A recombinant microorganism for producing nucleosides

Abstract

The present application relates to the technical field of microbial engineering, and particularly relates to a recombinant microorganism for producing nucleosides. The recombinant microorganism is obtained by reducing the expression level of spoVFA protein in the microorganism. The expression level of the spoVFA protein in the microorganism can be reduced by a gene mutation method, and the mutation mode can be selected from one or more of the following modes: (1) the first position is mutated into valine or leucine; (2) the 183rd position is mutated into lysine or arginine. The present application finds that the ability of the microorganism to produce nucleosides can be improved by reducing the expression level of the spoVFA protein in the microorganism, and provides multiple mutation modes for reducing the expression level of the spoVFA protein, which has important application value in the field of nucleoside production.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, and in particular to a recombinant microorganism that produces nucleosides. Background Technology

[0002] Nucleosides are a general term for a class of glycosides, which are components of nucleic acids and nucleotides. Nucleosides are formed by the condensation of D-ribose or D2-deoxyribose with pyrimidine or purine bases. They are generally colorless crystals, insoluble in common organic solvents, readily soluble in hot water, and have a melting point of 160–240℃. Nucleosides formed from D-ribose are called ribonucleosides, which participate in the composition of RNA; nucleosides formed 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 form the corresponding adenine ribonucleosides, guanine ribonucleosides, cytosine ribonucleosides, thymine ribonucleosides, and uracil ribonucleosides, which are abbreviated as adenosine (A), guanine (G), cytosine (C), thymine (T), and uridine (U), respectively.

[0003] Guanosine and inosine have wide applications in the food and pharmaceutical industries. In the food sector, guanosine and inosine are important precursors to disodium guanylate and disodium inosinate, respectively. Disodium guanylate and disodium inosinate are used in combination as flavor enhancers, widely applied in condiments such as chicken bouillon and soy sauce. In the pharmaceutical sector, guanosine and inosine serve as intermediates for various antiviral drugs, such as acyclic guanosine, triazole nucleoside, and guanosine triphosphate sodium, all of which require guanosine as a raw material for synthesis. Inosine is an important precursor to inosine monophosphate, which in turn serves as a precursor for the synthesis of adenosine monophosphate (AMP) and guanosine monophosphate (GMP). It is suitable for treating various causes of leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, cirrhosis, and can also be used to treat central retinitis and optic nerve atrophy.

[0004] Adenosine, or adenosine nucleoside, chemically named 6-amino-9-β-D-furanoribosyl-9-hydropurine, is a product of adenosine nucleotide dephosphorylation and an important nucleotide derivative. Adenosine is an endogenous nucleoside found throughout human cells. It can directly enter the myocardium, where it is phosphorylated to adenosine monophosphate, participating in myocardial energy metabolism and also contributing to coronary artery dilation and increased blood flow. Adenosine has physiological effects on the cardiovascular system and many other systems and tissues in the body. Besides being used as a specific drug for treating heart disease, it is also an important intermediate in the synthesis of adenosine triphosphate (ATP), adenine, adenosine monophosphate, and vidarabine, and is widely used in the pharmaceutical and other industries.

[0005] Currently, microbial fermentation is the main method for producing nucleosides, primarily using microorganisms such as Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus pumilus. In the selection and modification of growth strains, high-yielding nucleosides are selectively bred using ultraviolet mutagenesis and diethyl sulfate mutagenesis; alternatively, based on a thorough understanding of the metabolic pathways and regulatory mechanisms of nucleotides in bacteria, and by using metabolic engineering techniques to purposefully modify the strains, superior traits and high nucleosides can be obtained. However, the fermentation performance of current nucleoside strains remains relatively poor, and the conversion rate of nucleosides is still low, failing to meet the demands of large-scale industrial production. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a recombinant microorganism for producing nucleosides.

[0007] This invention is the first to discover the relationship between the expression level of spoVFA protein and the ability of microorganisms to produce nucleosides, namely, that the ability of microorganisms to produce nucleosides can be improved by reducing the expression level of spoVFA protein.

[0008] In a first aspect, the present invention provides a spoVFA mutant, wherein the spoVFA mutant is obtained by mutating the amino acid at position 1 or position 183 of the spoVFA protein.

[0009] Furthermore, the mutation includes any one or more of the following:

[0010] (1) The first position is mutated to valine or leucine;

[0011] (2) The 183rd position is mutated to lysine or arginine.

[0012] Furthermore, the spoVFA protein comprises the amino acid sequence shown in SEQ ID NO.1.

[0013] Furthermore, the gene encoding the spoVFA protein includes the nucleotide sequence shown in SEQ ID NO. 6.

[0014] After mutation, the amino acid sequence of the spoVFA protein becomes the amino acid sequence shown in SEQ ID NO.2-5, and correspondingly, its nucleotide sequence becomes the nucleotide sequence shown in SEQ ID NO.7-10.

[0015] Secondly, the present invention provides a nucleic acid for encoding the aforementioned spoVFA mutant.

[0016] Furthermore, the nucleic acid comprises a nucleotide sequence as described in any one of SEQ ID NO. 7-10.

[0017] The present invention further provides the application of the spoVFA mutant or the nucleic acid in improving the ability of microorganisms to produce nucleosides, or in constructing microorganisms with high nucleoside yield.

[0018] Thirdly, the present invention provides a method for improving the ability of microorganisms to produce nucleosides, comprising:

[0019] Reduce the expression level of spoVFA protein in the microorganism.

[0020] Furthermore, the spoVFA protein comprises the amino acid sequence shown in SEQ ID NO.1.

[0021] Furthermore, the expression level of spoVFA protein in the microorganism is reduced by one or more of the following methods: gene mutation, gene knockout, RNA interference, promoter replacement, or transcriptional repression.

[0022] Furthermore, the gene mutation includes: using mutagenesis, site-directed mutagenesis, or homologous recombination to reduce the expression level of the gene encoding the spoVFA protein in the microorganism.

[0023] Furthermore, the gene mutation includes any one or more of the following:

[0024] (1) The first position is mutated to valine or leucine;

[0025] (2) The 183rd position is mutated to lysine or arginine.

[0026] The present invention further provides recombinant microorganisms prepared by the method.

[0027] Furthermore, the microorganism can be any microorganism capable of producing nucleosides, whether it is a strain that inherently possesses nucleosides production capabilities, a strain that has acquired nucleosides production capabilities through gene editing technology, or a strain whose nucleosides production capabilities have been enhanced through gene editing.

[0028] For example, the strain may be Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus pumilus.

[0029] The present invention further provides a method for producing nucleosides, comprising: fermenting the recombinant microorganisms to obtain nucleosides from the fermentation products.

[0030] The nucleosides described in this invention include one or more of guanosine, adenosine, or inosine.

[0031] The present invention has the following beneficial effects:

[0032] This invention reveals that inhibiting the expression level of spoVFA protein in microorganisms can effectively enhance their ability to produce nucleosides. This invention also provides several spoVFA mutants that can significantly reduce the expression level of spoVFA protein, thereby improving the ability of microorganisms to produce nucleosides. This has significant application value in the field of microbial nucleoside production. Detailed Implementation

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended in the manufacturer's instructions.

[0034] The amino acid sequence of the wild-type Bacillus subtilis spore dipyridine synthase a subunit SpoVFA is shown in SEQ ID NO:1. The mutant SpoVFA... G183R The amino acid sequence is shown in SEQ ID NO:2 (the 183rd amino acid in SpoVFA is mutated from glycine to arginine). Mutant SpoVFA G183K The amino acid sequence is shown in SEQ ID NO:3 (the 183rd amino acid in SpoVFA is mutated from glycine to lysine). Mutant SpoVFA M1V The amino acid sequence is shown in SEQ ID NO:4 (the first amino acid in SpoVFA is mutated from methionine to valine). Mutant SpoVFA M1L The amino acid sequence is shown in SEQ ID NO:5 (the first amino acid of SpoVFA is mutated from methionine to leucine). The nucleotide sequences corresponding to the above wild-type SpoVFA and its mutants are shown in SEQ ID NO:6-10, respectively.

[0035] The DNA polymerase, DNA purification kit, restriction endonuclease, DNA ligase, and other molecular biology reagents used in this invention were purchased from TransGen Biotech Ltd. (https: / / www.transgen.com / ), and other biochemical reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0036] The primer names and primer sequences involved in the following examples are shown in Table 1. Primer synthesis was completed by Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0037] Table 1 Primer names and sequence information used in the embodiments of this invention.

[0038]

[0039]

[0040] Example 1: Construction of SpoVFA knockout mutant strain

[0041] This invention employs a scarless gene editing method to knock out the spoVFA gene encoding the α subunit of spore dipyridine synthase in the genome of the nucleoside-producing bacterium B. subtilis A5 (the construction method of strain B. subtilis A5 is described in patent document: CN110257315B), thereby constructing a SpoVFA knockout mutant strain. The specific process is as follows:

[0042] Using the genome of strain B. subtilis A5 as a template, the upstream and downstream homologous arms of the SpoVFA gene, SpoVFA-F and SpoVFA-R, were obtained by amplification with primer pairs SpoVFA-1f / SpoVFA-1r and SpoVFA-2f / SpoVFA-2r and Pfu high-fidelity DNA polymerase. The SpoVFA-FR and SpoVFA-AF fragments were recovered and fused using a gel, and amplified to obtain the SpoVFA-FR fragment. The fragment was then recovered again. The pKSU plasmid (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 double-digested with XbaI / PstI and recovered using a gel. The linearized plasmid and SpoVFA-FR fragment were assembled using an assembly kit and transformed into TransT1 competent cells. Subsequent identification and screening yielded the recombinant plasmid pKSU-SpoVFA. Transformed into B. subtilis A5 strain, transformants were screened on LB plates containing 2.5 μg / mL chloramphenicol at 30℃. The obtained transformants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h and passaged for one generation. The transformed transformants were then diluted and plated onto LB plates containing 5 μg / mL chloramphenicol to obtain primary recombinants. The primary recombinants were inoculated into 5 ml LB liquid medium, cultured at 42℃ and 200 rpm for 12 h and passaged for one generation. The primary recombinants were then diluted and plated onto LB plates containing 0.8 μM 5-fluorouracil (5-FU) to screen for secondary recombinants. The resulting Spo VFA knockout mutant strain was obtained and named A821.

[0043] Example 2: SpoVFA G183R Construction of mutant strains

[0044] This invention employs a traceless gene editing method to mutate the 183rd amino acid of the spore dipyridine synthase a subunit in the genome of the nucleoside-producing bacterium B. subtilis A5 from glycine to arginine, constructing a SpoVFA. G183R The specific process for mutant strains is as follows:

[0045] Using the genome of strain B. subtilis A5 as a template, SpoVFA was used. G183R -1f / SpoVFA G183R -1r, SpoVFA G183R -2f / SpoVFA G183R Using a -2r primer pair and Pfu high-fidelity DNA polymerase, the upstream and downstream homologous arms of the SpoVFA gene were obtained. The obtained fragments were then gel-cleaved and fused, and amplified to obtain SpoVFA. G183R The fragments were then subjected to gel recovery. The plasmid pKSU-SpoVFA was constructed according to the construction method described in Example 1. G183R The mutant strain was then transformed into B. subtilis A5 to obtain a mutant strain with the amino acid at position 183 of SpoVFA changed from glycine to arginine, which was named A824.

[0046] Example 3: SpoVFA G183K Construction of mutant strains

[0047] This invention employs a traceless gene editing method to mutate the 183rd amino acid of the spore dipyridine synthase a subunit in the genome of the nucleoside-producing bacterium B. subtilis A5 from glycine to lysine, constructing SpoVFA. G183K The specific process for mutant strains is as follows:

[0048] Using the genome of strain B. subtilis A5 as a template, SpoVFA was used. G183K -1f / SpoVFA G183K -1r、SpoVFA G183K -2f / SpoVFA G183K Using a -2r primer pair and Pfu high-fidelity DNA polymerase, the upstream and downstream homologous arms of the SpoVFA gene were obtained. The obtained fragments were then gel-cleaved and fused, and amplified to obtain SpoVFA. G183K The fragments were then subjected to gel recovery. The plasmid pKSU-SpoVFA was constructed according to the construction method described in Example 1. G183K The mutant strain was then transformed into B. subtilis A5 to obtain a mutant strain with the amino acid at position 183 of SpoVFA changed from glycine to lysine, and named A827.

[0049] Example 4: SpoVFA M1V Construction of mutant strains

[0050] This invention employs a traceless gene editing method to mutate the first amino acid of the subunit encoding spore dipyridine synthase a in the genome of the nucleoside-producing bacterium B. subtilis A5 from methionine to valine, constructing SpoVFA. M1VThe specific process for mutant strains is as follows:

[0051] Using the genome of strain B. subtilis A5 as a template, SpoVFA was used. M1V -1f / SpoVFA M1V -1r, SpoVFA M1V -2f / SpoVFA M1V Using a -2r primer pair and Pfu high-fidelity DNA polymerase, the upstream and downstream homologous arms of the SpoVFA gene were obtained. The obtained fragments were then gel-cleaved and fused, and amplified to obtain SpoVFA. M1V The fragments were then subjected to gel recovery. The plasmid pKSU-SpoVFA was constructed according to the construction method described in Example 1. M1V The strain was then transformed into B. subtilis A5 to obtain a mutant strain of SpoVFA with the first amino acid changed from methionine to valine, which was named A829.

[0052] Example 5: SpoVFA M1L Construction of mutant strains

[0053] This invention employs a traceless gene editing method to mutate the first amino acid of the subunit encoding spore dipyridine synthase a in the genome of the nucleoside-producing bacterium B. subtilis A5 from methionine to leucine, constructing SpoVFA. M1L The specific process for mutant strains is as follows:

[0054] Using the genome of strain B. subtilis A5 as a template, SpoVFA was used. M1L -1f / SpoVFA M1L -1r, SpoVFA M1L -2f / SpoVFA M1L Using a -2r primer pair and Pfu high-fidelity DNA polymerase, the upstream and downstream homologous arms of the SpoVFA gene were obtained. The obtained fragments were then gel-cleaved and fused, and amplified to obtain SpoVFA. M1L The fragments were then subjected to gel recovery. The plasmid pKSU-SpoVFA was constructed according to the construction method described in Example 1. M1L The strain was then transformed into B. subtilis A5 to obtain a mutant strain of SpoVFA with the first amino acid changed from methionine to leucine, which was named A833.

[0055] Example 6: Gene Transcription Level Test

[0056] In this invention, the mutant strains obtained in Examples 1-5 and their originating strain B. subtilis A5 were cultured overnight in LB medium, and the bacterial cells were collected. Total RNA was extracted according to the instructions of the TakaRa RNAiso Plus kit (Code No. 9108), and the RNA was extracted according to the instructions of the TakaRa PrimeScript kit. TM RT reagent Kit Code No. RR037A instructions describe reverse transcription to convert RNA into cDNA. Finally, follow the instructions for the TakaRa TB kit. Premix Ex Taq TM The PCR reaction system was prepared according to the IICodeNo.RR820A instruction manual. Real-time PCR was performed using the SpoVFA-F / SpoVFA-R primer pair. The data analysis results are shown in Table 2.

[0057] Table 2. Relative values ​​of transcription levels between mutant strains and the original strain.

[0058] Relative value of transcription level Decline B.subtilis A5 1.0 - A821 0 100％ A824 0.85 15％ A827 0.52 48％ A829 0.35 65％ A833 0.11 89％

[0059] The results show that the transcriptional level of SpoVFA changed significantly after modification, with the intensity in the following order: B. subtilis A5 > A824 > A827 > A829 > A833 > A821. That is, after modification by the methods described in Examples 1-5, the SpoVFA mutants were all weakened.

[0060] Example 7

[0061] This invention utilizes the mutant strains obtained in Examples 1-5 to conduct shake-flask fermentation experiments for nucleoside production, specifically including the following process:

[0062] 1. Take 50 μL of glycerol culture stored at -80℃ and inoculate it into a test tube containing 5 mL of LB medium (g / L: yeast extract 10, peptone 5, sodium chloride 10, pH 7.0-7.2, sterilized at 121℃ for 20 min). Incubate at 37℃ and 220 rpm for 16 h. Pick a piece of the bacterial culture from the test tube, streak it onto a sterile LB agar plate, and incubate at 37℃ for 36 h to grow single colonies.

[0063] 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, sterilize at 121℃ for 20 min), and incubate at 34℃ with shaking at 110 rpm for 5-6 h.

[0064] 3. Transfer 10% (v / v) of the inoculum to 30 ml of 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 lysine 13, corn steep liquor powder 15, calcium carbonate 25, pH 7.2-7.4, sterilized at 121℃ for 20 min), and incubate at 35℃ with shaking at 130 rpm for 48 h.

[0065] 4. The glycosides produced in the fermentation broth were detected using liquid chromatography. Each strain was repeated three times, and the average value of the results was taken. The final results are shown in Table 3.

[0066] Table 3 Results of shake-flask fermentation

[0067] strain Adenosine (g / L) Inosine (g / L) Guanosin (g / L) <![CDATA[OD 562 ]]> B.subtilis A5 7.9 1.0 0.2 26.3 A821 8.4 1.2 0.3 25.7 A824 8.3 1.1 0.2 25.9 A827 8.1 1.3 0.3 26.1 A829 8.4 1.0 0.2 26.5 A833 8.5 1.2 0.3 25.5

[0068] As can be seen from the shake-flask fermentation results in Table 3, the adenosine production of all mutant strains was significantly increased compared with the original strain B. subtilis A5, while the inosine production was also increased to some extent.

[0069] Example 8

[0070] This embodiment describes the construction of a SpoVFA mutant strain based on the Bacillus subtilis 168 model strain and the detection of its nucleoside production ability. The specific procedure is as follows:

[0071] This invention uses the genome of Bacillus subtilis 168 as a template and constructs SpoVFA knockout strain B350 and SpoVFA using the same methods as in Examples 1-5. G183R mutant strain B362, constructing SpoVFA G183K mutant strain B365, constructing SpoVFA M1V Mutant strain B367, constructing SpoVFA M1L Mutant strain B369.

[0072] Using the same method as in Example 7, the nucleoside production data from shake-flask fermentation are as follows.

[0073] Table 4 Results of shake-flask fermentation

[0074]

[0075]

[0076] As shown in Table 4, the shake-flask fermentation results indicate that the SpoVFA mutant strain of the B. subtilis 168 model strain exhibited varying degrees of increased adenosine and inosine production. These results demonstrate that both the nucleoside-producing strain and the model strain used in this application achieved increased nucleoside production after the SpoVFA gene mutation. This indicates that the increased nucleoside production resulting from the SpoVFA gene mutation provided by this invention does not depend on the various gene editing methods employed in the B. subtilis A5 strain. Those skilled in the art will understand that the various gene editing methods employed in the B. subtilis A5 strain were solely for the purpose of enabling it to possess significant nucleoside production capabilities. Therefore, it can be concluded that the increased nucleoside production resulting from the SpoVFA gene mutation provided by this invention is applicable to all strains in the art capable of nucleoside production.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions 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. Use of a spoVFA mutant or a nucleic acid encoding the spoVFA mutant in improving the ability of a microorganism to produce a nucleoside, or in constructing a recombinant microorganism with high nucleoside production. The amino acid sequence of the spoVFA mutant is shown in any one of SEQ ID NO. 2-5. The microorganism is Bacillus subtilis. The nucleoside is adenosine, inosine or guanosine.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid is shown in any one of SEQ ID NO. 7-10.

3. A method for improving the ability of a microorganism to produce a nucleoside, characterized in that, the expression level of a spoVFA protein in the microorganism is reduced; The microorganism is Bacillus subtilis, and the amino acid sequence of the spoVFA protein is shown in SEQ ID NO.

1. The nucleoside is adenosine, inosine or guanosine.

4. The method of claim 3, wherein, The expression level of the spoVFA protein in the microorganism is reduced by gene mutation or gene knockout.

5. The method of claim 4, wherein, The gene mutation is any one of the following: (1) the first mutation is valine or leucine; (2) the 183rd mutation is lysine or arginine.

6. The recombinant microorganism prepared by the method of claim 5.

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