Method for improving nucleoside production capacity of microorganisms

Abstract

The invention relates to the technical field of fermentation engineering, in particular to a method for improving nucleoside production capacity of microorganisms. The method comprises the step of improving the expression level of YqjM protein in a microorganism to be edited. The YqjM protein related to the nucleoside production capacity of microorganisms is obtained through research, and the nucleoside production capacity of the microorganisms can be improved by regulating the expression level of the encoding gene of the YqjM protein in the microorganisms. The YqjM protein and the coding gene thereof provided by the invention have important significance in the fields of increasing the nucleoside yield and developing high-nucleoside-yield microorganisms.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a method for improving the ability of microorganisms to produce nucleosides. Background Art

[0002] Nucleoside is a general term for a class of glycosides. It is a component of nucleic acids and nucleotides. Nucleoside is formed by the condensation of D-ribose or DZ-deoxyribose with pyrimidine base or purine base. Nucleoside is generally a colorless crystal, insoluble in common organic solvents, easily soluble in hot water, and has a melting point of 160-240°C. Nucleosides generated from D-ribose are called ribonucleosides, which participate in the composition of RNA, and nucleosides generated from D-α-deoxyribose are called deoxyribonucleosides, which participate in the composition of DNA. D-ribose condenses with adenine, guanine, cytosine, thymine or uracil to generate the corresponding adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, thymine ribonucleoside and uracil ribonucleoside, which are respectively referred to as adenosine (A), guanosine (G), cytidine (C), thymidine (T) and uridine (U).

[0003] Guanosine (guanosine) and inosine (inosine) play a wide range of roles in the food and pharmaceutical industries. In the food field, guanosine and inosine are important precursors of disodium guanylate and disodium inosinate, respectively. Disodium guanylate and disodium inosinate are used in combination as food flavor enhancers, and are widely used in condiments 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, triazole nucleoside, and sodium guanosine triphosphate, which all require guanosine as a synthetic raw material. Inosine is an important precursor of inosinic acid, and inosinic acid can be used as a precursor for the synthesis of adenosine (AMP) and guanylate (GMP), which is suitable for leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis or cirrhosis caused by various reasons, and can also be used to treat central retinitis, optic atrophy, etc.

[0004] Adenosine is adenine nucleoside, and its chemical name is 6-amino-9-β-D-ribofuranosyl-9-hydrogen purine. It is the product of adenine nucleotide dephosphorylation and is an important nucleotide derivative. Adenosine is an endogenous nucleoside that is found throughout human cells. It can directly enter the myocardium and generate adenosine acid through phosphorylation, and participate in myocardial energy metabolism. It also participates in dilating coronary vessels and increasing blood flow. Adenosine has 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 the heart, adenosine is also an important intermediate for the synthesis of adenosine triphosphate (ATP), adenine, adenosine acid, and adenosine arabinoside, and is widely used in the pharmaceutical and other industries.

[0005] At present, microbial fermentation is the main method for producing nucleosides, and the main microorganisms used include Bacillus subtilis, Bacillus amyloliquefaciens or Bacillus pumilus. In the process of selecting and transforming the growing strains, ultraviolet mutagenesis and diethyl sulfate mutagenesis can be used to selectively select high-yielding strains of nucleosides; or according to the metabolic pathways and regulatory mechanisms of nucleotides in bacteria, the genetic background and characteristics of the strains can be deeply understood, and the strains can be purposefully transformed through metabolic engineering methods to obtain production strains with excellent traits and high nucleoside production. However, the fermentation performance of 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

[0006] In order to solve the problems existing in the prior art, the present invention provides a method for improving the ability of microorganisms to produce nucleosides. The ability of microorganisms to produce nucleosides is improved by regulating the expression level of the gene encoding the YqjM protein in the microorganism.

[0007] At present, there is no patent or literature report on the modification of YqjM protein to improve the ability of microorganisms to produce adenosine. The present invention is the first to modify the YqjM protein in Bacillus subtilis or Bacillus amyloliquefaciens to obtain the corresponding mutant, improve the expression level of YqjM protein in microorganisms, so that microorganisms can efficiently produce adenosine, and successfully create microorganisms that can efficiently produce adenosine.

[0008] In a first aspect, the present invention provides a method for improving the ability of a microorganism to produce nucleosides, comprising:

[0009] Improve the expression level of YqjM protein in the microorganism to be edited.

[0010] Furthermore, the YqjM protein comprises the amino acid sequence shown in SEQ ID NO.1.

[0011] Furthermore, the gene encoding the YqjM protein includes the nucleotide sequence shown in SEQ ID NO.2.

[0012] Furthermore, the increasing the expression level of the YqjM protein in the microorganism to be edited is achieved by any one or more of the following methods:

[0013] i) replacing the promoter of the gene encoding the YqjM protein, or adding an enhancer;

[0014] ii) performing gene mutation on the gene encoding the YqjM protein, or changing the amino acid sequence of the YqjM protein;

[0015] iii) increasing the copy number of the gene encoding the YqjM protein.

[0016] Furthermore, the gene mutation includes mutagenesis, point mutation or homologous recombination.

[0017] Further, the promoter is a P43 promoter; and / or,

[0018] The gene mutation includes mutating the 96th amino acid sequence shown in SEQ ID NO.1 to other amino acids; preferably mutating to aspartic acid or glutamic acid.

[0019] The other amino acids described in the present invention are any other amino acids except the 96th amino acid itself.

[0020] Furthermore, the microorganism to be edited is a microorganism capable of producing nucleosides; and / or the nucleosides include one or more of adenosine, guanosine or inosine.

[0021] In a second aspect, the present invention provides a recombinant microorganism, which is edited by the method described above.

[0022] Furthermore, the recombinant microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

[0023] Furthermore, the present invention provides the use of the recombinant microorganism in nucleoside production or in increasing nucleoside yield.

[0024] In a third aspect, the present invention provides a YqjM protein mutant, wherein the YqjM protein mutant is obtained by mutating the 96th position of the amino acid sequence shown in SEQ ID NO.1 to aspartic acid or glutamic acid.

[0025] Furthermore, the nucleotide sequence corresponding to position 96 of the amino acid sequence shown in SEQ ID NO.1 was mutated to gat / gaa.

[0026] In a fourth aspect, the present invention provides a kit comprising the recombinant microorganism or the YqjM protein mutant.

[0027] The present invention further provides the use of the YqjM protein mutant in improving the ability of microorganisms to produce nucleosides.

[0028] The present invention has the following beneficial effects:

[0029] The present invention provides a YqjM protein related to the ability of microorganisms to produce nucleotides. Improving the expression level of the YqjM protein in the microorganism to be edited can effectively improve the ability of the microorganism to produce nucleosides. The present invention verifies this result through experiments. After introducing the artificially synthesized P43 promoter into the coding gene of the YqjM protein, the transcription level of the coding gene of the YqjM protein is increased by 32%, and the adenosine production capacity is increased by 10.4%; and when the 96th amino acid is mutated to aspartic acid and glutamic acid other than glycine, the adenosine production capacity is increased by 7.8% and 5.2%, respectively. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the examples of the present invention were performed according to conventional experimental conditions, such as Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001, or according to the conditions recommended by the manufacturer's instructions.

[0032] The DNA polymerase, DNA purification kit, restriction endonuclease, DNA ligase and other molecular biological reagents involved in this example were purchased from Beijing Quanshijin Company (https: / / www.transgen.com / ), and other biochemical reagents used were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0033] The amino acid sequence of the YqjM protein involved in this embodiment is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0034] The names and sequences of the primers involved in this example are shown in Table 1. The primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0035] Table 1 Primer names and sequence information used in the examples of the present invention

[0036]

[0037]

[0038] Example 1 Construction of YqjM promoter-enhanced strain

[0039] In this example, the genome of strain B. subtilis A5 (the strain is disclosed in patent CN110257315B) was used as a template, and primer pairs YqjM-1f / YqjM-1r, YqjM-2f / YqjM-2r and Pfu high-fidelity DNA polymerase were used to amplify the upstream and downstream homologous arms YqjM-F and YqjM-R of the YqjM gene.

[0040] Using PBE43 plasmid (PBE43 plasmid was fully gene synthesized, refer to Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens) as a template, the P43-YqjM-f / P43-YqjM-r primer pair and pfu high-fidelity DNA polymerase were used to amplify the P43 fragment.

[0041] The YqjM-F, YqjM-R, and P43 fragments were recovered by gel fusion and amplified to obtain the P43-YqjM fragment, which was recovered by gel fusion. The pKSU plasmid (pKSU plasmid was kindly donated 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 double-digested with XbaI / PstI and recovered by gel fusion. The linearized plasmid and P43-YqjM fragment after enzyme digestion were assembled using an assembly kit and transformed into the TransT1 competent medium. The recombinant plasmid pKSU-P43-YqjM was obtained by identification and screening later.

[0042] The recombinant plasmid pKSU-P43-YqjM was transformed into B. subtilis A5 strain, and 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 culture medium, cultured at 42°C 200 rpm for 12 h and passed 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 culture medium, cultured at 42°C 200 rpm for 12 h and passed for one generation, and diluted and spread on LB plates containing 0.8 μM 5-fluorouracil (5-FU) to screen secondary recombinants, and the mutant strain with enhanced YqjM promoter was obtained by screening and named A565.

[0043] Example 2 YqjM G96D Construction of mutant strains

[0044] In this example, the genome of strain B. subtilis A5 was used as a template and YqjM G96D -1f / YqjM G96D -1r,YqjM G96D -2f / YqjM G96D -2r primer pair, pfu high-fidelity DNA polymerase amplified the upstream and downstream homologous arms of the YqjM gene. The obtained fragments were recovered and fused, and the YqjM was amplified. G96D Fragments, for gel recovery.

[0045] The plasmid pKSU-YqjM was constructed according to the construction method in Example 1. G96D and transformed it into B. subtilis A5 strain to obtain a mutant strain in which the 96th amino acid of YqjM was mutated from glycine to aspartic acid, named A567.

[0046] Example 3 YqjM G96E Construction of mutant strains

[0047] In this example, the genome of strain B. subtilis A5 was used as a template and YqjM G96E -1f / YqjM G96E -1r, YqjM G96E -2f / YqjM G96E -2r primer pair, pfu high-fidelity DNA polymerase amplified the upstream and downstream homologous arms of the YqjM gene. The obtained fragments were recovered and fused, and the YqjM was amplified. G96E The fragments were recovered by gel recovery. The plasmid pKSU-YqjM was constructed according to the construction method in Example 1. G96Eand transformed it into B. subtilis A5 strain to obtain a mutant strain in which the 96th amino acid of YqjM was mutated from glycine to glutamate, named A568.

[0048] Example 4 Gene transcription level test

[0049] In this example, the mutant strains A565, A567, A568 obtained in the above examples 1-3 and the starting strain B. subtilis A5 were cultured overnight in LB medium, the cells were collected, and total RNA was extracted according to the instructions of the kit TakaRa RNAiso Plus Code No. 9108. TM RT reagent Kit Code No.RR037A instructions for reverse transcription, transcribe RNA into cDNA, and finally follow the kit TakaRa TB Premix Ex Taq TM The PCR reaction system was prepared according to the instructions of II Code No.RR820A, and the primer pair YqjM-F / YqjM-R was used to perform the Real Time PCR reaction. The data analysis results are shown in Table 2.

[0050] Table 2 Relative values ​​of transcription levels of mutant strains compared with the original strain

[0051]

[0052]

[0053] From the results, it can be seen that the transcription level of YqjM has changed significantly after modification, and the intensity is A565>A567>A568>B.subtilis A5, that is, after modification by the method described in Examples 1-3, the expression level of YqjM is enhanced.

[0054] Example 5: Shake flask fermentation to produce nucleosides

[0055] In this example, the aforementioned B. subtilis A5, A565, A567 and A568 were used for shake flask fermentation to produce nucleosides. The specific process is as follows:

[0056] 1. Take 50uL of glycerol culture stored at -80℃, inoculate it into a test tube containing 5mL LB medium (g / L: yeast powder 10, peptone 5, sodium chloride 10, pH7.0-7.2, sterilized at 121℃ for 20min), and culture it at 37℃ and 220r / min for 16h; pick the bacterial liquid from the test tube, streak it on an LB sterile plate, and culture it at 37℃ for 36h to grow a single colony.

[0057] 2. Pick a single colony and inoculate it into 30 mL of seed culture medium (g / L: glucose 20, yeast powder 6, corn steep liquor powder 5, potassium dihydrogen phosphate 2, magnesium sulfate 0.5, ferrous sulfate 0.02, manganese sulfate 0.01, pH 7.0-7.2, sterilized at 121°C for 20 min), and culture with reciprocating shaking at 34°C and 110 rpm for 5-6 h.

[0058] 3. Transfer the inoculum volume of 10% (volume ratio) to 30 ml fermentation medium (g / L: glucose 80, yeast powder 3.5, potassium dihydrogen phosphate 5, ammonium sulfate 20, manganese sulfate 0.01, magnesium sulfate 5, sodium glutamate 13, corn steep liquor powder 15, calcium carbonate 25, pH 7.2-7.4, sterilize at 121°C for 20 min), and culture with reciprocating shaking at 35°C and 130 rpm for 48 h.

[0059] 4. The glycosides produced in the fermentation broth were detected using a liquid chromatograph (Table 3). The results are shown in Table 3.

[0060] Table 3 Shake flask fermentation results

[0061] strain Adenosine (g / L) Inosine (g / L) Guanosine(g / L) <![CDATA[OD 562 ]]> B. subtilis A5 7.7 0.9 0.1 25.9 A565 8.5 1.1 0.3 24.7 A567 8.3 1.1 0.2 24.9 A568 8.1 1.0 0.3 25.1

[0062] From the results in Table 3, it can be seen that after modification of the YqjM protein (increased expression level), the levels of nucleosides (adenosine, inosine and guanosine) produced by the three recombinant strains were significantly increased at slightly lower OD values.

[0063] Example 6 Construction of YqjM mutant strain in Bacillus subtilis 168 model strain and production of nucleosides

[0064] In this example, the genome of the model strain of Bacillus subtilis 168 was used as a template, and the YqjM promoter-enhanced strain B263 and the YqjM promoter-enhanced strain B263 were constructed according to the same method as in Examples 1-3. G96D Mutant strain B265, construct YqjM G96E Mutant strain B266.

[0065] Using the same method as in Example 5, the data for nucleoside production by shake flask fermentation are as follows.

[0066] Table 4 Shake flask fermentation results

[0067] strain Adenosine (g / L) Inosine (g / L) Guanosine(g / L) <![CDATA[OD 562 ]]> B. subtilis 168 1.8 0.1 0.1 25.5 B263 2.3 0.2 0.2 24.4 B265 2.1 0.3 0.2 25.0 B266 2.1 0.2 0.2 25.2

[0068] From the results in Table 3, it can be seen that after modification of the YqjM protein (increased expression level), the production levels of nucleosides (adenosine, inosine and guanosine) by the three recombinant strains were significantly increased when the OD value was slightly lower than that of the starting strain.

[0069] The present invention respectively uses Bacillus subtilis (wild-type strain, genetically engineered production bacteria) of different genetic backgrounds as the starting strain to construct the above-mentioned modified microorganism. After verifying that the YqjM protein of these starting strains is replaced by the YqjM protein mutant, the nucleoside production efficiency of the strain is significantly high, which shows that the effect of the YqjM protein mutant on the improvement of the nucleoside production capacity of the strain is independent of other genetic modifications contained in the above-mentioned strain, and these genetic modifications are only for the purpose of making the strain have a certain nucleoside production capacity. Therefore, the effect of the YqjM protein mutant on the improvement of the nucleoside production capacity of the strain has universality for the starting strain that can produce nucleosides. Thus, it can be understood by those skilled in the art that the YqjM protein mutant is introduced into other nucleoside-producing strains and can also effectively promote the improvement of nucleoside production.

[0070] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the ability of a microorganism to produce nucleosides, characterized in that: include: Improve the expression level of YqjM protein in the microorganism to be edited.

2. The method according to claim 1, characterized in that The YqjM protein includes the amino acid sequence shown in SEQ ID NO.

1.

3. The method according to claim 1 or 2, characterized in that: The gene encoding the YqjM protein includes the nucleotide sequence shown in SEQ ID NO.

2.

4. The method according to claim 1, characterized in that: The increasing the expression level of the YqjM protein in the microorganism to be edited is achieved by any one or more of the following methods: i) replacing the promoter of the gene encoding the YqjM protein, or adding an enhancer; ii) performing gene mutation on the gene encoding the YqjM protein, or changing the amino acid sequence of the YqjM protein; iii) increasing the copy number of the gene encoding the YqjM protein.

5. The method according to claim 4, characterized in that The promoter is a P43 promoter; and / or, The gene mutation includes mutating the 96th amino acid sequence shown in SEQ ID NO.1 to other amino acids; preferably mutating to aspartic acid or glutamic acid.

6. The method according to any one of claims 1 to 5, characterized in that: The microorganism to be edited is a microorganism capable of producing nucleosides; and / or the nucleosides include one or more of adenosine, guanosine or inosine.

7. A recombinant microorganism, characterized in that The recombinant microorganism is edited by the method according to any one of claims 1 to 6.

8. The recombinant microorganism according to claim 7, characterized in that The recombinant microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

9. A YqjM protein mutant, characterized in that: The YqjM protein mutant is obtained by mutating the 96th position of the amino acid sequence shown in SEQ ID NO.1 to aspartic acid or glutamic acid.

10. Use of the YqjM protein mutant according to claim 9 in improving the ability of microorganisms to produce nucleosides.

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