Phosphoenolpyruvate carboxykinase mutants and uses thereof

Abstract

The application provides a phosphoenolpyruvate carboxykinase mutant and application thereof. After the 147th amino acid of phosphoenolpyruvate carboxykinase in a bacillus microorganism is mutated from lysine (K) to arginine (R) or histidine (H), the production of adenosine and inosine is improved, and the mutant pckA K147H Compared with the original strain A1, the production of adenosine is increased from 1.3 g / L to 1.8 g / L, and the production of inosine is increased from 0.6 g / L to 1.3 g / L. The application provides an effective means for large-scale production of nucleosides, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering technology, specifically, it relates to phosphoenolpyruvate carboxykinase mutants and their applications. 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°C. Nucleosides formed from D-ribose are called ribonucleosides and participate in the composition of RNA; nucleosides formed from D-α-deoxyribose are called deoxyribonucleosides and 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, respectively abbreviated as adenosine (A), guanine (G), cytosine (C), thymine (T), and uridine (U).

[0003] Guanosine and inosine have wide applications in the food and pharmaceutical industries. In the food sector, guanosine and inosine are important precursors of 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 of 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, and the main microorganisms used include Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus pumilus. In the selection and modification of growth strains, high-yielding nucleosides are selectively bred using ultraviolet mutagenesis and diethyl sulfate mutagenesis; or, based on a thorough understanding of the metabolic pathways and regulatory mechanisms of nucleotides in bacteria, the genetic background and characteristics of the strains are analyzed, and metabolic engineering is used to purposefully modify the strains to obtain high-yielding nucleosides with superior traits. However, the fermentation performance of current nucleoside strains remains relatively poor, and the conversion rate of nucleosides is still low, which cannot meet the needs of large-scale industrial production. Summary of the Invention

[0005] The purpose of this invention is to provide a phosphoenolpyruvate carboxykinase mutant and its applications.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a phosphoenolpyruvate carboxykinase mutant, said mutant comprising a mutation in the 147th amino acid of phosphoenolpyruvate carboxykinase from K to R or H.

[0007] In this invention, the reference sequence number of phosphoenolpyruvate carboxykinase on NCBI is NP_390934.2.

[0008] In a second aspect, the present invention provides a nucleic acid molecule encoding the phosphoenolpyruvate carboxykinase mutant.

[0009] Thirdly, the present invention provides biological materials containing the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0010] Fourthly, the present invention provides any of the following applications of the nucleic acid molecule or biological materials containing the nucleic acid molecule: (1) Used for the fermentation production of nucleosides; (2) Used to increase the fermentation yield of nucleosides; (3) Used to construct nucleoside-producing genetically engineered bacteria.

[0011] Fifthly, the present invention provides a method for constructing a nucleoside-producing strain, which uses genetic engineering to introduce mutations into the genome of a microorganism capable of nucleoside production, so that the encoded phosphoenolpyruvate carboxykinase contains a K147R or K147H mutation site.

[0012] or, Using genetic engineering techniques, mutations are introduced into the genomes of microorganisms capable of nucleoside production, so that the phosphoenolpyruvate carboxykinase they encode contains the K147R or K147H mutation site, and the pyruvate kinase they encode contains the T101K, T101R, or T101P mutation site.

[0013] or, Using genetic engineering techniques, mutations were introduced into the genomes of microorganisms capable of nucleoside production, causing their encoded phosphoenolpyruvate carboxylkinase to contain the K147R or K147H mutation site, and causing it to encode pyruvate carboxylase. pycA The gene contains a mutation in which the first base is changed from T to G or A.

[0014] or, Using genetic engineering techniques, mutations were introduced into the genomes of microorganisms capable of nucleoside production, causing their encoded phosphoenolpyruvate carboxylkinase to contain the K147R or K147H mutation site, and their encoded pyruvate kinase to contain the T101K, T101R, or T101P mutation site, and also causing their encoded pyruvate carboxylase... pycA The gene contains a mutation in which the first base is changed from T to G or A.

[0015] The microorganism is a species of Bacillus, such as Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus pumilus. Bacillus subtilis or Bacillus amyloliquefaciens are preferred, and Bacillus amyloliquefaciens is more preferred. B. subtilis A1 (see CN201910599510.8) or B.a 8333.

[0016] 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 reveals differences to plant-associated B. amyloliquefaciens Using the FZB42 genome as a template, and guaB-1f / 1r and guaB-2f / 3r as primers, two fragments were amplified using Phusion ultrafidelity 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... L454F 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) After SalI / PstI double digestion, assembly, transformation and other operations, the recombinant plasmid pKSU-guaB was obtained. L454FTransformed into Ba 836 strain (see CN112574934A), 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 transformed transformants were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, obtaining guaB. L454F The point mutation strain obtained was B.a837. The pBE43 plasmid was extracted (the pBE43 plasmid was synthesized as a whole gene; see reference: Effects of overexpression of key enzyme genes on guanosine accumulation in...). Bacillus amyloliquefaciens The plasmid was linearized using KpnI / SalI. A fusion PCR was performed on the mutated purR sequence (R2, SEQ ID NO:2) and the mutated tal sequence (L5, 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 the starting strain Ba 8333.

[0017] In this invention, the reference sequence number of pyruvate kinase on NCBI is NP_390796.1.

[0018] pycA The gene can originate from Bacillus subtilis, and it is: i) The nucleotide sequence shown in SEQ ID NO:8; ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:8 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:8 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the solution; iv) Nucleotide sequences that have more than 90% homology with the nucleotide sequences of i), ii) or iii) and express the same functional protein.

[0019] pycA The gene can originate from Bacillus amyloliquefaciens, which is: a) The nucleotide sequence shown in SEQ ID NO:11; b) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:11 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; c) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:11 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS, followed by washing the membrane with the solution. d) Nucleotide sequences that have more than 90% homology with the nucleotide sequences of a), b), or c) and express the same functional protein.

[0020] In a sixth aspect, the present invention provides a nucleoside-producing strain constructed according to the method.

[0021] In a seventh aspect, the present invention provides a method for producing nucleosides, the method comprising the following steps: 1) Cultivate the nucleoside-producing strain to obtain a microbial culture; 2) Collect the nucleosides produced from the culture obtained in step 1).

[0022] The nucleosides include adenosine, inosine, guanosine and their corresponding nucleoside derivatives, such as hypoxanthine, inosine, guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc.

[0023] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: Mutating the 101st amino acid of pyruvate kinase by changing threonine (T) to lysine (K), arginine (R), or proline (P) increases the production of adenosine and inosine, with the best effect observed after threonine (T) is mutated to lysine (K). Pyruvate kinase mutant strains pyk T101K Compared to the original strain A1, the adenosine production increased from 1.3 g / L to a maximum of 2.1 g / L; the pyruvate kinase mutant strain... pyk T101R Compared with the starting strain A1, the best result was an increase in inosine production from 0.6 g / L to 1.1 g / L.

[0024] Mutating the first base of the pyruvate carboxylase gene from T to G or A increases both adenosine and inosine production, with the T-to-A mutation showing the best effect. The mutant pycA has a positive effect on increasing adenosine and inosine production in Bacillus subtilis. t1a The optimal adenosine yield was increased from 1.3 g / L to 2.5 g / L by the engineered strain, with the mutant pycA showing the best yield. a1gThe optimal inosine yield of the engineered strain was increased from 0.6 g / L to 1.3 g / L.

[0025] After the amino acid at position 147 of phosphoenolpyruvate carboxykinase is mutated from lysine (K) to arginine (R) or histidine (H), the production of adenosine and inosine is increased. The mutant pckA... K147H Compared with the original strain A1, the engineered strain increased adenosine production from 1.3 g / L to 1.8 g / L and inosine production from 0.6 g / L to 1.3 g / L.

[0026] The superposition of pyruvate carboxylase mutant and pyruvate kinase mutant has a positive effect on increasing the production of adenosine and inosine. The mutant pycA t1a Overlay pyk T101K The engineered strain's adenosine production increased from 1.3 g / L to a maximum of 3.4 g / L, with the mutant pycA showing the highest yield. t1g Overlay pyk T101K The inosine yield of the engineered strain increased from 0.6 g / L to a maximum of 1.8 g / L.

[0027] The combined effects of pyruvate carboxylase mutant, pyruvate kinase mutant, and phosphoenolpyruvate carboxylkinase mutant significantly increased adenosine and inosine production. The mutant pycA... t1a , pyk T101K pckA K147R The superimposed engineered strains increased adenosine production from 1.3 g / L to 4.4 g / L and inosine production from 0.6 g / L to a maximum of 2.2 g / L.

[0028] The pyruvate carboxylase mutant has a positive effect on increasing the production of guanosine and inosine in Bacillus amyloliquefaciens. The mutant pycA t1a The optimal yield of guanosine from the engineered strain was increased from 9.8 g / L to 17.1 g / L, with the mutant pycA showing the best results. t1g The optimal inosine yield of the engineered strain was increased from 1.0 g / L to 1.6 g / L. Detailed Implementation

[0029] The present invention aims to provide a method for producing purine nucleosides using microorganisms, and a novel microorganism capable of producing purine nucleosides with high efficiency.

[0030] Studies have found that modified Bacillus subtilis or Bacillus amyloliquefaciens pyruvate kinase (by...) pyk Gene encoding) and / or pyruvate carboxylase (by pycA Gene-encoded) and / or phosphoenolpyruvate carboxykinase (by) pckA The invention utilizes gene coding to enable microorganisms to efficiently produce nucleosides, and successfully created new microorganisms capable of efficiently producing nucleosides, thus completing the present invention.

[0031] The present invention adopts the following technical solution: This invention provides a Bacillus subtilis strain, whose cells contain... pyk The 101st amino acid of the gene encoding pyruvate kinase (NCBI reference sequence number NP_390796.1) is mutated from threonine (T) to an amino acid other than threonine, preferably lysine (K), arginine (R), or proline (P), denoted as _____. pyk T101K , pyk T101R , pyk T101P ; and / or, its intracellular encoding pyruvate carboxylase (reference sequence number NP_389369.1 on NCBI). pycA A gene (reference sequence number on NCBI: Gene ID: 935920) whose first base is mutated from T to G or A, is denoted as pycA. t1g pycA t1a ; and / or, its cells are composed of pckA The 147th amino acid of the phosphoenolpyruvate carboxykinase (NCBI reference sequence number NP_390934.2), encoded by the gene (NCBI reference sequence number Gene ID: 937235), is mutated from lysine (K) to arginine (R) or histidine (H), respectively. pckA K147R , pckA K147H .

[0032] pyk The gene-encoded pyruvate kinase catalyzes the conversion of phosphoenolpyruvate to pyruvate, a process accompanied by the consumption of one molecule of ADP and the generation of one molecule of ATP. This invention, through... pyk Gene modification was used to mutate pyruvate kinase, which enhanced the microorganism's ability to produce nucleosides compared to the unmodified strain, ultimately increasing nucleoside production.

[0033] pycA The gene-encoded pyruvate carboxylase catalyzes the reaction of pyruvate and HCO3-. - The carboxylation reaction produces oxaloacetic acid, a process that consumes one molecule of ATP and generates one molecule of ADP. This invention, through... pycA Gene modification was used to mutate pyruvate carboxylase, which enhanced the microorganism's ability to produce nucleosides compared to the unmodified strain, ultimately increasing nucleoside production.

[0034] pckA The gene-encoded phosphoenolpyruvate carboxylkinase catalyzes the decarboxylation of oxaloacetate to produce phosphoenolpyruvate and carbon dioxide. This process is accompanied by the consumption of one molecule of ATP and the generation of one molecule of ADP. This invention, through... pckA Gene modification was used to mutate phosphoenolpyruvate carboxykinase, which enhanced the microorganism's ability to produce nucleosides compared to the unmodified strain, ultimately increasing nucleoside production.

[0035] The Bacillus subtilis strain used in this invention is Bacillus subtilis A1 (abbreviated as A1), see CN201910599510.8.

[0036] This invention also provides a Bacillus amyloliquefaciens, whose cells encode phosphoenolpyruvate carboxykinase (reference sequence number CBI42686.1 on NCBI). pycA The gene (reference sequence location on NCBI is BAMF_1560) has its first base mutated from T to G or A.

[0037] This invention, through the pycA The gene modification resulted in a mutation of pyruvate carboxylase, which enhanced the microorganism's ability to produce nucleosides compared to the unmodified strain, ultimately increasing nucleoside production.

[0038] The Bacillus amyloliquefaciens strain used in this invention is Bacillus amyloliquefaciens For details on how to construct 8333 (abbreviated as Ba 8333), please refer to CN202111266398.X.

[0039] The following examples are for illustrative purposes only and 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 in the manufacturer's instructions.

[0040] The amino acid sequence of pyruvate kinase involved in the following examples is shown in SEQ ID NO:4, pyruvate kinase mutant. pyk T101K The amino acid sequence is shown in SEQ ID NO:5, pyruvate kinase mutant. pyk T101R The amino acid sequence is shown in SEQ ID NO:6, pyruvate kinase mutant. pyk T101PThe amino acid sequence is shown in SEQ ID NO:7, and the nucleotide sequence encoding pyruvate carboxylase from Bacillus subtilis is shown in SEQ ID NO:8, encoding the pyruvate carboxylase mutant pycA from Bacillus subtilis. t1g The nucleotide sequence is shown in SEQ ID NO:9, encoding the pycA mutant pyruvate carboxylase from Bacillus subtilis. t1a The nucleotide sequence is shown in SEQ ID NO:10, encoding pyruvate carboxylase from Bacillus amyloliquefaciens, and the nucleotide sequence is shown in SEQ ID NO:11, encoding the pyruvate carboxylase mutant pycA from Bacillus amyloliquefaciens. t1g The nucleotide sequence is shown in SEQ ID NO:12, encoding the pycA mutant pyruvate carboxylase from Bacillus amyloliquefaciens. t1a The nucleotide sequence is shown in SEQ ID NO:13, the amino acid sequence of phosphoenolpyruvate carboxykinase is shown in SEQ ID NO:14, and the phosphoenolpyruvate carboxykinase mutant pckA K147R The amino acid sequence is shown in SEQ ID NO:15, phosphoenolpyruvate carboxykinase mutant pckA K147H The amino acid sequence is shown in SEQ ID NO:16.

[0041] The primers used in the following examples are shown in Table 1: Table 1

[0042] Example 1: Construction of pyruvate kinase mutant strain A1- pyk T101K strain B. subtilis Using the A1 genome as a template, primer pyk T101K -UP-1F / pyk T101K -UP-1R and pyk T101K -DN-2F / pyk T101K -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primers pyk were used. T101K -UP-1F / pyk T101K-DN-2R fuses two fragments to obtain a recombinant fragment. The recombinant fragment is then assembled with 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), transformed, and other operations to obtain the recombinant plasmid pKSU-pyk. T101K Transformed to B. subtilis In strain A1, transformants were screened on LB agar 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 agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then 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 agar plates containing 0.8 μM 5-FU to screen for secondary recombinants. pyk T101K The point mutant strain was named B. subtilis A1- pyk T101K Hereinafter referred to as A1- pyk T101K .

[0043] Example 2: Construction of pyruvate kinase mutant strain A1- pyk T101R strain B. subtilis Using the A1 genome as a template, primer pyk T101K -UP-1F / pyk T101R -UP-1R and pyk T101R -DN-2F / pyk T101K -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primers pyk were used. T101K -UP-1F / pyk T101K-DN-2R fuses two fragments to obtain a recombinant fragment. After assembling the recombinant fragment with the pKSU plasmid and performing transformation, the recombinant plasmid pKSU-pyk is obtained. T101R Transformed to B. subtilis In strain A1, transformants were screened on LB agar 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 agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then 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 agar plates containing 0.8 μM 5-FU to screen for secondary recombinants. pyk T101R The point mutant strain was named B. subtilis A1- pyk T101R Hereinafter referred to as A1- pyk T101R .

[0044] Example 3: Construction of pyruvate kinase mutant strain A1- pyk T101P strain B. subtilis Using the A1 genome as a template, primer pyk T101K -UP-1F / pyk T101P -UP-1R and pyk T101P -DN-2F / pyk T101K -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primers pyk were used. T101K -UP-1F / pyk T101K -DN-2R fuses two fragments to obtain a recombinant fragment. After assembling the recombinant fragment with the pKSU plasmid and performing transformation, the recombinant plasmid pKSU-pyk is obtained. T101P Transformed to B. subtilis In strain A1, transformants were screened on LB agar 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 agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then 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 agar plates containing 0.8 μM 5-FU to screen for secondary recombinants. pyk T101P The point mutant strain was namedB. subtilis A1- pyk T101P Hereinafter referred to as A1- pyk T101P .

[0045] Example 4: Construction of pyruvate carboxylase mutant strain A1- pycA t1g strain B. subtilis Using the A1 genome as a template, primer pycA t1g -UP-1F / pycA t1g -UP-1R and pycA t1g -DN-2F / pycA t1g -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pycA was used. t1g -UP-1F / pycA T1G -DN-2R fuses the two fragments to obtain a recombinant fragment. After assembling the recombinant fragment with the pKSU plasmid and performing transformation, the recombinant plasmid pKSU-pycA is obtained. t1g Transformed to B. subtilis In strain A1, 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. These primary recombinants were then 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 0.8 μM 5-FU to screen for secondary recombinants, obtaining pycA. t1g The point mutant strain was named B. subtilis A1-pycA t1g Hereinafter referred to as A1-pycA t1g .

[0046] Example 5: Construction of pyruvate carboxylase mutant strain A1- pycA t1a strain B. subtilis Using the A1 genome as a template, primer pycA t1g -UP-1F / pycA t1a -UP-1R and pycA t1a -DN-2F / pycA t1g-DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pycA was used. t1g -UP-1F / pycA t1g -DN-2R fuses the two fragments to obtain a recombinant fragment. After assembling the recombinant fragment with the pKSU plasmid and performing transformation, the recombinant plasmid pKSU-pycA is obtained. t1a Transformed to B. subtilis In strain A1, transformants were screened on LB agar 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 agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then 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 agar plates containing 0.8 μM 5-FU to screen for secondary recombinants. py cA t1a The point mutant strain was named B. subtilis A1- py cA t1a Hereinafter referred to as A1- py cA t1a .

[0047] Example 6: Construction of phosphoenolpyruvate carboxykinase mutant strain A1- pckA K147R strain B. subtilis Using the A1 genome as a template, primer pckA K147R -UP-1F / pckA K147R -UP-1R and pckA K147R -DN-2F / pckA K147R -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pckA was used. K147R -UP-1F / pckA K147R -DN-2R fuses the two fragments to obtain a recombinant fragment. After assembly and transformation with the pKSU plasmid, the recombinant fragment is obtained as the recombinant plasmid pKSU-pckA. K147R Transformed to B. subtilis In strain A1, 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. These primary recombinants were then 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 0.8 μM 5-FU to screen for secondary recombinants, obtaining pckA. K147R The point mutant strain was named B. subtilis A1-pckA K147R Hereinafter referred to as A1-pckA K147R .

[0048] Example 7: Construction of phosphoenolpyruvate carboxykinase mutant strain A1- pckA K147H strain B. subtilis Using the A1 genome as a template, primer pckA K147R -UP-1F / pckA K147H -UP-1R and pckA K147H -DN-2F / pckA K147R -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pckA was used. K147R -UP-1F / pckA K147R -DN-2R fuses the two fragments to obtain a recombinant fragment. After assembly and transformation with the pKSU plasmid, the recombinant fragment is obtained as the recombinant plasmid pKSU-pckA. K147H Transformed to B. subtilis In strain A1, 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. These primary recombinants were then 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 0.8 μM 5-FU to screen for secondary recombinants, obtaining pckA. K147H The point mutant strain was named B. subtilis A1- pckA K147H Hereinafter referred to as A1-pckA K147H .

[0049] Example 8: Construction of pyruvate carboxylase mutant strain A1- pyk T101K -pycA t1g strain B. subtilis Using the A1 genome as a template, primer pycA t1g -UP-1F / pycA t1g -UP-1R and pycA t1g -DN-2F / pycA t1g -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pycA was used. t1g -UP-1F / pycA t1g -DN-2R fuses the two fragments to obtain a recombinant fragment. After assembling the recombinant fragment with the pKSU plasmid and performing transformation, the recombinant plasmid pKSU-pycA is obtained. t1g Transformed to B. subtilis A1- pyk T101K 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, cultured at 42°C and 200 rpm for 12 h, and passaged for one generation. 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, 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 agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding pycA. t1g The point mutant strain was named B. subtilis A1- pyk T101K -pycA t1g Hereinafter referred to as A1- pyk T101K -pycA t1g .

[0050] Example 9: Construction of pyruvate carboxylase mutant strain A1- pyk T101K - py cA t1a strain B. subtilis Using the A1 genome as a template, primer pycA t1g -UP-1F / pycA t1a -UP-1R and pycA t1a -DN-2F / pycA t1g -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pycA was used. t1g-UP-1F / pycA t1g -DN-2R fuses the two fragments to obtain a recombinant fragment. After assembling the recombinant fragment with the pKSU plasmid and performing transformation, the recombinant plasmid pKSU-pycA is obtained. t1a Transformed to B. subtilis A1- pyk T101K Transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were then inoculated into 5 mL LB liquid medium and cultured at 42°C and 200 rpm for 12 h, followed by one generation. 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 and cultured at 42°C and 200 rpm for 12 h, followed by 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. py cA t1a The point mutant strain was named B. subtilis A1- pyk T101K - py cA t1a Hereinafter referred to as A1- pyk T101K - py cA t1a .

[0051] Example 10 Construction of pyruvate carboxylase mutant strain A1- pyk T101K -pycA t1a -pckA K147R strain B. subtilis Using the A1 genome as a template, primer pckA K147R -UP-1F / pckA K147R -UP-1R and pckA K147R -DN-2F / pckA K147R -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pckA was used. K147R -UP-1F / pckA K147R -DN-2R fuses the two fragments to obtain a recombinant fragment. After assembly and transformation with the pKSU plasmid, the recombinant fragment is obtained as the recombinant plasmid pKSU-pckA. K147R Transform to B. subtilisA1- pyk T101K -pycA t1aTransformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were then inoculated into 5 mL LB liquid medium, cultured at 42°C and 200 rpm for 12 h, and passaged for one generation. 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, 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 agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding pckA. K147R The point mutant strain was named B. subtilis A1- pyk T101K -pycA t1a -pckA K147R Hereinafter referred to as A1- pyk T101K -pycA t1a -pckA K147R .

[0052] Example 11 Construction of pyruvate carboxylase mutant strain A1- pyk T101K -pycA t1a -pckA K147H strain B. subtilis Using the A1 genome as a template, primer pckA K147R -UP-1F / pckA K147H -UP-1R and pckA K147H -DN-2F / pckA K147R -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pckA was used. K147R -UP-1F / pckA K147R -DN-2R fuses the two fragments to obtain a recombinant fragment. After assembly and transformation with the pKSU plasmid, the recombinant fragment is obtained as the recombinant plasmid pKSU-pckA. K147H Transform to B. subtilisA1- pyk T101K -pycA t1aTransformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were then inoculated into 5 mL LB liquid medium, cultured at 42°C and 200 rpm for 12 h, and passaged for one generation. 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, 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 agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding pckA. K147H The point mutant strain was named B. subtilis A1- pyk T101K -pycA t1a -pckA K147H Hereinafter referred to as A1- pyk T101K -pycA t1a -pckA K147H .

[0053] Example 12 Construction of pyruvate carboxylase mutant strain 8333-pycA t1g strain Bacillus amyloliquefaciens Using the 8333 genome as a template, primer pycA t1g -UP-3F / pycA t1g -UP-3R and pycA T1G -DN-4F / pycA T1G Two fragments of -DN-4R were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pycA was used to amplify the fragments. t1g -UP-3F / pycA t1g -DN-4R fuses the two fragments to obtain a recombinant fragment. After assembling the recombinant fragment with the pKSU plasmid and performing transformation, the recombinant plasmid pKSU-pycA is obtained. t1g Transformed to Bacillus amyloliquefaciens In strain 8333, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30℃. The transformed 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. These primary recombinants were then 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 0.8 μM 5-FU to screen for secondary recombinants, yielding pycA. t1g The point mutant strain was named Bacillus amyloliquefaciens 8333-pycA t1g Hereinafter referred to as 8333-pycAt1g .

[0054] Example 13 Construction of pyruvate carboxylase mutant strain 8333- pycA t1a strain Bacillus amyloliquefaciens Using the 8333 genome as a template, primer pycA t1g -UP-3F / pycA t1a -UP-3R and pycA t1a -DN-4F / pycA t1g Two fragments of -DN-4R were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer pycA was used to amplify the fragments. t1g -UP-3F / pycA t1g -DN-4R fuses the two fragments to obtain a recombinant fragment. After assembling the recombinant fragment with the pKSU plasmid and performing transformation, the recombinant plasmid pKSU-pycA is obtained. t1a Transformed to Bacillus amyloliquefaciens In strain 8333, 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 then 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. py cA t1a The point mutant strain was named Bacillus amyloliquefaciens 8333- py cA t1a hereinafter referred to as 8333- py cA t1a .

[0055] Example 14: Verification of Glycoside Production Performance of Bacillus subtilis Engineered Strains 1. Culture medium: (1) Seed culture medium formula (g / L): glucose 20, yeast powder 5, corn steep liquor powder 5, potassium dihydrogen phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.02, manganese sulfate 0.01, pH 7.0~7.2, sterilized at 121℃ for 20 min.

[0056] (2) Fermentation medium formula (g / L): glucose 60, yeast powder 3.5, potassium dihydrogen phosphate 3, ammonium sulfate 25, manganese sulfate 0.01, magnesium sulfate 5, monosodium glutamate 10, corn steep liquor powder 15, calcium carbonate 25, pH 7.0~7.2, sterilized at 121℃ for 20 min.

[0057] 2. Cultivation Methods (1) Streak the strain in three zones on LB plates and incubate overnight at 37°C; (2) Pick a single colony and inoculate it into 30 mL of seed culture medium, and incubate at 110 rpm and 36℃ for 7-8 h; (3) Transfer 10% (v / v) of the inoculum to 30 ml of fermentation medium, shake at 120 rpm, and incubate at 36°C for 36 h; 3. Detection and Results The nucleosides in the fermentation broth were detected using high performance liquid chromatography (HPLC), and the results are shown in Table 2.

[0058] Table 2. Results of shake-flask fermentation production capacity assessment (mean of three replicates)

[0059] Note: This indicates a significant difference compared to the original strain (P < 0.01).

[0060] The above experimental results show that the pyruvate kinase mutant has a positive effect on increasing the production of adenosine and inosine. T101K The optimal adenosine yield was increased from 1.3 g / L to 2.1 g / L by the engineered strain, with the mutant pyk... T101R The optimal inosine yield of the engineered strain was increased from 0.6 g / L to 1.1 g / L.

[0061] Pyruvate carboxylase mutants have a positive effect on increasing the production of adenosine and inosine. The mutant pycA t1a The optimal adenosine yield was increased from 1.3 g / L to 2.5 g / L by the engineered strain, with the mutant pycA showing the best yield. t1g The optimal inosine yield of the engineered strain was increased from 0.6 g / L to 1.3 g / L.

[0062] Phosphoenolpyruvate carboxykinase mutants have a positive effect on increasing adenosine and inosine production. The mutant pckA K147H The optimal increase in adenosine production from 1.3 g / L to 1.8 g / L and inosine production from 0.6 g / L to 1.3 g / L was achieved by engineered strains.

[0063] The superposition of pyruvate carboxylase mutant and pyruvate kinase mutant has a positive effect on increasing the production of adenosine and inosine. The mutant pycA t1a Overlay pyk T101K The engineered strain's adenosine production increased from 1.3 g / L to a maximum of 3.4 g / L, with the mutant pycA showing the highest yield. t1g Overlay pyk T101K The inosine yield of the engineered strain increased from 0.6 g / L to a maximum of 1.8 g / L.

[0064] The combined effects of pyruvate carboxylase mutant, pyruvate kinase mutant, and phosphoenolpyruvate carboxylkinase mutant significantly increased adenosine and inosine production. The mutant pycA... t1a , pyk T101K pckA K147R The superimposed engineered strains increased adenosine production from 1.3 g / L to 4.4 g / L and inosine production from 0.6 g / L to a maximum of 2.2 g / L.

[0065] Example 15: Verification of glycoside production performance of engineered Bacillus amyloliquefaciens strain 1. Culture medium: (1) Seed culture medium formula (g / L): glucose 20, yeast powder 5, corn steep liquor powder 5, potassium dihydrogen phosphate 3, magnesium sulfate 0.5, ferrous sulfate 0.02, manganese sulfate 0.01, pH 7.0~7.2, sterilized at 121℃ for 20 min.

[0066] (2) Fermentation medium formula (g / L): glucose 120, yeast powder 3.5, potassium dihydrogen phosphate 3, ammonium sulfate 25, manganese sulfate 0.01, magnesium sulfate 5, monosodium glutamate 10, corn steep liquor powder 15, calcium carbonate 25, pH 7.0~7.2, sterilized at 121℃ for 20 min.

[0067] 2. Cultivation Methods (1) Streak the glycerol-preserved bacterial strain on LB agar plates and culture at 37°C overnight to obtain single clones.

[0068] (2) Pick a single colony and inoculate it into 30 mL of seed culture medium. Incubate at 110 rpm and 37℃ for 7-8 h.

[0069] (3) Transfer 10% (v / v) of the inoculum to 30 ml of fermentation medium, shake at 130 rpm, and incubate at 35°C for 70 h.

[0070] 3. Detection and Results The nucleosides in the fermentation broth were detected using high performance liquid chromatography (HPLC), and the results are shown in Table 3.

[0071] Table 3. Evaluation results of guanosine and inosine production during shake-flask fermentation (mean of three replicates)

[0072] Note: This indicates a significant difference compared to the original strain (P < 0.01).

[0073] The above experimental results show that the pyruvate carboxylase mutant has a positive effect on increasing the production of guanosine and inosine. The mutant pycA... t1a The optimal yield of guanosine from the engineered strain was increased from 9.8 g / L to 17.1 g / L, with the mutant pycA showing the best results. t1g The optimal inosine yield of the engineered strain was increased from 1.0 g / L to 1.6 g / L.

[0074] Therefore, the pyruvate kinase mutant and / or pyruvate carboxylase mutant, and the pyruvate kinase and / or pyruvate carboxylase mutant strains provided by this invention have a significant promoting effect on the production of target products adenosine or guanosine, and inosine. These pyruvate kinase mutants and / or pyruvate carboxylase mutants, and their recombinant microorganisms, provide a reference for the construction of production strains that produce adenosine or guanosine, inosine, and derivatives using these as precursors.

[0075] The construction of the strain of the present invention is not limited in terms of the order of steps. Any person skilled in the art who achieves the purpose of the present invention by following the disclosure of the present invention shall fall within the protection scope of the present invention.

[0076] The strain designations in this invention are as follows: A1- pyk T101K A1- pyk T101R A1- pyk T101P The terms "etc." are used for ease of description and should not be construed as limiting the invention. The method described above constructs a product containing Bacillus subtilis pyruvate kinase mutant. pyk T101K , pyk T101R , pyk T101P And / or pyruvate carboxylase mutant pycA t1a pycA t1g Engineered bacteria, and / or phosphoenolpyruvate carboxykinase mutant pckA K147R pckA K147H Engineered bacteria, including those containing Bacillus amyloliquefaciens pyruvate carboxylase mutant pycA. t1a pycA t1g The uses of engineered bacteria include, but are not limited to, adenosine or guanosine, and inosine.

[0077] 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> Phosphoenolpyruvate carboxykinase mutants and their applications <130> KHP221111665.5 <160> 16 <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 tttttaacac 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 cggcaattttt 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 gaaagttat 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> 585 <212> PRT <213> Bacillus subtilis <400> 4 Met Arg Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Ala Ser Glu Ser 1 5 10 15 Ile Glu Met Leu Thr Lys Leu Met Glu Ser Gly Met Asn Val Ala Arg 20 25 30 Leu Asn Phe Ser His Gly Asp Phe Glu Glu His Gly Ala Arg Ile Lys 35 40 45 Asn Ile Arg Glu Ala Ser Lys Lys Leu Gly Lys Asn Val Gly Ile Leu 50 55 60 Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr His Thr Met Glu Asn Gly 65 70 75 80 Gly Ile Glu Leu Glu Thr Gly Lys Glu Leu Ile Ile Ser Met Asp Glu 85 90 95 Val Val Gly Thr Thr Asp Lys Ile Ser Val Thr Tyr Glu Gly Leu Val 100 105 110 His Asp Val Glu Gln Gly Ser Thr Ile Leu Leu Asp Asp Gly Leu Ile 115 120 125 Gly Leu Glu Val Leu Asp Val Asp Ala Ala Lys Arg Glu Ile Lys Thr 130 135 140 Lys Val Leu Asn Asn Gly Thr Leu Lys Asn Lys Lys Gly Val Asn Val 145 150 155 160 Pro Gly Val Ser Val Asn Leu Pro Gly Ile Thr Glu Lys Asp Ala Arg 165 170 175 Asp Ile Val Phe Gly Ile Glu Gln Gly Val Asp Phe Ile Ala Pro Ser 180 185 190 Phe Ile Arg Arg Ser Thr Asp Val Leu Glu Ile Arg Glu Leu Leu Glu 195 200 205 Glu His Asn Ala Gln Asp Ile Gln Ile Ile Pro Lys Ile Glu Asn Gln 210 215 220 Glu Gly Val Asp Asn Ile Asp Ala Ile Leu Glu Val Ser Asp Gly Leu 225 230 235 240 Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Ala Glu Glu Val 245 250 255 Pro Leu Val Gln Lys Glu Leu Ile Lys Lys Cys Asn Ala Leu Gly Lys 260 265 270 Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Gln Arg Asn Pro 275 280 285 Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala Ile Phe Asp 290 295 300 Gly Thr Asp Ala Ile Met Leu Ser Gly Glu Thr Ala Ala Gly Ser Tyr 305 310 315 320 Pro Val Glu Ala Val Gln Thr Met His Asn Ile Ala Ser Arg Ser Glu 325 330 335 Glu Ala Leu Asn Tyr Lys Glu Ile Leu Ser Lys Arg Arg Asp Gln Val 340 345 350 Gly Met Thr Ile Thr Asp Ala Ile Gly Gln Ser Val Ala His Thr Ala 355 360 365 Ile Asn Leu Asn Ala Ala Ala Ile Val Thr Pro Thr Glu Ser Gly His 370 375 380 Thr Ala Arg Met Ile Ala Lys Tyr Arg Pro Gln Ala Pro Ile Val Ala 385 390 395 400 Val Thr Val Asn Asp Ser Ile Ser Arg Lys Leu Ala Leu Val Ser Gly 405 410 415 Val Phe Ala Glu Ser Gly Gln Asn Ala Ser Ser Thr Asp Glu Met Leu 420 425 430 Glu Asp Ala Val Gln Lys Ser Leu Asn Ser Gly Ile Val Lys His Gly 435 440 445 Asp Leu Ile Val Ile Thr Ala Gly Thr Val Gly Glu Ser Gly Thr Thr 450 455 460 Asn Leu Met Lys Val His Thr Val Gly Asp Ile Ile Ala Lys Gly Gln 465 470 475 480 Gly Ile Gly Arg Lys Ser Ala Tyr Gly Pro Val Val Val Ala Gln Asn 485 490 495 Ala Lys Glu Ala Glu Gln Lys Met Thr Asp Gly Ala Val Leu Val Thr 500 505 510 Lys Ser Thr Asp Arg Asp Met Ile Ala Ser Leu Glu Lys Ala Ser Ala 515 520 525 Leu Ile Thr Glu Glu Gly Gly Leu Thr Ser His Ala Ala Val Val Gly 530 535 540 Leu Ser Leu Gly Ile Pro Val Ile Val Gly Leu Glu Asn Ala Thr Ser 545 550 555 560 Ile Leu Thr Asp Gly Gln Asp Ile Thr Val Asp Ala Ser Arg Gly Ala 565 570 575 Val Tyr Gln Gly Arg Ala Ser Val Leu 580 585 <210> 5 <211> 585 <212> PRT <213> Artificial Sequence <400> 5 Met Arg Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Ala Ser Glu Ser 1 5 10 15 Ile Glu Met Leu Thr Lys Leu Met Glu Ser Gly Met Asn Val Ala Arg 20 25 30 Leu Asn Phe Ser His Gly Asp Phe Glu Glu His Gly Ala Arg Ile Lys 35 40 45 Asn Ile Arg Glu Ala Ser Lys Lys Leu Gly Lys Asn Val Gly Ile Leu 50 55 60 Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr His Thr Met Glu Asn Gly 65 70 75 80 Gly Ile Glu Leu Glu Thr Gly Lys Glu Leu Ile Ile Ser Met Asp Glu 85 90 95 Val Val Gly Thr Lys Asp Lys Ile Ser Val Thr Tyr Glu Gly Leu Val 100 105 110 His Asp Val Glu Gln Gly Ser Thr Ile Leu Leu Asp Asp Gly Leu Ile 115 120 125 Gly Leu Glu Val Leu Asp Val Asp Ala Ala Lys Arg Glu Ile Lys Thr 130 135 140 Lys Val Leu Asn Asn Gly Thr Leu Lys Asn Lys Lys Gly Val Asn Val 145 150 155 160 Pro Gly Val Ser Val Asn Leu Pro Gly Ile Thr Glu Lys Asp Ala Arg 165 170 175 Asp Ile Val Phe Gly Ile Glu Gln Gly Val Asp Phe Ile Ala Pro Ser 180 185 190 Phe Ile Arg Arg Ser Thr Asp Val Leu Glu Ile Arg Glu Leu Leu Glu 195 200 205 Glu His Asn Ala Gln Asp Ile Gln Ile Ile Pro Lys Ile Glu Asn Gln 210 215 220 Glu Gly Val Asp Asn Ile Asp Ala Ile Leu Glu Val Ser Asp Gly Leu 225 230 235 240 Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Ala Glu Glu Val 245 250 255 Pro Leu Val Gln Lys Glu Leu Ile Lys Lys Cys Asn Ala Leu Gly Lys 260 265 270 Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Gln Arg Asn Pro 275 280 285 Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala Ile Phe Asp 290 295 300 Gly Thr Asp Ala Ile Met Leu Ser Gly Glu Thr Ala Ala Gly Ser Tyr 305 310 315 320 Pro Val Glu Ala Val Gln Thr Met His Asn Ile Ala Ser Arg Ser Glu 325 330 335 Glu Ala Leu Asn Tyr Lys Glu Ile Leu Ser Lys Arg Arg Asp Gln Val 340 345 350 Gly Met Thr Ile Thr Asp Ala Ile Gly Gln Ser Val Ala His Thr Ala 355 360 365 Ile Asn Leu Asn Ala Ala Ala Ile Val Thr Pro Thr Glu Ser Gly His 370 375 380 Thr Ala Arg Met Ile Ala Lys Tyr Arg Pro Gln Ala Pro Ile Val Ala 385 390 395 400 Val Thr Val Asn Asp Ser Ile Ser Arg Lys Leu Ala Leu Val Ser Gly 405 410 415 Val Phe Ala Glu Ser Gly Gln Asn Ala Ser Ser Thr Asp Glu Met Leu 420 425 430 Glu Asp Ala Val Gln Lys Ser Leu Asn Ser Gly Ile Val Lys His Gly 435 440 445 Asp Leu Ile Val Ile Thr Ala Gly Thr Val Gly Glu Ser Gly Thr Thr 450 455 460 Asn Leu Met Lys Val His Thr Val Gly Asp Ile Ile Ala Lys Gly Gln 465 470 475 480 Gly Ile Gly Arg Lys Ser Ala Tyr Gly Pro Val Val Val Ala Gln Asn 485 490 495 Ala Lys Glu Ala Glu Gln Lys Met Thr Asp Gly Ala Val Leu Val Thr 500 505 510 Lys Ser Thr Asp Arg Asp Met Ile Ala Ser Leu Glu Lys Ala Ser Ala 515 520 525 Leu Ile Thr Glu Glu Gly Gly Leu Thr Ser His Ala Ala Val Val Gly 530 535 540 Leu Ser Leu Gly Ile Pro Val Ile Val Gly Leu Glu Asn Ala Thr Ser 545 550 555 560 Ile Leu Thr Asp Gly Gln Asp Ile Thr Val Asp Ala Ser Arg Gly Ala 565 570 575 Val Tyr Gln Gly Arg Ala Ser Val Leu 580 585 <210> 6 <211> 585 <212> PRT <213> Artificial Sequence <400> 6 Met Arg Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Ala Ser Glu Ser 1 5 10 15 Ile Glu Met Leu Thr Lys Leu Met Glu Ser Gly Met Asn Val Ala Arg 20 25 30 Leu Asn Phe Ser His Gly Asp Phe Glu Glu His Gly Ala Arg Ile Lys 35 40 45 Asn Ile Arg Glu Ala Ser Lys Lys Leu Gly Lys Asn Val Gly Ile Leu 50 55 60 Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr His Thr Met Glu Asn Gly 65 70 75 80 Gly Ile Glu Leu Glu Thr Gly Lys Glu Leu Ile Ile Ser Met Asp Glu 85 90 95 Val Val Gly Thr Arg Asp Lys Ile Ser Val Thr Tyr Glu Gly Leu Val 100 105 110 His Asp Val Glu Gln Gly Ser Thr Ile Leu Leu Asp Asp Gly Leu Ile 115 120 125 Gly Leu Glu Val Leu Asp Val Asp Ala Ala Lys Arg Glu Ile Lys Thr 130 135 140 Lys Val Leu Asn Asn Gly Thr Leu Lys Asn Lys Lys Gly Val Asn Val 145 150 155 160 Pro Gly Val Ser Val Asn Leu Pro Gly Ile Thr Glu Lys Asp Ala Arg 165 170 175 Asp Ile Val Phe Gly Ile Glu Gln Gly Val Asp Phe Ile Ala Pro Ser 180 185 190 Phe Ile Arg Arg Ser Thr Asp Val Leu Glu Ile Arg Glu Leu Leu Glu 195 200 205 Glu His Asn Ala Gln Asp Ile Gln Ile Ile Pro Lys Ile Glu Asn Gln 210 215 220 Glu Gly Val Asp Asn Ile Asp Ala Ile Leu Glu Val Ser Asp Gly Leu 225 230 235 240 Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Ala Glu Glu Val 245 250 255 Pro Leu Val Gln Lys Glu Leu Ile Lys Lys Cys Asn Ala Leu Gly Lys 260 265 270 Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Gln Arg Asn Pro 275 280 285 Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala Ile Phe Asp 290 295 300 Gly Thr Asp Ala Ile Met Leu Ser Gly Glu Thr Ala Ala Gly Ser Tyr 305 310 315 320 Pro Val Glu Ala Val Gln Thr Met His Asn Ile Ala Ser Arg Ser Glu 325 330 335 Glu Ala Leu Asn Tyr Lys Glu Ile Leu Ser Lys Arg Arg Asp Gln Val 340 345 350 Gly Met Thr Ile Thr Asp Ala Ile Gly Gln Ser Val Ala His Thr Ala 355 360 365 Ile Asn Leu Asn Ala Ala Ala Ile Val Thr Pro Thr Glu Ser Gly His 370 375 380 Thr Ala Arg Met Ile Ala Lys Tyr Arg Pro Gln Ala Pro Ile Val Ala 385 390 395 400 Val Thr Val Asn Asp Ser Ile Ser Arg Lys Leu Ala Leu Val Ser Gly 405 410 415 Val Phe Ala Glu Ser Gly Gln Asn Ala Ser Ser Thr Asp Glu Met Leu 420 425 430 Glu Asp Ala Val Gln Lys Ser Leu Asn Ser Gly Ile Val Lys His Gly 435 440 445 Asp Leu Ile Val Ile Thr Ala Gly Thr Val Gly Glu Ser Gly Thr Thr 450 455 460 Asn Leu Met Lys Val His Thr Val Gly Asp Ile Ile Ala Lys Gly Gln 465 470 475 480 Gly Ile Gly Arg Lys Ser Ala Tyr Gly Pro Val Val Val Ala Gln Asn 485 490 495 Ala Lys Glu Ala Glu Gln Lys Met Thr Asp Gly Ala Val Leu Val Thr 500 505 510 Lys Ser Thr Asp Arg Asp Met Ile Ala Ser Leu Glu Lys Ala Ser Ala 515 520 525 Leu Ile Thr Glu Glu Gly Gly Leu Thr Ser His Ala Ala Val Val Gly 530 535 540 Leu Ser Leu Gly Ile Pro Val Ile Val Gly Leu Glu Asn Ala Thr Ser 545 550 555 560 Ile Leu Thr Asp Gly Gln Asp Ile Thr Val Asp Ala Ser Arg Gly Ala 565 570 575 Val Tyr Gln Gly Arg Ala Ser Val Leu 580 585 <210> 7 <211> 585 <212> PRT <213> Artificial Sequence <400> 7 Met Arg Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Ala Ser Glu Ser 1 5 10 15 Ile Glu Met Leu Thr Lys Leu Met Glu Ser Gly Met Asn Val Ala Arg 20 25 30 Leu Asn Phe Ser His Gly Asp Phe Glu Glu His Gly Ala Arg Ile Lys 35 40 45 Asn Ile Arg Glu Ala Ser Lys Lys Leu Gly Lys Asn Val Gly Ile Leu 50 55 60 Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr His Thr Met Glu Asn Gly 65 70 75 80 Gly Ile Glu Leu Glu Thr Gly Lys Glu Leu Ile Ile Ser Met Asp Glu 85 90 95 Val Val Gly Thr Pro Asp Lys Ile Ser Val Thr Tyr Glu Gly Leu Val 100 105 110 His Asp Val Glu Gln Gly Ser Thr Ile Leu Leu Asp Asp Gly Leu Ile 115 120 125 Gly Leu Glu Val Leu Asp Val Asp Ala Ala Lys Arg Glu Ile Lys Thr 130 135 140 Lys Val Leu Asn Asn Gly Thr Leu Lys Asn Lys Lys Gly Val Asn Val 145 150 155 160 Pro Gly Val Ser Val Asn Leu Pro Gly Ile Thr Glu Lys Asp Ala Arg 165 170 175 Asp Ile Val Phe Gly Ile Glu Gln Gly Val Asp Phe Ile Ala Pro Ser 180 185 190 Phe Ile Arg Arg Ser Thr Asp Val Leu Glu Ile Arg Glu Leu Leu Glu 195 200 205 Glu His Asn Ala Gln Asp Ile Gln Ile Ile Pro Lys Ile Glu Asn Gln 210 215 220 Glu Gly Val Asp Asn Ile Asp Ala Ile Leu Glu Val Ser Asp Gly Leu 225 230 235 240 Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Ala Glu Glu Val 245 250 255 Pro Leu Val Gln Lys Glu Leu Ile Lys Lys Cys Asn Ala Leu Gly Lys 260 265 270 Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Gln Arg Asn Pro 275 280 285 Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala Ile Phe Asp 290 295 300 Gly Thr Asp Ala Ile Met Leu Ser Gly Glu Thr Ala Ala Gly Ser Tyr 305 310 315 320 Pro Val Glu Ala Val Gln Thr Met His Asn Ile Ala Ser Arg Ser Glu 325 330 335 Glu Ala Leu Asn Tyr Lys Glu Ile Leu Ser Lys Arg Arg Asp Gln Val 340 345 350 Gly Met Thr Ile Thr Asp Ala Ile Gly Gln Ser Val Ala His Thr Ala 355 360 365 Ile Asn Leu Asn Ala Ala Ala Ile Val Thr Pro Thr Glu Ser Gly His 370 375 380 Thr Ala Arg Met Ile Ala Lys Tyr Arg Pro Gln Ala Pro Ile Val Ala 385 390 395 400 Val Thr Val Asn Asp Ser Ile Ser Arg Lys Leu Ala Leu Val Ser Gly 405 410 415 Val Phe Ala Glu Ser Gly Gln Asn Ala Ser Ser Thr Asp Glu Met Leu 420 425 430 Glu Asp Ala Val Gln Lys Ser Leu Asn Ser Gly Ile Val Lys His Gly 435 440 445 Asp Leu Ile Val Ile Thr Ala Gly Thr Val Gly Glu Ser Gly Thr Thr 450 455 460 Asn Leu Met Lys Val His Thr Val Gly Asp Ile Ile Ala Lys Gly Gln 465 470 475 480 Gly Ile Gly Arg Lys Ser Ala Tyr Gly Pro Val Val Val Ala Gln Asn 485 490 495 Ala Lys Glu Ala Glu Gln Lys Met Thr Asp Gly Ala Val Leu Val Thr 500 505 510 Lys Ser Thr Asp Arg Asp Met Ile Ala Ser Leu Glu Lys Ala Ser Ala 515 520 525 Leu Ile Thr Glu Glu Gly Gly Leu Thr Ser His Ala Ala Val Val Gly 530 535 540 Leu Ser Leu Gly Ile Pro Val Ile Val Gly Leu Glu Asn Ala Thr Ser 545 550 555 560 Ile Leu Thr Asp Gly Gln Asp Ile Thr Val Asp Ala Ser Arg Gly Ala 565 570 575 Val Tyr Gln Gly Arg Ala Ser Val Leu 580 585 <210> 8 <211> 3447 <212> DNA <213> Bacillus subtilis <400> 8 ttgtctcagc aatcgataca aaaagtatta gtagcaaaca ggggagaaat tgcaatccga 60 atattccggg cgtgtaccga gttgaatatt cgtacagttg cggtctattc aaaagaagat 120 tccggttcct accatcggta caaagcggat gaagcatact tggtcggtga agggaaaaa 180 ccgattgatg cttacctgga tattgaaggt atcattgata ttgcgaaaag aaacaaagtc 240 gatgcaattc atccgggata cggtttctta tctgaaaata ttcattttgc gagacgatgt 300 gaagaaag gcatcgtatt catagggcca aaatccgagc atctcgatat gtttggtgac 360 aaggtaaaag cgcgtgagca ggcagaaaaa gcgggaatcc ccgtgattcc gggaagcgac 420 ggtcctgccg aaacgcttga agccgtcgaa caatttggac aagctaacgg ttatccgatc 480 atcattaaag cctcgcttgg cggcggcggc cgcggtatgc ggattgtcag atctgaaagt 540 600 gaagtttatg tagaaaaatt aattgagaat ccgaaacata ttgaggttca ggtcattgga 660 gacaagcagg gcaatgtcgt ccatcttttt gagagggatt gctccgttca aagacgccat 720 caaaaagtca ttgaagtggc gccgagtgtc tcgctgtcac ctgaattaag ggaccaaatt 780 tgtgaggctg cagttgcgct tgccaaaaat gtaaactata taaatgcggg gacggtcgaa 840 ttccttgttg caaacaacga gttctacttt attgaagtaa atcctcgcgt acaagttgaa 900 cacacgataa cagaaatgat tactggtgtc gatattgttc aaactcagat ccttgttgcc 960 caagggcaca gccttcacag caaaaaagta aatattcctg agcaaaagga catttttaca 1020 atcggctatg ccattcagtc acgggttacg actgaggatc cgcaaaatga tttcatgcct 1080 gatacaggaa aaatcatggc ttaccgctca ggcggcggtt ttggtgtccg tcttgatacc 1140 ggaaacagct tccagggcgc cgtgatcaca ccatactatg attcacttct cgttaagctt 1200 tcaacttggg ctttaacgtt tgaacaggca gctgccaaaa tggtgcgaaa ccttcaggag 1260 tttagaatca gaggcataaa aacgaacatt ccgttccttg agaacgttgc aaagcatgag 1320 aagttcctga cagggcaata tgatacatct ttcattgata caacgcctga attatttaat 1380 ttccctaaac aaaaagaccg cggaacgaaa atgctcactt acatcggcaa tgtgacagtg 1440 aacggcttcc ctggaatcgg gaaaaaagaa aaaccggcgt ttgacaaacc gttaggcgta 1500 aaggtagacg ttgatcagca gcctgccaga ggaacaaagc aaattctcga tgaaaaaggt 1560 gcagaagggc ttgcaaattg ggttaaggag cagaaatctg tccttttaac tgatacgaca 1620 ttcagggatg cccaccaatc gttattggca actagaatca gatcgcatga tttgaaaaaa 1680 atcgcaaatc cgacggctgc gttatggcct gaactattca gtatggaaat gtggggaggc 1740 gcgaccttcg atgtagccta ccgattcctg aaagaagatc cgtggaaacg tttggaagat 1800 cttcgcaaag aagtgccgaa taccttattc cagatgttgc ttcgctcatc aaatgcggtc 1860 ggctatacga attatccgga caatgtgatt aaagaatttg tgaagcaatc agctcaatcc 1920 ggtattgatg tgtttcgtat tttcgacagc ttaaactggg taaaagggat gacgttagcc 1980 attgatgctg ttagggatac cggcaaagtg gcagaagctg cgatttgtta tacgggagat 2040 atccttgaca agaaccggac gaagtacgac cttgcatatt atacatcgat ggcgaaggag 2100 cttgaggcgg ccggagccca tattctcggg attaaagata tggcagggct gttaaaaccg 2160 caggctgcat atgagctcgt ttctgcgttg aaagaaacga tcgacattcc ggttcacctt 2220 catacgcatg atacgagcgg aaacggtatt tatatgtatg cgaaagctgt tgaagccggc 2280 gttgatatca tagacgtggc ggtcagctca atggcgggat taacgtcaca gcctagcgcg 2340 agcggatttt atcatgcgat ggaaggcaac gaccgccgtc cggaaatgaa tgtccaaggc 2400 gttgaattgc tgtcccaata ttgggagtcg gtgcgtaaat attatagtga atttgaaagc 2460 ggaatgaagt ctccgcatac tgaaatttat gaacacgaaa tgccaggggg ccaatacagc 2520 aacctgcagc agcaagccaa gggagtaggc cttggcgacc gctggaacga agtcaaggaa 2580 atgtacagac gcgtgaacga tatgttcggt gacatcgtca aggtaacgcc ttcctcaaaa 2640 gtagtcggag atatggcact ctacatggtg caaaacaatc tgactgaaaa agacgtttac 2700 gaaaaaggtg aatctttaga tttccctgat tctgtcgtgg agctttttaa aggaaatatc 2760 ggccagcctc atggcggatt cccagaaaaa ctgcaaaagc tgatcttaaa agggcaggag 2820 ccgattacag tcagaccggg cgaactgctt gagccggtgt catttgaagc gatcaaacag 2880 gaatttaaag agcagcataa cttggaaatt tcagatcagg atgctgtggc atatgccctt 2940 tatcctaaag tcttcactga ttatgtgaaa acgacagaaa gctatggaga catctcggta 3000 ttagatacac cgacattctt ctacggtatg acattaggtg aagagataga agttgaaatt 3060 gagcgcggca aaacgctgat cgttaagctg atttcaatcg gtgagcctca gcctgatgcc 3120 acccgcgtcg tttatttcga actcaacggg cagccgcgtg aagtagtcat taaagatgaa 3180 agcattaagt cttccgttca ggaaaggctg aaagcagacc ggacaaatcc aagccacatc 3240 gcagcttcca tgcctggaac agttattaag gtattggctg aagcaggcac aaaagtcaat 3300 aaaggtgatc atttgatgat taatgaagcg atgaaaatgg aaacaacggt tcaggcgcct 3360 ttctcaggaa caatcaagca ggttcatgtg aaaaatggtg agccgatcca aacgggagat 3420 ctgctccttg aaattgaaaa agcataa 3447 <210> 9 <211> 3447 <212> DNA <213> Artificial Sequence <400> 9 gtgtctcagc aatcgataca aaaagtatta gtagcaaaca ggggagaaat tgcaatccga 60 atattccggg cgtgtaccga gttgaatatt cgtacagttg cggtctattc aaaagaagat 120 tccggttcct accatcggta caaagcggat gaagcatact tggtcggtga agggaaaaaa 180 ccgattgatg cttacctgga tattgaaggt atcattgata ttgcgaaaag aaacaaagtc 240 gatgcaattc atccgggata cggtttctta tctgaaaata ttcattttgc gagacgatgt 300 gaagaaag gcatcgtatt catagggcca aaatccgagc atctcgatat gtttggtgac 360 aaggtaaaag cgcgtgagca ggcagaaaaa gcgggaatcc ccgtgattcc gggaagcgac 420 ggtcctgccg aaacgcttga agccgtcgaa caatttggac aagctaacgg ttatccgatc 480 atcattaaag cctcgcttgg cggcggcggc cgcggtatgc ggattgtcag atctgaaagt 540 600 gaagtttatg tagaaaaatt aattgagaat ccgaaacata ttgaggttca ggtcattgga 660 gacaagcagg gcaatgtcgt ccatcttttt gagagggatt gctccgttca aagacgccat 720 caaaaagtca ttgaagtggc gccgagtgtc tcgctgtcac ctgaattaag ggaccaaatt 780 tgtgaggctg cagttgcgct tgccaaaaat gtaaactata taaatgcggg gacggtcgaa 840 900 cacacgataa cagaaatgat tactggtgtc gatattgttc aaactcagat ccttgttgcc 960 caagggcaca gccttcacag caaaaaagta aatattcctg agcaaaagga catttttaca 1020 atcggctatg ccattcagtc acgggttacg actgaggatc cgcaaaatga tttcatgcct 1080 gatacaggaa aaatcatggc ttaccgctca ggcggcggtt ttggtgtccg tcttgatacc 1140 ggaaacagct tccagggcgc cgtgatcaca ccatactatg attcacttct cgttaagctt 1200 tcaacttggg ctttaacgtt tgaacaggca gctgccaaaa tggtgcgaaa ccttcaggag 1260 tttagaatca gaggcataaa aacgaacatt ccgttccttg agaacgttgc aaagcatgag 1320 aagttcctga cagggcaata tgatacatct ttcattgata caacgcctga attatttaat 1380 ttccctaaac aaaaagaccg cggaacgaaa atgctcactt acatcggcaa tgtgacagtg 1440 aacggcttcc ctggaatcgg gaaaaaagaa aaaccggcgt ttgacaaacc gttaggcgta 1500 aaggtagacg ttgatcagca gcctgccaga ggaacaaagc aaattctcga tgaaaaaggt 1560 gcagaagggc ttgcaaattg ggttaaggag cagaaatctg tccttttaac tgatacgaca 1620 ttcagggatg cccaccaatc gttattggca actagaatca gatcgcatga tttgaaaaaa 1680 atcgcaaatc cgacggctgc gttatggcct gaactattca gtatggaaat gtggggaggc 1740 gcgaccttcg atgtagccta ccgattcctg aaagaagatc cgtggaaacg tttggaagat 1800 cttcgcaaag aagtgccgaa taccttattc cagatgttgc ttcgctcatc aaatgcggtc 1860 ggctatacga attatccgga caatgtgatt aaagaatttg tgaagcaatc agctcaatcc 1920 ggtattgatg tgtttcgtat tttcgacagc ttaaactggg taaaagggat gacgttagcc 1980 attgatgctg ttagggatac cggcaaagtg gcagaagctg cgatttgtta tacgggagat 2040 atccttgaca agaaccggac gaagtacgac cttgcatatt atacatcgat ggcgaaggag 2100 cttgaggcgg ccggagccca tattctcggg attaaagata tggcagggct gttaaaaccg 2160 caggctgcat atgagctcgt ttctgcgttg aaagaaacga tcgacattcc ggttcacctt 2220 catacgcatg atacgagcgg aaacggtatt tatatgtatg cgaaagctgt tgaagccggc 2280 gttgatatca tagacgtggc ggtcagctca atggcgggat taacgtcaca gcctagcgcg 2340 agcggatttt atcatgcgat ggaggcaac gaccgccgtc cggaatgaa tgtccaggc 2400 gttgaattgc tgtcccaata ttgggagtcg gtgcgtaaat attatagtga atttgaaagc 2460 ggaatgaagt ctccgcatac tgaatttat gaacacgaaa tgccagggggg ccaatacagc 2520 aacctgcagc agcaagccaa gggagtaggc cttggcgacc gctgaacga agtcaggaa 2580 atgtacagac gcgtgaacga tatgttcggt gatacgtca aggtaacgcc ttcctcaaaa 2640 gtagtcggag atatggcact ctacatggtg caaacaatc tgactgaaa agacgtttac 2700 gaaaaaggtg aatctttaga ttccctgat tctgtcgtgg agctttaa aggaaatatc 2760 ggccagcctc atggcggatt cccagaaaaa ctgcaaagc tgatcttaa agggcaggag 2820 ccgattacag tcagaccgggg cgaactgctt gagccggtgt cattgaagc gatcaacag 2880 gatttaaag agcagcataa cttggaatt tcagatcagg atgctgtggc atatgccctt 2940 tatcctaaag tcttcactga ttatgtgaaa acgacagaaa gctatggaga catctcggta 3000 ttagatacac cgacattctt ctacggtag acattaggtg aagagataga agttgaatt 3060 gagcgcggca aaacgctgat cgttaagctg atttcaatcg gtgagcctca gcctgatgcc 3120 acccgcgtcg tttatttcga actcaacggg cagccgcgtg aagtagtcat taaagatgaa 3180 agcattaagt cttccgttca ggaaaggctg aaagcagacc ggacaaatcc aagccacatc 3240 gcagcttcca tgcctggaac agttattaag gtattggctg aagcaggcac aaaagtcaat 3300 aaaggtgatc atttgatgat taatgaagcg atgaaaatgg aaacaacggt tcaggcgcct 3360 ttctcaggaa caatcaagca ggttcatgtg aaaaatggtg agccgatcca aacgggagat 3420 ctgctccttg aaattgaaaa agcataa 3447 <210> 10 <211> 3447 DNA Artificial Sequence <400> 10 atgtctcagc aatcgataca aaaagtatta gtagcaaaca ggggagaaat tgcaatccga 60 atattccggg cgtgtaccga gttgaatatt cgtacagttg cggtctattc aaaagaagat 120 tccggttcct accatcggta caaagcggat gaagcatact tggtcggtga agggaaaaaa 180 ccgattgatg cttacctgga tattgaaggt atcattgata ttgcgaaaag aaacaaagtc 240 gatgcaattc atccgggata cggtttctta tctgaaaata ttcattttgc gagacgatgt 300 gaagaaag gcatcgtatt catagggcca aaatccgagc atctcgatat gtttggtgac 360 aaggtaaaag cgcgtgagca ggcagaaaaa gcgggaatcc ccgtgattcc gggaagcgac 420 ggtcctgccg aaacgcttga agccgtcgaa caatttggac aagctaacgg ttatccgatc 480 atcattaaag cctcgcttgg cggcggcggc cgcggtatgc ggattgtcag atctgaaagt 540 600 gaagtttatg tagaaaaatt aattgagaat ccgaaacata ttgaggttca ggtcattgga 660 gacaagcagg gcaatgtcgt ccatcttttt gagagggatt gctccgttca aagacgccat 720 caaaaagtca ttgaagtggc gccgagtgtc tcgctgtcac ctgaattaag ggaccaaatt 780 tgtgaggctg cagttgcgct tgccaaaaat gtaaactata taaatgcggg gacggtcgaa 840 900 cacacgataa cagaaatgat tactggtgtc gatatgttc aaactcagat ccttgttgcc 960 caagggcaca gccttcacag caaaaaagta aatattcctg agcaaaagga catttttaca 1020 atcggctatg ccattcagtc acgggttacg actgaggatc cgcaaaatga tttcatgcct 1080 gatacaggaa aaatcatggc ttaccgctca ggcggcggtt ttggtgtccg tcttgatacc 1140 ggaaacagct tccagggcgc cgtgatcaca ccatactatg attcacttct cgttaagctt 1200 tcaacttggg ctttaacgtt tgaacaggca gctgccaaaa tggtgcgaaa ccttcaggag 1260 tttagaatca gaggcataaa aacgaacatt ccgttccttg agaacgttgc aaagcatgag 1320 aagttcctga cagggcaata tgatacatct ttcattgata caacgcctga attatttaat 1380 ttccctaaac aaaaagaccg cggaacgaaa atgctcactt acatcggcaa tgtgacagtg 1440 aacggcttcc ctggaatcgg gaaaaaagaa aaaccggcgt ttgacaaacc gttaggcgta 1500 aaggtagacg ttgatcagca gcctgccaga ggaacaaagc aaattctcga tgaaaaaggt 1560 gcagaagggc ttgcaaattg ggttaaggag cagaaatctg tccttttaac tgatacgaca 1620 ttcagggatg cccaccaatc gttattggca actagaatca gatcgcatga tttgaaaaaa 1680 atcgcaaatc cgacggctgc gttatggcct gaactattca gtatggaaat gtggggaggc 1740 gcgaccttcg atgtagccta ccgattcctg aaagaagatc cgtggaaacg tttggaagat 1800 cttcgcaaag aagtgccgaa taccttattc cagatgttgc ttcgctcatc aaatgcggtc 1860 ggctatacga attatccgga caatgtgatt aaagaatttg tgaagcaatc agctcaatcc 1920 ggtattgatg tgtttcgtat tttcgacagc ttaaactggg taaaagggat gacgttagcc 1980 attgatgctg ttagggatac cggcaaagtg gcagaagctg cgatttgtta tacgggagat 2040 atccttgaca agaaccggac gaagtacgac cttgcatatt atacatcgat ggcgaaggag 2100 cttgaggcgg ccggagccca tattctcggg attaaagata tggcagggct gttaaaaccg 2160 caggctgcat atgagctcgt ttctgcgttg aaagaaacga tcgacattcc ggttcacctt 2220 catacgcatg atacgagcgg aaacggtatt tatatgtatg cgaaagctgt tgaagccggc 2280 gttgatatca tagacgtggc ggtcagctca atggcgggat taacgtcaca gcctagcgcg 2340 agcggatttt atcatgcgat ggaaggcaac gaccgccgtc cggaaatgaa tgtccaaggc 2400 gttgaattgc tgtcccaata ttgggagtcg gtgcgtaaat attatagtga atttgaaagc 2460 ggaatgaagt ctccgcatac tgaaatttat gaacacgaaa tgccaggggg ccaatacagc 2520 aacctgcagc agcaagccaa gggagtaggc cttggcgacc gctggaacga agtcaaggaa 2580 atgtacagac gcgtgaacga tatgttcggt gacatcgtca aggtaacgcc ttcctcaaaa 2640 gtagtcggag atatggcact ctacatggtg caaaacaatc tgactgaaaa agacgtttac 2700 gaaaaaggtg aatctttaga tttccctgat tctgtcgtgg agctttttaa aggaaatatc 2760 ggccagcctc atggcggatt cccagaaaaa ctgcaaaagc tgatcttaaa agggcaggag 2820 ccgattacag tcagaccggg cgaactgctt gagccggtgt catttgaagc gatcaaacag 2880 gaatttaaag agcagcataa cttggaaatt tcagatcagg atgctgtggc atatgccctt 2940 tatcctaaag tcttcactga ttatgtgaaa acgacagaaa gctatggaga catctcggta 3000 ttagatacac cgacattctt ctacggtatg acattaggtg aagagataga agttgaaatt 3060 gagcgcggca aaacgctgat cgttaagctg atttcaatcg gtgagcctca gcctgatgcc 3120 acccgcgtcg tttatttcga actcaacggg cagccgcgtg aagtagtcat taaagatgaa 3180 agcattaagt cttccgttca ggaaaggctg aaagcagacc ggacaaatcc aagccacatc 3240 gcagcttcca tgcctggaac agttattaag gtattggctg aagcaggcac aaaagtcaat 3300 aaaggtgatc atttgatgat taatgaagcg atgaaaatgg aaacaacggt tcaggcgcct 3360 ttctcaggaa caatcaagca ggttcatgtg aaaaatggtg agccgatcca aacgggagat 3420 ctgctccttg aaattgaaaa agcataa 3447 <210> 11 <211> 3447 <212> DNA <213> Bacillus amyloliquefaciens <400> 11 ttgtcacaac aatccatcca aaaagtgtta gtagcaaaca ggggagaaat tgcgatccgg 60 attttccggg cgtgcacaga attgaacatc agaacggtag ccgtttattc aaaagaagat 120 tcaggttcct accaccgcta taaagcggat gaagcctatc ttgtcggaga aggcaaaaaa 180 ccgattgacg cgtatcttga tattgaaggg attattgaga tcgcgaaaag aaacggcgtt 240 gatgccattc atccgggcta tggatttctg tctgaaaata tccaatttgc gagacgctgt 300 gaagaagg gcatcgtatt tatcggaccg acgtcagcgc atttggatat gttcggagat 360 aaggtcaagg ccagagagca ggcgggaaaaa gccggcatcc ccgttattcc ggggagtgac 420 gggcctgccg aaacattaaa agatgtcgaa caattcgcaa aagtacacgg atttccgttt 480 attattaaag cgtcgctcgg aggcggcggc cgcggaatga gaatcgtcag gaacgaaaac 540 gaattgaaag attcgtttga gcgggccaaa tccgaagcga aagccgcatt cggaaacgac 600 gaagtgtatg ttgaaaaact gatcgaaaat cctaagcata ttgaagttca ggtgatcgga 660 gataaaag gaatgtcgt ccatctgtat gagcgggact gttccgttca aagacggcat 720 caaaaggtca ttgaggtggc gccgagcgtg tccttacagc ctgaattaag agacgagatc 780 tgtgaagcgg ctgtcgcact tgcgaaaaat gtcggctata tcaacgctgg aacagtggaa 840 tttctcgtag cggacggtga attttacttt attgaagtga atccgcgcgt aaagtggag 900 catacgatta cggaaatgat tacggggggtt gatatcgttc agacgcaaat cctcgtcgcg 960 cagggccacg gtctgcacag ccgtgccgtc aacatccctc atcaagaaga catttttaca 1020 aacgggtatg cgatacagtc acgcgtgacc actgaagatc cgctgaatga ctttatgcct 1080 gacacgggta aaattatggc ttaccgctca ggcggcggct tcggcgtgcg tcttgatact 1140 gggaacagct tccaaggcgc cgtcatcacg ccttattatg attcactgct cgtcaagctg 1200 tcgacatggg cgctgacgtt tgaacaggca tccgcaaaaa tggtccgcaa cctgcaggaa 1260 ttcagaatca ggggcattaa gaccaatatc ccgtttcttg aaaacgtggc aaagcatgaa 1320 aaattcctga cgggacaata cgatacatcg tttatcgaca caacgcctga gctgtttgtt 1380 tttcctaaac agaaggaccg cggaacaaaa atgctttcct atatcggcaa tgtaacggtg 1440 aacggttttc cgggaatcgg aaaaaaagaa aaacctgcgt ttgataagcc ccagactgtt 1500 acgttaggta tcggcgaaaa gccggcaagc ggcacgaagc agattcttga tgaaagaggc 1560 gctgaagggc ttgctgactg ggtgaaagag cagaaatccg tacttctgac cgatacaacg 1620 ttcagggatg cccatcagtc cctgcttgca acccggatca gatccaatga cttgaaaaaa 1680 atcgcaaacc ctacggctgc tctgtggcct gaactgttca gccttgaaat gtggggtggg 1740 gcgacatttg atgtagcgta ccgtttctta aaagaatc cttggaaacg ccttgaagat 1800 ctccgcaagg aagtgccgaa cacattgttc cagatgctgc ttcgttcatc aaatgccgtc 1860 ggctatacaa actatccgga caatgtcatt caaaaatttg tccggcagtc agcagcatca 1920 ggtattgacg tattccggat ctttgacagc ctgaactggg tgaaagggat gacgctggcc 1980 atcgatgccg tccgtgaaac cggaaaagtg gcggaggctg cgatttgtta tacgggagat 2040 attcttgata agagccggac gaagtatgat ctgaaatact atctttctat ggcaaaagag 2100 cttgaggcat cgggagcgca tattctcggc attaaagaca tggccggtct tttgaaacct 2160 caagccgcat acgaacttgt ctcagcgctg aaggaaacga ttgatattcc cgttcatctt 2220 cacagcatg atacgagcgg caacggcgtc tttctctatg ccaaagccgt tgaagcgggt 2280 gtggatatcg tagatgtcgc cgtcagctca atggcgggtc tgacttcaca gccgagtgca 2340 agcggatttt atcatgcgct tgaaggaac agccgccgtc ctgaaatgga tgtccggcag 2400 gttgaaagac tgtcacaata ttgggaatcc gtccgtcaat attacagtga atttgaaagc 2460 gggatgaagt ctccgcatac tgagattat aatcacgaaa tgccgggcgg acagtacagc 2520 aatctgcagc agcaggcaaa aggagtcggc ctcggcgacc gctgggaatga agtgaaagac 2580 atgtacagcg tcgtcaaccg catgttcggc gacgtcgtta aagtaacgcc ttcttccaaa 2640 gtcgtcggag atatggcgct ttacatggtg caaaacaatt taacggaaca agatgtttat 2700 gataaaggcg aaactcttga ttttccggat tctgttgttg agctctttaa aggacagatc 2760 ggccagccgc acggcggatt tccggaaaaa ctgcaaaagc tcgttctaaa aggacagacg 2820 ccgattaagg taagaccggg tgaactgctt gaaccggtat cttttgaagg catcaaagaa 2880 gaatggaaag aaactcatca gatggaattg agtgatcagg acgcaatcgc gtatgctctt 2940 tatccgaagg tattcactga atacgttaaa acggctgaac gcttcggtga tatttccgta 3000 ttggacacac cgactttctt ctacggcatg aggctcggtg aagaaatcga agtggaaatc 3060 gagcgaggaa aaacgctgat cgtaaagctt gtgtctatcg gtgaaccgca gcctgatgcg 3120 acacgtgtcg tttattttga gttgaacggc cagcctcgtg aggtggtcat taaagatgaa 3180 agcattaaat catccgtaca ggaaaaatta aaggccgatc ggacaaaccc aagccacatc 3240 gcagcatcaa tgccgggaac cgttattaaa ttgttaacgc aaaccggtgc gaaggtgaac 3300 aaaggggatc atctgatgat taatgaagcg atgaaaatgg agacgacggt gcaggcgccg 3360 ttctccggga cgattcagca gattcatgtg aaaaacggtg aaccgattca gacaggcgat 3420 ttactgattg agattgaaaa agcgtag 3447 <210> 12 <211> 3447 <212> DNA <213> Artificial Sequence <400> 12 gtgtcacaac aatccatcca aaaagtgtta gtagcaaaca ggggagaaat tgcgatccgg 60 attttccggg cgtgcacaga attgaacatc agaacggtag ccgtttattc aaaagaagat 120 tcaggttcct accaccgcta taaagcggat gaagcctatc ttgtcggaga aggcaaaaaa 180 ccgattgacg cgtatcttga tattgaaggg attattgaga tcgcgaaaag aaacggcgtt 240 gatgccattc atccgggcta tggatttctg tctgaaaata tccaatttgc gagacgctgt 300 gaagaagagg gcatcgtatt tatcggaccg acgtcagcgc atttggatat gttcggagat 360 aaggtcaagg ccagagagca ggcggaaaaa gccggcatcc ccgttattcc ggggagtgac 420 gggcctgccg aaacattaaa agatgtcgaa caattcgcaa aagtacacgg atttccgttt 480 attattaaag cgtcgctcgg aggcggcggc cgcggaatga gaatcgtcag gaacgaaaac 540 gaattgaaag attcgtttga gcgggccaaa tccgaagcga aagccgcatt cggaaacgac 600 gaagtgtatg ttgaaaaact gatcgaaaat cctaagcata ttgaagttca ggtgatcgga 660 gataaagaag gaaatgtcgt ccatctgtat gagcgggact gttccgttca aagacggcat 720 caaaaggtca ttgaggtggc gccgagcgtg tccttacagc ctgaattaag agacgagatc 780 tgtgaagcgg ctgtcgcact tgcgaaaaat gtcggctata tcaacgctgg aacagtggaa 840 tttctcgtag cggacggtga attttacttt attgaagtga atccgcgcgt acaagtggag 900 catacgatta cggaaatgat tacgggggtt gatatcgttc agacgcaaat cctcgtcgcg 960 cagggccacg gtctgcacag ccgtgccgtc aacatccctc atcagaaaga catttttaca 1020 aacgggtatg cgatacagtc acgcgtgacc actgaagatc cgctgaatga ctttatgcct 1080 gacacgggta aaattatggc ttaccgctca ggcggcggct tcggcgtgcg tcttgatact 1140 gggaacagct tccaaggcgc cgtcatcacg ccttattatg attcactgct cgtcaagctg 1200 tcgacatggg cgctgacgtt tgaacaggca tccgcaaaaa tggtccgcaa cctgcaggaa 1260 ttcagaatca ggggcattaa gaccaatatc ccgtttcttg aaaacgtggc aaagcatgaa 1320 aaattcctga cgggacaata cgatacatcg tttatcgaca caacgcctga gctgtttgtt 1380 tttcctaaac agaaggaccg cggaacaaaa atgctttcct atatcggcaa tgtaacggtg 1440 aacggttttc cgggaatcgg aaaaaaaagaa aaacctgcgt ttgataagcc ccagactgtt 1500 1560 gctgaagggc ttgctgactg ggtgaaagag cagaaatccg tacttctgac cgatacaacg 1620 ttcagggatg cccatcagtc cctgcttgca acccggatca gatccaatga cttgaaaaaa 1680 atcgcaaacc ctacggctgc tctgtggcct gaactgttca gccttgaaat gtggggtggg 1740 gcgacatttg atgtagcgta ccgtttctta aaagaatc cttggaaacg ccttgaagat 1800 ctccgcaagg aagtgccgaa cacattgttc cagatgctgc ttcgttcatc aaatgccgtc 1860 ggctatacaa actatccgga caatgtcatt caaaaatttg tccggcagtc agcagcatca 1920 ggtattgacg tattccggat ctttgacagc ctgaactggg tgaaagggat gacgctggcc 1980 atcgatgccg tccgtgaaac cggaaaagtg gcggaggctg cgatttgtta tacgggagat 2040 attcttgata agagccggac gaagtatgat ctgaaatact atctttctat ggcaaaagag 2100 cttgaggcat cgggagcgca tattctcggc attaaagaca tggccggtct tttgaaacct 2160 caagccgcat acgaacttgt ctcagcgctg aaggaaacga ttgatattcc cgttcatctt 2220 cacagcatg atacgagcgg caacggcgtc tttctctatg ccaaagccgt tgaagcgggt 2280 gtggatatcg tagatgtcgc cgtcagctca atggcgggtc tgacttcaca gccgagtgca 2340 agcggatttt atcatgcgct tgaaggaac agccgccgtc ctgaaatgga tgtccggcag 2400 gttgaaagac tgtcacaata ttgggaatcc gtccgtcaat attacagtga atttgaaagc 2460 gggaatgaagt ctccgcatac tgagattat aatcacgaaa tgccgggcgg aagtacagc 2520 aatctgcagc agcaggcaaa aggagtcggc ctcggcgacc gctgggaatga agtgaaagac 2580 atgtacagcg tcgtcaaccg catgttcggc gacgtcgtta aagtaacgcc ttcttccaaa 2640 gtcgtcggag atatggcgct ttacatggtg caaaacaatt taacggaaca agatgtttat 2700 gataaaggcg aaactcttga ttttccggat tctgttgttg agctctttaa aggacagatc 2760 ggccagccgc acggcggatt tccggaaaaa ctgcaaaagc tcgttctaaa aggacagacg 2820 ccgattaagg taagaccggg tgaactgctt gaaccggtat cttttgaagg catcaaagaa 2880 gaatggaaag aaactcatca gatggaattg agtgatcagg acgcaatcgc gtatgctctt 2940 tatccgaagg tattcactga atacgttaaa acggctgaac gcttcggtga tatttccgta 3000 ttggacacac cgactttctt ctacggcatg aggctcggtg aagaaatcga agtggaaatc 3060 gagcgaggaa aaacgctgat cgtaaagctt gtgtctatcg gtgaaccgca gcctgatgcg 3120 acacgtgtcg tttattttga gttgaacggc cagcctcgtg aggtggtcat taaagatgaa 3180 agcattaaat catccgtaca ggaaaaatta aaggccgatc ggacaaaccc aagccacatc 3240 gcagcatcaa tgccgggaac cgttattaaa ttgttaacgc aaaccggtgc gaaggtgaac 3300 aaaggggatc atctgatgat taatgaagcg atgaaaatgg agacgacggt gcaggcgccg 3360 ttctccggga cgattcagca gattcatgtg aaaaacggtg aaccgattca gacaggcgat 3420 ttactgattg agattgaaaa agcgtag 3447 <210> 13 <211> 3447 <212> DNA <213> Artificial Sequence <400> 13 atgtcacaac aatccatcca aaaagtgtta gtagcaaaca ggggagaaat tgcgatccgg 60 attttccggg cgtgcacaga attgaacatc agaacggtag ccgtttattc aaaagaagat 120 tcaggttcct accaccgcta taaagcggat gaagcctatc ttgtcggaga aggcaaaaaa 180 ccgattgacg cgtatcttga tattgaaggg attattgaga tcgcgaaaag aaacggcgtt 240 gatgccattc atccgggcta tggatttctg tctgaaaata tccaatttgc gagacgctgt 300 gaagaagagg gcatcgtatt tatcggaccg acgtcagcgc atttggatat gttcggagat 360 aaggtcaagg ccagagagca ggcggaaaaa gccggcatcc ccgttattcc ggggagtgac 420 gggcctgccg aaacattaaa agatgtcgaa caattcgcaa aagtacacgg atttccgttt 480 attattaaag cgtcgctcgg aggcggcggc cgcggaatga gaatcgtcag gaacgaaaac 540 gaattgaaag attcgtttga gcgggccaaa tccgaagcga aagccgcatt cggaaacgac 600 gaagtgtatg ttgaaaaact gatcgaaaat cctaagcata ttgaagttca ggtgatcgga 660 gataaagaag gaaatgtcgt ccatctgtat gagcgggact gttccgttca aagacggcat 720 caaaaggtca ttgaggtggc gccgagcgtg tccttacagc ctgaattaag agacgagatc 780 tgtgaagcgg ctgtcgcact tgcgaaaaat gtcggctata tcaacgctgg aacagtggaa 840 tttctcgtag cggacggtga attttacttt attgaagtga atccgcgcgt acaagtggag 900 catacgatta cggaaatgat tacgggggtt gatatcgttc agacgcaaat cctcgtcgcg 960 cagggccacg gtctgcacag ccgtgccgtc aacatccctc atcagaaaga catttttaca 1020 aacgggtatg cgatacagtc acgcgtgacc actgaagatc cgctgaatga ctttatgcct 1080 gacacgggta aaattatggc ttaccgctca ggcggcggct tcggcgtgcg tcttgatact 1140 gggaacagct tccaaggcgc cgtcatcacg ccttattatg attcactgct cgtcaagctg 1200 tcgacatggg cgctgacgtt tgaacaggca tccgcaaaaa tggtccgcaa cctgcaggaa 1260 ttcagaatca ggggcattaa gaccaatatc ccgtttcttg aaaacgtggc aaagcatgaa 1320 aaattcctga cgggacaata cgatacatcg tttatcgaca caacgcctga gctgtttgtt 1380 tttcctaaac agaaggaccg cggaacaaaa atgctttcct atatcggcaa tgtaacggtg 1440 aacggttttc cgggaatcgg aaaaaaaagaa aaacctgcgt ttgataagcc ccagactgtt 1500 1560 gctgaagggc ttgctgactg ggtgaaagag cagaaatccg tacttctgac cgatacaacg 1620 ttcagggatg cccatcagtc cctgcttgca acccggatca gatccaatga cttgaaaaaa 1680 atcgcaaacc ctacggctgc tctgtggcct gaactgttca gccttgaaat gtggggtggg 1740 gcgacatttg atgtagcgta ccgtttctta aaagaatc cttggaaacg ccttgaagat 1800 ctccgcaagg aagtgccgaa cacattgttc cagatgctgc ttcgttcatc aaatgccgtc 1860 ggctatacaa actatccgga caatgtcatt caaaaatttg tccggcagtc agcagcatca 1920 ggtattgacg tattccggat ctttgacagc ctgaactggg tgaaagggat gacgctggcc 1980 atcgatgccg tccgtgaaac cggaaaagtg gcggaggctg cgatttgtta tacgggagat 2040 attcttgata agagccggac gaagtatgat ctgaaatact atctttctat ggcaaaagag 2100 cttgaggcat cgggagcgca tattctcggc attaaagaca tggccggtct tttgaaacct 2160 caagccgcat acgaacttgt ctcagcgctg aaggaaacga ttgatattcc cgttcatctt 2220 cacagcatg atacgagcgg caacggcgtc tttctctatg ccaaagccgt tgaagcgggt 2280 gtggatatcg tagatgtcgc cgtcagctca atggcgggtc tgacttcaca gccgagtgca 2340 agcggatttt atcatgcgct tgaaggaac agccgccgtc ctgaaatgga tgtccggcag 2400 gttgaaagac tgtcacaata ttgggaatcc gtccgtcaat attacagtga atttgaaagc 2460 gggaatgaagt ctccgcatac tgagattat aatcacgaaa tgccgggcgg aagtacagc 2520 aatctgcagc agcaggcaaa aggagtcggc ctcggcgacc gctggaatga agtgaaagac 2580 atgtacagcg tcgtcaaccg catgttcggc gacgtcgtta aagtaacgcc ttcttccaaa 2640 gtcgtcggag atatggcgct ttacatggtg caaaacaatt taacggaaca agatgtttat 2700 gataaaggcg aaactcttga ttttccggat tctgttgttg agctctttaa aggacagatc 2760 ggccagccgc acggcggatt tccggaaaaa ctgcaaaagc tcgttctaaa aggacagacg 2820 ccgattaagg taagaccggg tgaactgctt gaaccggtat cttttgaagg catcaaagaa 2880 gaatggaaag aaactcatca gatggaattg agtgatcagg acgcaatcgc gtatgctctt 2940 tatccgaagg tattcactga atacgttaaa acggctgaac gcttcggtga tatttccgta 3000 ttggacacac cgactttctt ctacggcatg aggctcggtg aagaaatcga agtggaaatc 3060 gagcgaggaa aaacgctgat cgtaaagctt gtgtctatcg gtgaaccgca gcctgatgcg 3120 acacgtgtcg tttattttga gttgaacggc cagcctcgtg aggtggtcat taaagatgaa 3180 agcattaaat catccgtaca ggaaaaatta aaggccgatc ggacaaaccc aagccacatc 3240 gcagcatcaa tgccgggaac cgttattaaa ttgttaacgc aaaccggtgc gaaggtgaac 3300 aaaggggatc atctgatgat taatgaagcg atgaaaatgg agacgacggt gcaggcgccg 3360 ttctccggga cgattcagca gattcatgtg aaaaacggtg aaccgattca gacaggcgat 3420 ttactgattg agattgaaaa agcgtag 3447 <210> 14 <211> 527 <212> PRT <213> Bacillus <400> 14 Met Asn Ser Val Asp Leu Thr Ala Asp Leu Gln Ala Leu Leu Thr Cys 1 5 10 15 Pro Asn Val Arg His Asn Leu Ser Ala Ala Gln Leu Thr Glu Lys Val 20 25 30 Leu Ser Arg Asn Glu Gly Ile Leu Thr Ser Thr Gly Ala Val Arg Ala 35 40 45 Thr Thr Gly Ala Tyr Thr Gly Arg Ser Pro Lys Asp Lys Phe Ile Val 50 55 60 Glu Glu Glu Ser Thr Lys Asn Lys Ile Asp Trp Gly Pro Val Asn Gln 65 70 75 80 Pro Ile Ser Glu Glu Ala Phe Glu Arg Leu Tyr Thr Lys Val Val Ser 85 90 95 Tyr Leu Lys Glu Arg Asp Glu Leu Phe Val Phe Glu Gly Phe Ala Gly 100 105 110 Ala Asp Glu Lys Tyr Arg Leu Pro Ile Thr Val Val Asn Glu Phe Ala 115 120 125 Trp His Asn Leu Phe Ala Arg Gln Leu Phe Ile Arg Pro Glu Gly Asn 130 135 140 Asp Lys Lys Thr Val Glu Gln Pro Phe Thr Ile Leu Ser Ala Pro His 145 150 155 160 Phe Lys Ala Asp Pro Lys Thr Asp Gly Thr His Ser Glu Thr Phe Ile 165 170 175 Ile Val Ser Phe Glu Lys Arg Thr Ile Leu Ile Gly Gly Thr Glu Tyr 180 185 190 Ala Gly Glu Met Lys Lys Ser Ile Phe Ser Ile Met Asn Phe Leu Leu 195 200 205 Pro Glu Arg Asp Ile Leu Ser Met His Cys Ser Ala Asn Val Gly Glu 210 215 220 Lys Gly Asp Val Ala Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr 225 230 235 240 Thr Leu Ser Ala Asp Ala Asp Arg Lys Leu Ile Gly Asp Asp Glu His 245 250 255 Gly Trp Ser Asp Thr Gly Val Phe Asn Ile Glu Gly Gly Cys Tyr Ala 260 265 270 Lys Cys Ile His Leu Ser Glu Glu Lys Glu Pro Gln Ile Phe Asn Ala 275 280 285 Ile Arg Phe Gly Ser Val Leu Glu Asn Val Val Val Asp Glu Asp Thr 290 295 300 Arg Glu Ala Asn Tyr Asp Asp Ser Phe Tyr Thr Glu Asn Thr Arg Ala 305 310 315 320 Ala Tyr Pro Ile His Met Ile Asn Asn Ile Val Thr Pro Ser Met Ala 325 330 335 Gly His Pro Ser Ala Ile Val Phe Leu Thr Ala Asp Ala Phe Gly Val 340 345 350 Leu Pro Pro Ile Ser Lys Leu Thr Lys Glu Gln Ala Met Tyr His Phe 355 360 365 Leu Ser Gly Tyr Thr Ser Lys Leu Ala Gly Thr Glu Arg Gly Val Thr 370 375 380 Ser Pro Glu Thr Thr Phe Ser Thr Cys Phe Gly Ser Pro Phe Leu Pro 385 390 395 400 Leu Pro Ala His Val Tyr Ala Glu Met Leu Gly Lys Lys Ile Asp Glu 405 410 415 His Gly Ala Asp Val Phe Leu Val Asn Thr Gly Trp Thr Gly Gly Gly 420 425 430 Tyr Gly Thr Gly Glu Arg Met Lys Leu Ser Tyr Thr Arg Ala Met Val 435 440 445 Lys Ala Ala Ile Glu Gly Lys Leu Glu Asp Ala Glu Met Ile Thr Asp 450 455 460 Asp Ile Phe Gly Leu His Ile Pro Ala His Val Pro Gly Val Pro Asp 465 470 475 480 His Ile Leu Gln Pro Glu Asn Thr Trp Thr Asn Lys Glu Glu Tyr Lys 485 490 495 Glu Lys Ala Val Tyr Leu Ala Asn Glu Phe Lys Glu Asn Phe Lys Lys 500 505 510 Phe Ala His Thr Asp Ala Ile Ala Gln Ala Gly Gly Pro Leu Val 515 520 525 <210> 15 <211> 527 <212> PRT <213> Artificial Sequence <400> 15 Met Asn Ser Val Asp Leu Thr Ala Asp Leu Gln Ala Leu Leu Thr Cys 1 5 10 15 Pro Asn Val Arg His Asn Leu Ser Ala Ala Gln Leu Thr Glu Lys Val 20 25 30 Leu Ser Arg Asn Glu Gly Ile Leu Thr Ser Thr Gly Ala Val Arg Ala 35 40 45 Thr Thr Gly Ala Tyr Thr Gly Arg Ser Pro Lys Asp Lys Phe Ile Val 50 55 60 Glu Glu Glu Ser Thr Lys Asn Lys Ile Asp Trp Gly Pro Val Asn Gln 65 70 75 80 Pro Ile Ser Glu Glu Ala Phe Glu Arg Leu Tyr Thr Lys Val Val Ser 85 90 95 Tyr Leu Lys Glu Arg Asp Glu Leu Phe Val Phe Glu Gly Phe Ala Gly 100 105 110 Ala Asp Glu Lys Tyr Arg Leu Pro Ile Thr Val Val Asn Glu Phe Ala 115 120 125 Trp His Asn Leu Phe Ala Arg Gln Leu Phe Ile Arg Pro Glu Gly Asn 130 135 140 Asp Lys Arg Thr Val Glu Gln Pro Phe Thr Ile Leu Ser Ala Pro His 145 150 155 160 Phe Lys Ala Asp Pro Lys Thr Asp Gly Thr His Ser Glu Thr Phe Ile 165 170 175 Ile Val Ser Phe Glu Lys Arg Thr Ile Leu Ile Gly Gly Thr Glu Tyr 180 185 190 Ala Gly Glu Met Lys Lys Ser Ile Phe Ser Ile Met Asn Phe Leu Leu 195 200 205 Pro Glu Arg Asp Ile Leu Ser Met His Cys Ser Ala Asn Val Gly Glu 210 215 220 Lys Gly Asp Val Ala Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr 225 230 235 240 Thr Leu Ser Ala Asp Ala Asp Arg Lys Leu Ile Gly Asp Asp Glu His 245 250 255 Gly Trp Ser Asp Thr Gly Val Phe Asn Ile Glu Gly Gly Cys Tyr Ala 260 265 270 Lys Cys Ile His Leu Ser Glu Glu Lys Glu Pro Gln Ile Phe Asn Ala 275 280 285 Ile Arg Phe Gly Ser Val Leu Glu Asn Val Val Val Asp Glu Asp Thr 290 295 300 Arg Glu Ala Asn Tyr Asp Asp Ser Phe Tyr Thr Glu Asn Thr Arg Ala 305 310 315 320 Ala Tyr Pro Ile His Met Ile Asn Asn Ile Val Thr Pro Ser Met Ala 325 330 335 Gly His Pro Ser Ala Ile Val Phe Leu Thr Ala Asp Ala Phe Gly Val 340 345 350 Leu Pro Pro Ile Ser Lys Leu Thr Lys Glu Gln Ala Met Tyr His Phe 355 360 365 Leu Ser Gly Tyr Thr Ser Lys Leu Ala Gly Thr Glu Arg Gly Val Thr 370 375 380 Ser Pro Glu Thr Thr Phe Ser Thr Cys Phe Gly Ser Pro Phe Leu Pro 385 390 395 400 Leu Pro Ala His Val Tyr Ala Glu Met Leu Gly Lys Lys Ile Asp Glu 405 410 415 His Gly Ala Asp Val Phe Leu Val Asn Thr Gly Trp Thr Gly Gly Gly 420 425 430 Tyr Gly Thr Gly Glu Arg Met Lys Leu Ser Tyr Thr Arg Ala Met Val 435 440 445 Lys Ala Ala Ile Glu Gly Lys Leu Glu Asp Ala Glu Met Ile Thr Asp 450 455 460 Asp Ile Phe Gly Leu His Ile Pro Ala His Val Pro Gly Val Pro Asp 465 470 475 480 His Ile Leu Gln Pro Glu Asn Thr Trp Thr Asn Lys Glu Glu Tyr Lys 485 490 495 Glu Lys Ala Val Tyr Leu Ala Asn Glu Phe Lys Glu Asn Phe Lys Lys 500 505 510 Phe Ala His Thr Asp Ala Ile Ala Gln Ala Gly Gly Pro Leu Val 515 520 525 <210> 16 <211> 527 <212> PRT <213> Artificial Sequence <400> 16 Met Asn Ser Val Asp Leu Thr Ala Asp Leu Gln Ala Leu Leu Thr Cys 1 5 10 15 Pro Asn Val Arg His Asn Leu Ser Ala Ala Gln Leu Thr Glu Lys Val 20 25 30 Leu Ser Arg Asn Glu Gly Ile Leu Thr Ser Thr Gly Ala Val Arg Ala 35 40 45 Thr Thr Gly Ala Tyr Thr Gly Arg Ser Pro Lys Asp Lys Phe Ile Val 50 55 60 Glu Glu Glu Ser Thr Lys Asn Lys Ile Asp Trp Gly Pro Val Asn Gln 65 70 75 80 Pro Ile Ser Glu Glu Ala Phe Glu Arg Leu Tyr Thr Lys Val Val Ser 85 90 95 Tyr Leu Lys Glu Arg Asp Glu Leu Phe Val Phe Glu Gly Phe Ala Gly 100 105 110 Ala Asp Glu Lys Tyr Arg Leu Pro Ile Thr Val Val Asn Glu Phe Ala 115 120 125 Trp His Asn Leu Phe Ala Arg Gln Leu Phe Ile Arg Pro Glu Gly Asn 130 135 140 Asp Lys His Thr Val Glu Gln Pro Phe Thr Ile Leu Ser Ala Pro His 145 150 155 160 Phe Lys Ala Asp Pro Lys Thr Asp Gly Thr His Ser Glu Thr Phe Ile 165 170 175 Ile Val Ser Phe Glu Lys Arg Thr Ile Leu Ile Gly Gly Thr Glu Tyr 180 185 190 Ala Gly Glu Met Lys Lys Ser Ile Phe Ser Ile Met Asn Phe Leu Leu 195 200 205 Pro Glu Arg Asp Ile Leu Ser Met His Cys Ser Ala Asn Val Gly Glu 210 215 220 Lys Gly Asp Val Ala Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr 225 230 235 240 Thr Leu Ser Ala Asp Ala Asp Arg Lys Leu Ile Gly Asp Asp Glu His 245 250 255 Gly Trp Ser Asp Thr Gly Val Phe Asn Ile Glu Gly Gly Cys Tyr Ala 260 265 270 Lys Cys Ile His Leu Ser Glu Glu Lys Glu Pro Gln Ile Phe Asn Ala 275 280 285 Ile Arg Phe Gly Ser Val Leu Glu Asn Val Val Val Asp Glu Asp Thr 290 295 300 Arg Glu Ala Asn Tyr Asp Asp Ser Phe Tyr Thr Glu Asn Thr Arg Ala 305 310 315 320 Ala Tyr Pro Ile His Met Ile Asn Asn Ile Val Thr Pro Ser Met Ala 325 330 335 Gly His Pro Ser Ala Ile Val Phe Leu Thr Ala Asp Ala Phe Gly Val 340 345 350 Leu Pro Pro Ile Ser Lys Leu Thr Lys Glu Gln Ala Met Tyr His Phe 355 360 365 Leu Ser Gly Tyr Thr Ser Lys Leu Ala Gly Thr Glu Arg Gly Val Thr 370 375 380 Ser Pro Glu Thr Thr Phe Ser Thr Cys Phe Gly Ser Pro Phe Leu Pro 385 390 395 400 Leu Pro Ala His Val Tyr Ala Glu Met Leu Gly Lys Lys Ile Asp Glu 405 410 415 His Gly Ala Asp Val Phe Leu Val Asn Thr Gly Trp Thr Gly Gly Gly 420 425 430 Tyr Gly Thr Gly Glu Arg Met Lys Leu Ser Tyr Thr Arg Ala Met Val 435 440 445 Lys Ala Ala Ile Glu Gly Lys Leu Glu Asp Ala Glu Met Ile Thr Asp 450 455 460 Asp Ile Phe Gly Leu His Ile Pro Ala His Val Pro Gly Val Pro Asp 465 470 475 480 His Ile Leu Gln Pro Glu Asn Thr Trp Thr Asn Lys Glu Glu Tyr Lys 485 490 495 Glu Lys Ala Val Tyr Leu Ala Asn Glu Phe Lys Glu Asn Phe Lys Lys 500 505 510 Phe Ala His Thr Asp Ala Ile Ala Gln Ala Gly Gly Pro Leu Val 515 520 525

Claims

1. Engineered bacteria containing nucleic acid molecules encoding a phosphoenolpyruvate carboxykinase mutant; The amino acid sequences of the phosphoenolpyruvate carboxykinase mutants are shown in SEQ ID NO:15 or 16, respectively. The starting strain of the engineered bacteria is Bacillus subtilis (B. subtilis). Bacillus subtilis For the construction method of Bacillus subtilis A1, please refer to CN201910599510.

8.

2. Any of the following applications of the engineered bacteria described in claim 1: (1) Used for the fermentation production of nucleosides; (2) Used to increase the fermentation yield of nucleosides; The nucleosides are adenosine and inosine.

3. A method for constructing a nucleoside-producing strain, characterized in that, Using genetic engineering techniques, mutations are introduced into the genomes of microorganisms capable of nucleoside production, causing their encoded phosphoenolpyruvate carboxykinase to contain either the K147R or K147H mutation site; wherein the amino acid sequences of the phosphoenolpyruvate carboxykinase containing the K147R or K147H mutation site are shown in SEQ ID NO:15 or 16, respectively; or, Using genetic engineering techniques, mutations are introduced into the genomes of microorganisms capable of nucleoside production, causing their encoded phosphoenolpyruvate carboxykinase to contain the K147R or K147H mutation site, and their encoded pyruvate kinase to contain the T101K, T101R, or T101P mutation site; wherein the amino acid sequence of the phosphoenolpyruvate carboxykinase containing the K147R or K147H mutation site is shown in SEQ ID NO:15 or 16, respectively, and the amino acid sequence of the pyruvate kinase containing the T101K, T101R, or T101P mutation site is shown in SEQ ID NO:5, 6, or 7, respectively; or, Using genetic engineering techniques, mutations were introduced into the genomes of microorganisms capable of nucleoside production, causing their encoded phosphoenolpyruvate carboxylkinase to contain the K147R or K147H mutation site, and causing it to encode pyruvate carboxylase. pycA The gene contains a mutation in which the first base is changed from T to G or A; wherein, the amino acid sequence of the phosphoenolpyruvate carboxykinase containing the K147R or K147H mutation site is shown in SEQ ID NO:15 or 16, respectively, and contains a mutation in which the first base is changed from T to G or A. pycA The nucleotide sequences of the genes are shown in SEQ ID NO:9 or 10, respectively; or, Using genetic engineering techniques, mutations were introduced into the genomes of microorganisms capable of nucleoside production, causing their encoded phosphoenolpyruvate carboxylkinase to contain the K147R or K147H mutation site, and their encoded pyruvate kinase to contain the T101K, T101R, or T101P mutation site, and also causing their encoded pyruvate carboxylase... pycA The gene contains a mutation in which the first base changes from T to G or A; wherein, the amino acid sequences of phosphoenolpyruvate carboxykinase containing the K147R or K147H mutation site are shown in SEQ ID NO:15 or 16, respectively, and the amino acid sequences of pyruvate kinase containing the T101K, T101R, or T101P mutation sites are shown in SEQ ID NO:5, 6, or 7, respectively, and the gene contains a mutation in which the first base changes from T to G or A. pycA The nucleotide sequences of the genes are shown in SEQ ID NO:9 or 10, respectively; The microorganism is Bacillus subtilis (B. subtilis) Bacillus subtilis For the construction method of Bacillus subtilis A1, please refer to CN201910599510.8; The nucleosides are adenosine and inosine.

4. The nucleoside-producing strain constructed according to the method of claim 3; The nucleosides are adenosine and inosine.

5. A method for producing nucleosides, characterized in that, The method includes the following steps: 1) Cultivate the strain according to claim 4 to obtain a culture of the microorganism; 2) Collect the nucleosides produced from the culture obtained in step 1); The nucleosides are adenosine and inosine.

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