In the field of medicine, guanosine and inosine can be used as medicine intermediates of various antiviral medicines, such as acyclovir, ribavirin, guanosine triphosphate sodium and the like, and guanosine is required to be used as a synthetic raw material. Inosine is an important precursor of inosinic acid, and inosinic acid can be used as a precursor for synthesizing Adenylate (AMP) and Guanylate (GMP), and is suitable for treating leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, liver cirrhosis and the like caused by various reasons, and can also treat central retinitis, optic atrophy and the like.

At present, microbial fermentation is a main method for producing nucleosides, and mainly used microorganisms include bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and the like. In the breeding and transformation process of the growing strain, the nucleoside high-yield strain is directionally bred by using ultraviolet mutagenesis and diethyl sulfate for mutation breeding; or based on the metabolic path and regulation mechanism of nucleotide in bacteria, the genetic background and the characteristics of the strain are deeply known, and the strain is purposefully modified by metabolic engineering means to obtain the production strain with excellent properties and high nucleoside yield. However, the fermentation performance of the current nucleoside strain is still poor, the conversion rate of the nucleoside is still low, and the requirement of large-scale industrial production cannot be met.

Disclosure of Invention

The invention aims to provide a construction method of nucleoside producing engineering bacteria and a method for producing nucleosides.

To achieve the object of the present invention, in a first aspect, the present invention provides a modified microorganism which has an enhanced activity of glucokinase (encoded by glcK gene) as compared to an unmodified microorganism and which has an enhanced nucleoside producing ability as compared to an unmodified microorganism. Also, these unmodified microorganisms have nucleoside producing ability themselves.

In the present invention, the microorganism is a Bacillus (Bacillus) or Escherichia) species, preferably Bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus), escherichia coli (Escherichia coli). More preferably B.subtilis or B.amyloliquefaciens, such as strain B.a8333.

The construction process of the a8333 strain is as follows (see cn202111266398. X): with strain DSM7 (ATCC 23350, see Genome sequence of B. Amyloquefaciens type strain DSM 7) ^T reveals differences to plant-associated B.amyloquefaciens FZB 42) genome as template, guaB-1f/1r and guaB-2f/3r as primers, 2 pieces were amplified using Phusion super Fidelity polymerase (New England BioLabs)Segments. 2 fragments were fused by using the primer guaB-1f/3r to obtain a recombinant fragment (the nucleotide sequence of the ORF region is shown as SEQ ID NO: 1). Will guaB ^L454F Fragment and pKSU plasmid (pKSU plasmid is given by university of south opening Wang Shufang, see A markerless gene replacement method for B. Amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-gamma-glutamic acid production [ J)]The recombinant plasmid pKSU-guaB is obtained after operations such as SalI/PstI double digestion, assembly, transformation and the like of 8963-8973.Zhang W,Gao W,Feng J,et al DOI:10.1007/s00253-014-5824-2 (21), applied Microbiology and Biotechnology,2014,98 (21) ^L454F . Transformants were selected at 30℃with LB plates containing 2.5. Mu.g/mL chloramphenicol, transferred to B.a 836 strain (see CN 112574934A), inoculated into 5mL LB liquid medium, cultured at 42℃at 200rpm for 12 hours and transferred to a generation, and diluted and spread to LB plates containing 5. Mu.g/mL chloramphenicol to obtain primary recombinants; inoculating the primary recombinant into 5ml LB liquid medium, culturing at 42 deg.C and 200rpm for 12 hr, transferring to primary, diluting and coating LB plate containing 0.8 μm 5-FU for screening secondary recombinant, and screening to obtain guaB ^L454F The strain obtained was b.a 837. The plasmid pBE43 was extracted (PBE 43 plasmid was total gene synthesis, reference Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens), linearized using KpnI/SalI, fusion PCR of the mutated purR sequence (abbreviated as R2, SEQ ID NO: 2) and mutated tal sequence (abbreviated as L5, SEQ ID NO: 3) to obtain a fragment R2+L5, and ligated into the linearized PBE43 plasmid using an assembly kit to construct the plasmid PBE43-R2+L5. This plasmid was transformed into strain b.a 837 to give the starting strain b.a8333.

The enhancement of glucokinase activity is achieved by a member selected from the following 1) to 6), or an optional combination:

1) Enhanced by introducing a plasmid having a gene encoding the enzyme;

2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;

3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;

4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;

5) Enhancement by modification of the amino acid sequence of the enzyme;

6) Enhanced by altering the nucleotide sequence of the gene encoding the enzyme.

Further, the enhancement of glucokinase activity was achieved by inserting a P43 promoter upstream of the start codon of the glcK gene.

Further, the enhancement of glucokinase activity is achieved by mutating amino acid 105 of glucokinase from I to M.

Further, the enhancement of glucokinase activity is achieved by the ectopic insertion of one, two or more copies of the glcK gene (preferably two copies of the glcK gene) driven by the P43 promoter.

Further, the enhancement of glucokinase activity is achieved by inserting a P43 promoter upstream of the start codon of the glcK gene, and inserting one, two or more copies of the glcK gene (preferably two copies of the glcK gene) driven by the P43 promoter ectopic, and mutating amino acid 105 of glucokinase from I to M.

Preferably, the microorganism is bacillus subtilis or bacillus amyloliquefaciens.

In a second aspect, the invention provides a modified microorganism having an increased activity of glucokinase (encoded by the glcK gene) and glucose transporter and/or mannose transporter (encoded by the glcP gene) compared to an unmodified microorganism, and having an increased nucleoside production capacity compared to an unmodified microorganism. Also, these unmodified microorganisms have nucleoside producing ability themselves.

Enhancement of glucokinase, glucose transporter and/or mannose transporter activity is achieved by a member selected from the following 1) to 6), or an optional combination:

1) Enhanced by introducing a plasmid having a gene encoding the enzyme or protein;

2) Enhancement by increasing the copy number of the gene encoding the enzyme or protein on the chromosome;

3) Enhancement by altering the promoter sequence of the gene encoding the enzyme or protein on the chromosome;

4) Enhanced by operably linking a strong promoter to a gene encoding the enzyme or protein;

5) Enhancement by modification of the amino acid sequence of the enzyme or protein;

6) Enhanced by altering the nucleotide sequence of the gene encoding the enzyme or protein.

Further, the enhancement of glucokinase activity was achieved by inserting a P43 promoter upstream of the start codon of the glcK gene.

Further, the enhancement of glucokinase activity is achieved by mutating amino acid 105 of glucokinase from I to M.

Further, the enhancement of glucokinase activity is achieved by the ectopic insertion of one, two or more copies of the glcK gene (preferably two copies of the glcK gene) driven by the P43 promoter.

Further, the enhancement of glucokinase activity is achieved by inserting a P43 promoter upstream of the start codon of the glcK gene, and inserting one, two or more copies of the glcK gene (preferably two copies of the glcK gene) driven by the P43 promoter ectopic, and mutating amino acid 105 of glucokinase from I to M.

Further, the enhancement of glucose transporter and/or mannose transporter activity is achieved by inserting a P43 promoter upstream of the glcP gene start codon.

Further, enhancement of glucose transporter and/or mannose transporter activity is achieved by insertion of a two-copy glcP gene driven by the P43 promoter at the alpha amylase gene.

Further, 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, and inserting two copies of the glcP gene driven by the P43 promoter at the alpha amylase gene.

Further, the enhancement of the glucose transporter and/or mannose transporter activity 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 alpha amylase gene, and mutating amino acid 206 of the glucose transporter and/or mannose transporter from H to R.

Preferably, the microorganism is bacillus subtilis or bacillus amyloliquefaciens.

In the present invention, the reference sequence number of glucokinase at NCBI is KS08_12550, or the amino acid sequence with similarity of 90%. The glucose/mannose transporter GlcP has a reference sequence number ks08_00900 at NCBI, or an amino acid sequence that is 90% similar thereto. The alpha amylase gene has a reference sequence number of K08_ 07940, or a nucleotide sequence with 97% similarity thereto, at NCBI.

In a third aspect, the present invention provides a method for constructing a nucleoside producing engineering bacterium, the method comprising: the gene glcK in the microorganism with nucleoside production capacity is enhanced by utilizing a genetic engineering means to obtain a strain with enhanced glucokinase activity; alternatively, genes glcK and glcP in a microorganism having nucleoside producing ability are enhanced to obtain a strain having enhanced glucose kinase and glucose transporter and/or mannose transporter activity.

In a fourth aspect, the present invention provides a method of producing nucleosides, the method comprising the steps of:

a) Culturing the microorganism to obtain a culture of the microorganism;

b) Collecting the produced nucleosides from the culture obtained in step a).

The nucleoside includes inosine, guanosine, etc. or corresponding nucleoside derivatives such as inosine, inosinic acid, guanosine, guanylic acid, riboflavin, diacetylguanylic acid, etc.

In a fifth aspect, the present invention provides the use of the modified microorganism or the engineered nucleoside producing bacterium constructed according to the above method in the fermentative production of nucleosides or in improving the fermentative production of nucleosides.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

mutant strain B.a 8338 (containing glcK) ^I105M Point mutation, P43-glcP two-copy in which the in situ glcP H206R point mutant strain), B.a 8339 (glcK promoter-enhanced, P43-glcP two-copy in which the in situ glcP H206R point mutant strain) and B.a 8340 (glcK) ^I105M The two-copy strain and the P43-glcP two-copy strain in which the in-situ glcP is subjected to H206R point mutation) have the guanosine yield increased from 15.0g/L, 15.5g/L, 16.2g/L to 15.5g/L, 16.2g/L and 17g/L respectively compared with the respective original strain. The glycoside conversion rate is respectively improved by 1%, 1% and 0.7%.

Detailed Description

The present invention aims to provide a method for producing purine nucleosides by using a microorganism, and a novel microorganism capable of efficiently producing purine nucleosides and a method for constructing the same, which are used in the method.

It has been found that the GlcK (encoded by the GlcK gene) of the glucokinase of bacillus subtilis or bacillus amyloliquefaciens is modified so that the GlcK protein activity is enhanced, so that a microorganism can efficiently and rapidly produce guanosine or inosine, and a novel microorganism capable of efficiently producing nucleoside is successfully created, thereby completing the present invention.

Alternatively, the ability of the strain to produce nucleosides is further enhanced by modifying the glucose/mannose transporter GlcP (encoded by the GlcP gene) such that GlcP protein activity is enhanced.

The invention firstly changes the amino acid sequence in bacillus amyloliquefaciens by mutating the gene, or inserts a strong promoter in front of an original promoter or increases the copy number in an ectopic way, so that the corresponding mutant can be used for producing the nucleoside with high efficiency, and a microorganism capable of producing the nucleoside with high efficiency is successfully created.

The invention adopts the following technical scheme:

the present invention provides a Bacillus amyloliquefaciens, wherein the intracellular coding glucokinase gene has changed amino acid sequence, and/or a P43 strong promoter is inserted before the glcK initiation codon, and/or gl containing the P43 promoter is inserted in an ectopic mannerThe cK gene or glcK ^I105M One or more copies such that the ability of the strain to produce nucleosides is enhanced compared to an unmodified strain.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

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

TABLE 1

EXAMPLE 1 construction of glcP promoter-enhanced Strain

Using Bacillus amyloliquefaciens ATCC13952 genome as a template, and using a P43-glcP-1f/P43-glcP-1r, P43-glcP-2f/P43-glcP-2r primer pair to obtain upstream and downstream homology arms of glcP by pfu high-fidelity DNA polymerase amplification; the P43 fragment was obtained by amplification using the P43-F1/P43-R1 primer and the PBE43 plasmid (PBE 43 plasmid is total gene synthesis, reference Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens) as a template. The fragments obtained above were subjected to gel recovery. The three fragments obtained above were fused together with the primer P43-glcP-1f/P43-glcP-2r to obtain a full-length fragment of P43-glcP, and the gel was recovered. The pKSU plasmid (pKSU plasmid is given by the university of south opening Wang Shufang, see A markerless gene replacement method for B. Amyloquefascians LL3 and its use in genome reduction and improvement of poly-. Gamma. -glutamic acid production [ J ], applied Microbiology and Biotechnology,2014,98 (21): 8963-8973.Zhang W,Gao W,Feng J,et al DOI:10.1007/s 00253-014-5824-2) was double digested with XbaI/PstI and subjected to gel recovery. And assembling the digested linearized plasmid and the P43-glcP fragment by using an assembly kit, converting the linearized plasmid and the P43-glcP fragment into a TransT1 competence, and performing identification and screening at a later stage to obtain the recombinant plasmid pKSU-P43-glcP. Transferring to B.a8333 strain, screening transformant with LB plate containing 2.5 mug/mL chloramphenicol at 30 deg.C, inoculating obtained transformant into 5mL LB liquid medium, culturing at 42 deg.C and 200rpm for 12h and transferring to generation, diluting and coating onto LB plate containing 5 mug/mL chloramphenicol to obtain primary recombinant; the primary recombinants were inoculated into 5ml of LB liquid medium, cultured at 42℃at 200rpm for 12 hours and transferred to the first generation, and the secondary recombinants were screened by diluting and coating with LB plates containing 0.8. Mu.M 5-FU, and the glcP promoter-enhanced strain was obtained by screening and named B.a 8334.

EXAMPLE 2 construction of glcP two-copy Gene-enriched Strain

The primer pairs glcP2nd-1f/glcP2nd-1R, P43-glcP-2f/glcP2nd-2R, glcP2nd-3f/glcP2nd-3R and ATCC13952 genome are used as templates, left homology arms glcP2nd-L, glcP and glcP2nd-R fragments are obtained by amplification, and the fragments are subjected to gel recovery. The 4 fragments of glcP2nd-L, the P43-glcP fragment obtained in example 1, glcP and glcP2nd-R were fused using the glcP2nd-1f/glcP2nd-3R primers to obtain a full length fragment of glcP2 nd. The plasmid pKSU-glcP2nd was constructed and transformed into strain B.a8333 according to the construction method of the plasmid in example 1, and the strain containing two copies of glcP was obtained by screening according to the method of the strain screening in example 1, and designated as B.a8335.

Example 3 construction of glcP two-copy Gene and in situ promoter-enhanced Strain

The plasmid pKSU-P43-glcP obtained in example 1 was transformed using the strain B.a8335 as starting strain, and the positive strain obtained by screening in the method of screening strain in example 1 was designated as B.a 8336.

EXAMPLE 4 construction of glcP Point mutant Strain

The primer pairs glcP-1f/1R and glcP-2f/2R are used, the B.a 8336 genome is used as a template, and the left homology arm glcP-L and the right homology arm glcP-R are obtained through amplification, so that glue recovery is carried out. Using the gel fraction as template, and carrying out fragment fusion on glcP-1f and glcP-2r primers to obtain full-length fragment containing point mutationThe sequence of the ORF frame of the gene is shown in SEQ ID NO. 4, and the 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 Plasmid was transformed into B.a 8336 strain, and the resultant strain was selected to contain glcP according to the strain selection method in example 1 ^H206R The strain with point mutation was designated b.a 8337.

EXAMPLE 5 construction of glcP-enhanced Strain superimposed glcK Point mutant Strain

The upstream and downstream homology arms of glcK were obtained by amplification with pfu high fidelity DNA polymerase using the DSM7 (wild type strain) genome as template (ATCC 23350, see Genome sequence of B. Amyloliquefaciens type strain DSM7T reveals differences to plant-associated B. Amyloliquefaciens FZB 42), using the glcK-1f/glcK-1r, glcK-2f/glcK-2r primer pair; the fragments obtained above were subjected to gel recovery. Then uses the primer glcK-1f/glcK-2r and uses the homologous arms of glcK as templates to fuse two fragments to obtain the product containing glcK ^I105M The full-length fragment of the point mutation (the nucleic acid sequence is shown as SEQ ID NO:6, the coded amino acid sequence is shown as SEQ ID NO: 7) is subjected to gel recovery. Recombinant plasmid pKSU-glcK was obtained according to the plasmid construction method in example 1 ^I105M . The strain was transformed into strain B.a 8337 and the strain obtained by screening was designated as B.a 8338.

EXAMPLE 6 construction of glcP-enhanced Strain superimposed with glcK Strong promoter Strain

Using bacillus amyloliquefaciens DSM7 genome as a template, and using a P43-glcK-1f/P43-glcK-1r primer pair and a P43-glcK-3f/P43-glcK-3r primer pair to obtain upstream and downstream homology arms of the glcK through pfu high-fidelity DNA polymerase amplification; the P43 fragment was obtained by amplification using the P43-F1/P43-R1 primer and the PBE43 plasmid as a template. The fragments obtained above were subjected to gel recovery. The three fragments obtained above were fused together with the primer P43-glcK-1f/P43-glcK-3r to obtain the fragment corresponding to P43-glcK ^I105M Full length fragment, plasmid PKSU-P43-glcK was obtained as constructed in example 1 ^I105M . The strain was transformed into B.a 8338 strain, and the glcK promoter-enhanced strain was obtained by screening and designated as B.a 8339.

EXAMPLE 7 construction of glcP-enhanced Strain superimposed glcK two-copy Strain

Respectively using leadsThe pair of glcP2nd-1F/glcP2nd-1R, glcK2nd-3F/glcK2nd-3R, DSM7 genome as template, the left homology arm glcK2nd-L, glcP nd-R fragment was amplified, the B.a 8339 strain genome as template, and the P43-F1/glcK2nd-2R primer was used for amplification to obtain P43-glcK2nd ^I105M Fragments. The 3-fragment gel was recovered. The construction of the plasmid according to example 1 gave pKSU-glcK2nd ^I105M Plasmid, which was transformed into B.a 8339 strain, was selected to obtain a strain containing glcK according to the method for selecting a strain in example 1 ^I105M The two-copy strain was designated b.a 8340.

EXAMPLE 8 construction of glcK mutant strains

To verify whether the single engineering of glcK would increase the glycoside production of the strain, the pKSU-glcK obtained in example 5 was used as starting strain with the B.a8333 strain ^I105M Plasmid transformation wherein strain b.a 8341 was obtained by screening; the plasmid PKSU-P43-glcK obtained in example 7 was obtained using the B.a 8341 strain as starting material ^I105M Transferring, wherein the strain obtained by screening is named B.a 8342; the plasmid pKSU-glcK2nd obtained in example 7 was prepared by using the B.a 8342 strain as starting material ^I105M Into which the strain obtained by screening was b.a 8343.

EXAMPLE 9 verification of glycoside production Performance of mutant Strain

1. The strain stored in glycerol was cultured overnight at 37℃to score a monoclonal.

2. The single colony is inoculated into 30mL seed culture medium (20 g/L glucose, 5g/L yeast powder, 5g/L corn steep liquor dry powder, 3g/L monopotassium phosphate, 0.5g/L magnesium sulfate, 0.02g/L ferrous sulfate, 0.01g/L manganese sulfate, pH 7.0-7.2) and cultured for 7-8 h at 37 ℃ at 110 rpm.

3. Transferring the mixture into 30ml fermentation culture medium (120 g/L of glucose, 3.5g/L of yeast powder, 3g/L of monopotassium phosphate, 25g/L of ammonium sulfate, 0.01g/L of manganese sulfate, 5g/L of magnesium sulfate, 10g/L of sodium glutamate, 15g/L of corn steep liquor dry powder, 25g/L of calcium carbonate and pH 7.0-7.2) according to the inoculation amount of 10% v/v, and culturing at 35 ℃ for 70h at the rotation speed of a shaking table of 130 rpm.

4. The fermentation broth was tested for glycoside production using a liquid chromatograph (table 2).

Table 2 mutant strains were shake-flask fermented to guanosine and inosine evaluation results (average of three replicates)

Strain	Total glycoside yield (g/L)	Guanosine yield (g/L)	Inosine yield (g/L)	OD 562	Total glycoside lifting ratio%
						B.a 8333	11.2	9.8	1.0	28.3	Control strain
B.a 8334	13.2	11.5	1.3	28.5	17.9％
						B.a 8335	13.8	12.0	1.3	27.6	23.2％
B.a 8336	14.5	13.1	1.0	27.6	29.5％
						B.a 8337	15.9	15.0	0.5	29.0	42.0
B.a 8338	16.4	15.5	0.3	29.8	46.4
						B.a 8339	16.9	16.2	0.6	30.1	51.0
B.a 8340	17.5	17	0.3	31.4	56.3
						B.a 8341	11.7	10.2	1.2	28.3	4.5
B.a 8342	12.3	10.7	1.5	27.6	9.8
						B.a 8343	12.7	11	1.5	28.6	13.4

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

<110> gallery plum blossom biotechnology development Co., ltd

<120> construction method of nucleoside producing engineering bacterium and method for producing nucleoside

<130> KHP221111667.7

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1467

<212> DNA

<213> Bacillus amyloliquefaciens (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

tttttaacac ctgatcatca agtattcgat gcggagcatt tgatggggaa atacagaatt 360

tccggtgttc cgatcgtaga taataaagac gatcaaaagc tggtcggtat cattacaaac 420

cgcgatcttc gctttatctc tgattattca atgaaaatca gtgatgttat gacaaaagaa 480

gagctggtta cggctcctgt gggaaccaca ttagacgaag cggaaaaaat cttgcagaag 540

cataaaattg aaaaacttcc attagtggat gaccaaaaca aattaaaagg 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 (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 (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 (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 cgggcgcaaa aacgtcgtta tctttggcct ggcactcatc 240

agctgtgagg ttttcatcac cttcctgccg ccctggccgc tgctctttct tctcgtcagt 300

atagcaggac tcggtgctgg attggtggaa tcctgcgtag gcacgattat cctcactgct 360

attaaagaaa gacaagccgt ggctatgagc aagatggaag tggcttacgg attaggggcg 420

ctgttcatgc ctctgctttc aggctttctt atcaacagcc acatgtggac aattgctttt 480

ttagtattag ggttatccag ttttgcactt ttaatcgcat ggaagcaaat gagttttggg 540

agcattgatc agcttctcat acgcaaagat gtctcttccg acggcactaa aaaagaaagc 600

accggctatc ggtcacgtgg attgctgttt attgccctgg cggctgctta cttcttcttt 660

tacggaggca gtgaagtttc aattgtacat tttatcccct ccatcttcgc tgagaaatgg 720

gatatcccca actccttagc aacgataaca gtcaccgtgt attggactgg gatgattatc 780

ggcaggttat taacaggtcc ggtatctgaa aagctgacat atcaccgtta tctccgtatt 840

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 (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

<210> 6

<211> 963

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

atggaagaga catggtttgc ggggattgat cttggcggaa caaccatcaa gctggccttt 60

atcaacatgt acggtgaaat tcagcacaaa tgggaggttc cgaccgataa atcaggaaac 120

acaattacgg tcacgatcgc taaagcactt gaccagaaac tcgaagaatt gaataagccg 180

aaacggatcg taaaatggat cggcatggga gcgcccggac ccgtggaaat ggcgacgggg 240

atggtttatg agacgacaaa tctggggtgg aaaaactatc ccttgaaaga ccatctcgag 300

gcggaaacgg gtatgccggc cgtcattgaa aacgatgcca atatcgcggc gctcggagaa 360

atgtggaaag gtgcaggcga cggagctaaa gatgtcattt tagtgacgct tggaaccgga 420

gtcggcgggg gcatcatcgt caacggagaa atcgttcacg gtaaaaacgg ggcaggcgga 480

gaaatcggtc atatttgcag tatcccggaa ggcggggctc cgtgcaactg cggaaaatcc 540

ggctgtattg aaacgattgc gtcagcaacc ggcatcgtcc gcatcgcgaa agaaaagctt 600

gcagcggttt ccgactcttc gctcctgcaa gtgcgcgatc tcacggcacg tgacgtgttc 660

gaagcggcaa aacagcagga taagacagcg ctggaagtcg tcgattatgt cgcaaaacat 720

ttaggccttg tgctcggaaa cctggcaagc gccatgaacc cgactaaaat cgtgctcggc 780

ggaggcgtct cgaaagcagg agaaatactg cggtcaaaag tggaagagac cttcaaaatc 840

accgcgttcc cgcgttctgc ggaagcggcc gatatttcga ttgcggcact tggaaatgac 900

gccggagtca tcggcggggc gtggattgcc aaaaatgaat ggcttaaata tcaaaactgc 960

tag 963

<210> 7

<211> 320

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Met Glu Glu Thr Trp Phe Ala Gly Ile Asp Leu Gly Gly Thr Thr Ile

1 5 10 15

Lys Leu Ala Phe Ile Asn Met Tyr Gly Glu Ile Gln His Lys Trp Glu

20 25 30

Val Pro Thr Asp Lys Ser Gly Asn Thr Ile Thr Val Thr Ile Ala Lys

35 40 45

Ala Leu Asp Gln Lys Leu Glu Glu Leu Asn Lys Pro Lys Arg Ile Val

50 55 60

Lys Trp Ile Gly Met Gly Ala Pro Gly Pro Val Glu Met Ala Thr Gly

65 70 75 80

Met Val Tyr Glu Thr Thr Asn Leu Gly Trp Lys Asn Tyr Pro Leu Lys

85 90 95

Asp His Leu Glu Ala Glu Thr Gly Met Pro Ala Val Ile Glu Asn Asp

100 105 110

Ala Asn Ile Ala Ala Leu Gly Glu Met Trp Lys Gly Ala Gly Asp Gly

115 120 125

Ala Lys Asp Val Ile Leu Val Thr Leu Gly Thr Gly Val Gly Gly Gly

130 135 140

Ile Ile Val Asn Gly Glu Ile Val His Gly Lys Asn Gly Ala Gly Gly

145 150 155 160

Glu Ile Gly His Ile Cys Ser Ile Pro Glu Gly Gly Ala Pro Cys Asn

165 170 175

Cys Gly Lys Ser Gly Cys Ile Glu Thr Ile Ala Ser Ala Thr Gly Ile

180 185 190

Val Arg Ile Ala Lys Glu Lys Leu Ala Ala Val Ser Asp Ser Ser Leu

195 200 205

Leu Gln Val Arg Asp Leu Thr Ala Arg Asp Val Phe Glu Ala Ala Lys

210 215 220

Gln Gln Asp Lys Thr Ala Leu Glu Val Val Asp Tyr Val Ala Lys His

225 230 235 240

Leu Gly Leu Val Leu Gly Asn Leu Ala Ser Ala Met Asn Pro Thr Lys

245 250 255

Ile Val Leu Gly Gly Gly Val Ser Lys Ala Gly Glu Ile Leu Arg Ser

260 265 270

Lys Val Glu Glu Thr Phe Lys Ile Thr Ala Phe Pro Arg Ser Ala Glu

275 280 285

Ala Ala Asp Ile Ser Ile Ala Ala Leu Gly Asn Asp Ala Gly Val Ile

290 295 300

Gly Gly Ala Trp Ile Ala Lys Asn Glu Trp Leu Lys Tyr Gln Asn Cys

305 310 315 320

Claims (10)

1. A modified microorganism, characterized in that the microorganism has an increased activity of glucokinase as compared to an unmodified microorganism and the microorganism has an increased nucleoside production capacity as compared to an unmodified microorganism;

wherein the microorganism is a Bacillus or Escherichia species, preferably Bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus), escherichia coli (Escherichia coli).

2. The microorganism of claim 1, wherein the enhancement of glucokinase activity is achieved by a member selected from the group consisting of 1) to 6), or an optional combination of:

1) Enhanced by introducing a plasmid having a gene encoding the enzyme;

2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;

3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;

4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;

5) Enhancement by modification of the amino acid sequence of the enzyme;

6) Enhanced by altering the nucleotide sequence of the gene encoding the enzyme.

3. The microorganism according to claim 1 or 2, characterized in that the enhancement of the glucokinase activity is achieved by inserting a P43 promoter upstream of the start codon of the glcK gene; or alternatively, the process may be performed,

enhancement of glucokinase activity is achieved by mutating amino acid 105 of glucokinase from I to M; or alternatively, the process may be performed,

enhancement of glucokinase activity is achieved by ectopic insertion of one, two or more copies of the glcK gene driven by the P43 promoter; or alternatively, the process may be performed,

the enhancement of glucokinase activity is achieved by inserting a P43 promoter upstream of the start codon of the glcK gene, and ectopic insertion of one, two or more copies of the glcK gene driven by the P43 promoter, and mutating amino acid 105 of glucokinase from I to M;

preferably, the microorganism is bacillus subtilis or bacillus amyloliquefaciens.

4. A modified microorganism, characterized in that the microorganism has an increased activity of glucokinase and glucose transporter and/or mannose transporter compared to an unmodified microorganism and the microorganism has an increased nucleoside production capacity compared to an unmodified microorganism;

wherein the microorganism is a Bacillus or Escherichia species, preferably Bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus), escherichia coli (Escherichia coli).

5. The microorganism of claim 1, wherein the enhancement of the activity of glucokinase, glucose transporter and/or mannose transporter is achieved by a member selected from the group consisting of 1) to 6), or an optional combination of:

1) Enhanced by introducing a plasmid having a gene encoding the enzyme or protein;

2) Enhancement by increasing the copy number of the gene encoding the enzyme or protein on the chromosome;

3) Enhancement by altering the promoter sequence of the gene encoding the enzyme or protein on the chromosome;

4) Enhanced by operably linking a strong promoter to a gene encoding the enzyme or protein;

5) Enhancement by modification of the amino acid sequence of the enzyme or protein;

6) Enhanced by altering the nucleotide sequence of the gene encoding the enzyme or protein.

6. The microorganism according to claim 4, wherein the enhancement of the glucokinase activity is achieved by inserting a P43 promoter upstream of the start codon of the glcK gene; or alternatively, the process may be performed,

enhancement of glucokinase activity is achieved by mutating amino acid 105 of glucokinase from I to M; or alternatively, the process may be performed,

enhancement of glucokinase activity is achieved by ectopic insertion of one, two or more copies of the glcK gene driven by the P43 promoter; or alternatively, the process may be performed,

the enhancement of glucokinase activity is achieved by inserting a P43 promoter upstream of the start codon of the glcK gene and inserting ectopic one, two or more copies of the glcK gene driven by the P43 promoter and mutating amino acid 105 of glucokinase from I to M.

7. The microorganism according to any of claims 4 to 6, characterized in that the enhancement of the glucose transporter and/or mannose transporter activity is achieved by inserting a P43 promoter upstream of the glcP gene start codon; or alternatively, the process may be performed,

the enhancement of glucose transporter and/or mannose transporter activity is achieved by inserting a two-copy glcP gene driven by the P43 promoter at the alpha amylase gene; or alternatively, the process may be performed,

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, and inserting a two copy glcP gene driven by the P43 promoter at the alpha amylase gene; or alternatively, the process may be performed,

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 and inserting a double copy of the glcP gene driven by the P43 promoter at the alpha amylase gene and mutating amino acid 206 of the glucose transporter and/or mannose transporter from H to R;

preferably, the microorganism is bacillus subtilis or bacillus amyloliquefaciens.

8. The construction method of the nucleoside producing engineering bacteria is characterized by comprising the following steps: the gene glcK in the microorganism with nucleoside production capacity is enhanced by utilizing a genetic engineering means to obtain a strain with enhanced glucokinase activity; alternatively, genes glcK and glcP in a microorganism having nucleoside producing ability are enhanced to obtain a strain having enhanced glucose kinase and glucose transporter and/or mannose transporter activity;

the enhanced pathway is selected from the following 1) to 6), or an optional combination:

1) Enhanced by introducing a plasmid having a gene encoding the enzyme or protein;

2) Enhancement by increasing the copy number of the gene encoding the enzyme or protein on the chromosome;

3) Enhancement by altering the promoter sequence of the gene encoding the enzyme or protein on the chromosome;

4) Enhanced by operably linking a strong promoter to a gene encoding the enzyme or protein;

5) Enhancement by modification of the amino acid sequence of the enzyme or protein;

6) Enhancement by altering the nucleotide sequence of the gene encoding the enzyme or protein;

wherein the microorganism is a Bacillus or Escherichia species, preferably Bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus), escherichia coli (Escherichia coli).

9. A method of producing nucleosides, said method comprising the steps of:

a) Culturing the microorganism of any one of claims 1-7 to obtain a culture of the microorganism;

b) Collecting the produced nucleosides from the culture obtained in step a).

10. The method of claim 9, wherein the nucleoside comprises inosine, guanosine, and corresponding nucleoside derivatives thereof.