Recombinant microorganism reinforced by sulfur-containing amino acid ABC transporter or homologous protein thereof and application of recombinant microorganism in nucleoside production

Abstract

The invention relates to the technical field of microorganisms, in particular to a recombinant microorganism strengthened by sulfur-containing amino acid ABC transporter or homologous protein thereof and application of the recombinant microorganism in nucleoside production. By enhancing the sulfur-containing amino acid ABC transporter TcyM or the homologous protein thereof, the nucleoside production capacity and production efficiency of microorganisms are remarkably improved, and a new modification target and strategy are provided for construction of nucleoside production strains. The sulfur-containing amino acid ABC transporter TcyM or the homologous protein variant thereof provided by the invention can effectively promote microorganisms to accumulate nucleoside and remarkably improve the nucleoside production capacity of the microorganisms. The nucleoside yield of the recombinant microorganism provided by the invention is remarkably improved compared with that of an original strain, the production efficiency of nucleoside is improved, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a recombinant microorganism enhanced with a sulfur amino acid ABC transporter or its homologous protein and its application in nucleoside production. Background Art

[0002] Nucleosides are glycosides formed by the condensation of D-ribose or D-2-deoxyribose with pyrimidine bases or purine bases. The condensation of D-ribose with adenine, guanine, hypoxanthine, cytosine, thymine or uracil generates the corresponding adenosine ribonucleoside, guanosine ribonucleoside, inosine ribonucleoside, cytidine ribonucleoside, thymidine ribonucleoside and uridine ribonucleoside, which are abbreviated as adenosine (A), guanosine (G), inosine (I), cytidine (C), thymidine (T) and uridine (U) respectively.

[0003] Guanosine, inosine and adenosine have wide applications in the food and pharmaceutical industries. In the food field, guanosine and inosine are important precursors of disodium guanylate and disodium inosinate respectively, and the food flavor enhancer formed by the combination of disodium guanylate and disodium inosinate is widely used in seasonings such as chicken essence and soy sauce. In the pharmaceutical field, guanosine and inosine can be used as pharmaceutical intermediates for a variety of antiviral drugs, such as acyclovir, ribavirin, sodium guanosine triphosphate, etc. Inosine is an important precursor of inosinic acid, and inosinic acid can be used as a precursor for the synthesis of adenylic acid (AMP) and guanylic acid (GMP), and is applicable to leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, cirrhosis, etc. caused by various reasons. In addition, it can also treat central retinitis, optic atrophy, etc. Adenosine is an endogenous nucleoside distributed throughout human cells, can directly enter the myocardium and be phosphorylated to generate adenylic acid, participate in myocardial energy metabolism, and at the same time also participate in dilating coronary blood vessels and increasing blood flow. Adenosine has important physiological effects on the cardiovascular system and many other systems and tissues of the body. In addition to being used as a specific drug for treating heart diseases, it is also an important intermediate for the synthesis of adenosine triphosphate (ATP), adenine, adenylic acid, vidarabine, and is widely used in industries such as medicine.

[0004] At present, the microbial fermentation method is the main method for producing nucleosides, and the main microorganisms used include Bacillus subtilis, Bacillus amyloliquefaciens or Bacillus pumilus, etc. At present, the fermentation performance of nucleoside-producing strains is still poor and cannot meet the requirements of large-scale industrial production. Therefore, it is still necessary to develop new metabolic engineering transformation targets and strains related to nucleoside production. Summary of the Invention

[0005] The present invention provides a recombinant microorganism enhanced with a sulfur amino acid ABC transporter or its homologous protein and its application in nucleoside production.

[0006] The present invention discovers that the sulfur-containing amino acid ABC transporter (L-cystine transportsystem permease protein) TcyM of Bacillus subtilis promotes the accumulation of nucleosides, and strengthening the sulfur-containing amino acid ABC transporter TcyM can significantly increase the nucleoside production of the strain. Further discovery shows that the homologous protein of the sulfur-containing amino acid ABC transporter TcyM also has the above effect, and by strengthening the homologous protein of the sulfur-containing amino acid ABC transporter TcyM, the nucleoside production of the strain can also be increased.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides any one of the following enhanced applications of the sulfur-containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein:

[0009] (1) Application in improving the ability of microorganisms to produce nucleosides or their derivatives;

[0010] (2) Application in constructing microorganisms for producing nucleosides or their derivatives.

[0011] In the above applications, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Escherichia, or a bacterium of the genus Corynebacterium.

[0012] Among them, the bacteria of the genus Bacillus include Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus megaterium; the bacteria of the genus Corynebacterium include Corynebacterium glutamicum; the bacteria of the genus Escherichia include Escherichia coli.

[0013] In some embodiments of the present invention, the microorganism is Bacillus subtilis or Bacillus amyloliquefaciens.

[0014] In the above applications, the improvement of the ability of microorganisms to produce nucleosides or their derivatives includes increasing the production and / or conversion rate of nucleosides or their derivatives by microorganisms.

[0015] In the present invention, the nucleosides include purine nucleosides and pyrimidine nucleosides. Preferably purine nucleosides. The purine nucleosides include adenosine, guanosine, inosine.

[0016] In the present invention, the nucleoside derivatives include but are not limited to hypoxanthine, guanine, guanylic acid, riboflavin, diacetyl guanylic acid, inosinic acid, adenylic acid, adenosine triphosphate (ATP), adenine, vidarabine, etc.

[0017] Preferably, the enhancement is to increase the expression level of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein, and / or to increase the activity of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein.

[0018] The increase in the expression level includes an increase at the transcriptional level and / or the translational level.

[0019] In the present invention, by enhancing the expression of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein in the strain, the nucleoside production capacity of the strain is significantly improved, and the yield of nucleosides is increased. The enhancement of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein can be used to construct a recombinant strain for producing nucleosides to increase the nucleoside yield of the strain.

[0020] Specifically, the enhancement can be achieved by any one or more of the following ways (1)-(7):

[0021] (1) Enhancement by introducing a plasmid containing the coding gene;

[0022] (2) Enhancement by increasing the copy number of the coding gene on the chromosome;

[0023] (3) Enhancement by enhancing the activity of the promoter of the coding gene;

[0024] (4) Enhancement by enhancing the activity of the RBS sequence of the coding gene;

[0025] (5) Enhancement by changing the amino acid sequence of the protein;

[0026] (6) Enhancement by replacing the start codon of the coding gene with ATG;

[0027] (7) Enhancement by changing the nucleotide sequence of the coding gene.

[0028] In some embodiments of the present invention, the enhancement is achieved by enhancing the activity of the promoter of the coding gene. Preferably, the original promoter of the coding gene is replaced with a strong promoter (such as the P43 promoter), and / or the enhancement is achieved by mutating the 49th position of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein from K to E and / or mutating the 58th position from T to F, and / or the enhancement is achieved by mutating the start codon of the coding gene from TTG to ATG.

[0029] In the present invention, the homologous protein is a protein that is derived from different strains or different species of microorganisms than TcyM, has a similar function, and has a high similarity. Preferably, the amino acid sequence similarity of the homologous protein to TcyM is not less than 60%, more preferably not less than 70%, and even more preferably not less than 75%.

[0030] For the sequence of the sulfur amino acid ABC transporter TcyM or its homologous protein, those skilled in the art can obtain it through publicly available databases. Among them, the reference sequence number of the wild-type sulfur amino acid ABC transporter TcyM of Bacillus subtilis is CAB14895.1, its amino acid sequence is shown in SEQ ID NO.2, the nucleotide sequence of the encoding gene (tcyM gene) is shown in SEQ ID NO.1, the reference sequence number of the homologous protein of TcyM in Bacillus amyloliquefaciens is CBI44333.1, its amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0031] Preferably, the amino acid sequence of the sulfur amino acid ABC transporter TcyM is shown in SEQ ID NO.2, and / or the amino acid sequence of the homologous protein is shown in SEQ ID NO.4.

[0032] Based on the above new uses of the sulfur amino acid ABC transporter TcyM or its homologous protein, the present invention has also developed variants of the sulfur amino acid ABC transporter TcyM or its homologous protein, and these variants have enhanced functions of the sulfur amino acid ABC transporter TcyM or its homologous protein.

[0033] In a second aspect, the present invention provides variants of the sulfur amino acid ABC transporter TcyM or its homologous protein. Compared with the wild-type protein of Bacillus corresponding to their source, the variants of the sulfur amino acid ABC transporter TcyM or its homologous protein contain any one or more of the following mutations: K49E, T58F.

[0034] The present invention has found that mutating the 49th position of the sulfur amino acid ABC transporter TcyM or its homologous protein from K to E and the 58th position from T to F can enhance the sulfur amino acid ABC transporter TcyM or its homologous protein, significantly improve the nucleoside production capacity of the strain, and thus increase the yield of nucleosides.

[0035] Specifically, the amino acid sequence of the wild-type protein of Bacillus described above is shown in SEQ ID NO.2 or 4.

[0036] Preferably, the amino acid sequence of the variant of the sulfur amino acid ABC transporter TcyM is shown in SEQ ID NO.6 or 8.

[0037] The amino acid sequence of the variant of the homologous protein is as shown in SEQ ID NO.11 or 13.

[0038] The above-mentioned variant can significantly improve the nucleoside (especially purine nucleoside) production ability of microorganisms (especially bacteria of the genus Bacillus), and significantly promote the increase in nucleoside production.

[0039] In a third aspect, the present invention provides a nucleic acid molecule encoding the variant of the sulfur-containing amino acid ABC transporter TcyM or its homologous protein described above.

[0040] According to the amino acid sequence of the protein variant and codon rules, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the protein variant. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding a protein variant is not unique, and all nucleic acid molecules capable of encoding the above-mentioned protein variant are within the protection scope of the present invention.

[0041] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.5, 7, 10 or 12.

[0042] In a fourth aspect, the present invention provides a biological material comprising the nucleic acid molecule or expressing the variant of the sulfur-containing amino acid ABC transporter TcyM or its homologous protein.

[0043] The above-mentioned biological materials include expression cassettes, vectors or host cells.

[0044] Wherein, the expression cassette is a recombinant nucleic acid molecule obtained by operably connecting the nucleic acid molecule with transcriptional or translational regulatory elements.

[0045] The vectors include but are not limited to plasmid vectors, viral vectors, transposons.

[0046] The host cells include microbial cells. Preferably, they are bacteria of the genus Bacillus, bacteria of the genus Corynebacterium, and bacteria of the genus Escherichia.

[0047] In a fifth aspect, the present invention provides a recombinant microorganism, which is modified such that the sulfur-containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein therein is enhanced.

[0048] Preferably, the recombinant microorganism is a nucleoside-producing recombinant microorganism.

[0049] Compared with its parental strain, the recombinant microorganism has increased nucleoside production.

[0050] Preferably, compared with its parental strain, the recombinant microorganism has at least 10%, at least 20%, at least 30%, at least 50%, at least 80% or at least 100% higher nucleoside production.

[0051] Preferably, the microorganism is a bacterium of the genus Bacillus, Escherichia or Corynebacterium.

[0052] Among them, the bacteria of the genus Bacillus include Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus megaterium; the bacteria of the genus Corynebacterium include Corynebacterium glutamicum; the bacteria of the genus Escherichia include Escherichia coli.

[0053] In some embodiments of the present invention, the recombinant microorganism is recombinant Bacillus subtilis or recombinant Bacillus amyloliquefaciens.

[0054] The above enhancement is to increase the expression level of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein, and / or to increase the activity of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein.

[0055] Preferably, the enhancement is achieved by any one or more of the following (1) - (7):

[0056] (1) Enhancement by introducing a plasmid containing the coding gene;

[0057] (2) Enhancement by increasing the copy number of the coding gene on the chromosome;

[0058] (3) Enhancement by enhancing the activity of the promoter of the coding gene;

[0059] (4) Enhancement by enhancing the activity of the RBS sequence of the coding gene;

[0060] (5) Enhancement by changing the amino acid sequence of the protein;

[0061] (6) Enhancement by replacing the start codon of the coding gene with ATG;

[0062] (7) Enhancement by changing the nucleotide sequence of the coding gene.

[0063] In some embodiments of the present invention, the enhancement is achieved by enhancing the activity of the promoter of the coding gene. Preferably, the original promoter of the coding gene is replaced with a strong promoter (such as the P43 promoter), and / or the enhancement is achieved by mutating the 49th position of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein from K to E and / or mutating the 58th position from T to F, and / or the enhancement is achieved by mutating the start codon of the coding gene from TTG to ATG.

[0064] Preferably, the recombinant microorganism is modified to express a variant of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein as described above, or the recombinant microorganism is modified to enhance the activity of the promoter of the coding gene, or the recombinant microorganism is modified to replace the start codon of the coding gene with ATG.

[0065] In some embodiments of the present invention, the recombinant microorganism uses Bacillus subtilis 168 or A5 (for the construction method of B. subtilis A5, see patent CN110257315B, which has certain adenosine and inosine production capabilities) as the starting strain, and is modified to enhance the sulfur - containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein therein.

[0066] In some embodiments of the present invention, the recombinant microorganism uses Bacillus amyloliquefaciens DSM7, B.s833 or B.a 836 (for the construction methods of B.s833 and B.a 836, see patent application CN112574934A, which has certain guanosine or inosine production capabilities) as the starting strain, and is modified to enhance the sulfur - containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein therein.

[0067] In the present invention, by enhancing the sulfur - containing amino acid ABC transporter TcyM or its homologous protein in wild - type Bacillus subtilis, wild - type Bacillus amyloliquefaciens, and Bacillus subtilis and Bacillus amyloliquefaciens with nucleoside production capabilities respectively, the nucleoside production capacity of the strains is significantly improved, enabling the strains to accumulate nucleosides more efficiently and rapidly, demonstrating that the enhancement of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein has universality for promoting nucleoside accumulation in different strains.

[0068] In the sixth aspect, the present invention provides a method for constructing the above - described recombinant microorganism, the method comprising: modifying a microorganism to enhance the sulfur - containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein therein.

[0069] In the seventh aspect, the present invention provides any one of the following applications of the variant of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein, or the nucleic acid molecule, or the biological material, or the recombinant microorganism as described above:

[0070] (1) Application in the production of nucleosides or their derivatives;

[0071] (2) Use in constructing a microorganism for producing a nucleoside or its derivative.

[0072] The present invention also provides the use of the sulfur-containing amino acid ABC transporter TcyM or a variant of its homologous protein, or the nucleic acid molecule, or the biological material, or the recombinant microorganism as described above in improving the nucleoside production ability of a microorganism.

[0073] Preferably, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Corynebacterium, or a bacterium of the genus Escherichia. Among them, the bacteria of the genus Bacillus include Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium; the bacteria of the genus Corynebacterium include Corynebacterium glutamicum; and the bacteria of the genus Escherichia include Escherichia coli.

[0074] In the eighth aspect, the present invention provides a method for constructing a recombinant microorganism producing a nucleoside, the method comprising: modifying a microorganism so that the sulfur-containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein is strengthened;

[0075] Preferably, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Escherichia, or a bacterium of the genus Corynebacterium.

[0076] In the ninth aspect, the present invention provides a method for fermentatively producing a nucleoside or a nucleoside derivative, the method comprising: culturing the recombinant microorganism to obtain a culture, and collecting the nucleoside or the nucleoside derivative from the culture.

[0077] In some embodiments of the present invention, the method comprises: inoculating the recombinant microorganism into a seed medium for seed culture to obtain a seed liquid, inoculating the seed liquid into a fermentation medium for culture to obtain a fermentation liquid, and separating and extracting the nucleoside or its derivative from the fermentation liquid.

[0078] In the tenth aspect, the present invention provides a method for increasing the yield of a nucleoside or its derivative, the method comprising: modifying a microorganism so that the sulfur-containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein is strengthened.

[0079] The beneficial effects of the present invention at least include: by strengthening the sulfur-containing amino acid ABC transporter TcyM or its homologous protein, the present invention significantly improves the nucleoside production ability and production efficiency of a microorganism, providing new transformation targets and strategies for the construction of nucleoside-producing strains. The sulfur-containing amino acid ABC transporter TcyM or its homologous protein variant provided by the present invention can effectively promote the accumulation of nucleosides by microorganisms, significantly enhancing the nucleoside production ability of microorganisms. The nucleoside yield of the recombinant microorganism provided by the present invention is significantly higher than that of the starting strain, which is beneficial to improving the production efficiency of nucleosides and reducing production costs. Detailed Embodiments

[0080] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0081] In the following embodiments, the protein shown in SEQ ID NO.4 in Bacillus amyloliquefaciens is also named TcyM, which represents the homologous protein of Bacillus subtilis TcyM.

[0082] The primer names and primer sequences involved in the following embodiments are shown in Table 1.

[0083] Table 1 Primer names and sequence information used in the embodiments of the present invention

[0084]

[0085]

[0086] Example 1: TcyM K49E Construction of point-mutated Bacillus subtilis

[0087] Using the genome of adenosine-producing strain Bacillus subtilis (B. subtilis) A5 as a template, the left and right homologous arms were amplified using the primer pairs A5-TcyM49-1f / 1r and A5-TcyM49-2f / 2r, and A5-TcyM K49E Full-length fragment (TcyM K49EThe coding gene sequence is as shown in SEQ ID NO.5, and the amino acid sequence is as shown in SEQ ID NO.6). The pKSU plasmid (the pKSU plasmid was kindly provided by Professor Wang Shufang of Nankai University. See A markerless gene replacement method for B. amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production [J], Applied Microbiology and Biotechnology, 2014, 98(21): 8963-8973. Zhang W, Gao W, Feng J, et al DOI: 10.1007 / s00253-014-5824-2) was digested with XbaI / PstI and recovered by gel extraction. The digested linearized plasmid and the A5-TcyM K49E fragment were assembled using an assembly kit and transformed into TransT1 competent cells. Subsequently, identification and screening were carried out to obtain the recombinant plasmid pKSU-A5-TcyM K49E , which was respectively transformed into the wild-type Bacillus subtilis 168 and the B. subtilis A5 strain. Strains with TcyM K49E point mutations were screened and named B. subtilis A0142 (the starting strain was wild-type 168) and B. subtilis A0143 (the starting strain was B. subtilis A5).

[0088] Example 2: Construction of B. subtilis with TcyM Y58F point mutation

[0089] Using the genome of the adenosine-producing strain B. subtilis A5 as a template, the left and right homologous arms were amplified using the primer pairs A5-TcyM49-1f / A5-TcyM58-1r and A5-TcyM58-2f / A5-TcyM49-2r, and the A5-TcyM Y58F full-length fragment (the coding gene sequence of TcyM Y58F is as shown in SEQ ID NO.7, and the amino acid sequence is as shown in SEQ ID NO.8) was obtained by fusion. The recombinant plasmid pKSU-A5-TcyM Y58F was obtained according to the construction method in Example 1 and was respectively transformed into the wild-type 168 and the B. subtilis A5 strain. Strains with TcyM Y58FPoint mutant strains, named B. subtilis A0144 (the starting strain is wild strain 168) and B. subtilis A0145 (the starting strain is B. subtilis A5) respectively.

[0090] Example 3: Construction of Bacillus subtilis with enhanced start codon of tcyM gene

[0091] Using the genome of adenosine-producing strain B. subtilis A5 as a template, amplify the left and right homologous arms with the primer pairs A5-TcyM1-1f / 1r and A5-TcyM1-2f / 2r, and fuse to obtain A5-tcyM T1A Full-length fragment (the start codon TTG of the tcyM gene is mutated to ATG, and the mutated nucleotide sequence is shown in SEQ ID NO.9). Obtain the recombinant plasmid pKSU-A5-tcyM according to the construction method in Example 1 T1A , and transform it into wild strain 168 and B. subtilis A5 strains respectively, and screen to obtain strains with the start codon TTG of the tcyM gene mutated to ATG. The strains are named B. subtilis A0146 (the starting strain is wild strain 168) and B. subtilis A0147 (the starting strain is B. subtilis A5) respectively.

[0092] Example 4: Construction of Bacillus subtilis with P43 promoter inserted into tcyM gene

[0093] Using the genome of adenosine-producing strain B. subtilis A5 as a template, the left and right homologous arms were amplified using the primer pairs A5-P43-TcyM-1f / 1r and A5-P43-TcyM-3f / 3r. The P43 promoter fragment was amplified using P43-F / P43-R with the PBE43 plasmid (the PBE43 plasmid was synthesized by total gene synthesis, reference: Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens) as a template. Using the three fragments obtained above as templates and A5-P43-TcyM-1f / 3r as primers, the three fragments were subjected to fusion PCR to obtain the full-length fragment A5-P43-TcyM. The recombinant plasmid pKSU-A5-P43-TcyM was obtained according to the construction method in Example 1 and was transformed into the wild strain 168 and B. subtilis A5 strain respectively. Strains in which the transcription of the tcyM gene was initiated by the P43 promoter were screened and named B. subtilis A0148 (the starting strain was wild strain 168) and B. subtilis A0149 (the starting strain was B. subtilis A5).

[0094] Example 5: TcyM K49E Construction of point-mutated Bacillus amyloliquefaciens

[0095] Using the genome of the Bacillus amyloliquefaciens model strain DSM7 as a template, the left and right homologous arms were amplified using the primer pairs TcyM49-1f / 1r and TcyM49-2f / 2r, and TcyM was obtained by fusion K49E Full-length fragment (the coding gene sequence of TcyM K49E is shown in SEQ ID NO.10, and the amino acid sequence is shown in SEQ ID NO.11). The recombinant plasmid pKSU-TcyM was obtained according to the construction method in Example 1 K49E and was transformed into the DSM7 strain and two guanosine-producing strains B.s833 and B.a836 respectively. Strains with TcyM K49E point mutations were screened, and the obtained strains were named B.a8483, B.s8484, and B.a8485 respectively.

[0096] Example 6: TcyM Y58F Construction of point-mutated Bacillus amyloliquefaciens

[0097] Using the genome of the Bacillus amyloliquefaciens type strain DSM7 as a template, the left and right homologous arms were amplified using the primer pairs TcyM49-1f / TcyM58-1r and TcyM58-2f / TcyM49-2r, and TcyM was obtained by fusion. Y58F The full-length fragment (the coding gene sequence of TcyM Y58F is shown in SEQ ID NO.12, and the amino acid sequence is shown in SEQ ID NO.13). The recombinant plasmid pKSU-TcyM Y58F was obtained according to the construction method in Example 1 and was respectively transformed into the DSM7 strain and two guanosine-producing strains B.s833 and B.a 836, and strains with TcyM Y58F point mutations were screened, and the obtained strains were named B.a8486, B.s8487, and B.a8488 respectively.

[0098] Example 7: Construction of Bacillus amyloliquefaciens with enhanced tcyM gene start codon

[0099] Using the genome of the Bacillus amyloliquefaciens type strain DSM7 as a template, the left and right homologous arms were amplified using the primer pairs TcyM1-1f / 1r and TcyM1-2f / 2r, and the full-length tcyM T1A fragment was obtained by fusion (the start codon TTG of the Bacillus amyloliquefaciens tcyM gene was mutated to ATG, and the mutated nucleotide sequence is shown in SEQ ID NO.14). The recombinant plasmid pKSU-tcyM T1A was obtained according to the construction method in Example 1 and was respectively transformed into the DSM7 strain and two guanosine-producing strains B.s833 and B.a836, and strains with the start codon TTG of the tcyM gene mutated to ATG were screened. The strains were named B.a8489, B.s8490, and B.a8491 respectively.

[0100] Example 8: Construction of Bacillus amyloliquefaciens with the P43 promoter inserted into the tcyM gene

[0101] Using the genome of Bacillus amyloliquefaciens type strain DSM7 as a template, the left and right homologous arms were amplified using the primer pairs P43-TcyM-1f / 1r and P43-TcyM-3f / 3r, and the P43 promoter fragment obtained in Example 4 was used; using the above three obtained fragments as templates and P43-TcyM-1f / 3r as primers, the three fragments were subjected to fusion PCR to obtain the full-length fragment P43-TcyM. The recombinant plasmid pKSU-P43-TcyM was obtained according to the construction method in Example 1 and transformed into the DSM7 strain and two guanosine-producing strains B.s833 and B.a836. Strains in which the transcription of the tcyM gene was initiated by the P43 promoter were screened and obtained, and the obtained strains were named B.a8492, B.s8493, and B.a8494, respectively.

[0102] Example 9: Verification of tcyM expression levels in each mutant strain by real-time quantitative fluorescence PCR

[0103] All mutant strains with tcyM modification constructed in Examples 1-8 and the control strains Bacillus subtilis 168, Bacillus subtilis A5 (the control strain corresponding to the Bacillus subtilis mutant strain), and DSM7, B.s833, B.a836 (the control strain corresponding to the Bacillus amyloliquefaciens mutant strain) were cultured in LB medium until the logarithmic growth phase. 1 mL of the bacterial solution was taken, treated with an appropriate amount of lysozyme, and then total RNA was extracted for reverse transcription. Real-time quantitative PCR reaction was carried out using cDNA as a template. Reaction conditions: pre-denaturation at 95 °C for 10 min; 95 °C for 15 s, 55 °C for 1 min, 40 cycles. After the reaction was completed, using bacterial 16S rRNA as a reference, the transcription levels of related genes were calculated according to the 2 -ΔΔCT method. The results are shown in Table 2.

[0104] Table 2 tcyM transcription levels of each strain

[0105]

[0106]

[0107] The above results show that compared with their corresponding starting strains, the transcription levels of tcyM in each modified strain have been improved to varying degrees, indicating that the above modifications have all achieved the effect of enhancing the transcription level of the tcyM gene.

[0108] Example 10: Verification of the nucleoside fermentation performance of mutant strains

[0109] All the tcyM - modified mutant strains constructed in Examples 1 - 8 and the control strains B. subtilis 168, B. subtilis A5 (the control strain corresponding to the B. subtilis mutant strain), and DSM7, B.s833, B.a836 (the control strains corresponding to the B. amyloliquefaciens mutant strain) were subjected to fermentation experiments to test their nucleoside production performance. The specific steps are as follows:

[0110] 1. The strains preserved in glycerol were cultured overnight at 37°C, and single colonies were streaked out.

[0111] 2. Single colonies were picked and inoculated into 30 mL of seed medium (glucose 20 g / L, yeast powder 5 g / L, corn steep liquor dry powder 5 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 0.02 g / L, manganese sulfate 0.01 g / L, pH 7.0 - 7.2, sterilized at 121°C for 20 min), and cultured at a shaker speed of 110 rpm and 37°C for 7 - 8 h to obtain a seed solution.

[0112] 3. The seed solution was transferred to 30 mL of fermentation medium (glucose 120 g / L, yeast powder 3.5 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 25 g / L, manganese sulfate 0.01 g / L, magnesium sulfate 5 g / L, sodium glutamate 10 g / L, corn steep liquor dry powder 15 g / L, calcium carbonate 25 g / L, pH 7.0 - 7.2, sterilized at 121°C for 20 min) at an inoculation amount of 10% v / v, and cultured at a shaker speed of 130 rpm and 35°C. Among them, the B.s / a8483 - 8494 strain was fermented for 72 h, and the B. subtilis A0142 - 0149 strain was fermented for 48 h.

[0113] 4. The content of nucleosides in the fermentation broth was detected using a liquid chromatograph, and the cell biomass of the fermentation broth was measured simultaneously. The results are shown in Table 3.

[0114] Table 3 Detection results of guanosine, inosine, and adenosine production by mutant strains in shake - flask fermentation (average value of three replicates)

[0115]

[0116] The above results show that for each mutant strain obtained by mutating K at position 49 of TcyM to E, or mutating Y at position 58 to F, or enhancing the start codon, or inserting the P43 strong promoter, the corresponding nucleoside yields have all increased to varying degrees; it shows that the enhancement of TcyM is effective in improving the nucleoside production ability of the strain.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Any of the following enhanced applications of the sulfur - containing amino acid ABC transporter TcyM, its homologous protein, or the coding gene of said protein: (1) Application in improving the ability of microorganisms to produce nucleosides or their derivatives; (2) Application in constructing microorganisms for producing nucleosides or their derivatives.

2. The application according to claim 1, wherein, the microorganism is a bacterium of the genus Bacillus, Escherichia, or Corynebacterium; preferably, the enhancement is to increase the expression level of the sulfur - containing amino acid ABC transporter TcyM, its homologous protein, or the coding gene of said protein, and / or to increase the activity of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein; more preferably, the amino acid sequence of the sulfur - containing amino acid ABC transporter TcyM is as shown in SEQ ID NO.2, and / or the amino acid sequence of the homologous protein is as shown in SEQ ID NO.

4.

3. A variant of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein, wherein, compared with the Bacillus wild - type protein from which it is derived, the variant of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein contains any one or more of the following mutations: K49E, T58F; preferably, the amino acid sequence of the variant of the sulfur - containing amino acid ABC transporter TcyM is as shown in SEQ ID NO.6 or 8; and / or the amino acid sequence of the variant of the homologous protein is as shown in SEQ ID NO.11 or 13.

4. A nucleic acid molecule, wherein, the nucleic acid molecule encodes the variant of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein according to claim 3.

5. A biological material, wherein, the biological material contains the nucleic acid molecule according to claim 4 or expresses the variant of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein according to claim 3.

6. A recombinant microorganism, wherein, the recombinant microorganism is modified such that the sulfur - containing amino acid ABC transporter TcyM, its homologous protein, or the coding gene of said protein therein is enhanced; preferably, the microorganism is a bacterium of the genus Bacillus, Escherichia, or Corynebacterium.

7. The recombinant microorganism according to claim 6, wherein, the enhancement is to increase the expression level of the sulfur - containing amino acid ABC transporter TcyM, its homologous protein, or the coding gene of said protein, and / or to increase the activity of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein; preferably, the enhancement is achieved by any one or more of the following methods (1) - (7): (1) Enhanced by introducing a plasmid containing the coding gene; (2) Enhanced by increasing the copy number of the coding gene on the chromosome; (3) Enhanced by enhancing the activity of the promoter of the coding gene; (4) Enhanced by enhancing the activity of the RBS sequence of the coding gene; (5) Enhanced by changing the amino acid sequence of the protein; (6) Enhanced by replacing the start codon of the coding gene with ATG; (7) Enhanced by altering the nucleotide sequence of the coding gene; More preferably, the recombinant microorganism is modified to express a variant of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein as claimed in claim 3, or the recombinant microorganism is modified to enhance the activity of the promoter of the coding gene, or the recombinant microorganism is modified to replace the start codon of the coding gene with ATG.

8. Any one of the following applications of the sulfur - containing amino acid ABC transporter TcyM or its homologous protein variant as claimed in claim 3, the nucleic acid molecule as claimed in claim 4, the biological material as claimed in claim 5, or the recombinant microorganism as claimed in claim 6 or 7: (1) Application in the production of nucleosides or their derivatives; (2) Application in constructing a microorganism for producing nucleosides or their derivatives; Preferably, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Corynebacterium or a bacterium of the genus Escherichia.

9. A method for constructing a recombinant microorganism for producing nucleosides, characterized in that the method comprises: modifying a microorganism so that the sulfur - containing amino acid ABC transporter TcyM or its homologous protein or the coding gene of the protein is enhanced; Preferably, the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Escherichia or a bacterium of the genus Corynebacterium.

10. A method for fermentatively producing nucleosides or nucleoside derivatives, characterized in that the method comprises: culturing the recombinant microorganism as claimed in claim 6 or 7 to obtain a culture, and collecting nucleosides or nucleoside derivatives from the culture.

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