Nucleoside-producing genetically engineered bacteria and their construction method and application

Abstract

The present invention provides a genetically engineered bacterium that produces nucleosides, as well as a method for constructing and using the same. By modifying the guanylate dehydrogenase (encoded by the guaB gene) of Bacillus subtilis or Bacillus amyloliquefaciens to enhance its activity and / or weakening or inactivating the hypothetical protein YeaC (encoded by the yeaC gene), the present invention enables the microorganism to efficiently and rapidly produce guanosine or inosine nucleotides. The invention successfully constructs a genetically engineered bacterium capable of efficiently producing nucleosides, such as inosine and guanosine, or their corresponding nucleoside derivatives. The present invention provides a powerful tool for the large-scale industrial production of nucleosides.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a nucleoside-producing genetic engineering bacterium and a construction method and application thereof. Background Art

[0002] Nucleoside is a general term for a class of glycosides. Nucleosides are components of nucleic acids and nucleotides. Nucleosides are formed by the condensation of D-ribose or D-α-deoxyribose with a pyrimidine or purine base. Nucleosides are generally colorless crystals, insoluble in common organic solvents but readily soluble in hot water, with 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 uridine ribonucleosides, which are abbreviated as adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U), respectively.

[0003] Guanosine (guanosine) and inosine (inosine) play a wide range of roles in the food and pharmaceutical industries. In the food industry, guanosine and inosine are important precursors of disodium guanylate and disodium inosinate, respectively. Disodium guanylate and disodium inosinate are used together as food flavor enhancers, widely used in condiments such as chicken bouillon and soy sauce. In the pharmaceutical field, guanosine and inosine can be used as pharmaceutical intermediates for various antiviral drugs, such as acyclovir, triazole ribavirin, and sodium guanosine triphosphate, all of which require guanosine as a synthetic raw material. Inosine is an important precursor of inosinic acid, which can be used as a precursor for the synthesis of adenosine monophosphate (AMP) and guanylate monophosphate (GMP). It is suitable for treating leukopenia and thrombocytopenia caused by various causes, various heart diseases, acute and chronic hepatitis, cirrhosis, etc., and can also treat central retinitis and optic atrophy.

[0004] Currently, microbial fermentation is the primary method for producing nucleosides, and the main microorganisms used include Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus pumilus. During the selection and modification of growing strains, high-nucleoside-producing strains are selected through the use of ultraviolet mutagenesis and diethyl sulfate mutagenesis breeding. Alternatively, based on the metabolic pathways and regulatory mechanisms of nucleotides in bacteria, the genetic background and characteristics of the strains are deeply understood, and through metabolic engineering methods, the strains are purposefully modified to obtain production strains with excellent traits and the ability to produce high nucleosides. However, the fermentation performance of current nucleoside strains is still 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 the present invention is to provide a genetically engineered bacterium for producing nucleosides and a construction method and application thereof.

[0006] The present invention is conceived as follows: by modifying the guanylate dehydrogenase (encoded by the guaB gene) of Bacillus subtilis or Bacillus amyloliquefaciens to enhance its activity, and / or weakening or inactivating the hypothetical protein YeaC (encoded by the yeaC gene), the microorganism can efficiently and rapidly produce guanosine or inosine nucleotides, and successfully construct a genetically engineered bacterium capable of efficiently producing nucleosides.

[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a guanylate dehydrogenase mutant, wherein the mutant comprises at least one of the following mutation sites ① to ⑦:

[0008] ① The 454th amino acid of guanylate dehydrogenase mutated from L to F;

[0009] ② The 454th amino acid of guanylate dehydrogenase mutated from L to P;

[0010] ③ The 454th amino acid of guanylate dehydrogenase mutated from L to K;

[0011] ④ The 481st amino acid of guanylate dehydrogenase mutated from E to K;

[0012] ⑤ The 481st amino acid of guanylate dehydrogenase mutated from E to R;

[0013] ⑥ The 481st amino acid of guanylate dehydrogenase mutated from E to H;

[0014] ⑦ The 444th amino acid of guanylate dehydrogenase mutated from G to E;

[0015] In the present invention, the guanylate dehydrogenase is from Bacillus, and its reference sequence number on NCBI is WP_003150717.1.

[0016] In a second aspect, the present invention provides a nucleic acid molecule encoding the guanylate dehydrogenase mutant or a biological material containing the nucleic acid molecule, wherein the biological material includes but is not limited to recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

[0017] In a third aspect, the present invention provides a YeaC protein mutant, wherein the mutant comprises a mutation of the 71st amino acid of the YeaC protein from Q to a terminator.

[0018] In the present invention, the YeaC protein is from Bacillus subtilis, and its reference sequence number on NCBI is WP_013351265.1.

[0019] In a fourth aspect, the present invention provides any of the following uses of a nucleic acid molecule encoding the guanylate dehydrogenase mutant or a biological material containing the nucleic acid molecule, or a nucleic acid molecule encoding the YeaC protein mutant or a biological material containing the nucleic acid molecule:

[0020] (1) For the fermentation production of nucleosides or nucleoside-related derivatives;

[0021] (2) Used to increase the fermentation yield of nucleosides or nucleoside-related derivatives;

[0022] (3) Used to construct genetically engineered bacteria that produce nucleosides or nucleoside-related derivatives.

[0023] Preferably, the nucleoside or nucleoside-related derivatives can be selected from guanosine, inosine, adenosine, diacetylguanine, riboflavin, and the like.

[0024] In a fifth aspect, the present invention provides a method for constructing a genetically engineered bacterium that produces nucleosides or nucleoside-related derivatives, enhancing the guanylate dehydrogenase gene in the original strain to obtain a gene-enhanced strain; wherein the reference sequence number of guanylate dehydrogenase on NCBI is WP_003150717.1.

[0025] The enhanced approach is selected from the following 1) to 6), or an optional combination:

[0026] 1) Enhanced by introducing a plasmid carrying the gene;

[0027] 2) Enhanced by increasing the copy number of the gene on the chromosome;

[0028] 3) Enhancement by changing the promoter sequence of the gene on the chromosome;

[0029] 4) Enhanced by operably linking a strong promoter to the gene;

[0030] 5) Enhancement by introducing enhancers;

[0031] 6) Enhancement by using genes or alleles encoding high activity of the corresponding enzymes or proteins.

[0032] In the present invention, the original strain is a bacterium capable of producing nucleosides, preferably a species of Bacillus or Escherichia, more preferably Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus pumilus, and most preferably Bacillus amyloliquefaciens 836. Bacillus amyloliquefaciens 836 can be found in CN112574934A. Bacillus amyloliquefaciens 836 contains guaA V234I and guaC E8* Double point mutation.

[0033] Preferably, the method comprises: using genetic engineering means to introduce mutations into the genome of bacteria having nucleoside production ability, so that the guanylate dehydrogenase encoded by the bacteria contains at least one of the following mutation sites ① to ⑦:

[0034] ① A mutation in the 454th amino acid of guanylate dehydrogenase from L to F;

[0035] ② A mutation in the 454th amino acid of guanylate dehydrogenase from L to P;

[0036] ③ A mutation in amino acid 454 of guanylate dehydrogenase from L to K;

[0037] ④ A mutation in amino acid 481 of guanylate dehydrogenase from E to K;

[0038] ⑤ A mutation in the 481st amino acid of guanylate dehydrogenase from E to R;

[0039] ⑥ A mutation in the 481st amino acid of guanylate dehydrogenase from E to H;

[0040] ⑦ Mutation of amino acid 444 of guanylate dehydrogenase from G to E.

[0041] In a sixth aspect, the present invention provides a method for constructing a genetically engineered bacterium that produces nucleosides or nucleoside-related derivatives, weakening the gene encoding the YeaC protein in the original strain to obtain a genetically weakened strain; wherein the reference sequence number of the YeaC protein on NCBI is WP_013351265.1.

[0042] The attenuation includes knocking out or reducing the expression of a gene.

[0043] Preferably, the method comprises: using genetic engineering means to introduce a mutation into the genome of a bacterium having nucleoside production capability, so that the YeaC protein encoded thereby contains a Q71* mutation site; wherein * is a terminator.

[0044] In a seventh aspect, the present invention provides a method for constructing a genetically engineered bacterium that produces nucleosides or nucleoside-related derivatives, including Scheme I and Scheme II;

[0045] Option I:

[0046] A. weakening the gene encoding the YeaC protein in the original strain to obtain a genetically weakened strain; the weakening includes knocking out or reducing the expression of the gene; and

[0047] B. Enhance the guanylate dehydrogenase gene in the genetically weakened strain in step A to obtain a genetically enhanced strain.

[0048] Option II:

[0049] A. enhancing the guanylate dehydrogenase gene in the original strain to obtain a gene-enhanced strain; and

[0050] B. Attenuation Step A: The gene encoding the YeaC protein in the gene-enhanced strain is removed to obtain a gene-attenuated strain; the attenuation includes knocking out or reducing the expression of the gene.

[0051] The weakening method comprises: using genetic engineering means to introduce a mutation into the genome of a bacterium with nucleoside production ability, so that the YeaC protein encoded by the bacterium contains a Q71* mutation site; wherein * is a terminator.

[0052] The enhancement method comprises: using genetic engineering means to introduce mutations into the genome of bacteria with nucleoside production ability, so that the guanylate dehydrogenase encoded by the bacteria contains at least one of the following mutation sites ① to ⑦:

[0053] ① A mutation in the 454th amino acid of guanylate dehydrogenase from L to F;

[0054] ② A mutation in the 454th amino acid of guanylate dehydrogenase from L to P;

[0055] ③ A mutation in amino acid 454 of guanylate dehydrogenase from L to K;

[0056] ④ A mutation in amino acid 481 of guanylate dehydrogenase from E to K;

[0057] ⑤ A mutation in the 481st amino acid of guanylate dehydrogenase from E to R;

[0058] ⑥ A mutation in the 481st amino acid of guanylate dehydrogenase from E to H;

[0059] ⑦ Mutation of amino acid 444 of guanylate dehydrogenase from G to E.

[0060] In one embodiment of the present invention, the constructed nucleoside-producing genetically engineered bacteria (strain Ba 839) contains a mutation of the 454th amino acid of guanylate dehydrogenase from L to F and a mutation of the 481st amino acid from E to K. Compared with the starting strain Ba 836, the guanosine production was increased from 1.1 g / L to 4.9 g / L, and the sugar (guanosine) conversion rate was increased by 4.1%.

[0061] In another specific embodiment of the present invention, the constructed nucleoside-producing genetically engineered bacteria (strain B.a8310) contains a mutation of the 454th amino acid of guanylate dehydrogenase from L to F, a mutation of the 481st amino acid from E to K, and a mutation of the 71st amino acid of the YeaC protein from Q to a terminator. Compared with the starting strain Ba 836, the guanosine production increased from 1.1 g / L to 7.3 g / L, and the glycoside conversion rate increased by 6.6%.

[0062] In another embodiment of the present invention, the constructed nucleoside-producing genetically engineered bacteria (strain B.a8311) contains a mutation from G to E at amino acid position 444 of guanylate dehydrogenase. Compared with the starting strain Ba 836, the inosine production increased from 0.6 g / L to 4.5 g / L, and the glycoside conversion rate increased by 4.1%.

[0063] In an eighth aspect, the present invention provides a genetically engineered bacterium that produces nucleosides or nucleoside-related derivatives constructed according to the above method.

[0064] In a ninth aspect, the present invention provides the use of the genetically engineered bacteria in the fermentative production of nucleosides or nucleoside-related derivatives or in increasing the fermentative yield of nucleosides or nucleoside-related derivatives.

[0065] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0066] The present invention uses Bacillus amyloliquefaciens 836 as a starting strain, and transforms its genome by introducing point mutations into the guanylate dehydrogenase gene guaB (thus generating GuaB protein variants: L454F, L454P, L454K, E481K, E481R, E481H, G444E) and / or introducing point mutations into the yeaC gene (a gene possibly related to ATPase) (thus generating YeaC protein variant: Q71*). Bacillus amyloliquefaciens containing GuaB and YeaC variants was subjected to shake flask fermentation, and the fermentation results showed that the mutant strain guaB L454F Compared with the original strain Ba 836, the guanosine production of the strain increased from 1.1 g / L to 4.9 g / L, and the sugar (guanosine) conversion rate increased by 4.1%. E481KCompared to the original strain Ba 836, the strain increased guanosine production from 1.1 g / L to 3.2 g / L, and the sugar (guanosine) conversion rate increased by 2.4%. When the 454th amino acid of guanylate dehydrogenase was mutated from leucine (L) to proline (P) or lysine (K), guanosine production increased (the effect was even greater when leucine was mutated to proline). Guanosine production also increased when the 481st amino acid of guanylate dehydrogenase was mutated from glutamic acid (E) to lysine (K), arginine (R), or histidine (H). When the 444th amino acid of guanylate dehydrogenase was mutated from glycine (G) to glutamic acid (E), guanosine production decreased, while inosine production increased from 0.6 g / L to 4.5 g / L, indicating that this mutation site is beneficial for increasing inosine production. The above mutation sites can also be applied to host bacteria such as Bacillus subtilis and Bacillus pumilus to produce nucleosides such as inosine and guanosine, or their corresponding nucleoside derivatives, such as hypoxanthine, inosinic acid, guanine, guanylic acid, riboflavin, and diacetylguanylic acid. The present invention provides a powerful tool for the large-scale industrial production of nucleosides. DETAILED DESCRIPTION

[0067] The present invention provides a Bacillus amyloliquefaciens strain, wherein the leucine (L) at position 454 of the guaB gene encoding guanylate dehydrogenase in the strain is mutated to phenylalanine (F), specifically CTT mutates to TTT; the amino acid at position 481 is mutated from glutamic acid (E) to lysine (K), specifically GAA mutates to AAA; the glycine (G) at position 444 is mutated to glutamic acid (E), specifically GGA mutates to GAA. The glutamine at position 71 of the yeaC gene encoding a hypothetical protein undergoes a termination mutation, specifically CAG mutates to TAG.

[0068] GuaB encodes guanylate dehydrogenase, which catalyzes the conversion of inosinic acid (IMP) to xanthosine (XMP). The present invention mutates guanylate dehydrogenase, ultimately increasing GMP production. The microorganism is modified by the guaB gene, resulting in an enhanced ability to produce nucleosides compared to unmodified strains. YeaC is a hypothetical protein, presumably related to ATPase. The strain obtained by inactivating amino acid position 71 in the present invention exhibits enhanced guanosine production compared to the non-inactivated strain.

[0069] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0070] The primer sequences used in the following examples are shown in Table 1.

[0071] Table 1

[0072]

[0073]

[0074] Example 1 Bacillus amyloliquefaciens guaB L454F Construction of mutant strains

[0075] Take strain DSM7 (see reference Genome sequence of B. amyloliquefaciens type strain DSM7 T The genome of B. amyloliquefaciens FZB42 was used as a template, and primers guaB-1f / 1r and guaB-2f / 3r were used to amplify two fragments using Phusion high-fidelity polymerase (New England BioLabs). The two fragments were fused using primers guaB-1f / 3r to obtain a recombinant fragment (the nucleotide sequence of the ORF region is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2). L454F The fragment was combined with 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) to obtain the recombinant plasmid pKSU-guaB after double digestion with SalI / PstI, assembly, and transformation. L454FThe strain was transformed into Ba 836 (preliminary constructed strain), and the transformants were screened at 30°C using LB plates containing 2.5 μg / mL chloramphenicol. The obtained transformants were inoculated into 5 ml LB liquid medium, cultured at 42°C and 200 rpm for 12 h and propagated for one generation, and diluted and spread on LB plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. The primary recombinants were inoculated into 5 ml LB liquid medium, cultured at 42°C and 200 rpm for 12 h and propagated for one generation, and diluted and spread on LB plates containing 0.8 μM 5-FU to screen secondary recombinants, and guaB was obtained by screening. L454F The point mutation strain was named Ba837.

[0076] Example 2 Bacillus amyloliquefaciens guaB E481K Construction of mutant strains

[0077] The specific construction process is the same as in Example 1, the primers used are guaB-1f / 2r and guaB-3f / 3r, the nucleotide sequence of the ORF region is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The plasmid obtained by construction is pKSU-guaB E481K , transformed into the Ba 836 strain, and the obtained strain was named B.a838.

[0078] Example 3 Bacillus amyloliquefaciens guaB L454F and guaB E481K Construction of stacked mutant strains

[0079] Using the DSM7 genome as a template, the primers used were guaB-1f / 1r, guaB-2f / 2r, and guaB-3f / 3r. The three fragments were fused to obtain a recombinant fragment. The plasmid and strain construction process were the same as in Example 1. The nucleotide sequence of the ORF region is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6. The constructed plasmid is pKSU-guaB L454F E481K , transformed into the Ba 836 strain, and the obtained strain containing the above two mutations was named Ba 839.

[0080] Example 4 Bacillus amyloliquefaciens yeaC Q71* Strain construction

[0081] Using the DSM7 genome as a template, primers yeaC-1f / 1r and yeaC-2f / 2r were used to amplify the left and right homology arm fragments to obtain yeaC Q71* The nucleotide sequence of the ORF region is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. The plasmid pKSU-yeaC was obtained according to the method of plasmid construction in Example 1. Q71*, transformed into Ba 836 strain, and the yeaC Q71* The mutant strain was named Ba 8310. It was transformed into Ba 839 strain and the yeaC Q71* The mutant strain was named Ba 8311.

[0082] Example 5 Bacillus amyloliquefaciens guaB G444E Construction of mutant strains

[0083] Using the DSM7 genome as a template, the primers used were guaB-1f / 4r and guaB-4f / 3r, and two fragments were amplified. The recombinant fragment was then obtained by fusion PCR. The nucleotide sequence of the ORF region is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10. The constructed plasmid is pKSU-guaB G444E The specific construction method was the same as that in Example 1, and the strain was transformed into the Ba 836 strain. The strain containing the above mutation obtained by screening was named Ba 8312.

[0084] Example 6 Bacillus amyloliquefaciens guaB L454P ,guaB L454K 、guaB E481R 、guaB E481H Construction of mutant strains

[0085] Referring to the method of Example 1, guaB was amplified and obtained. L454P (guaB-1f / 5r, guaB-5f / 3r), guaB L454K (guaB-1f / 6r, guaB-6f / 3r), guaB E481R (guaB-1f / 7r, guaB-7f / 3r), guaB E481H The strain construction method was the same as in Example 1, and the strains were transformed into the Ba 836 strain, and the strains containing the above mutations were screened and named Ba 8313-Ba 8316 respectively.

[0086] Example 7 Verification of the nucleoside production performance of the mutant strain

[0087] 1. Culture the bacteria stored in glycerol at 37°C overnight and isolate a single colony.

[0088] 2. Pick a single colony and inoculate it into 30 mL of seed culture medium (20 g / L glucose, 5 g / L yeast powder, 5 g / L corn steep liquor powder, 3 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.02 g / L ferrous sulfate, 0.01 g / L manganese sulfate, pH 7.0-7.2), shake at 110 rpm, and culture at 37°C for 7-8 h.

[0089] 3. Transfer the inoculum at 10% v / v to 30 ml of fermentation medium (120 g / L glucose, 3.5 g / L yeast extract, 3 g / L potassium dihydrogen phosphate, 25 g / L ammonium sulfate, 0.01 g / L manganese sulfate, 5 g / L magnesium sulfate, 10 g / L sodium glutamate, 15 g / L corn steep liquor powder, 25 g / L calcium carbonate, pH 7.0-7.2) and culture at 35°C for 70 h on a shaker at 130 rpm.

[0090] 4. Liquid chromatography was used to detect the nucleosides in the fermentation broth (Table 2).

[0091] Table 2 Evaluation results of guanosine and inosine production by shake flask fermentation of engineered bacteria (mean of three replicates)

[0092]

[0093]

[0094] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Sequence Listing <110> Meihua (Shanghai) Biotechnology Co., Ltd. <120> Nucleoside-producing genetically engineered bacteria and their construction method and application <130> KHP211117789.7 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1467 <212> DNA <213> Artificial Sequence <400> 1 atgtgggaaa gtaaattttc aaaagaaggc ttaacgttcg atgatgtact gctcgtacca 60 gctcaatcag acgtacttcc gcgtgatgtg gatttgtctg ttgaactgac aaaaacgtta 120 aagcttaata ttcctgtcat cagtgcagga atggatacag taacagaatc agcaatggcg 180 attgcgatgg cccgacaagg cggcttgggc attattcata aaaacatgtc catcgaacag 240 caggctgaac atgttgacaa agtcaaacgt tctgaacggg gcgttattac aaatcccttc 300 ttttaacac ctgatcatca agtattcgat gcggagcatt tgatggggaa atacagaatt 360 tccggtgtc cgatcgtaga tataaagac gatcaaaagc tggtcggtat cattacaaac 420 cgcgatcttc gctttatctc tgattattca atgaaaatca gtgatgttat gacaaaagaa 480 gagctggtta cggctcctgt gggaaccaca ttagacgaag cggaaaaaat cttgcagaag 540 cataaaattg aaaaacttcc attagtggat gaccaaaaca attaaaagg tcttatcacg 600 atcaaagata ttgaaaaggt tatcgaattc ccgaattcat ctaaagatga acacggacgc 660 ctgatcgtcg gcgctgcggt aggcgtgaca ggtgatacaa tgactcgtgt cagcaagctt 720 gttgaagcga atgtcgacgt tatcgtggtt gatacggctc acggacattc cagaggcgta 780 ctgaacacag ttgcgaaaat ccgtgagaca tatcctgaat tgaacattat cgcaggaaat 840 gttgctacgg ctgaagcgac aaaggctttg attgaagccg gagcaaacat tgtaaaagtg 900 ggaatcggac ctggatctat ctgtacgaca cgcgtcgttg caggcgtagg tgtaccgcaa 960 atcactgcga tttatgattg tgccactgaa gcgagaaaac acggcgcaac aattatcgcg 1020 gacggcggta ttaaattctc cggagatatt acgaaagcat tggcatccgg cggacatgct 1080 gtcatgcttg gaagcctgct tgccggtact tcagaaagcc cgggcgaaac tgaaatctat 1140 caaggcagaa gatttaaagt gtatcgcggt atgggttctg tcgctgccat ggaaaaaggc 1200 agtaaagacc gatatttcca agaagaaaat aagaaattcg tccctgaagg tatcgaagga 1260 cggactccgt acaaaggtcc tgtagaagaa acagtgtatc agcttgtcgg cggtcttcgt 1320 tcaggtatgg gatattgcgg ttcaaaagac ttgcgcgctt ttagagaaga agctcaattt 1380 atccgtatga caggagcagg tcttcgcgaa agccatccgc atgatgtcca aatcacgaag 1440 gaatcaccaa actacacaat ctcataa 1467 <210> 2 <211> 488 <212> PRT <213> Artificial Sequence <400> 2 Met Trp Glu Ser Lys Phe Ser Lys Glu Gly Leu Thr Phe Asp Asp Val 1 5 10 15 [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​Lys Asp Asp Gln Lys Leu Val Gly Ile Ile Thr Asn Arg Asp Leu Arg 130 135 140 Phe Ile Ser Asp Tyr Ser Met Lys Ile Ser Asp Val Met Thr Lys Glu 145 150 155 160 Glu Leu Val Thr Ala Pro Val Gly Thr Thr Leu Asp Glu Ala Glu Lys 165 170 175 Ile Leu Gln Lys His Lys Ile Glu Lys Leu Pro Leu Val Asp Asp Gln 180 185 190 Asn Lys Leu Lys Gly Leu Ile Thr Ile Lys Asp Ile Glu Lys Val Ile 195 200 205 Glu Phe Pro Asn Ser Ser Lys Asp Glu His Gly Arg Leu Ile Val Gly 210 215 220 Ala Ala Val Gly Val Thr Gly Asp Thr Met Thr Arg Val Ser Lys Leu 225 230 235 240 Val Glu Ala Asn Val Asp Val Ile Val Val Asp Thr Ala His Gly His 245 250 255 Ser Arg Gly Val Leu Asn Thr Val Ala Lys Ile Arg Glu Thr Tyr Pro 260 265 270 Glu Leu Asn Ile Ile Ala Gly Asn Val Ala Thr Ala Glu Ala Thr Lys 275 280 285 Ala Leu Ile Glu Ala Gly Ala Asn Ile Val Lys Val Gly Ile Gly Pro 290 295 300 Gly Ser Ile Cys Thr Thr Arg Val Val Ala Gly Val Gly Val Pro Gln 305 310 315 320 Ile Thr Ala Ile Tyr Asp Cys Ala Thr Glu Ala Arg Lys His Gly Ala 325 330 335 Thr Ile Ile Ala Asp Gly Gly Ile Lys Phe Ser Gly Asp Ile Thr Lys 340 345 350 Ala Leu Ala Ser Gly Gly His Ala Val Met Leu Gly Ser Leu Leu Ala 355 360 365 Gly Thr Ser Glu Ser Pro Gly Glu Thr Glu Ile Tyr Gln Gly Arg Arg 370 375 380 Phe Lys Val Tyr Arg Gly Met Gly Ser Val Ala Ala Met Glu Lys Gly 385 390 395 400 Ser Lys Asp Arg Tyr Phe Gln Glu Glu Asn Lys Lys Phe Val Pro Glu 405 410 415 Gly Ile Glu Gly Arg Thr Pro Tyr Lys Gly Pro Val Glu Glu Thr Val 420 425 430 Tyr Gln Leu Val Gly Gly Leu Arg Ser Gly Met Gly Tyr Cys Gly Ser 435 440 445 Lys Asp Leu Arg Ala Phe Arg Glu Glu Ala Gln Phe Ile Arg Met Thr 450 455 460 Gly Ala Gly Leu Arg Glu Ser His Pro His Asp Val Gln Ile Thr Lys 465 470 475 480 Glu Ser Pro Asn Tyr Thr Ile Ser 485 <210> 3 <211> 1467 <212> DNA <213> Artificial Sequence <400> 3 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 ggaaaaaggc 1200 caaggcagaa gatttaaagt gtatcgcggt atgggttctg tcgctgccat ggaaaaaggc 1200 agtaaagacc gatatttcca agaagaaaat aagaaattcg tccctgaagg tatcgaagga 1260 agtaaagacc gatatttcca agaagaaaat aagaaattcg tccctgaagg tatcgaagga 1260 cggactccgt acaaaggtcc tgtagaagaa acagtgtatc agcttgtcgg cggtcttcgt 1320 cggactccgt acaaaggtcc tgtagaagaa acagtgtatc agcttgtcgg cggtcttcgt 1320 tcaggtatgg gatattgcgg ttcaaaagac ttgcgcgctt taagagaaga agctcaattt 1380 tcaggtatgg gatattgcgg ttcaaaagac ttgcgcgctt taagagaaga agctcaattt 1380 atccgtatga caggagcagg tcttcgcgaa agccatccgc atgatgtcca aatcacgaag 1440 atccgtatga caggagcagg tcttcgcgaa agccatccgc atgatgtcca aatcacgaag 1440 aaatcaccaa actacacaat ctcataa 1467 aaatcaccaa actacacaat ctcataa 1467 <210> 4<210> 4 <211> 488<211> 488 <212> PRT<212> PRT <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <400> 4<400> 4 Met Trp Glu Ser Lys Phe Ser Lys Glu Gly Leu Thr Phe Asp Asp Val Met Trp Glu Ser Lys Phe Ser Lys Glu Gly Leu Thr Phe Asp Asp Val 1 5 10 15 1 5 10 15 Leu Leu Val Pro Ala Gln Ser Asp Val Leu Pro Arg Asp Val Asp Leu Leu Leu Val Pro Ala Gln Ser Asp Val Leu Pro Arg Asp Val Asp Leu 20 25 30 20 25 30 Ser Val Glu Leu Thr Lys Thr Leu Lys Leu Asn Ile Pro Val Ile Ser Ser Val Glu Leu Thr Lys Thr Leu Lys Leu Asn Ile Pro Val Ile Ser 35 40 45 35 40 45 Ala Gly Met Asp Thr Val Thr Glu Ser Ala Met Ala Ile Ala Met Ala Ala Gly Met Asp Thr Val Thr Glu Ser Ala Met Ala Ile Ala Met Ala 50 55 60 50 55 60 Arg Gln Gly Gly Leu Gly Ile Ile His Lys Asn Met Ser Ile Glu Gln 65 70 75 80 Gln Ala Glu His Val Asp Lys Val Lys Arg Ser Glu Arg Gly Val Ile 85 90 95 Thr Asn Pro Phe Phe Leu Thr Pro Asp His Gln Val Phe Asp Ala Glu 100 105 110 His Leu Met Gly Lys Tyr Arg Ile Ser Gly Val Pro Ile Val Asp Asn 115 120 125 Lys Asp Asp Gln Lys Leu Val Gly Ile Ile Thr Asn Arg Asp Leu Arg 130 135 140 Phe Ile Ser Asp Tyr Ser Met Lys Ile Ser Asp Val Met Thr Lys Glu 145 150 155 160 Glu Leu Val Thr Ala Pro Val Gly Thr Thr Leu Asp Glu Ala Glu Lys 165 170 175 Ile Leu Gln Lys His Lys Ile Glu Lys Leu Pro Leu Val Asp Asp Gln 180 185 190 Asn Lys Leu Lys Gly Leu Ile Thr Ile Lys Asp Ile Glu Lys Val Ile 195 200 205 Glu Phe Pro Asn Ser Ser Lys Asp Glu His Gly Arg Leu Ile Val Gly 210 215 220 Ala Ala Val Gly Val Thr Gly Asp Thr Met Thr Arg Val Ser Lys Leu 225 230 235 240 Val Glu Ala Asn Val Asp Val Ile Val Val Asp Thr Ala His Gly His 245 250 255 Ser Arg Gly Val Leu Asn Thr Val Ala Lys Ile Arg Glu Thr Tyr Pro 260 265 270 Glu Leu Asn Ile Ile Ala Gly Asn Val Ala Thr Ala Glu Ala Thr Lys 275 280 285 Ala Leu Ile Glu Ala Gly Ala Asn Ile Val Lys Val Gly Ile Gly Pro 290 295 300 Gly Ser Ile Cys Thr Thr Arg Val Val Ala Gly Val Gly Val Pro Gln 305 310 315 320 Ile Thr Ala Ile Tyr Asp Cys Ala Thr Glu Ala Arg Lys His Gly Ala 325 330 335 Thr Ile Ile Ala Asp Gly Gly Ile Lys Phe Ser Gly Asp Ile Thr Lys 340 345 350 Ala Leu Ala Ser Gly Gly His Ala Val Met Leu Gly Ser Leu Leu Ala 355 360 365 Gly Thr Ser Glu Ser Pro Gly Glu Thr Glu Ile Tyr Gln Gly Arg Arg 370 375 380 Phe Lys Val Tyr Arg Gly Met Gly Ser Val Ala Ala Met Glu Lys Gly 385 390 395 400 Ser Lys Asp Arg Tyr Phe Gln Glu Glu Asn Lys Lys Phe Val Pro Glu 405 410 415 Gly Ile Glu Gly Arg Thr Pro Tyr Lys Gly Pro Val Glu Glu Thr Val 420 425 430 Tyr Gln Leu Val Gly Gly Leu Arg Ser Gly Met Gly Tyr Cys Gly Ser 435 440 445 Lys Asp Leu Arg Ala Leu Arg Glu Glu Ala Gln Phe Ile Arg Met Thr 450 455 460 Gly Ala Gly Leu Arg Glu Ser His Pro His Asp Val Gln Ile Thr Lys 465 470 475 480 Lys Ser Pro Asn Tyr Thr Ile Ser 485 <210> 5 <211> 1467 <212> DNA <213> Artificial Sequence <400> 5 atgtgggaaa gtaaattttc aaaagaaggc ttaacgttcg atgatgtact gctcgtacca 60 gctcaatcag acgtacttcc gcgtgatgtg gatttgtctg ttgaactgac aaaaacgtta 120 aagcttaata ttcctgtcat cagtgcagga atggatacag taacagaatc agcaatggcg 180 attgcgatgg cccgacaagg cggcttgggc attattcata aaaacatgtc catcgaacag 240 caggctgaac atgttgacaa agtcaaacgt tctgaacggg gcgttattac aaatcccttc 300 ttttaacac ctgatcatca agtattcgat gcggagcatt tgatggggaa atacagaatt 360 tccggtgtc cgatcgtaga tataaagac gatcaaaagc tggtcggtat cattacaaac 420 cgcgatcttc gctttatctc tgattattca atgaaaatca gtgatgttat gacaaaagaa 480 gagctggtta cggctcctgt gggaaccaca ttagacgaag cggaaaaaat cttgcagaag 540 cataaaattg aaaaacttcc attagtggat gaccaaaaca attaaaagg tcttatcacg 600 atcaaagata ttgaaaaggt tatcgaattc ccgaattcat ctaaagatga acacggacgc 660 ctgatcgtcg gcgctgcggt aggcgtgaca ggtgatacaa tgactcgtgt cagcaagctt 720 gttgaagcga atgtcgacgt tatcgtggtt gatacggctc acggacattc cagaggcgta 780 ctgaacacag ttgcgaaaat ccgtgagaca tatcctgaat tgaacattat cgcaggaaat 840 gttgctacgg ctgaagcgac aaaggctttg attgaagccg gagcaaacat tgtaaaagtg 900 ggaatcggac ctggatctat ctgtacgaca cgcgtcgttg caggcgtagg tgtaccgcaa 960 atcactgcga tttatgattg tgccactgaa gcgagaaaac acggcgcaac aattatcgcg 1020 gacggcggta ttaaattctc cggagatatt acgaaagcat tggcatccgg cggacatgct 1080 gtcatgcttg gaagcctgct tgccggtact tcagaaagcc cgggcgaaac tgaaatctat 1140 caaggcagaa gatttaaagt gtatcgcggt atgggttctg tcgctgccat ggaaaaaggc 1200 agtaaagacc gatatttcca agaagaaaat aagaaattcg tccctgaagg tatcgaagga 1260 cggactccgt acaaaggtcc tgtagaagaa acagtgtatc agcttgtcgg cggtcttcgt 1320 tcaggtatgg gatattgcgg ttcaaaagac ttgcgcgctt ttagagaaga agctcaattt 1380 atccgtatga caggagcagg tcttcgcgaa agccatccgc atgatgtcca aatcacgaag 1440 aaatcaccaa actacacaat ctcataa 1467 <210> 6 <211> 488 <212> PRT <213> Artificial Sequence <400> 6 Met Trp Glu Ser Lys Phe Ser Lys Glu Gly Leu Thr Phe Asp Asp Val 1 5 10 15 Leu Leu Val Pro Ala Gln Ser Asp Val Leu Pro Arg Asp Val Asp Leu 20 25 30 Ser Val Glu Leu Thr Lys Thr Leu Lys Leu Asn Ile Pro Val Ile Ser 35 40 45 Ala Gly Met Asp Thr Val Thr Glu Ser Ala Met Ala Ile Ala Met Ala 50 55 60 Arg Gln Gly Gly Leu Gly Ile Ile His Lys Asn Met Ser Ile Glu Gln 65 70 75 80 Gln Ala Glu His Val Asp Lys Val Lys Arg Ser Glu Arg Gly Val Ile 85 90 95 Thr Asn Pro Phe Phe Leu Thr Pro Asp His Gln Val Phe Asp Ala Glu 100 105 110 His Leu Met Gly Lys Tyr Arg Ile Ser Gly Val Pro Ile Val Asp Asn 115 120 125 Lys Asp Asp Gln Lys Leu Val Gly Ile Ile Thr Asn Arg Asp Leu Arg 130 135 140 Phe Ile Ser Asp Tyr Ser Met Lys Ile Ser Asp Val Met Thr Lys Glu 145 150 155 160 Glu Leu Val Thr Ala Pro Val Gly Thr Thr Leu Asp Glu Ala Glu Lys 165 170 175 Ile Leu Gln Lys His Lys Ile Glu Lys Leu Pro Leu Val Asp Asp Gln 180 185 190 Asn Lys Leu Lys Gly Leu Ile Thr Ile Lys Asp Ile Glu Lys Val Ile 195 200 205 Glu Phe Pro Asn Ser Ser Lys Asp Glu His Gly Arg Leu Ile Val Gly 210 215 220 Ala Ala Val Gly Val Thr Gly Asp Thr Met Thr Arg Val Ser Lys Leu 225 230 235 240 Val Glu Ala Asn Val Asp Val Ile Val Val Asp Thr Ala His Gly His 245 250 255 Ser Arg Gly Val Leu Asn Thr Val Ala Lys Ile Arg Glu Thr Tyr Pro 260 265 270 Glu Leu Asn Ile Ile Ala Gly Asn Val Ala Thr Ala Glu Ala Thr Lys 275 280 285 Ala Leu Ile Glu Ala Gly Ala Asn Ile Val Lys Val Gly Ile Gly Pro 290 295 300 Gly Ser Ile Cys Thr Thr Arg Val Val Ala Gly Val Gly Val Pro Gln 305 310 315 320 Ile Thr Ala Ile Tyr Asp Cys Ala Thr Glu Ala Arg Lys His Gly Ala 325 330 335 Thr Ile Ile Ala Asp Gly Gly Ile Lys Phe Ser Gly Asp Ile Thr Lys 340 345 350 Ala Leu Ala Ser Gly Gly His Ala Val Met Leu Gly Ser Leu Leu Ala 355 360 365 Gly Thr Ser Glu Ser Pro Gly Glu Thr Glu Ile Tyr Gln Gly Arg Arg 370 375 380 Phe Lys Val Tyr Arg Gly Met Gly Ser Val Ala Ala Met Glu Lys Gly 385 390 395 400 Ser Lys Asp Arg Tyr Phe Gln Glu Glu Asn Lys Lys Phe Val Pro Glu 405 410 415 Gly Ile Glu Gly Arg Thr Pro Tyr Lys Gly Pro Val Glu Glu Thr Val 420 425 430 Tyr Gln Leu Val Gly Gly Leu Arg Ser Gly Met Gly Tyr Cys Gly Ser 435 440 445 Lys Asp Leu Arg Ala Phe Arg Glu Glu Ala Gln Phe Ile Arg Met Thr 450 455 460 Gly Ala Gly Leu Arg Glu Ser His Pro His Asp Val Gln Ile Thr Lys 465 470 475 480 Lys Ser Pro Asn Tyr Thr Ile Ser 485 <210> 7 <211> 963 <212> DNA <213> Artificial Sequence <400> 7 atggcttata aagaagagct gcatccgctg ctggagaaag ccgttgagca tattgaaaac 60 atcatcatag gaaaacggga tatcgccata ctcagcttgg ccgccattct cgcaaaaggg 120 catgtgctgc tggaagacgt gccgggagtc ggaaaaacga tgatggtccg cgctttggcc 180 aagctgatcg gagctgactt taagaggatt tagttcactc cggatcttct gccgtcagat 240 gtgacgggtg tctctattta taatacgaaa acgatggaat ttgaataccg cccgggaccg 300 attatgggga atatcgtgct ggccgacgaa attaacagaa cctctccgaa aacgcagtct 360 tctttgcttg aggcgatgga agagggaaat gtcacgattg acggaaagac aatgcggctg 420 cttcctgaag cccagcttga ccggttttta tttaagctga agatgggcta tccgactgcc 540 ​gaggaggagc ttaaggttct gtctttgcag gaagggcgca atccgcttga aacaattgag 600 cccgtcattt ctaaagaaca atttatcagt cttcagcaaa agctggagca ggtgcgggtg 660 gatgatggca ttaaagccta tatcgtcggc atcactcagc atacccgccg gcatccgtcc 720 gtacatttgg gcgtaagtcc gcgcggttcg atttcattaa tgaaagccgc acaggcatac 780 gcgctgcttc atgatcgcga ctacgtcatt cctgatgacg tccagtattt ggctccgtat 840 acgctgcctc acagaatgat cttaacggcc gaggcgaaat ttaatgatgt cacgccggaa 900 gctgtgatcg aagacatcat gcagacggaa aaagttcccg ttcaaaggat gtcggtccga 960 taa 963 <210> 8 <211> 319 <212> PRT <213> Artificial Sequence <400> 8 Met Ala Tyr Lys Glu Glu Leu His Pro Leu Leu Glu Lys Ala Val Glu 1 5 10 15 His Ile Glu Asn Ile Ile Ile Gly Lys Arg Asp Ile Ala Ile Leu Ser 20 25 30 Leu Ala Ala Ile Leu Ala Lys Gly His Val Leu Leu Glu Asp Val Pro 35 40 45 Gly Val Gly Lys Thr Met Met Val Arg Ala Leu Ala Lys Leu Ile Gly 50 55 60 Ala Asp Phe Lys Arg Ile Phe Thr Pro Asp Leu Leu Pro Ser Asp Val 65 70 75 80 Thr Gly Val Ser Ile Tyr Asn Thr Lys Thr Met Glu Phe Glu Tyr Arg 85 90 95 Pro Gly Pro Ile Met Gly Asn Ile Val Leu Ala Asp Glu Ile Asn Arg 100 105 110 Thr Ser Pro Lys Thr Gln Ser Ser Leu Leu Glu Ala Met Glu Glu Gly 115 120 125 Asn Val Thr Ile Asp Gly Lys Thr Met Arg Leu Ala Glu Pro Phe Phe 130 135 140 Val Met Ala Thr Gln Asn Pro Val Glu Tyr Glu Gly Thr Tyr Pro Leu 145 150 155 160 Pro Glu Ala Gln Leu Asp Arg Phe Leu Phe Lys Leu Lys Met Gly Tyr 165 170 175 Pro Thr Ala Glu Glu Glu Leu Lys Val Leu Ser Leu Gln Glu Gly Arg 180 185 190 Asn Pro Leu Glu Thr Ile Glu Pro Val Ile Ser Lys Glu Gln Phe Ile 195 200 205 Ser Leu Gln Gln Lys Leu Glu Gln Val Arg Val Asp Asp Gly Ile Lys 210 215 220 Ala Tyr Ile Val Gly Ile Thr Gln His Thr Arg Arg His Pro Ser Val 225 230 235 240 His Leu Gly Val Ser Pro Arg Gly Ser Ile Ser Leu Met Lys Ala Ala 245 250 255 Gln Ala Tyr Ala Leu Leu His Asp Arg Asp Tyr Val Ile Pro Asp Asp 260 265 270 Val Gln Tyr Leu Ala Pro Tyr Thr Leu Pro His Arg Met Ile Leu Thr 275 280 285 Ala Glu Ala Lys Phe Asn Asp Val Thr Pro Glu Ala Val Ile Glu Asp 290 295 300 Ile Met Gln Thr Glu Lys Val Pro Val Gln Arg Met Ser Val Arg 305 310 315 <210> 9 <211> 1467 <212> DNA <213> 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 ttttaacac ctgatcatca agtattcgat gcggagcatt tgatggggaa atacagaatt 360 tccggtgtc cgatcgtaga tataaagac gatcaaaagc tggtcggtat cattacaaac 420 cgcgatcttc gctttatctc tgattattca atgaaaatca gtgatgttat gacaaaagaa 480 gagctggtta cggctcctgt gggaaccaca ttagacgaag cggaaaaaat cttgcagaag 540 cataaaattg aaaaacttcc attagtggat gaccaaaaca attaaaagg tcttatcacg 600 atcaaagata ttgaaaaggt tatcgaattc ccgaattcat ctaaagatga acacggacgc 660 ctgatcgtcg gcgctgcggt aggcgtgaca ggtgatacaa tgactcgtgt cagcaagctt 720 gttgaagcga atgtcgacgt tatcgtggtt gatacggctc acggacattc cagaggcgta 780 ctgaacacag ttgcgaaaat ccgtgagaca tatcctgaat tgaacattat cgcaggaaat 840 gttgctacgg ctgaagcgac aaaggctttg attgaagccg gagcaaacat tgtaaaagtg 900 ggaatcggac ctggatctat ctgtacgaca cgcgtcgttg caggcgtagg tgtaccgcaa 960 atcactgcga tttatgattg tgccactgaa gcgagaaaac acggcgcaac aattatcgcg 1020 gacggcggta ttaaattctc cggagatatt acgaaagcat tggcatccgg cggacatgct 1080 gtcatgcttg gaagcctgct tgccggtact tcagaaagcc cgggcgaaac tgaaatctat 1140 caaggcagaa gatttaaagt gtatcgcggt atgggttctg tcgctgccat ggaaaaaggc 1200 agtaaagacc gatatttcca agaagaaaat aagaaattcg tccctgaagg tatcgaagga 1260 cggactccgt acaaaggtcc tgtagaagaa acagtgtatc agcttgtcgg cggtcttcgt 1320 tcaggtatgg aatattgcgg ttcaaaagac ttgcgcgctt taagagaaga agctcaattt 1380 atccgtatga caggagcagg tcttcgcgaa agccatccgc atgatgtcca aatcacgaag 1440 gaatcaccaa actacacaat ctcataa 1467 <210> 10 <211> 488 <212> PRT <213> Artificial Sequence <400> 10 Met Trp Glu Ser Lys Phe Ser Lys Glu Gly Leu Thr Phe Asp Asp Val 1 5 10 15 Leu Leu Val Pro Ala Gln Ser Asp Val Leu Pro Arg Asp Val Asp Leu 20 25 30 Ser Val Glu Leu Thr Lys Thr Leu Lys Leu Asn Ile Pro Val Ile Ser 35 40 45 Ala Gly Met Asp Thr Val Thr Glu Ser Ala Met Ala Ile Ala Met Ala 50 55 60 Arg Gln Gly Gly Leu Gly Ile Ile His Lys Asn Met Ser Ile Glu Gln 65 70 75 80 Gln Ala Glu His Val Asp Lys Val Lys Arg Ser Glu Arg Gly Val Ile 85 90 95 Thr Asn Pro Phe Phe Leu Thr Pro Asp His Gln Val Phe Asp Ala Glu 100 105 110 His Leu Met Gly Lys Tyr Arg Ile Ser Gly Val Pro Ile Val Asp Asn 115 120 125 Lys Asp Asp Gln Lys Leu Val Gly Ile Ile Thr Asn Arg Asp Leu Arg 130 135 140 Phe Ile Ser Asp Tyr Ser Met Lys Ile Ser Asp Val Met Thr Lys Glu 145 150 155 160 Glu Leu Val Thr Ala Pro Val Gly Thr Thr Leu Asp Glu Ala Glu Lys 165 170 175 Ile Leu Gln Lys His Lys Ile Glu Lys Leu Pro Leu Val Asp Asp Gln 180 185 190 Asn Lys Leu Lys Gly Leu Ile Thr Ile Lys Asp Ile Glu Lys Val Ile 195 200 205 Glu Phe Pro Asn Ser Ser Lys Asp Glu His Gly Arg Leu Ile Val Gly 210 215 220 Ala Ala Val Gly Val Thr Gly Asp Thr Met Thr Arg Val Ser Lys Leu 225 230 235 240 Val Glu Ala Asn Val Asp Val Ile Val Val Asp Thr Ala His Gly His 245 250 255 Ser Arg Gly Val Leu Asn Thr Val Ala Lys Ile Arg Glu Thr Tyr Pro 260 265 270 Glu Leu Asn Ile Ile Ala Gly Asn Val Ala Thr Ala Glu Ala Thr Lys 275 280 285 Ala Leu Ile Glu Ala Gly Ala Asn Ile Val Lys Val Gly Ile Gly Pro 290 295 300 Gly Ser Ile Cys Thr Thr Arg Val Val Ala Gly Val Gly Val Pro Gln 305 310 315 320 Ile Thr Ala Ile Tyr Asp Cys Ala Thr Glu Ala Arg Lys His Gly Ala 325 330 335 Thr Ile Ile Ala Asp Gly Gly Ile Lys Phe Ser Gly Asp Ile Thr Lys 340 345 350 Ala Leu Ala Ser Gly Gly His Ala Val Met Leu Gly Ser Leu Leu Ala 355 360 365 Gly Thr Ser Glu Ser Pro Gly Glu Thr Glu Ile Tyr Gln Gly Arg Arg 370 375 380 Phe Lys Val Tyr Arg Gly Met Gly Ser Val Ala Ala Met Glu Lys Gly 385 390 395 400 Ser Lys Asp Arg Tyr Phe Gln Glu Glu Asn Lys Lys Phe Val Pro Glu 405 410 415 Gly Ile Glu Gly Arg Thr Pro Tyr Lys Gly Pro Val Glu Glu Thr Val 420 425 430 Tyr Gln Leu Val Gly Gly Leu Arg Ser Gly Met Glu Tyr Cys Gly Ser 435 440 445 Lys Asp Leu Arg Ala Leu Arg Glu Glu Ala Gln Phe Ile Arg Met Thr 450 455 460 Gly Ala Gly Leu Arg Glu Ser His Pro His Asp Val Gln Ile Thr Lys 465 470 475 480 Glu Ser Pro Asn Tyr Thr Ile Ser 485

Claims

1. A nucleic acid molecule encoding a YeaC protein mutant or a biological material containing the nucleic acid molecule, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, or an engineered bacterium; The YeaC protein mutant is a mutant obtained by mutating the 71st amino acid of the YeaC protein from Q to a terminator based on the YeaC protein having an amino acid sequence of WP_013351265.

1.

2. Any of the following uses of the nucleic acid molecule according to claim 1 or the biological material 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 genetically engineered bacteria that produce nucleosides; The nucleosides are guanosine and inosine.

3. A method for constructing a genetically engineered bacterium producing nucleosides, characterized in that: By using genetic engineering, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) introduces mutations into the genome, so that it contains the following mutation sites: ① The mutation of amino acid 71 of YeaC protein from Q to terminator; ②YeaC protein amino acid 71 mutation from Q to terminator, guanylate dehydrogenase amino acid 454 mutation from L to F, and amino acid 481 mutation from E to K; Among them, the reference sequence number of guanylate dehydrogenase on NCBI is WP_003150717.1, and the reference sequence number of YeaC protein on NCBI is WP_013351265.1; The nucleosides are guanosine and inosine.

4. The method according to claim 3, characterized in that The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens 836.

5. A nucleoside-producing genetically engineered bacterium constructed according to the method of claim 3 or 4; The nucleosides are guanosine and inosine.

6. Use of the genetically engineered bacteria according to claim 5 in the fermentative production of nucleosides or in increasing the fermentative yield of nucleosides; The nucleosides are guanosine and inosine.

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