Construction method and application of adenosine producing strain

Abstract

The invention provides a construction method and application of a strain for producing adenosine. After an iolF gene for coding inositol transporter in bacillus subtilis or bacillus amyloliquefaciens is modified (weakened or inactivated), the adenosine production capacity of the strain is enhanced compared with that of an unmodified strain. IolF gene deletion mutation, iolF gene initiation codon modification mutation and iolF gene 1111-1113 site mutation from TGG to TAA mutation all cause reduction of the transcription level of the iolF gene, meanwhile, the adenosine production capacity is improved to different degrees, and the improvement effect of the adenosine production capacity caused by the fact that the iolF gene 1111-1113 site mutation from TGG to TAA is the best.

Description

Technical Field

[0001] The invention relates to the fields of bioengineering and microbial fermentation, and in particular to a construction method for an adenosine-producing strain and its application. Background Art

[0002] Nucleoside is a general term for a class of glycosides. Nucleoside is a component of nucleic acid and nucleotide. 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, with 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, cirrhosis, etc. caused by various reasons. In addition, it can also treat central retinitis and optic atrophy.

[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 breeding are 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 are deeply understood, and the strains are 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] The purpose of the present invention is to provide a method for constructing an adenosine-producing strain and its application.

[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a modified microorganism, wherein the activity of inositol transporter of the microorganism is reduced or lost compared to an unmodified microorganism, and the microorganism has enhanced adenosine production capacity compared to an unmodified microorganism.

[0008] In the present invention, the reference sequence number of the inositol transporter in NCBI is NP_391850.2.

[0009] Further, the activity reduction or loss of the inositol transporter in the microorganism is achieved by selecting from the following 1)-4), or an optional combination:

[0010] 1) reducing or losing the inositol transporter by changing the promoter sequence of the gene encoding the inositol transporter;

[0011] 2) reducing or losing the amino acid sequence of the inositol transporter;

[0012] 3) reducing or losing the inositol transporter by changing the nucleotide sequence encoding the inositol transporter;

[0013] 4) Loss by knocking out the coding sequence of the inositol transporter.

[0014] Furthermore, the expression of the gene encoding the inositol transporter protein is reduced or the gene encoding the inositol transporter protein is knocked out by using mutagenesis, site-directed mutagenesis or homologous recombination.

[0015] In a specific embodiment of the present invention, the reduction or loss of inositol transporter activity is achieved by the following A or B:

[0016] A. The start codon of the gene encoding the inositol transporter was mutated from atg to gtg or ttg;

[0017] B. The bases 1111-1113 of the gene encoding inositol transporter mutated from tgg to taa.

[0018] The microorganism can be Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli, etc. For example, the starting strain is B. subtilis 168, B. subtilis A5 (the construction method of strain B. subtilis A5 is shown in CN110257315B).

[0019] In a second aspect, the present invention provides a method for constructing an adenosine-producing strain, the method comprising: utilizing genetic engineering means to inactivate or weaken the gene encoding the inositol transporter in a microorganism having the ability to produce nucleosides.

[0020] The weakening is selected from the following a)-d), or an optional combination thereof:

[0021] a) reducing or losing the inositol transporter by changing the promoter sequence of the gene encoding the inositol transporter;

[0022] b) reducing or losing the inositol transporter by changing the amino acid sequence of the inositol transporter;

[0023] c) reducing or losing the inositol transporter by changing the nucleotide sequence encoding the inositol transporter;

[0024] d) Loss by knocking out the coding sequence of the inositol transporter.

[0025] The weakening method can be selected from at least one of mutagenesis, site-directed mutagenesis, homologous recombination, and the like.

[0026] Preferably, the weakening is achieved by ① or ② as follows:

[0027] ① The start codon of the gene encoding the inositol transporter protein mutated from atg to gtg or ttg;

[0028] ②The 1111-1113 bases of the gene encoding the inositol transporter mutated from tgg to taa.

[0029] In a third aspect, the present invention provides a recombinant bacterium constructed according to the method.

[0030] In a fourth aspect, the present invention provides the use of the microorganism or the recombinant bacteria constructed according to the method in the fermentation production of adenosine or adenosine derivatives or in increasing the fermentation yield of adenosine or adenosine derivatives.

[0031] The adenosine derivatives of the present invention include but are not limited to adenosine triphosphate (ATP), adenine, adenylic acid, and adenosine arabinoside.

[0032] In a fifth aspect, the present invention provides a method for producing adenosine, the method comprising the following steps:

[0033] i) culturing the microorganism or the recombinant bacteria constructed according to the method to obtain a culture;

[0034] ii) collecting the produced adenosine from the culture obtained in step i).

[0035] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0036] The present invention provides a Bacillus subtilis or Bacillus amyloliquefaciens, wherein the iolF gene encoding an inositol transporter is modified (weakened or inactivated) so that the ability of the strain to produce adenosine is enhanced compared with that of an unmodified strain. The iolF gene deletion mutation, the iolF gene start codon modification mutation, and the iolF gene 1111-1113 position mutation from TGG to TAA all cause the iolF gene transcription level to decrease, and the adenosine production ability is improved to varying degrees, wherein the iolF gene 1111-1113 position mutation from TGG to TAA brings the best effect of improving the adenosine production ability. DETAILED DESCRIPTION

[0037] The present invention aims to provide a microorganism for producing nucleosides, wherein the inositol transporter IolF of the microorganism is weakened or inactivated, so that the ability to produce adenosine is enhanced.

[0038] The IolF weakening includes, but is not limited to, promoter replacement, start codon replacement, point mutation, knockout, and the like.

[0039] The weakened IolF can be used to produce adenosine and adenosine-related derivatives, such as adenosine triphosphate (ATP), adenine, adenylic acid, adenosine arabinoside, etc.

[0040] The weakened IolF is applied to Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Escherichia coli, etc.

[0041] The study found that by modifying the inositol transporter (Protein ID: NP_391850.2, encoded by the iolF gene, Gene ID: 937616) of Bacillus subtilis or Bacillus amyloliquefaciens, the microorganism can efficiently generate adenine nucleoside, guanine nucleotide or inosine nucleoside, and successfully created a new microorganism that can efficiently produce adenosine, thereby completing the present invention.

[0042] The present invention is the first to modify the gene in Bacillus subtilis or Bacillus amyloliquefaciens to obtain corresponding mutants capable of efficiently producing adenosine, and successfully create a microorganism capable of efficiently producing adenosine.

[0043] The present invention also provides a method for producing adenosine by using the microorganism of the inositol transporter IolF mutant.

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0045] The amino acid sequence of the wild-type Bacillus subtilis inositol transporter IolF is shown in SEQ ID NO: 1. M1V The amino acid sequence of the mutant IolF is shown in SEQ ID NO: 2 (the start codon of the iolF gene is mutated from atg to gtg). M1L The amino acid sequence of the mutant IolF is shown in SEQ ID NO: 3 (the start codon of the iolF gene is mutated from atg to ttg). W371* The amino acid sequence is shown in SEQ ID NO: 4 (the bases at positions 1111-1113 of the iolF gene mutate from tgg to taa, and the amino acid at position 371 of the IolF protein mutates from tryptophan to nonsense mutation). The nucleotide sequences corresponding to the wild-type IolF protein and the IolF protein mutant are shown in SEQ ID NO: 5-8, respectively.

[0046] The DNA polymerase, DNA purification kit, restriction endonuclease, DNA ligase and other molecular biological reagents used in the present invention 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 / ).

[0047] The names and sequences of the primers involved in the following examples are shown in Table 1. The primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0048] Table 1 Primer names and sequence information

[0049]

[0050] Example 1 Construction of iolF gene deletion mutant strain

[0051] The genome of strain B. subtilis A5 (for the construction method of strain B. subtilis A5, see CN110257315B) was used as a template, and primer pairs △iolF-1f / △iolF-1r, △iolF-2f / △iolF-2r were used, and pfu high-fidelity DNA polymerase was used to amplify the upstream and downstream homologous arms of the iolF gene. The obtained fragments were recovered by gel and fused, and the △iolF fragment was amplified and recovered by gel. 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. The linearized plasmid and △iolF fragment after restriction digestion were assembled using an assembly kit and transformed into TransT1 competent medium, and the recombinant plasmid pKSU-△iolF was obtained by identification and screening later. The strain was transformed into B. subtilis A5, and transformants were screened at 30°C using an LB plate 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 propagated for one generation, and diluted and spread on an LB plate containing 5 μg / mL chloramphenicol to obtain a primary recombinant. The primary recombinant was inoculated into 5 ml LB liquid culture medium, cultured at 42°C 200 rpm for 12 h and propagated for one generation, and diluted and spread on an LB plate containing 0.8 μM 5-FU to screen for a secondary recombinant, and the iolF gene deletion mutant strain was obtained by screening and named A510.

[0052] Example 2 Construction of iolF gene start codon modified mutant strain

[0053] The genome of strain B. subtilis A5 was used as template and iolF A1G -1f / iolF A1G -1r,iolF A1G -2f / iolF A1G-2r primer pair, pfu high-fidelity DNA polymerase amplified the upstream and downstream homologous arms of the iolF gene. The obtained fragments were recovered by gel recovery and fused, and the iolF was amplified. A1G The fragments were recovered by gel recovery. The plasmid pKSU-iolF was constructed according to the construction method in Example 1. A1G and transformed it into B. subtilis A5 strain to obtain a strain in which the start codon of the iolF gene was mutated from ATG to GTG, named A521.

[0054] According to the above method, primer pair iolF A1G -1f / iolF A1G -1r、iolF A1G -2f / iolF A1G -2r is replaced by iolF A1T -1f / iolF A1T -1r、iolF A1T -2f / iolF A1T -2r, a strain in which the start codon of the iolF gene was mutated from ATG to TTG was constructed and named A522.

[0055] Example 3 Construction of a mutant strain in which positions 1111-1113 of the iolF gene were mutated from TGG to TAA

[0056] The genome of strain B. subtilis A5 was used as template and iolF G1113A -1f / iolF G1113A -1r,iolF G1113A -2f / iolF G1113A -2r primer pair, pfu high-fidelity DNA polymerase amplified the upstream and downstream homologous arms of the iolF gene. The obtained fragments were recovered by gel recovery and fused, and the iolF was amplified. G1113A The fragments were recovered by gel recovery. The plasmid pKSU-iolF was constructed according to the construction method in Example 1. G1113A and transformed it into B. subtilis A5 strain to obtain a mutant strain in which the 1111-1113 positions of the iolF gene were mutated from TGG to TAA, which was named A530.

[0057] Example 4 Test of iolF gene transcription level

[0058] The mutant strains A510, A521, A522, A530 obtained in 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 IICode No.RR820A instruction manual, and the primers were iolF-F / iolF-R, and Real Time PCR reaction was performed. The experimental results are shown in Table 2.

[0059] Table 2 Relative values ​​of transcription levels of mutant strains compared with the starting strains

[0060] Relative value of transcription level Decline B. subtilis A5 1 - A510 0 100％ A521 0.52 48％ A522 0.27 72％ A530 0.33 67％

[0061] It can be seen that the transcription level of iolF changed significantly after modification, and the decrease was in the order of B. subtilisA510>A522>A530>A521, that is, after modification by the method described in Examples 1-3, iolF was weakened.

[0062] Example 5 Shake flask fermentation to produce nucleosides

[0063] 1. Take 50uL of glycerol culture stored at -80℃, inoculate it into a test tube containing 5mL LB medium (yeast powder 10g / L, peptone 5g / L, sodium chloride 10g / L, 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 the LB sterile plate, and culture it at 37℃ for 36h to grow a single colony.

[0064] 2. Pick a single colony and inoculate it into 30 mL of seed culture medium (20 g / L glucose, 6 g / L yeast powder, 5 g / L corn steep liquor powder, 2 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, sterilized at 121°C for 20 min), and culture with reciprocating shaking at 34°C and 110 rpm for 5-6 h.

[0065] 3. Transfer the inoculum volume to 30 ml fermentation medium (glucose 80 g / L, yeast powder 3.5 g / L, potassium dihydrogen phosphate 5 g / L, ammonium sulfate 20 g / L, manganese sulfate 0.01 g / L, magnesium sulfate 5 g / L, sodium glutamate 13 g / L, corn steep liquor powder 15 g / L, calcium carbonate 25 g / L, pH 7.2-7.4, sterilize at 121°C for 20 min) at a 10% (volume ratio) inoculum volume, and culture with reciprocating shaking at 35°C and 130 rpm for 48 h.

[0066] 4. Liquid chromatography was used to detect the glycosides produced in the fermentation broth. The experimental results are shown in Table 3.

[0067] Table 3 Shake flask fermentation results

[0068] strains Adenosine (g / L) Inosine (g / L) Guanosine(g / L) OD562 B. subtilis A5 7.6 0.9 0.1 24.9 A510 8.1 1.0 0.2 25.3 A521 8.3 1.1 0.2 24.1 A522 8.2 1.2 0.2 24.2 A530 8.5 1.1 0.1 24.5

[0069] Example 6 Construction of IolF mutant strain in Bacillus subtilis 168 model strain and production of nucleosides

[0070] Using the genome of the model strain B. subtilis 168 as a template, the same method as in Examples 1-3 was used to construct the iolF gene deletion mutant strain B110 (△iolF), the iolF gene start codon modification mutant strain B211 (iolF A1G )、B213(iolF A1T ), and constructed the mutant strain B227 in which the 1111-1113 position of the iolF gene was mutated from TGG to TAA.

[0071] The same method as in Example 5 was used to produce nucleosides by shake flask fermentation. The experimental results are shown in Table 4.

[0072] Table 4 Shake flask fermentation results

[0073]

[0074]

[0075] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A modified microorganism, characterized in that Compared with an unmodified microorganism, the activity of the inositol transporter of the microorganism is reduced or lost, and the microorganism has an enhanced adenosine production capacity compared with an unmodified microorganism; The reference sequence number of the inositol transporter in NCBI is NP_391850.

2.

2. The microorganism according to claim 1, characterized in that The activity reduction or loss of the inositol transporter in the microorganism is achieved by selecting from the following 1)-4), or an optional combination: 1) reducing or losing the inositol transporter by changing the promoter sequence of the gene encoding the inositol transporter; 2) reducing or losing the amino acid sequence of the inositol transporter; 3) reducing or losing the inositol transporter by changing the nucleotide sequence encoding the inositol transporter; 4) Loss by knocking out the coding sequence of the inositol transporter.

3. The microorganism according to claim 2, characterized in that The method of mutagenesis, site-directed mutagenesis or homologous recombination is adopted to reduce the expression of the gene encoding the inositol transporter or knock out the gene encoding the inositol transporter.

4. The microorganism according to claim 1, characterized in that The reduction or loss of inositol transporter activity is achieved by A or B as follows: A. The start codon of the gene encoding the inositol transporter was mutated from atg to gtg or ttg; B. The bases 1111-1113 of the gene encoding inositol transporter mutated from tgg to taa.

5. The microorganism according to any one of claims 1 to 4, characterized in that The microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

6. A method for constructing an adenosine-producing strain, characterized in that: The method comprises: using genetic engineering means to inactivate or weaken the coding gene of the inositol transporter in a microorganism having the ability to produce nucleosides; The weakening is selected from the following a)-d), or an optional combination thereof: a) reducing or losing the inositol transporter by changing the promoter sequence of the gene encoding the inositol transporter; b) reducing or losing the inositol transporter by changing the amino acid sequence of the inositol transporter; c) reducing or losing the inositol transporter by changing the nucleotide sequence encoding the inositol transporter; d) lost by knocking out the coding sequence of the inositol transporter; The reference sequence number of the inositol transporter in NCBI is NP_391850.

2.

7. The method according to claim 6, characterized in that The weakening method is selected from at least one of mutagenesis, site-directed mutagenesis, and homologous recombination; Preferably, the weakening is achieved by ① or ② as follows: ① The start codon of the gene encoding the inositol transporter protein mutated from atg to gtg or ttg; ② The 1111-1113 bases of the gene encoding the inositol transporter mutated from tgg to taa; The microorganism is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus or Escherichia coli.

8. The recombinant bacteria constructed according to the method of claim 6 or 7.

9. Use of the microorganism according to any one of claims 1 to 5 or the recombinant bacterium according to claim 8 in the fermentation production of adenosine or adenosine derivatives or in increasing the fermentation yield of adenosine or adenosine derivatives; The adenosine derivatives include adenosine triphosphate, adenine, adenylic acid, and adenosine arabinoside.

10. A method for producing adenosine, characterized in that: The method comprises the following steps: i) culturing the microorganism according to any one of claims 1 to 5 or the recombinant bacterium according to claim 8 to obtain a culture; ii) collecting the produced adenosine from the culture obtained in step i).

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