CN116790454A

CN116790454A - Construction method of nucleoside producing strain and method for producing nucleoside

Info

Publication number: CN116790454A
Application number: CN202210261865.8A
Authority: CN
Inventors: 薛婷莉; 吴涛; 胡丹; 栾明月; 姚嘉琪; 张庆帅
Original assignee: Langfang Meihua Bio Technology Development Co Ltd
Current assignee: Langfang Meihua Bio Technology Development Co Ltd
Priority date: 2022-03-16
Filing date: 2022-03-16
Publication date: 2023-09-22

Abstract

The invention provides a construction method of a nucleoside producing strain and a method for producing nucleosides. The glucose/mannose transporter GlcP (coded by the glcP gene) of the bacillus subtilis or the bacillus amyloliquefaciens is modified, so that the activity of the GlcP protein is enhanced, and the microorganism can efficiently and rapidly produce guanosine or inosine, thereby providing an effective means for mass production of the nucleoside and having wide application prospect.

Description

Construction method of nucleoside producing strain and method for producing nucleoside

Technical Field

The invention belongs to the technical field of microbial engineering, and particularly relates to a construction method of a nucleoside production strain and a method for producing nucleosides.

Background

Nucleosides are a generic term for a class of glycosides. Nucleosides are constituents of nucleic acids and nucleotides. The nucleoside is formed by condensing D-ribose or D-Z-deoxyribose with pyrimidine base or purine base. The nucleoside is generally colorless crystals, insoluble in common organic solvents, soluble in hot water and having a melting point of 160-240 ℃. Nucleosides produced from D-ribose are called ribonucleosides, participate in RNA composition, nucleosides produced from D- α -deoxyribose are called deoxyribonucleosides, participate in DNA composition. D-ribose condenses with adenine, guanine, cytosine, thymine, or uracil to form the corresponding adenine ribonucleoside, guanine ribonucleoside, cytosine ribonucleoside, thymine ribonucleoside, and uracil ribonucleoside, which are abbreviated as adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U), respectively.

Guanosine (guanosine) and inosine (inosine) have a wide variety 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, and the combination of disodium guanylate and disodium inosinate is used as a food flavor enhancer, and is widely applied to condiments such as chicken essence, soy sauce and the like. In the field of medicine, guanosine and inosine can be used as medicine intermediates of various antiviral medicines, such as acyclovir, ribavirin, guanosine triphosphate sodium and the like, and guanosine is required to be used as a synthetic raw material. Inosine is an important precursor of inosinic acid, and inosinic acid can be used as a precursor for synthesizing Adenylate (AMP) and Guanylate (GMP), and is suitable for treating leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, liver cirrhosis and the like caused by various reasons, and can also treat central retinitis, optic atrophy and the like.

At present, microbial fermentation is a main method for producing nucleosides, and mainly used microorganisms include bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and the like. In the breeding and transformation process of the growing strain, the nucleoside high-yield strain is directionally bred by using ultraviolet mutagenesis and diethyl sulfate for mutation breeding; or based on the metabolic path and regulation mechanism of nucleotide in bacteria, the genetic background and the characteristics of the strain are deeply known, and the strain is purposefully modified by metabolic engineering means to obtain the production strain with excellent properties and high nucleoside yield. However, the fermentation performance of the current nucleoside strains is still poor, the conversion rate of the nucleosides is still low, and the requirement of large-scale industrial production cannot be met.

Disclosure of Invention

The invention aims to provide a construction method of a nucleoside producing strain and a method for producing nucleosides.

To achieve the object of the present invention, in a first aspect, the present invention provides a modified microorganism whose activity of glucose transporter and/or mannose transporter GlcP (encoded by a GlcP gene) is enhanced as compared to an unmodified microorganism, and which has an enhanced nucleoside production ability as compared to an unmodified microorganism. In the present invention, the microorganism is a Bacillus (Bacillus) or Escherichia) strain, preferably Bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus), escherichia coli (Escherichia coli) or the like, more preferably Bacillus subtilis or Bacillus amyloliquefaciens, such as B.a 8333 strain.

The construction process of the a 8333 strain is as follows (see cn202111266398. X): with strain DSM7 (ATCC 23350, see Genome sequence of B. Amyloquefaciens type strain DSM 7) ^T reveals differences to plant-associated B.amyloquefaciens FZB 42) genome as template, guaB-1f/1r and guaB-2f/3r as primers, 2 fragments were amplified using Phusion super Fidelity polymerase (New England BioLabs). 2 fragments were fused by using the primer guaB-1f/3r to obtain a recombinant fragment (the nucleotide sequence of the ORF region is shown as SEQ ID NO: 1). Will guaB ^L454F Fragment and pKSU plasmid (pKSU plasmid is given by university of south opening Wang Shufang, see A markerless gene replacement method for B. Amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-gamma-glutamic acid production [ J)]，Applied Microbiology and Biotechnology,2014,98 (21): 8963-8973.Zhang W,Gao W,Feng J,et al DOI:10.1007/s 00253-014-5824-2) by SalI/PstI double digestion, assembly, transformation and the like to obtain recombinant plasmid pKSU-guaB ^L454F . Transformants were selected at 30℃with LB plates containing 2.5. Mu.g/mL chloramphenicol, transferred to B.a 836 strain (see CN 112574934A), inoculated into 5mL LB liquid medium, cultured at 42℃at 200rpm for 12 hours and transferred to a generation, and diluted and spread to LB plates containing 5. Mu.g/mL chloramphenicol to obtain primary recombinants; inoculating the primary recombinant into 5ml LB liquid medium, culturing at 42 deg.C and 200rpm for 12 hr, transferring to primary, diluting and coating LB plate containing 0.8 μm 5-FU for screening secondary recombinant, and screening to obtain guaB ^L454F The strain obtained was b.a 837. The plasmid pBE43 was extracted (PBE 43 plasmid was total gene synthesis, reference Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens), linearized using KpnI/SalI, fusion PCR of the mutated purR sequence (abbreviated as R2, SEQ ID NO: 2) and mutated tal sequence (abbreviated as L5, SEQ ID NO: 3) to obtain a fragment R2+L5, and ligated into the linearized PBE43 plasmid using an assembly kit to construct the plasmid PBE43-R2+L5. This plasmid was transformed into strain b.a 837 to give the starting strain b.a 8333.

The enhancement of glucose transporter and/or mannose transporter activity is achieved by a member selected from the group consisting of 1) to 6), or an optional combination of:

1) Enhanced by introducing a plasmid having a gene encoding the protein;

2) Enhancement by increasing the copy number of the gene encoding the protein on the chromosome;

3) Enhanced by altering the promoter sequence of the gene encoding the protein on the chromosome;

4) Enhanced by operably linking a strong promoter to a gene encoding said protein;

5) Enhancement by modification of the amino acid sequence of the protein;

6) Enhanced by altering the nucleotide sequence of the gene encoding the protein.

Further, the enhancement of glucose transporter and/or mannose transporter activity is achieved by inserting a P43 promoter upstream of the glcP gene start codon.

Further, enhancement of glucose transporter and/or mannose transporter activity is achieved by insertion of a two-copy glcP gene driven by the P43 promoter at the alpha amylase gene.

Further, the enhancement of glucose transporter and/or mannose transporter activity is achieved by inserting a P43 promoter upstream of the start codon of the glcP gene, and inserting two copies of the glcP gene driven by the P43 promoter at the alpha amylase gene.

Further, the enhancement of the glucose transporter and/or mannose transporter activity is achieved by inserting a P43 promoter upstream of the start codon of the glcP gene and inserting two copies of the glcP gene driven by the P43 promoter at the alpha amylase gene, and mutating amino acid 206 of the glucose transporter and/or mannose transporter from H to R.

Preferably, the microorganism is bacillus subtilis or bacillus amyloliquefaciens.

In the present invention, the glucose/mannose transporter GlcP has the reference sequence number ks08_00900 at NCBI, or an amino acid sequence having a 90% similarity thereto. The alpha amylase gene has a reference sequence number of K08_ 07940, or a nucleotide sequence with 97% similarity thereto, at NCBI.

In a second aspect, the present invention provides a method of constructing a nucleoside producing strain, the method comprising: the gene glcP in the microorganism having nucleoside producing ability is enhanced by genetic engineering means to obtain a strain having enhanced glucose transporter and/or mannose transporter activity.

The enhanced pathway is selected from the following 1) to 6), or an optional combination:

1) Enhanced by introducing a plasmid having a gene encoding the protein;

5) Enhancement by modification of the amino acid sequence of the protein;

In a third aspect, the present invention provides a method of producing nucleosides, the method comprising the steps of:

a) Culturing the microorganism to obtain a culture of the microorganism;

b) Collecting the produced nucleosides from the culture obtained in step a).

The nucleoside includes inosine, guanosine, etc. or corresponding nucleoside derivatives such as inosine, inosinic acid, guanosine, guanylic acid, riboflavin, diacetylguanylic acid, etc.

In a fourth aspect, the present invention provides the use of the modified microorganism or nucleoside producing strain constructed according to the method described above in the fermentative production of nucleosides or in improving the yield of nucleoside fermentation.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

compared with the original strain B.a 8333, the guanosine yield of the engineering strain B.a 8334 (the strong promoter P43 is inserted before the glcP gene initiation codon ATG) is improved from 9.8g/L to 11.5g/L, and the glycoside conversion rate is improved by 1.5%. Compared with the original strain B.a 8333, the yield of guanosine of the engineering strain B.a8335 (the glcP gene double copy containing the P43 promoter is inserted at the KS08_07940 gene) is improved from 9.8g/L to 12.0g/L, and the glycoside conversion rate is improved by 2.6%. And the two strengthening modes are overlapped to obtain a mutant strain B.a 8336, the guanosine yield of the mutant strain is improved from 12.0g/L to 13.0g/L, and the glucoside conversion rate is improved by 1%.

Detailed Description

The present invention aims to provide a method for producing purine nucleosides by using a microorganism, and a novel microorganism capable of efficiently producing purine nucleosides and a method for constructing the same, which are used in the method.

It has been found that the glucose/mannose transporter GlcP (encoded by the glcP gene) of Bacillus subtilis or Bacillus amyloliquefaciens is modified so that the GlcP protein activity is enhanced, enabling a microorganism to produce guanosine or inosine with high efficiency and rapidity, and a novel microorganism capable of producing nucleoside with high efficiency has been successfully created, thereby completing the present invention.

According to the invention, the corresponding mutant can be obtained by inserting a strong promoter in front of an original promoter or ectopic increasing the copy number in the bacillus amyloliquefaciens for the first time, so that the nucleoside can be efficiently produced, and a microorganism capable of efficiently producing the nucleoside is successfully constructed.

The invention adopts the following technical scheme:

the invention provides bacillus amyloliquefaciens, which is characterized in that a P43 strong promoter is inserted before a glcP start codon of a gene encoding glucose/mannose transporter in cells, and/or two copies of the glcP gene containing the P43 promoter are inserted at a KS08_07940 gene. The introduction of the corresponding promoter sequences into the nucleoside producing strain by genetic engineering means allows the ability of the strain to produce nucleosides to be enhanced compared to the unmodified strain.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

The primers used in the following examples are shown in Table 1:

TABLE 1

EXAMPLE 1 construction of glcP promoter-enhanced Strain

Using Bacillus amyloliquefaciens ATCC13952 genome as a template, and using a P43-glcP-1f/P43-glcP-1r, P43-glcP-2f/P43-glcP-2r primer pair to obtain upstream and downstream homology arms of glcP by pfu high-fidelity DNA polymerase amplification; the P43 fragment was obtained by amplification using the P43-F1/P43-R1 primer and the PBE43 plasmid (PBE 43 plasmid is total gene synthesis, reference Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens) as a template. The fragments obtained above were subjected to gel recovery. The three fragments obtained above were fused together with the primer P43-glcP-1f/P43-glcP-2r to obtain a full-length fragment of P43-glcP, and the gel was recovered. The pKSU plasmid (pKSU plasmid is given by the university of south opening Wang Shufang, see A markerless gene replacement method for B. Amyloquefascians LL3 and its use in genome reduction and improvement of poly-. Gamma. -glutamic acid production [ J ], applied Microbiology and Biotechnology,2014,98 (21): 8963-8973.Zhang W,Gao W,Feng J,et al DOI:10.1007/s 00253-014-5824-2) was double digested with XbaI/PstI and subjected to gel recovery. And assembling the digested linearized plasmid and the P43-glcP fragment by using an assembly kit, converting the linearized plasmid and the P43-glcP fragment into a TransT1 competence, and performing identification and screening at a later stage to obtain the recombinant plasmid pKSU-P43-glcP. Transferring to B.a 8333 strain, screening transformant with LB plate containing 2.5 mug/mL chloramphenicol at 30 deg.C, inoculating obtained transformant into 5mL LB liquid medium, culturing at 42 deg.C and 200rpm for 12h and transferring to generation, diluting and coating onto LB plate containing 5 mug/mL chloramphenicol to obtain primary recombinant; the primary recombinants were inoculated into 5ml of LB liquid medium, cultured at 42℃at 200rpm for 12 hours and transferred to the first generation, and the secondary recombinants were screened by diluting and coating with LB plates containing 0.8. Mu.M 5-FU, and the glcP promoter-enhanced strain was obtained by screening and named B.a 8334.

EXAMPLE 2 construction of glcP two-copy Gene-enriched Strain

The primer pairs glcP2nd-1f/glcP2nd-1R, P43-glcP-2f/glcP2nd-2R, glcP2nd-3f/glcP2nd-3R and ATCC13952 genome are used as templates, left homology arms glcP2nd-L, glcP and glcP2nd-R fragments are obtained by amplification, and the fragments are subjected to gel recovery. The 4 fragments of glcP2nd-L, the P43-glcP fragment obtained in example 1, glcP and glcP2nd-R were fused using the glcP2nd-1f/glcP2nd-3R primers to obtain a full length fragment of glcP2 nd. The plasmid pKSU-glcP2nd was constructed and transformed into strain B.a 8333 according to the construction method of the plasmid in example 1, and the strain containing two copies of glcP was obtained by screening according to the method of the strain screening in example 1, and designated as B.a8335.

Example 3 construction of glcP two-copy Gene and in situ promoter-enhanced Strain

The plasmid pKSU-P43-glcP obtained in example 1 was transformed using the strain B.a8335 as starting strain, and the positive strain obtained by screening in the method of screening strain in example 1 was designated as B.a 8336.

EXAMPLE 4 construction of glcP Point mutant Strain

The primer pairs glcP-1f/1R and glcP-2f/2R are used, the B.a 8336 genome is used as a template, and the left homology arm glcP-L and the right homology arm glcP-R are obtained through amplification, so that glue recovery is carried out. And (3) taking the obtained gel collection fragment as a template, carrying out fragment fusion on the glcP-1f and the glcP-2r primers to obtain a full-length fragment, wherein the sequence of the ORF frame of the gene containing the point mutation is shown as SEQ ID NO. 4, and the translated amino acid sequence is shown as SEQ ID NO. 5. pKSU-glcP was obtained according to the plasmid construction method in example 1 ^H206R Plasmid was transformed into B.a 8336 strain, and the resultant strain was selected to contain glcP according to the strain selection method in example 1 ^H206R The strain with point mutation was designated b.a 8337.

Example 5 test of Performance of engineering bacteria to produce nucleosides

1. The strain stored in glycerol was cultured overnight at 37℃to score a monoclonal.

2. The single colony is inoculated into 30mL seed culture medium (20 g/L glucose, 5g/L yeast powder, 5g/L corn steep liquor dry powder, 3g/L monopotassium phosphate, 0.5g/L magnesium sulfate, 0.02g/L ferrous sulfate, 0.01g/L manganese sulfate, pH 7.0-7.2) and cultured for 7-8 h at 37 ℃ and 110 rpm.

3. Transferring the mixture into 30mL of fermentation medium (120 g/L of glucose, 3.5g/L of yeast powder, 3g/L of monopotassium phosphate, 25g/L of ammonium sulfate, 0.01g/L of manganese sulfate, 5g/L of magnesium sulfate, 10g/L of sodium glutamate, 15g/L of corn steep liquor dry powder, 25g/L of calcium carbonate and pH 7.0-7.2) according to the inoculation amount of 10% v/v, and culturing at 35 ℃ for 70h at 130rpm of shaking table.

4. The fermentation broth was tested for glycoside production using a liquid chromatograph (table 2).

Table 2 mutant strains were shake-flask fermented to guanosine and inosine evaluation results (average of three replicates)

Strain	Total glycoside yield (g/L)	Guanosine yield (g/L)	Inosine yield (g/L)	OD ₅₆₂	Glycoside production elevation
						B.a 8333	11.2	9.8	1.0	28.3	-
B.a 8334	13.2	11.5	1.3	28.5	17.9％
						B.a 8335	13.8	12.0	1.3	27.6	23.2％
B.a 8336	14.5	13.1	1.0	27.6	29.5％
						B.a 8337	15.9	15.0	0.5	29.0	42.0％

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

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Val Gly Thr Ile Ile Leu Thr Ala Ile Lys Glu Arg Gln Ala Val Ala

115 120 125

Met Ser Lys Met Glu Val Ala Tyr Gly Leu Gly Ala Leu Phe Met Pro

130 135 140

Leu Leu Ser Gly Phe Leu Ile Asn Ser His Met Trp Thr Ile Ala Phe

145 150 155 160

Leu Val Leu Gly Leu Ser Ser Phe Ala Leu Leu Ile Ala Trp Lys Gln

165 170 175

Met Ser Phe Gly Ser Ile Asp Gln Leu Leu Ile Arg Lys Asp Val Ser

180 185 190

Ser Asp Gly Thr Lys Lys Glu Ser Thr Gly Tyr Arg Ser Arg Gly Leu

195 200 205

Leu Phe Ile Ala Leu Ala Ala Ala Tyr Phe Phe Phe Tyr Gly Gly Ser

210 215 220

Glu Val Ser Ile Val His Phe Ile Pro Ser Ile Phe Ala Glu Lys Trp

225 230 235 240

Asp Ile Pro Asn Ser Leu Ala Thr Ile Thr Val Thr Val Tyr Trp Thr

245 250 255

Gly Met Ile Ile Gly Arg Leu Leu Thr Gly Pro Val Ser Glu Lys Leu

260 265 270

Thr Tyr His Arg Tyr Leu Arg Ile Ile Ser Val Gly Gly Leu Ala Ala

275 280 285

Leu Ala Val Leu Ala Leu Ser Lys Ser Val Trp Phe Gly Phe Ala Leu

290 295 300

Cys Phe Phe Leu Gly Leu Phe Met Ala Gly Met Phe Ala Ile Ala Leu

305 310 315 320

Ile Ile Thr Asn His Phe Tyr Pro Gly Lys Thr Glu Thr Thr Thr Ser

325 330 335

Ile Leu Leu Ala Ser Asn Gly Leu Gly Gly Ser Leu Leu Pro Ile Ala

340 345 350

Val Gly Trp Ser Leu Asp Glu Tyr Pro Ala Gln Thr Ala Phe Trp Leu

355 360 365

Phe Thr Ala Leu Met Leu Leu Met Leu Leu Ile Val Phe Ser Leu Arg

370 375 380

Met Leu Glu Thr Ile Lys Ser Asn Ser Leu Gln Asn His Ser Ser Lys

385 390 395 400

Ala Lys Ser Ile

Claims

1. A modified microorganism, characterized in that the microorganism has an increased activity of glucose transporter and/or mannose transporter compared to an unmodified microorganism and the microorganism has an increased nucleoside production capacity compared to an unmodified microorganism;

wherein the microorganism is a Bacillus or Escherichia species, preferably Bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus), escherichia coli (Escherichia coli).

2. The microorganism according to claim 1, characterized in that the enhancement of glucose transporter and/or mannose transporter activity is achieved by a protein selected from the following 1) to 6), or optionally in combination:

1) Enhanced by introducing a plasmid having a gene encoding the protein;

5) Enhancement by modification of the amino acid sequence of the protein;

3. The microorganism of claim 1, wherein the enhancement of glucose transporter and/or mannose transporter activity is achieved by inserting a P43 promoter upstream of the glcP gene start codon.

4. The microorganism of claim 1, wherein the enhancement of glucose transporter and/or mannose transporter activity is achieved by inserting a two copy glcP gene driven by a P43 promoter at the alpha amylase gene;

5. The microorganism of claim 1, wherein the enhancement of glucose transporter and/or mannose transporter activity is achieved by inserting a P43 promoter upstream of the glcP gene start codon and inserting a two copy glcP gene driven by the P43 promoter at the alpha amylase gene;

6. The microorganism according to claim 1, characterized in that the enhancement of the glucose transporter and/or the mannose transporter activity is achieved by inserting a P43 promoter upstream of the start codon of the glcP gene and inserting a double copy of the glcP gene driven by the P43 promoter at the alpha amylase gene and mutating the amino acid at position 206 of the glucose transporter and/or the mannose transporter from H to R;

7. A method of constructing a nucleoside producing strain, comprising: using genetic engineering means to enhance the gene glcP in a microorganism having nucleoside producing ability to obtain a strain having enhanced glucose transporter and/or mannose transporter activity;

1) Enhanced by introducing a plasmid having a gene encoding the protein;

5) Enhancement by modification of the amino acid sequence of the protein;

6) Enhancement by altering the nucleotide sequence of the gene encoding the protein;

8. The method of claim 7, wherein the microorganism is bacillus subtilis or bacillus amyloliquefaciens.

9. A method of producing nucleosides, said method comprising the steps of:

a) Culturing the microorganism of any one of claims 1-6 to obtain a culture of the microorganism;

b) Collecting the produced nucleosides from the culture obtained in step a).

10. The method of claim 9, wherein the nucleoside comprises inosine, guanosine, and corresponding nucleoside derivatives thereof.