Fructose 1,6-bisphosphatase ii mutants and uses thereof

Abstract

The present application provides a fructose 1,6-bisphosphatase II mutant and application thereof. After the 175th amino acid of fructose 1,6-bisphosphatase II in a bacillus microorganism is mutated from arginine (R) to histidine (H) or proline (P), the microorganism can efficiently produce nucleosides. The present 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 fructose 1,6-bisphosphatase II 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℃. 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, which are abbreviated as adenosine (A), guanine (G), cytosine (C), thymine (T), and uridine (U), respectively.

[0003] Guanosine and inosine have wide applications in the food and pharmaceutical industries. In the food sector, guanosine and inosine are important precursors 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 is still relatively poor, and the conversion rate of nucleosides remains 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 fructose 1,6-bisphosphatase II mutant and its applications.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides a fructose 1,6-bisphosphatase II mutant, said mutant comprising a mutation in the 175th amino acid of fructose 1,6-bisphosphatase II from R to H or P.

[0007] In this invention, the reference sequence number of fructose 1,6-bisphosphatase II on NCBI can be NP_391590.1 or CBI44672.1.

[0008] In a second aspect, the present invention provides a nucleic acid molecule encoding the fructose 1,6-bisphosphatase II 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:

[0011] (1) Used for the fermentation production of nucleosides;

[0012] (2) Used to increase the fermentation yield of nucleosides;

[0013] (3) Used to construct genetically engineered bacteria that produce nucleosides.

[0014] 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 fructose-1,6-bisphosphatase II it encodes contains an R175H or R175P mutation site.

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

[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) TUsing the genome of *B. amyloliquefaciens* FZB42 (which reveals differences to plant-associated B. amyloliquefaciens FZB42) as a template, and guaB-1f / 1r and guaB-2f / 3r as primers, two fragments were amplified using Phusion high-fidelity polymerase (New England BioLabs). The two fragments were then fused using primer guaB-1f / 3r to obtain the recombinant fragment (ORF region nucleotide sequence shown in SEQ ID NO:9). The guaB... L454F The fragment and pKSU plasmid (pKSU plasmid was kindly provided by Professor Wang Shufang of Nankai University, see Amarkerless gene replacement method for B. amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production[J], Applied Microbiology and Biotechnology, 2014, 98(21):8963-8973. Zhang W, Gao W, Feng J, et al DOI:10.1007 / s00253-014-5824-2) were digested with SalI / PstI, assembled, and transformed to obtain the recombinant plasmid pKSU-guaB. L454F Transformed into Ba 836 strain (see CN112574934A), transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, thus obtaining guaB. L454FThe point mutation strain obtained was Ba 837. The pBE43 plasmid (the PBE43 plasmid was synthesized in its entirety; see reference: Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens) was extracted and linearized using KpnI / SalI. The mutated purR sequence (R2, SEQ ID NO:10) and the mutated tal sequence (L5, SEQ ID NO:11) were fused by PCR to obtain the R2+L5 fragment. This fragment was then ligated into the linearized PBE43 plasmid using an assembly kit to construct the plasmid PBE43-R2+L5. This plasmid was transformed into B. a837 strain to obtain the starting strain B. a8333.

[0017] Sixthly, the present invention provides a method for constructing a nucleoside-producing engineered bacterium, wherein the nucleoside-producing strain constructed by the above method is used as the starting strain, and at least one of the following methods ① to ④ is used to genetically engineer the starting strain:

[0018] ① Using genetic engineering techniques, mutations are introduced into the starting strain so that the pyruvate kinase it encodes contains T101K, T101R or T101P mutation sites.

[0019] ② Using genetic engineering techniques, a mutation was introduced into the starting strain so that the pycA gene encoding pyruvate carboxylase contained a mutation in which the first base was changed from T to G or A.

[0020] ③ Using genetic engineering techniques, a mutation is introduced into the starting strain so that the phosphoenolpyruvate carboxykinase it encodes contains the K147R or K147H mutation site.

[0021] ④ Using genetic engineering techniques, a mutation is introduced into the starting strain so that the encoded glyceraldehyde-3-phosphate dehydrogenase contains the G169D or G169E mutation site.

[0022] Preferably, the starting strain is genetically engineered using a combination of methods ① to ④.

[0023] In this invention, the reference sequence number for pyruvate kinase on NCBI can be NP_390796.1, and the reference sequence number for phosphoenolpyruvate carboxykinase on NCBI can be NP_390934.2. The reference sequence number for glyceraldehyde-3-phosphate dehydrogenase on NCBI can be NP_390780.1 or CBI43831.1;

[0024] The pycA gene can originate from Bacillus subtilis, and it is as follows:

[0025] i) The nucleotide sequence shown in SEQ ID NO:7;

[0026] ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:7 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function;

[0027] iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:7 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;

[0028] iv) Nucleotide sequences that have more than 90% homology with the nucleotide sequences of i), ii) or iii) and express the same functional protein.

[0029] The pycA gene can originate from Bacillus amyloliquefaciens, and it is as follows:

[0030] a) The nucleotide sequence shown in SEQ ID NO:8;

[0031] b) 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;

[0032] c) 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.

[0033] d) A nucleotide sequence that has more than 90% homology with the nucleotide sequences of a), b), or c) and expresses the same functional protein.

[0034] In a seventh aspect, the present invention provides a nucleoside-producing strain or engineered strain constructed according to the method.

[0035] Eighthly, the present invention provides a method for producing nucleosides, the method comprising the following steps:

[0036] 1) Cultivate the nucleoside-producing strain or engineered bacteria to obtain a microbial culture;

[0037] 2) Collect the nucleosides produced from the culture obtained in step 1).

[0038] The nucleosides include adenosine, inosine, guanosine and their corresponding nucleoside derivatives, such as hypoxanthine, inosine, guanine, guanylic acid, riboflavin, diacetylguanylic acid, etc.

[0039] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0040] Fructose 1,6-bisphosphatase II mutants and / or glyceraldehyde-3-phosphate dehydrogenase mutants have a positive effect on increasing adenosine and inosine production in Bacillus subtilis. Mutating the 175th amino acid of fructose 1,6-bisphosphatase II from arginine (R) to histidine (H) or proline (P) increases the production of both adenosine and inosine, with the most significant effect observed after the mutation from arginine (R) to histidine (H). The mutant glpX... R175H The optimal adenosine yield of engineered strain A368 was increased from 4.4 g / L to 5.6 g / L.

[0041] The superposition of fructose 1,6-bisphosphatase II mutant and glyceraldehyde-3-phosphate dehydrogenase mutant has a positive effect on increasing the production of adenosine and inosine. The mutant glpX R175H superposition mutant gapB G169E The adenosine yield of engineered strain A372 increased from 4.4 g / L to 6.8 g / L, and the inosine yield increased from 2.2 g / L to 2.9 g / L.

[0042] Fructose-1,6-bisphosphatase II mutants and / or glyceraldehyde-3-phosphate dehydrogenase mutants have a positive effect on increasing the production of guanosine and inosine in Bacillus amyloliquefaciens. Mutating the 175th amino acid of fructose-1,6-bisphosphatase II from arginine (R) to histidine (H) or proline (P) increases the production of both guanosine and inosine, with the mutation from arginine (R) to histidine (H) showing the best effect. The mutant glpX R175H The optimal results were achieved by increasing the yield of guanosine from 17.1 g / L to 18.9 g / L and the yield of inosine from 1.5 g / L to 1.9 g / L using the engineered strain 8461.

[0043] The superposition of fructose 1,6-bisphosphatase II mutant and glyceraldehyde-3-phosphate dehydrogenase mutant has a positive effect on increasing the production of guanosine and inosine. The mutant glpX R175H superposition mutant gapB G169E The yield of guanosine from engineered strain 8471 increased from 17.1 g / L to 20.6 g / L, and the yield of inosine increased from 1.5 g / L to 2.0 g / L. Detailed Implementation

[0044] 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.

[0045] Research has found that modifying Bacillus subtilis or Bacillus amyloliquefaciens with fructose-1,6-bisphosphatase II and / or glyceraldehyde-3-phosphate dehydrogenase enables microorganisms to efficiently produce nucleosides, and successfully creates new microorganisms capable of producing nucleosides efficiently, thus completing this invention.

[0046] The present invention adopts the following technical solution:

[0047] This invention provides a Bacillus subtilis strain in which the 169th amino acid of the glyceraldehyde-3-phosphate dehydrogenase (NCBI reference sequence number NP_390780.1), encoded by the gapB gene (NCBI reference sequence number Gene ID: 937393), is mutated from glycine (G) to aspartic acid (D) or glutamic acid (E).

[0048] The gapB gene encodes glyceraldehyde-3-phosphate dehydrogenase, which participates in gluconeogenesis and catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate (G3P) to glycerol-1,3-diphosphate (BPG). This process can be divided into two steps, requiring the cofactor NADP (or NAD). The first step involves the formation of a hemiacetal intermediate between G3P and cysteine ​​residues, followed by the oxidation of this intermediate to a thioester, while simultaneously reducing NADP (or NAD) to NADPH (or NADH). The second step involves the exchange of reduced NADPH (or NADH) with a second NADP (or NAD), and the thioester is attacked by a nucleophilic inorganic phosphate to generate BPG. The overall reaction equation is: G3P + phosphate + NAD(P)(+) <=> BPG + NAD(P)H.

[0049] This invention modifies the gapB gene to mutate glyceraldehyde-3-phosphate dehydrogenase, thereby enhancing the ability of the microorganism to produce nucleosides compared to the unmodified strain, ultimately increasing the yield of nucleosides.

[0050] This invention also provides a *Bacillus subtilis* strain in which the 175th amino acid of fructose-1,6-bisphosphatase II (NCBI reference sequence number NP_391590.1), encoded by the glpX gene (NCBI reference sequence number Gene ID: 937035), is mutated from arginine (R) to histidine (H) or proline (P). The wild-type fructose-1,6-bisphosphatase II and its mutant glpX from *Bacillus subtilis* are also described. R175H glpX R175P The amino acid sequences are shown in SEQ ID NO:1-3, respectively.

[0051] The glpX gene encodes fructose-1,6-bisphosphatase II, which participates in gluconeogenesis and catalyzes the hydrolysis of fructose-1,6-bisphosphate (FBP) to produce fructose-6-phosphate (F6P) and phosphate. The overall reaction equation is: FBP + H₂O <=> F6P + phosphate.

[0052] This invention modifies the glpX gene to mutate fructose 1,6-bisphosphatase II, thereby enhancing the ability of the microorganism to produce nucleosides compared to the unmodified strain, ultimately increasing nucleoside production.

[0053] The Bacillus subtilis strain used in this invention is Bacillus subtilis A1 (hereinafter referred to as A1), and its construction method can be found in CN201910599510.8.

[0054] The Bacillus subtilis mutant strain A342-pckA used in this invention K147R (hereinafter referred to as A358) is self-constructed, and its construction method can also be found in CN202210255870.8.

[0055] The present invention also provides a Bacillus amyloliquefaciens, wherein the 169th amino acid of the glyceraldehyde-3-phosphate dehydrogenase (reference sequence number CBI43831.1 on NCBI) encoded by the gapB gene (reference sequence number BAMF_2705 on NCBI) is mutated from glycine (G) to aspartic acid (D) or glutamic acid (E).

[0056] This invention modifies the gapB gene from Bacillus amyloliquefaciens to mutate glyceraldehyde-3-phosphate dehydrogenase, thereby enhancing the ability of the microorganism to produce nucleosides compared to the unmodified strain, ultimately increasing the yield of nucleosides.

[0057] This invention also provides a *Bacillus amyloliquefaciens* strain in which the 175th amino acid of fructose-1,6-bisphosphatase II (reference sequence number CBI44672.1 on NCBI), encoded by the glpX gene (reference sequence number BAMF_3546 on NCBI), is mutated from arginine (R) to histidine (H) or proline (P). The wild-type fructose-1,6-bisphosphatase II and its mutant glpX from *Bacillus amyloliquefaciens* are described. R175H glpX R175P The amino acid sequences are shown in SEQ ID NO:4 to 6, respectively.

[0058] This invention modifies the glpX gene from Bacillus amyloliquefaciens to mutate fructose 1,6-bisphosphatase II, thereby enhancing the ability of the microorganism to produce nucleosides compared to the unmodified strain, ultimately increasing nucleoside production.

[0059] The Bacillus amyloliquefaciens strain used in this invention is Bacillus amyloliquefaciens 8333 (hereinafter referred to as Ba 8333), and its construction method can be found in CN202111266398.X.

[0060] The Bacillus amyloliquefaciens mutant strain 8333-pycA used in this invention T1A (hereinafter referred to as 8441) is self-constructed, and its construction method can also be found in CN202210255870.8.

[0061] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0062] The amino acid sequences of pyruvate kinase involved in the following examples are shown in SEQ ID NO:12; the nucleotide sequences encoding pyruvate carboxylase from Bacillus subtilis are shown in SEQ ID NO:7; the nucleotide sequences encoding pyruvate carboxylase from Bacillus amyloliquefaciens are shown in SEQ ID NO:8; and the amino acid sequences of phosphoenolpyruvate carboxylkinase are shown in SEQ ID NO:13. The amino acid sequences of glyceraldehyde-3-phosphate dehydrogenase from Bacillus subtilis are shown in SEQ ID NO:14, and the amino acid sequences of glyceraldehyde-3-phosphate dehydrogenase from Bacillus amyloliquefaciens are shown in SEQ ID NO:15.

[0063] The primers used in the following examples are shown in Table 1:

[0064] Table 1

[0065]

[0066]

[0067] Example 1: Construction of pyruvate kinase mutant strain A1-pyk T101K

[0068] Using the genome of strain B. subtilis A1 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 into *B. subtilis* A1 strain, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding pyk... T101K The point mutant strain was named A1-pyk T101K Hereinafter referred to as A325.

[0069] Example 2: Construction of pyruvate carboxylase mutant strain A325-pycA t1a

[0070] Using the genome of strain B. subtilis A1 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 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 into strain A325, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding pycA. t1A The point mutant strain was named A325-pycA t1a Hereinafter referred to as A342.

[0071] Example 3: Construction of a phosphoenolpyruvate carboxykinase mutant strain A342-pckA K147R

[0072] Using the genome of strain B. subtilis A1 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 into strain A342, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding pckA. K147R The point mutant strain was named A342-pckA K147R Hereinafter referred to as A358.

[0073] Example 4: Construction of glyceraldehyde-3-phosphate dehydrogenase mutant strain A358-gapB G169E

[0074] Using the genome of strain B. subtilis A1 as a template, primer gapB G169D -UP-1F / gapB G169E -UP-1R and gapB G169E -DN-2F / gapB G169D -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primer gapB was used. G169D -UP-1F / gapB G169D -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-gapB is obtained. G169E Transformed into strain A358, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding gapB. G169E The point mutant strain was named A358-gapB G169E Hereinafter referred to as A365.

[0075] Example 5: Construction of fructose 1,6-bisphosphatase II mutant strain A358-glpX R175H

[0076] Using the genome of strain B. subtilis A1 as a template, primer glpX R175H -UP-1F / glpX R175H -UP-1R and glpX R175H -DN-2F / glpX R175H -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primers glpX were used. R175H -UP-1F / glpX R175H -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-glpX is obtained. R175HTransformed into strain A358, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding glpX. R175H The point mutant strain was named A358-glpX R175H Hereinafter referred to as A368.

[0077] Example 6: Construction of fructose 1,6-bisphosphatase II mutant strain A358-glpX R175P

[0078] Using the genome of strain B. subtilis A1 as a template, primer glpX R175H -UP-1F / glpX R175P -UP-1R and glpX R175P -DN-2F / glpX R175H -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primers glpX were used. R175H -UP-1F / glpX R175H -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-glpX is obtained. R175P Transformed into strain A358, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding glpX. R175P The point mutant strain was named A358-glpX R175P Hereinafter referred to as A369.

[0079] Example 7: Construction of fructose 1,6-bisphosphatase II mutant strain A365-glpX R175H

[0080] Using the genome of strain B. subtilis A1 as a template, primer glpX R175H-UP-1F / glpX R175H -UP-1R and glpX R175H -DN-2F / glpX R175H -DN-2R, two fragments were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primers glpX were used. R175H -UP-1F / glpX R175H -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-glpX is obtained. R175H Transformed into strain A365, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed transformants were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. 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, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed transformants were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding glpX. R175H The point mutant strain was named A365-glpX R175H Hereinafter referred to as A372.

[0081] Example 8: Construction of pyruvate carboxylase mutant strain 8333-pycA t1a

[0082] Using the genome of strain Bacillus amyloliquefaciens 8333 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. T1ATransformed into *Bacillus amyloliquefaciens* strain 8333, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding pycA. t1a The point mutant strain was named 8333-pycA t1a Hereinafter referred to as 8441.

[0083] Example 9: Construction of glyceraldehyde-3-phosphate dehydrogenase mutant strain 8441-gapB G169E

[0084] Using the genome of strain Bacillus amyloliquefaciens 8333 as a template, primer gapB G169D -UP-3F / gapB G169E -UP-3R and gapB G169E -DN-4F / gapB G169D Two fragments of -DN-4R were amplified using Phusion ultrafidelity polymerase (New England BioLabs). The amplification was performed using primer gapB. G169D -UP-3F / gapB G169D -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-gapB is obtained. G169E Transformed into strain 8441, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding gapB. G169E The point mutant strain was named 8441-gapB G169E Hereinafter referred to as 8453.

[0085] Example 10: Construction of fructose 1,6-bisphosphatase II mutant strain 8441-glpX R175H

[0086] Using the genome of strain Bacillus amyloliquefaciens 8333 as a template, primer glpX R175H -UP-3F / glpX R175H -UP-3R and glpX R175H -DN-4F / glpX R175H Two fragments of -DN-4R were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primers glpX were used. R175H -UP-3F / glpX R175H -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-glpX is obtained. R175H Transformed into strain 8441, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding glpX. R175H The point mutant strain was named 8441-glpX R175H Hereinafter referred to as 8461.

[0087] Example 11 Construction of fructose 1,6-bisphosphatase II mutant strain 8441-glpX R175P

[0088] Using the genome of strain Bacillus amyloliquefaciens 8333 as a template, primer glpX R175H -UP-3F / glpX R175P -UP-3R and glpX R175P -DN-4F / glpX R175H Two fragments of -DN-4R were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primers glpX were used. R175H -UP-3F / glpX R175H -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-glpX is obtained. R175PTransformed into strain 8441, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding glpX. R175P The point mutant strain was named 8441-glpX R175P Hereinafter referred to as 8463.

[0089] Example 12 Construction of fructose 1,6-bisphosphatase II mutant strain 8453-glpX R175H

[0090] Using the genome of strain Bacillus amyloliquefaciens 8333 as a template, primer glpX R175H -UP-3F / glpX R175H -UP-3R and glpX R175H -DN-4F / glpX R175H Two fragments of -DN-4R were amplified using Phusion ultrafidelity polymerase (New England BioLabs). Primers glpX were used. R175H -UP-3F / glpX R175H -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-glpX is obtained. R175H Transformed into strain 8453, transformants were screened on LB agar plates containing 2.5 μg / mL chloramphenicol at 30°C. The transformed strains were inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. These primary recombinants were then inoculated into 5 mL LB liquid medium, incubated at 42°C and 200 rpm for 12 h, and passaged. The transformed strains were then diluted and plated onto LB agar plates containing 0.8 μM 5-FU to screen for secondary recombinants, yielding glpX. R175H The point mutant strain was named 8453-glpX R175H Hereinafter referred to as 8471.

[0091] Example 13: Verification of Glycoside Production Performance of Bacillus subtilis Engineered Strains

[0092] 1. Culture medium:

[0093] (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 20min.

[0094] (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 20min.

[0095] 2. Cultivation Methods

[0096] (1) Streak the strain in three zones on LB plates and incubate overnight at 37°C.

[0097] (2) Pick a single colony and inoculate it into 30 mL of seed culture medium. Incubate at 110 rpm and 36 °C for 7–8 h.

[0098] (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.

[0099] 3. Detection and Results

[0100] The nucleosides in the fermentation broth were detected using high performance liquid chromatography (HPLC), and the results are shown in Table 2.

[0101] Table 2. Results of shake-flask fermentation production capacity assessment (mean of three replicates)

[0102] A1 1.3 0.6 A325 2.1* 1.0* A342 3.4* 1.5* A358 4.4* 2.2* A365 5.5* 2.6* A368 5.6* 2.6* A369 5.2* 2.5* A372 6.8* 2.9*

[0103] Note: * indicates a significant difference compared to the starting strain (P < 0.01).

[0104] The above experimental results show that the fructose 1,6-bisphosphatase II mutant has a positive effect on increasing the production of adenosine and inosine. The mutant glpX... R175H The optimal adenosine yield was increased from 4.4 g / L to 5.6 g / L in engineered strain A368. The superposition of fructose-1,6-bisphosphatase II mutant and glyceraldehyde-3-phosphate dehydrogenase mutant had a positive effect on increasing adenosine and inosine yields. The mutant glpX... R175H superposition mutant gapB G169E The adenosine yield of engineered strain A372 increased from 4.4 g / L to 6.8 g / L, and the inosine yield increased from 2.2 g / L to 2.9 g / L.

[0105] Example 14 Verification of glycoside production performance of engineered strains of Bacillus amyloliquefaciens

[0106] 1. Culture medium:

[0107] (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 20min.

[0108] (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 20min.

[0109] 2. Cultivation Methods

[0110] (1) Streak the glycerol-preserved bacterial strain on LB agar plates and culture at 37°C overnight to obtain single clones.

[0111] (2) Pick a single colony and inoculate it into 30 mL of seed culture medium. Incubate at 110 rpm and 37 °C for 7–8 h.

[0112] (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.

[0113] 3. Detection and Results

[0114] The nucleosides in the fermentation broth were detected using high performance liquid chromatography (HPLC), and the results are shown in Table 3.

[0115] Table 3. Evaluation results of guanosine and inosine production during shake-flask fermentation (mean of three replicates)

[0116]

[0117]

[0118] Note: * indicates a significant difference compared to the starting strain (P < 0.01).

[0119] The above experimental results show that the fructose 1,6-bisphosphatase II mutant has a positive effect on increasing the production of guanosine and inosine. The mutant glpX... R175H The engineered strain 8461 showed an optimal increase in guanosine production from 17.1 g / L to 18.9 g / L and inosine production from 1.5 g / L to 1.9 g / L. The superposition of the fructose-1,6-bisphosphatase II mutant and the glyceraldehyde-3-phosphate dehydrogenase mutant had a positive effect on increasing guanosine and inosine production. The mutant glpX... R175H superposition mutant gapB G169EThe yield of guanosine from engineered strain 8471 increased from 17.1 g / L to 20.6 g / L, and the yield of inosine increased from 1.5 g / L to 2.0 g / L.

[0120] Therefore, the fructose 1,6-bisphosphatase II mutant and / or glyceraldehyde-3-phosphate dehydrogenase mutant, and their mutant strains provided by this invention, have a significant promoting effect on the production of target products adenosine or guanosine, and inosine. These fructose 1,6-bisphosphatase II mutants and / or glyceraldehyde-3-phosphate dehydrogenase mutants, and their recombinant microorganisms, provide a reference for the construction of production strains that produce adenosine or guanosine, inosine, and their precursor derivatives.

[0121] The construction of the strain of the present invention is not limited in the order of steps. Anyone skilled in the art who achieves the purpose of the present invention by following the disclosure of the present invention is within the protection scope of the present invention.

[0122] The strain designation in this invention is, for example, A1-pyk. T101K A325-pycA t1a A342-pckA K147R A358-gapB G169D A358-gapB G169E The terms "pyk" are used for ease of description and should not be construed as limiting the invention. The method described above constructs a pyk gene containing Bacillus subtilis pyruvate kinase mutant pyk. T101K pyruvate carboxylase mutant pycA T1A Engineered bacteria, phosphoenolpyruvate carboxykinase mutant pckA K147R Engineered bacteria, and glyceraldehyde-3-phosphate dehydrogenase mutant gapB G169D gapB G169E Engineered bacteria and those containing Bacillus amyloliquefaciens glyceraldehyde-3-phosphate dehydrogenase mutant GAPB G169D gapB G169E The uses of engineered bacteria include, but are not limited to, adenosine or guanosine, and inosine.

[0123] 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> Fructose-1,6-bisphosphatase II mutant and its application <130> KHP221113207.9 <160> 15 <170> SIPOSequenceListing 1.0 <210> 1 <211> 321 <212> PRT <213> Bacillus subtilis <400> 1 Met Glu Arg Ser Leu Ser Met Glu Leu Val Arg Val Thr Glu Ala Ala 1 5 10 15 Ala Leu Ala Ser Ala Arg Trp Met Gly Arg Gly Lys Lys Asp Glu Ala 20 25 30 Asp Glu Ala Ala Thr Ser Ala Met Arg Asp Val Phe Asp Thr Val Pro 35 40 45 Met Lys Gly Thr Val Val Ile Gly Glu Gly Glu Met Asp Glu Ala Pro 50 55 60 Met Leu Tyr Ile Gly Glu Lys Leu Gly Asn Gly Tyr Gly Pro Arg Val 65 70 75 80 Asp Val Ala Val Asp Pro Leu Glu Gly Thr Asn Ile Leu Ala Ser Gly 85 90 95 Gly Trp Asn Ala Leu Thr Val Ile Ala Val Ala Asp His Gly Thr Leu 100 105 110 Leu Asn Ala Pro Asp Met Tyr Met Gln Lys Ile Ala Val Gly Pro Glu 115 120 125 Ala Val Gly Cys Ile Asp Ile Glu Ala Pro Val Ile Asp Asn Leu Lys 130 135 140 Ala Val Ala Lys Ala Lys Asn Lys Asp Val Glu Asp Val Val Ala Thr 145 150 155 160 Ile Leu Asn Arg Glu Arg His Ala Lys Ile Ile Ser Glu Leu Arg Glu 165 170 175 Ala Gly Ala Arg Ile Lys Leu Ile Asn Asp Gly Asp Val Ala Gly Ala 180 185 190 Ile Asn Thr Ala Phe Asp His Thr Gly Val Asp Ile Leu Phe Gly Ser 195 200 205 Gly Gly Ala Pro Glu Gly Val Leu Ser Ala Val Ala Leu Lys Ala Leu 210 215 220 Gly Gly Glu Ile Ile Gly Lys Leu Leu Pro Gln Ser Glu Glu Glu Ile 225 230 235 240 Thr Arg Cys His Lys Met Gly Leu Asp Leu Ser Lys Val Leu Arg Met 245 250 255 Glu Asp Leu Val Lys Gly Asp Asp Ala Ile Phe Ala Ala Thr Gly Val 260 265 270 Thr Asp Gly Glu Leu Leu Lys Gly Val Gln Phe Lys Gly Ser Val Gly 275 280 285 Thr Thr Glu Ser Leu Val Ile Arg Ala Lys Ser Gly Thr Val Arg Phe 290 295 300 Val Asp Gly Arg His Ser Leu Lys Lys Lys Pro Asn Leu Val Ile Arg 305 310 315 320 Pro <210> 2 <211> 321 <212> PRT <213> Artificial Sequence <400> 2 Met Glu Arg Ser Leu Ser Met Glu Leu Val Arg Val Thr Glu Ala Ala 1 5 10 15 Ala Leu Ala Ser Ala Arg Trp Met Gly Arg Gly Lys Lys Asp Glu Ala 20 25 30 Asp Glu Ala Ala Thr Ser Ala Met Arg Asp Val Phe Asp Thr Val Pro 35 40 45 Met Lys Gly Thr Val Val Ile Gly Glu Gly Glu Met Asp Glu Ala Pro 50 55 60 Met Leu Tyr Ile Gly Glu Lys Leu Gly Asn Gly Tyr Gly Pro Arg Val 65 70 75 80 Asp Val Ala Val Asp Pro Leu Glu Gly Thr Asn Ile Leu Ala Ser Gly 85 90 95 Gly Trp Asn Ala Leu Thr Val Ile Ala Val Ala Asp His Gly Thr Leu 100 105 110 Leu Asn Ala Pro Asp Met Tyr Met Gln Lys Ile Ala Val Gly Pro Glu 115 120 125 Ala Val Gly Cys Ile Asp Ile Glu Ala Pro Val Ile Asp Asn Leu Lys 130 135 140 Ala Val Ala Lys Ala Lys Asn Lys Asp Val Glu Asp Val Val Ala Thr 145 150 155 160 Ile Leu Asn Arg Glu Arg His Ala Lys Ile Ile Ser Glu Leu His Glu 165 170 175 Ala Gly Ala Arg Ile Lys Leu Ile Asn Asp Gly Asp Val Ala Gly Ala 180 185 190 Ile Asn Thr Ala Phe Asp His Thr Gly Val Asp Ile Leu Phe Gly Ser 195 200 205 Gly Gly Ala Pro Glu Gly Val Leu Ser Ala Val Ala Leu Lys Ala Leu 210 215 220 Gly Gly Glu Ile Ile Gly Lys Leu Leu Pro Gln Ser Glu Glu Glu Ile 225 230 235 240 Thr Arg Cys His Lys Met Gly Leu Asp Leu Ser Lys Val Leu Arg Met 245 250 255 Glu Asp Leu Val Lys Gly Asp Asp Ala Ile Phe Ala Ala Thr Gly Val 260 265 270 Thr Asp Gly Glu Leu Leu Lys Gly Val Gln Phe Lys Gly Ser Val Gly 275 280 285 Thr Thr Glu Ser Leu Val Ile Arg Ala Lys Ser Gly Thr Val Arg Phe 290 295 300 Val Asp Gly Arg His Ser Leu Lys Lys Lys Pro Asn Leu Val Ile Arg 305 310 315 320 Pro <210> 3 <211> 321 <212> PRT <213> Artificial Sequence <400> 3 Met Glu Arg Ser Leu Ser Met Glu Leu Val Arg Val Thr Glu Ala Ala 1 5 10 15 Ala Leu Ala Ser Ala Arg Trp Met Gly Arg Gly Lys Lys Asp Glu Ala 20 25 30 Asp Glu Ala Ala Thr Ser Ala Met Arg Asp Val Phe Asp Thr Val Pro 35 40 45 Met Lys Gly Thr Val Val Ile Gly Glu Gly Glu Met Asp Glu Ala Pro 50 55 60 Met Leu Tyr Ile Gly Glu Lys Leu Gly Asn Gly Tyr Gly Pro Arg Val 65 70 75 80 Asp Val Ala Val Asp Pro Leu Glu Gly Thr Asn Ile Leu Ala Ser Gly 85 90 95 Gly Trp Asn Ala Leu Thr Val Ile Ala Val Ala Asp His Gly Thr Leu 100 105 110 Leu Asn Ala Pro Asp Met Tyr Met Gln Lys Ile Ala Val Gly Pro Glu 115 120 125 Ala Val Gly Cys Ile Asp Ile Glu Ala Pro Val Ile Asp Asn Leu Lys 130 135 140 Ala Val Ala Lys Ala Lys Asn Lys Asp Val Glu Asp Val Val Ala Thr 145 150 155 160 Ile Leu Asn Arg Glu Arg His Ala Lys Ile Ile Ser Glu Leu Pro Glu 165 170 175 Ala Gly Ala Arg Ile Lys Leu Ile Asn Asp Gly Asp Val Ala Gly Ala 180 185 190 Ile Asn Thr Ala Phe Asp His Thr Gly Val Asp Ile Leu Phe Gly Ser 195 200 205 Gly Gly Ala Pro Glu Gly Val Leu Ser Ala Val Ala Leu Lys Ala Leu 210 215 220 Gly Gly Glu Ile Ile Gly Lys Leu Leu Pro Gln Ser Glu Glu Glu Ile 225 230 235 240 Thr Arg Cys His Lys Met Gly Leu Asp Leu Ser Lys Val Leu Arg Met 245 250 255 Glu Asp Leu Val Lys Gly Asp Asp Ala Ile Phe Ala Ala Thr Gly Val 260 265 270 Thr Asp Gly Glu Leu Leu Lys Gly Val Gln Phe Lys Gly Ser Val Gly 275 280 285 Thr Thr Glu Ser Leu Val Ile Arg Ala Lys Ser Gly Thr Val Arg Phe 290 295 300 Val Asp Gly Arg His Ser Leu Lys Lys Lys Pro Asn Leu Val Ile Arg 305 310 315 320 Pro <210> 4 <211> 321 <212> PRT <213> Bacillus amyloliquefaciens <400> 4 Met Glu Arg Ser Leu Ser Met Glu Leu Val Arg Val Thr Glu Ala Ala 1 5 10 15 Ala Leu Ala Ser Ala Arg Trp Met Gly Arg Gly Lys Lys Asp Glu Ala 20 25 30 Asp Asp Ala Ala Thr Ser Ala Met Arg Asp Val Phe Asp Thr Val Pro 35 40 45 Met Lys Gly Thr Val Val Ile Gly Glu Gly Glu Met Asp Glu Ala Pro 50 55 60 Met Leu Tyr Ile Gly Glu Lys Leu Gly Asn Gly Tyr Gly Pro Arg Val 65 70 75 80 Asp Val Ala Val Asp Pro Leu Glu Gly Thr Asn Ile Val Ala Ser Gly 85 90 95 Gly Trp Asn Ala Leu Thr Val Ile Ala Val Ala Asp His Gly Thr Leu 100 105 110 Leu Asn Ala Pro Asp Met Tyr Met Glu Lys Ile Ala Val Gly Pro Glu 115 120 125 Ala Val Gly Tyr Ile Asp Ile Glu Ala Pro Val Ile Asp Asn Leu Lys 130 135 140 Ala Val Ala Lys Ala Lys Asn Lys Asp Ile Glu Asp Val Val Ala Thr 145 150 155 160 Ile Leu Asn Arg Asp Arg His Glu Arg Ile Ile Ser Glu Leu Arg Glu 165 170 175 Ala Gly Ala Arg Ile Lys Leu Ile Asn Asp Gly Asp Val Ala Gly Ala 180 185 190 Ile Asn Thr Ala Phe Asp His Thr Gly Val Asp Ile Leu Phe Gly Ser 195 200 205 Gly Gly Ala Pro Glu Gly Val Leu Ser Ala Val Ala Leu Lys Ala Leu 210 215 220 Gly Gly Glu Ile Ile Gly Arg Leu Arg Pro Gln Ser Ala Glu Glu Gln 225 230 235 240 Ala Arg Cys Glu Arg Met Gly Leu Asp Val Ser Lys Val Leu Arg Leu 245 250 255 Glu Asp Leu Val Arg Gly Asp Asp Ala Ile Phe Ala Ala Thr Gly Val 260 265 270 Thr Asp Gly Glu Leu Leu Lys Gly Val Gln Phe Lys Gly Ser Val Gly 275 280 285 Thr Thr Glu Ser Leu Val Ile Arg Ala Lys Ser Gly Thr Val Arg Phe 290 295 300 Val Asp Gly Arg His Ser Leu Lys Lys Lys Pro Asn Leu Val Ile Arg 305 310 315 320 Pro <210> 5 <211> 321 <212> PRT <213> Artificial Sequence <400> 5 Met Glu Arg Ser Leu Ser Met Glu Leu Val Arg Val Thr Glu Ala Ala 1 5 10 15 Ala Leu Ala Ser Ala Arg Trp Met Gly Arg Gly Lys Lys Asp Glu Ala 20 25 30 Asp Asp Ala Ala Thr Ser Ala Met Arg Asp Val Phe Asp Thr Val Pro 35 40 45 Met Lys Gly Thr Val Val Ile Gly Glu Gly Glu Met Asp Glu Ala Pro 50 55 60 Met Leu Tyr Ile Gly Glu Lys Leu Gly Asn Gly Tyr Gly Pro Arg Val 65 70 75 80 Asp Val Ala Val Asp Pro Leu Glu Gly Thr Asn Ile Val Ala Ser Gly 85 90 95 Gly Trp Asn Ala Leu Thr Val Ile Ala Val Ala Asp His Gly Thr Leu 100 105 110 Leu Asn Ala Pro Asp Met Tyr Met Glu Lys Ile Ala Val Gly Pro Glu 115 120 125 Ala Val Gly Tyr Ile Asp Ile Glu Ala Pro Val Ile Asp Asn Leu Lys 130 135 140 Ala Val Ala Lys Ala Lys Asn Lys Asp Ile Glu Asp Val Val Ala Thr 145 150 155 160 Ile Leu Asn Arg Asp Arg His Glu Arg Ile Ile Ser Glu Leu His Glu 165 170 175 Ala Gly Ala Arg Ile Lys Leu Ile Asn Asp Gly Asp Val Ala Gly Ala 180 185 190 Ile Asn Thr Ala Phe Asp His Thr Gly Val Asp Ile Leu Phe Gly Ser 195 200 205 Gly Gly Ala Pro Glu Gly Val Leu Ser Ala Val Ala Leu Lys Ala Leu 210 215 220 Gly Gly Glu Ile Ile Gly Arg Leu Arg Pro Gln Ser Ala Glu Glu Gln 225 230 235 240 Ala Arg Cys Glu Arg Met Gly Leu Asp Val Ser Lys Val Leu Arg Leu 245 250 255 Glu Asp Leu Val Arg Gly Asp Asp Ala Ile Phe Ala Ala Thr Gly Val 260 265 270 Thr Asp Gly Glu Leu Leu Lys Gly Val Gln Phe Lys Gly Ser Val Gly 275 280 285 Thr Thr Glu Ser Leu Val Ile Arg Ala Lys Ser Gly Thr Val Arg Phe 290 295 300 Val Asp Gly Arg His Ser Leu Lys Lys Lys Pro Asn Leu Val Ile Arg 305 310 315 320 Pro <210> 6 <211> 321 <212> PRT <213> Artificial Sequence <400> 6 Met Glu Arg Ser Leu Ser Met Glu Leu Val Arg Val Thr Glu Ala Ala 1 5 10 15 Ala Leu Ala Ser Ala Arg Trp Met Gly Arg Gly Lys Lys Asp Glu Ala 20 25 30 Asp Asp Ala Ala Thr Ser Ala Met Arg Asp Val Phe Asp Thr Val Pro 35 40 45 Met Lys Gly Thr Val Val Ile Gly Glu Gly Glu Met Asp Glu Ala Pro 50 55 60 Met Leu Tyr Ile Gly Glu Lys Leu Gly Asn Gly Tyr Gly Pro Arg Val 65 70 75 80 Asp Val Ala Val Asp Pro Leu Glu Gly Thr Asn Ile Val Ala Ser Gly 85 90 95 Gly Trp Asn Ala Leu Thr Val Ile Ala Val Ala Asp His Gly Thr Leu 100 105 110 Leu Asn Ala Pro Asp Met Tyr Met Glu Lys Ile Ala Val Gly Pro Glu 115 120 125 Ala Val Gly Tyr Ile Asp Ile Glu Ala Pro Val Ile Asp Asn Leu Lys 130 135 140 Ala Val Ala Lys Ala Lys Asn Lys Asp Ile Glu Asp Val Val Ala Thr 145 150 155 160 Ile Leu Asn Arg Asp Arg His Glu Arg Ile Ile Ser Glu Leu Pro Glu 165 170 175 Ala Gly Ala Arg Ile Lys Leu Ile Asn Asp Gly Asp Val Ala Gly Ala 180 185 190 Ile Asn Thr Ala Phe Asp His Thr Gly Val Asp Ile Leu Phe Gly Ser 195 200 205 Gly Gly Ala Pro Glu Gly Val Leu Ser Ala Val Ala Leu Lys Ala Leu 210 215 220 Gly Gly Glu Ile Ile Gly Arg Leu Arg Pro Gln Ser Ala Glu Glu Gln 225 230 235 240 Ala Arg Cys Glu Arg Met Gly Leu Asp Val Ser Lys Val Leu Arg Leu 245 250 255 Glu Asp Leu Val Arg Gly Asp Asp Ala Ile Phe Ala Ala Thr Gly Val 260 265 270 Thr Asp Gly Glu Leu Leu Lys Gly Val Gln Phe Lys Gly Ser Val Gly 275 280 285 Thr Thr Glu Ser Leu Val Ile Arg Ala Lys Ser Gly Thr Val Arg Phe 290 295 300 Val Asp Gly Arg His Ser Leu Lys Lys Lys Pro Asn Leu Val Ile Arg 305 310 315 320 Pro <210> 7 <211> 3447 <212> DNA <213> Bacillus subtilis <400> 7 ttgtctcagc 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 gaagaagaag 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 gaagttaaag aagcatatga gcgtgctaaa tcagaggcga aagcagcctt tggcaatgat 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 ttcgacagc ttaaactggg taaaagggat gacgttagcc 1980 attgatgctg ttagggatac cggcaaagtg gcagaagctg cgatttgtta tacggggagat 2040 atccttgaca agaaccggac gaagtacgac cttgcatatt atacatcgat ggcgaaggag 2100 cttgaggcgg ccggagccca tattctcggg attaaagata tggcagggct gttaaaaccg 2160 caggctgcat atgagctcgt ttctgcgttg aaagaaacga tcgacattc 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 tttgtgaaa 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> 8 <211> 3447 <212> DNA <213> Bacillus amyloliquefaciens <400> 8 ttgtcacaac aatccatcca aaaagtgtta gtagcaaaca ggggagaaat tgcgatccgg 60 atttccggg 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 cacacgcatg atacgagcgg caacggcgtc tttctctatg ccaaagccgt tgaagcgggt 2280 gtggatatcg tagatgtcgc cgtcagctca atggcgggtc tgacttcaca gccgagtgca 2340 agcggatttt atcatgcgct tgaaggaaac 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> 9 <211> 1467 DNA Artificial Sequence <400> 9 atgtgggaaa gtaaattttc aaaagaaggc ttaacgttcg atgatgtact gctcgtacca 60 gctcaatcag acgtacttcc gcgtgatgtg gatttgtctg ttgaactgac aaaaacgtta 120 aagcttaata ttcctgtcat cagtgcagga atggatacag taacagaatc agcaatggcg 180 attgcgatgg cccgacaagg cggcttgggc attattcata aaaacatgtc catcgaacag 240 caggctgaac atgttgacaa agtcaaacgt tctgaacggg gcgttattac aaatcccttc 300 tttttaacac ctgatcatca agtattcgat gcggagcatt tgatggggaa atacagaatt 360 tccggtgttc cgatcgtaga taataaagac gatcaaaagc tggtcggtat cattacaaac 420 cgcgatcttc gctttatctc tgattattca atgaaaatca gtgatgttat gacaaaagaa 480 gagctggtta cggctcctgt gggaaccaca ttagacgaag cggaaaaaat cttgcagaag 540 cataaaattg aaaaacttcc attagtggat gaccaaaaca 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 ggaaaaggc 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> 10 <211> 931 <212> DNA <213> Artificial Sequence <400> 10 acagaatgct cttgattaaa tccgtatgtt aagttatatt ttttttaatt tttcggattt 60 tgggggtaag ttcatgaagt ttcgtcgcag cggcagattg gtggacttaa caaattattt 120 gttaacccat ccgcatgaat taataccgtt aacgtttttc tcagaacggt atcaatcagc 180 aaagtcatca atcagtgaag atttaacaat tattaaacag accttcgaac agcaaggcat 240 tgggacgctg cttacagtgc cgggagctgc cggaggcgtc aaatatattc cgaaagtaaa 300 acaggctgaa gcagaagcgt ttatacagga gctgggacag tctttagtaa atcctgagcg 360 tatccttccg ggcggttatg tatatttaac ggatatctta ggcaaacctt ctgtcctctc 420 taatgcaggc aggctttttg cttccgtttt tgcggagcgg gagattgatg tggtgatgac 480 cgttgcgaca aaaggaatcc ctcttgctta cgcagcggcc agttatctga acgttccggt 540 tgtcatcgta cgaaaagaca ataaagtgac ggaaggctct acagtgagca tcaattatgt 600 atcagggtcg tctaaccgca ttcaaacaat gtcgcttgcg aaaagaagtt tggccacggg 660 gtcgaacgtt ttgattattg acgactttat gaaagccggc ggcacaatta acggcatgat 720 cagcctgctt gatgagttta atgcgaacgt cgcgggtata ggcgtcttgg ttgaagctga 780 gggagtgaat gaacggcttg tcgatgaata catgtcgctg cttacccttt caaccatcaa 840 catgaaagac aaaacgattg agattcaaaa cggcaatttt ctgcgatttt ttaaagaaca 900 gcatttaaag aatggggaga cagaaaaatg a 931 <210> 11 <211> 839 <212> DNA <213> Artificial Sequence <400> 11 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 ctgtttttt ataatgtcct tgtattcttt ctcttcgaa 839 <210> 12 <211> 585 <212> PRT <213> Bacillus subtilis <400> 12 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> 13 <211> 527 <212> PRT <213> Bacillus <400> 13 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> 14 <211> 340 <212> PRT <213> Bacillus subtilis <400> 14 Met Lys Val Lys Val Ala Ile Asn Gly Phe Gly Arg Ile Gly Arg Met 1 5 10 15 Val Phe Arg Lys Ala Met Leu Asp Asp Gln Ile Gln Val Val Ala Ile 20 25 30 Asn Ala Ser Tyr Ser Ala Glu Thr Leu Ala His Leu Ile Lys Tyr Asp 35 40 45 Thr Ile His Gly Arg Tyr Asp Lys Glu Val Val Ala Gly Glu Asp Ser 50 55 60 Leu Ile Val Asn Gly Lys Lys Val Leu Leu Leu Asn Ser Arg Asp Pro 65 70 75 80 Lys Gln Leu Pro Trp Arg Glu Tyr Asp Ile Asp Ile Val Val Glu Ala 85 90 95 Thr Gly Lys Phe Asn Ala Lys Asp Lys Ala Met Gly His Ile Glu Ala 100 105 110 Gly Ala Lys Lys Val Ile Leu Thr Ala Pro Gly Lys Asn Glu Asp Val 115 120 125 Thr Ile Val Met Gly Val Asn Glu Asp Gln Phe Asp Ala Glu Arg His 130 135 140 Val Ile Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Val 145 150 155 160 Val Lys Val Leu Asp Glu Glu Phe Gly Ile Glu Ser Gly Leu Met Thr 165 170 175 Thr Val His Ala Tyr Thr Asn Asp Gln Lys Asn Ile Asp Asn Pro His 180 185 190 Lys Asp Leu Arg Arg Ala Arg Ala Cys Gly Glu Ser Ile Ile Pro Thr 195 200 205 Thr Thr Gly Ala Ala Lys Ala Leu Ser Leu Val Leu Pro His Leu Lys 210 215 220 Gly Lys Leu His Gly Leu Ala Leu Arg Val Pro Val Pro Asn Val Ser 225 230 235 240 Leu Val Asp Leu Val Val Asp Leu Lys Thr Asp Val Thr Ala Glu Glu 245 250 255 Val Asn Glu Ala Phe Lys Arg Ala Ala Lys Thr Ser Met Tyr Gly Val 260 265 270 Leu Asp Tyr Ser Asp Glu Pro Leu Val Ser Thr Asp Tyr Asn Thr Asn 275 280 285 Pro His Ser Ala Val Ile Asp Gly Leu Thr Thr Met Val Met Glu Asp 290,295,300 Arg Lys Val Lys Val Leu Ala Trp Tyr Asp Asn Glu Trp Gly Tyr Ser 305 310 315 320 Cys Arg Val Val Asp Leu Ile Arg His Val Ala Ala Arg Met Lys His 325 330 335 Pro Being Wing Val 340 <210> 15 <211> 340 <212> PRT <213> Bacillus amyloliquefaciens <400> 15 Met Lys Val Lys Val Ala Ile Asn Gly Phe Gly Arg Ile Gly Arg Met 1 5 10 15 Val Phe Arg Lys Ala Met Glu Asp Asp Gln Ile Gln Ile Thr Ala Ile 20 25 30 Asn Ala Ser Tyr Pro Glu Thr Leu Ala His Leu Ile Lys Tyr Asp 35 40 45 Thr Ile His Gly Arg Tyr Asp Gln Glu Val Glu Ala Ala Glu Asp Ser 50 55 60 Leu Ile Val Asn Gly Lys His Ile Met Leu Phe Asn Arg Arg Asp Pro 65 70 75 80 Arg Glu Leu Pro Trp Lys Glu Cys Gly Ile Asp Ile Val Val Glu Ala 85 90 95 Thr Gly Lys Phe Asn Ser Lys Glu Lys Ala Met Ser His Ile Glu Ala 100 105 110 Gly Ala Lys Lys Val Ile Leu Thr Ala Pro Gly Lys Asn Glu Asp Val 115 120 125 Thr Ile Val Met Gly Val Asn Glu Glu Gln Phe Asn Pro Asp Glu His 130 135 140 Val Ile Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Val 145 150 155 160 Val Lys Val Leu Asp Gln Glu Phe Gly Ile Glu Ser Gly Leu Met Thr 165 170 175 Thr Val His Ala Tyr Thr Asn Asp Gln Lys Asn Ile Asp Asn Pro His 180 185 190 Lys Asp Leu Arg Arg Ala Arg Ala Cys Gly Glu Ser Ile Ile Pro Thr 195 200 205 Ser Thr Gly Ala Ala Lys Ala Leu Ser Leu Val Leu Pro His Leu Lys 210 215 220 Gly Lys Leu His Gly Leu Ala Leu Arg Val Pro Val Pro Asn Val Ser 225 230 235 240 Leu Val Asp Leu Val Cys Asp Leu Lys Thr Asp Val Ser Ala Glu Gln 245 250 255 Val Asn Ala Ala Phe Gln Arg Ala Ala Lys Thr Ser Met Tyr Gly Ile 260 265 270 Leu Asp Tyr Ser Asp Glu Pro Leu Val Ser Ser Asp Tyr Asn Thr Asn 275 280 285 Ser His Ser Ala Ile Ile Asp Gly Leu Thr Thr Met Val Met Glu Asp 290 295 300 Arg Lys Val Lys Val Leu Ala Trp Tyr Asp Asn Glu Trp Gly Tyr Ser 305 310 315 320 Cys Arg Val Val Asp Leu Ile Arg His Val Ala Ala Arg Met Lys His 325 330 335 Pro Ser Ala Val 340

Claims

1. Engineered bacteria containing nucleic acid molecules encoding a fructose-1,6-bisphosphatase II mutant; The amino acid sequence of the fructose 1,6-bisphosphatase II mutant is shown in SEQ ID NO:2 or 3, and the starting strain of the engineered bacteria is Bacillus subtilis. Bacillus subtilis A1; or, The amino acid sequence of the fructose 1,6-bisphosphatase II mutant is shown in SEQ ID NO:5 or 6, and the starting strain of the engineered bacteria is Bacillus amyloliquefaciens. Bacillus amyloliquefaciens 8333.

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; When the starting strain of the engineered bacteria is Bacillus subtilis Bacillus subtilis In A1, the nucleosides are adenosine and inosine; When the starting strain of the engineered bacteria is Bacillus amyloliquefaciens Bacillus amyloliquefaciens At 8333, the nucleosides are guanosine and inosine.

3. A method for constructing a nucleoside-producing strain, characterized in that, Using genetic engineering techniques, mutations were introduced into the genomes of microorganisms capable of nucleoside production, causing their encoded fructose 1,6-bisphosphatase II to contain R175H or R175P mutation sites. The amino acid sequence of fructose 1,6-bisphosphatase II containing the R175H or R175P mutation site is shown in SEQ ID NO:2 or 3, and the originating strain of the engineered bacteria is Bacillus subtilis. Bacillus subtilis A1; or, The amino acid sequence of fructose 1,6-bisphosphatase II containing the R175H or R175P mutation site is shown in SEQ ID NO:5 or 6, and the starting strain of the engineered bacteria is Bacillus amyloliquefaciens. Bacillus amyloliquefaciens 8333.

4. A method for constructing nucleoside-producing engineered bacteria, characterized in that, Using the nucleoside-producing strain obtained by the method described in claim 3 as the starting strain, the starting strain is genetically engineered using at least one of the following methods ① to ④: ① Using genetic engineering techniques, a mutation was introduced into the starting strain so that the amino acid sequence it encodes, as shown in SEQ ID NO:12, contains the T101K mutation site in pyruvate kinase; the starting strain is Bacillus subtilis. Bacillus subtilis A1; ② Using genetic engineering techniques, a mutation is introduced into the starting strain to encode pyruvate carboxylase. pycA The first base of the gene is mutated from T to A; pycA The nucleotide sequence of the gene is shown in SEQ ID NO:7; the originating strain is Bacillus subtilis. Bacillus subtilis A1; or, The pycA The nucleotide sequence of the gene is shown in SEQ ID NO:8; the originating strain is Bacillus amyloliquefaciens. Bacillus amyloliquefaciens 8333; ③ Using genetic engineering techniques, a mutation was introduced into the starting strain so that the encoded amino acid sequence, as shown in SEQ ID NO:13, contains the K147R mutation site in phosphoenolpyruvate carboxykinase; the starting strain is Bacillus subtilis. Bacillus subtilis A1; ④ Using genetic engineering techniques, a mutation was introduced into the starting strain so that the amino acid sequence it encodes, as shown in SEQ ID NO:14, contains the G169E mutation site in glyceraldehyde-3-phosphate dehydrogenase; the starting strain is Bacillus subtilis. Bacillus subtilis A1; or, Using genetic engineering techniques, a mutation was introduced into the starting strain so that the encoded amino acid sequence, as shown in SEQ ID NO:15, contains the G169E mutation site in glyceraldehyde-3-phosphate dehydrogenase; the starting strain is *Bacillus amyloliquefaciens*. Bacillus amyloliquefaciens 8333.

5. The nucleoside-producing strain or engineered strain constructed according to the method of claim 4.

6. A method for producing nucleosides, characterized in that, The method includes the following steps: 1) Cultivate the strain or engineered bacteria described in claim 5 to obtain a culture of the microorganism; 2) Collect the nucleosides produced from the culture obtained in step 1); When the starting strain of the engineered bacteria is Bacillus subtilis Bacillus subtilis In A1, the nucleosides are adenosine and inosine; When the starting strain of the engineered bacteria is Bacillus amyloliquefaciens Bacillus amyloliquefaciens At 8333, the nucleosides are guanosine and inosine.

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