CN114317386B

CN114317386B - Genetic engineering strain for producing inosine and construction method and application thereof

Info

Publication number: CN114317386B
Application number: CN202111481366.1A
Authority: CN
Inventors: 谢希贤; 朱彦凯; 刘铁重; 吴鹤云
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2021-12-06
Filing date: 2021-12-06
Publication date: 2023-12-08
Anticipated expiration: 2041-12-06
Also published as: CN114317386A

Abstract

The invention belongs to the technical field of genetic engineering, relates to breeding of industrial microorganisms, and in particular relates to a genetic engineering strain for producing inosine, and a construction method and application thereof. The gene engineering strain heterogenous over-expressed nucleoside transporter gene pfuE and purine operon mutant gene purEKBCSQLF ^K316Q MNCD and PRPP transamidase mutant gene purF ^K316Q And the gene purA is mutated by heterologous adenosine succinate synthase ^P242N The purA gene was replaced, and purine nucleoside phosphorylase genes deoD, ppnP and nucleoside hydrolase gene rihA, rihB, rihC were not expressed. The invention starts from the genome level of escherichia coli, and mainly carries out systematic comprehensive combination optimization on all modules of an inosine decomposition pathway, an inosine synthesis pathway, an inosine transport system and a branch metabolic pathway by metabolic engineering technical means, thereby improving the inosine fermentation performance of the strain.

Description

Genetic engineering strain for producing inosine and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, relates to breeding of industrial microorganisms, and in particular relates to a genetic engineering strain for producing inosine, and a construction method and application thereof.

Background

Inosine is purine nucleoside, participates in organism substance metabolism and energy metabolism, and has important application value. Microbial fermentation is the main method for industrial production of inosine at present, but the production level of inosine produced by the fermentation method is still lower due to the poor fermentation performance of the existing strain, and high-performance strain is needed to meet the industrial requirement of inosine. The traditional inosine producing strain is obtained by screening structural analogue resistant strains through mutagenesis, however, the continuous improvement of the performance of the strain obtained through mutagenesis is not facilitated due to unclear genetic background and accumulation of a large number of negative mutations. With the development of system biology, a method for rationally constructing metabolic engineering becomes a preferred method for constructing and continuously improving inosine producing strains from scratch. The prior research results provide important references for the rational construction of inosine engineering strains, but a plurality of problems still exist and are not solved. The specific expression is as follows: (1) At present, the inosine production performance of a rationally constructed strain is generally low, the highest yield is only 14g/L, the production period is longer, and the production period is generally as long as 72 hours or even longer. (2) The strain carries plasmids, the strain is heavy in load, and the plasmids are easy to lose, so that production is unstable. Antibiotics are added to maintain the stable existence of plasmids, so that the production cost is increased, and potential safety hazards such as drug resistance are increased. (3) In terms of construction strategies, the existing strains are limited to modification of individual nodes such as degradation pathways, de novo synthesis pathways, precursor synthesis pathways and branch metabolic pathways, and lack of systematic combination of all modules of the whole inosine metabolic network. (4) Most of the strains are defective in adenine, adenine needs to be added in the fermentation process, the cost of raw materials is high, the fermentation process is complex, and the production stability is reduced.

Common strategies for de novo construction of inosine producing strains mainly include blocking the degradation pathway, enhancing the de novo synthesis pathway, enhancing the precursor synthesis pathway, and blocking the branching metabolic pathway. Shimaoka M et al (Effect of amplification of desensitized purF and prs on inosine accumulation in Escherichia coll. Journal of bioscience and bioengineering,2007,103 (3): 255-261.) block the inosine degradation pathway by knocking out deoD and xapA genes with E.coli W3110 as the starting strain; by knocking out purR gene and over-expressing purF ^K326Q,P410W Genes, which enhance the pathway of de novo synthesis; by knocking out pgi and edd genes and over-expressing prs ^D128A Genes, which enhance the precursor synthesis pathway; by knocking out purA gene, the adenosine synthesis branch is blocked. The obtained strain IAfter 72h fermentation, the yield of the inosine reaches 7.5g/L. Asahara T et al (Accumulation of gene-targeted Bacillus subtilis mutations that enhance fermentative inosine production. Appl Microbiol Biotechnol,2010,87 (6): 2195-2207.) by taking Bacillus subtilis W168 as an initial strain, blocking the inosine degradation pathway by knocking out deoD and punA genes; the de novo synthesis pathway is enhanced by knocking out purR gene, 5' -UTR region of purine operon and optimizing-10 region of purine operon promoter; by knocking out purA and guaB genes, the adenosine synthesis branch and the guanosine synthesis branch are blocked. The obtained strain KMBS375 is fermented for 72 hours, and the yield of inosine reaches 6g/L. Lijian et al (De novo engineering and metabolic flux analysis of inosine biosynthesis in Bacillus subtilis. Biotechnol Lett,2011,33 (8): 1575-1580.) blocked the inosine degradation pathway by knocking out the deoD gene with Bacillus subtilis W168 as the starting strain; by knocking out purA gene, the adenosine synthesis branch is blocked. The strain BS019 is fermented for 72 hours, and the inosine yield reaches 7.6g/L. Wen Tingyi et al (CN 106906174A) blocked the adenosine synthesis branch by knocking out purA gene with Bacillus subtilis W168 as the starting strain; by knocking out the drm gene, the degradation path from ribose 1-phosphate to ribose 5-phosphate is blocked. The obtained strain IR-2 was fermented for 72 hours, and the inosine yield reached 14g/L, which was the highest yield of inosine in the rational construction strain so far.

Disclosure of Invention

Aiming at the problems, the invention aims to construct an engineering strain for efficiently and stably producing inosine and utilize the strain to ferment and produce the inosine.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect, the present invention provides a genetically engineered E.coli strain that heterologously overexpresses the nucleoside transporter gene pfuE, the purine operon mutant genepurEKBCSQLF ^K316Q MNCD and PRPP transamidase mutant gene purF ^K316Q And the gene purA is mutated by heterologous adenosine succinate synthase ^P242N The purA gene was replaced, and purine nucleoside phosphorylase genes deoD, ppnP and nucleoside hydrolase gene rihA, rihB, rihC were not expressed.

In a second aspect, the invention provides a method for constructing the escherichia coli genetic engineering strain, which comprises the following steps: introducing nucleoside transporter gene pfue, purine operon mutant gene purEKBCSQLF into original strain escherichia coli ^K316Q MNCD and PRPP transamidase mutant gene purF ^K316Q And the gene purA is mutated by heterologous adenosine succinate synthase ^P242N The purA gene was replaced, and purine nucleoside phosphorylase genes deoD, ppnP and nucleoside hydrolase gene rihA, rihB, rihC were knocked out or inactivated.

In a third aspect, the invention provides the use of the genetically engineered strain of E.coli as described above in high inosine production.

The beneficial effects of the invention are as follows:

the invention obtains the engineering strain which has clear genetic background, does not contain plasmid and efficiently produces inosine by utilizing a rational metabolic engineering modification method. From the production phenotype, the strain is fermented for 48 hours on a 5L tank, the inosine yield reaches 20.16g/L, and compared with the highest yield of the prior art rationally constructed strain, the production period is shortened by 1/3, and the strain does not contain plasmids. From the construction strategy, the invention carries out systematic and comprehensive combination optimization on the inosine decomposition pathway, the inosine synthesis pathway, the inosine transport system and the branch metabolic pathway by the metabolic engineering technical means through the split modules, and the strain has clear genetic background and higher and more stable production performance. In particular, compared with other inosine producing strains, the method of the invention not only ensures enough inosine branch flux, but also maintains a certain growth level of the strain by weakening rather than directly blocking the adenosine synthesis branch. The strain obtained finally can stably and efficiently produce inosine and has good industrial application prospect.

Drawings

Fig. 1A: construction of deoD gene knockout fragment and verification of electrophoresis pattern. Wherein M: a Marker;1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping the segments; 4: positive bacteria identification fragment 5: negative control.

Fig. 1B: construction of ppnP knockout fragment and verification of electrophoresis pattern. Wherein M: a Marker;1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping the segments; 4: positive bacteria identification fragment 5: negative control.

Fig. 1C: construction and verification of an rihA gene knockout fragment. Wherein M: a Marker;1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping the segments; 4: positive bacteria identification fragment 5: negative control.

Fig. 1D: construction of a rihB gene knockout fragment and verification of an electrophoresis pattern. Wherein M: a Marker;1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping the segments; 4: positive bacteria identification fragment 5: negative control.

Fig. 1E: construction and verification of an rihC gene knockout fragment and an electrophoresis chart. Wherein M: a Marker;1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping the segments; 4: positive bacteria identification fragment 5: negative control.

Fig. 2A: and (3) constructing and verifying an electrophoresis chart by pur1 integration fragment. Wherein: m: a marker;1: an upstream homology arm; 2: pur1 fragment; 3: a downstream homology arm; 4: overlapping the segments; 5: identifying fragments of positive bacteria; 6: negative control.

Fig. 2B: and (3) constructing and verifying an electrophoresis chart by pur2 integration fragments. Wherein: m: a marker;1: : pur2 upstream fragment-pur 2 fragment; 2: a downstream homology arm; 3: overlapping the segments; 4: identifying fragments of positive bacteria; 5: negative control.

Fig. 2C: and (3) constructing and verifying an electrophoresis chart by pur3 integration fragments. Wherein: m: a marker;1: : pur3 upstream fragment-pur 3 fragment; 2: a downstream homology arm; 3: overlapping the segments; 4: identifying fragments of positive bacteria; 5: negative control.

Fig. 2D: and (3) constructing and verifying an electrophoresis chart by pur4 integration fragment. Wherein: m: a marker;1: : pur4 upstream fragment-pur 4 fragment; 2: a downstream homology arm; 3: overlapping the segments; 4: identifying fragments of positive bacteria; 5: negative control.

Fig. 2E: and (5) constructing and verifying an electrophoresis chart by pur5 integration fragments. Wherein: m: a marker;1: : pur5 upstream fragment-pur 5 fragment; 2: a downstream homology arm; 3: overlapping the segments; 4: identifying fragments of positive bacteria; 5: negative control.

Fig. 2F: and (3) constructing and verifying an electrophoresis chart by pur6 integration fragment. Wherein: m: a marker;1: : pur6 upstream fragment-pur 6 fragment; 2: a downstream homology arm; 3: overlapping the segments; 4: identifying fragments of positive bacteria; 5: negative control.

Fig. 2G: purF (PurF) ^K326Q,P410W And (5) integrating fragment construction and verification of an electrophoresis chart. Wherein: m: a marker;1: an upstream homology arm; 2: purF (PurF) ^K326Q,P410W A gene fragment; 3: a downstream homology arm; 4: overlapping the segments; 5: identifying fragments of positive bacteria; 6: negative control.

Fig. 2H: purF (PurF) ^{D293V,K316Q,S400W} And (5) integrating fragment construction and verification of an electrophoresis chart. Wherein: m: a marker;1: an upstream homology arm; 2: purF (PurF) ^{D293V,K316Q,S400W} A gene fragment; 3: a downstream homology arm; 4: overlapping the segments; 5: identifying fragments of positive bacteria; 6: negative control.

Fig. 2I: purF (PurF) ^K316Q And (5) integrating fragment construction and verification of an electrophoresis chart. Wherein: m: a marker;1: an upstream homology arm; 2: purF (PurF) ^K316Q A gene fragment; 3: a downstream homology arm; 4: overlapping the segments; 5: identifying fragments of positive bacteria; 6: negative control.

Fig. 3: construction of the pbuE integration fragment and verification of the electropherogram. Wherein: m: a marker;1: an upstream homology arm; 2: a pbuE gene fragment; 3: a downstream homology arm; 4: overlapping the segments; 5: identifying fragments of positive bacteria; 6: negative control.

Fig. 4: purA (PurA) ^P242N And (5) integrating fragment construction and verification of an electrophoresis chart. Wherein: m: a marker;1: an upstream homology arm; 2: purA (PurA) ^P242N A gene fragment; 3: a downstream homology arm; 4: overlapping the segments; 5: identifying fragments of positive bacteria; 6: negative control.

Fig. 5: overexpression of the different PurF mutants effect on inosine yield.

Fig. 6: strain INO4 fed-batch fermentation curve in a 5L fermenter.

Fig. 7: the technical scheme principle of the invention is schematically shown.

Detailed Description

The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

In a first aspect, the present invention provides an E.coli genetically engineered strain that heterologously overexpresses the nucleoside transporter gene pfue, the purine operon mutant gene purEKBCSQLF ^K316Q MNCD and PRPP transamidase mutant gene purF ^K316Q Wherein purF ^K316Q Is prepared through substituting glutamine for 316 th lysine in the coding amino acid sequence of purF gene and mutating purA with adenosine succinic acid synthetase ^P242N The purA gene was replaced, and purine nucleoside phosphorylase genes deoD, ppnP and nucleoside hydrolase gene rihA, rihB, rihC were not expressed.

Preferably, the nucleotide sequence of the nucleoside transporter gene pfue is shown in SEQ ID NO:1, NCBI-GeneID:12132461.

preferably, the PRPP transamidase mutant gene purF ^K316Q The nucleotide sequence of (2) is shown as SEQ ID NO: 2. Wherein purF ^K316Q Is the substitution of lysine (K) at position 316 of the amino acid sequence encoded by purF gene with glutamine (Q). The numbering of the positions corresponds to the positions of the amino acid sequence of the parent PRPP transamidase.

Preferably, the purine operon mutant gene purEKBCSQLF ^K316Q The nucleotide sequence of MNCD is shown as SEQ ID NO: 3. Wherein purF is contained ^K316Q Is a mutant purF gene, and represents that lysine (K) at position 316 of the amino acid sequence encoded by purF gene is replaced by glutamine (Q).

Preferably, the method comprises the steps of,the mutant gene purA of the adenosine succinate synthetase ^P242N The nucleotide sequence of (2) is shown as SEQ ID NO: 4. Wherein purA ^P242N Is the substitution of proline (P) at position 128 of the amino acid sequence encoded by the purA gene with asparagine (N). The numbering of positions corresponds to the amino acid sequence of the parent adenosine succinate synthase.

The manner in which the above-described gene is not expressed according to the present invention may be by conventional means in the art, for example, by inactivating the gene or knocking out the gene by conventional means in the art.

According to the invention, non-expression means that the amount of the gene expression product is significantly lower than the original level, e.g. significantly reduced by at least 50%, 60%, 70%, 80%, 90%, 100%.

The manner in which the above-described genes are overexpressed according to the present invention may be by conventional means in the art, for example, by increasing the copy number of the genes or by ligating the genes to a strong promoter.

According to the invention, the overexpression means that the amount of the gene expression product is significantly higher than the original level.

According to a preferred embodiment of the invention, the nucleoside transporter gene pfue is linked to a promoter P _trc The method comprises the steps of carrying out a first treatment on the surface of the And/or said purine operon mutant gene purEKBCSQLF ^K316Q MNCD is connected with promoter P _trc The method comprises the steps of carrying out a first treatment on the surface of the And/or said PRPP transamidase mutant gene purF ^K316Q Is connected with a promoter P _trc The method comprises the steps of carrying out a first treatment on the surface of the Preferably, the promoter P _trc The nucleotide sequence of (2) is shown as SEQ ID NO: shown at 5.

According to the present invention, the starting strain used for constructing the E.coli genetically engineered strain may be any E.coli, and according to a preferred embodiment of the present invention, the starting strain is E.coli MG1655.

In a second aspect, the invention provides a method for constructing the escherichia coli genetic engineering strain, which comprises the following steps: introducing nucleoside transporter gene pfue, purine operon mutant gene purEKBCSQLF into original strain escherichia coli ^K316Q MNCD and PRPP transamidase mutant gene purF ^K316Q Wherein purF ^K316Q Is the substitution of lysine at position 316 of amino acid sequence with glutamine, and the mutation of gene purA with adenosine succinate synthetase ^P242N The purA gene was replaced, and purine nucleoside phosphorylase genes deoD, ppnP and nucleoside hydrolase gene rihA, rihB, rihC were knocked out or inactivated.

The selection of the above genes, the selection of the promoter, the selection of the starting strain, etc. have been described in detail in the first aspect of the present invention, and the details of the description of the first aspect will not be repeated here.

According to a specific embodiment of the invention, the method comprises:

(1) Blocking inosine decomposition pathway

Starting from the genome of the strain E.coli MG1655, knocking out purine nucleoside phosphorylase genes deoD and ppnP and knocking out nucleoside hydrolase gene rihA, rihB, rihC; this step blocked the degradation of inosine;

(2) Enhancing inosine synthesis pathway

The purine operon purEKBCSQLF of Bacillus amyloliquefaciens TA208 ^K316Q MNCD and promoter P _trc Fusion fragment P of (C) _trc -purEKBCSQLF ^K316Q MNHD is integrated at the yghE pseudogene locus; this step enhances the de novo purine anabolic flux while simultaneously relieving feedback inhibition of PRPP transamidase by AMP and GMP;

The PRPP transamidase mutant gene purF of Bacillus amyloliquefaciens TA208 ^K316Q And promoter P _trc Fusion fragment P of (C) _trc -purF ^K316Q Integration at the yeeP pseudogene locus; the step further strengthens the transcriptional expression of PRPP transamidase and promotes the synthesis of inosine;

(3) Modifying inosine transport systems

The nucleotide transporter gene pfue of Bacillus amyloliquefaciens TA and the promoter P are added _trc Fusion fragment P of (C) _trc -pbuE integration at the yjiT pseudogene locus; this step enhances the ability of inosine to migrate extracellular;

(4) Weakening an adenosine synthesis branch

Substitution of the purA Gene of the adenylsuccinic acid synthase with B.subilis W168 mutant purA of 168 ^P242N The method comprises the steps of carrying out a first treatment on the surface of the This step weakens the adenosine synthesis branch in the inosine competition pathway.

The principle of the above construction process of the present invention can be illustrated with reference to fig. 7.

In a third aspect, the invention provides an application of the escherichia coli genetic engineering strain in high-yield inosine, comprising the following steps: culturing the genetically engineered strain under suitable conditions and collecting inosine from the culture thereof.

According to a preferred embodiment of the invention, the suitable conditions are a culture temperature of 35 ℃, a pH of around 7.0, a dissolved oxygen of between 25 and 35% and a culture medium composition of: 15-25g/L glucose, 1-4g/L yeast powder, 1-5g/L peptone, 0.1-2g/L sodium citrate, 0.1-0.3g/L adenine and KH ₂ PO ₄ ·3H ₂ O 0.1-2g/L，MgSO ₄ ·7H ₂ O 0.1-2g/L，FeSO ₄ ·7H ₂ O 5-20mg/L，MnSO ₄ ·H ₂ O 5-20mg/L，V _B1 、V _B3 、V _B5 、V _B12 And V _H 0.1-2mg/L each, 2 drops of defoamer, the balance of water, and pH 7.0-7.2.

The present invention will be described in more detail with reference to specific examples. In the following examples:

unless otherwise indicated, the methods of gene editing according to the examples of the present invention are described in reference to the literature (Li Y, lin Z, huang C, et al, metal engineering of Escherichia coli using CRISPR-Cas9 mediated genome engineering,2015, 31:13-21.) and other specific procedures for molecular biology, genetic engineering, etc. may be carried out according to technical manuals, textbooks or literature reports readily available to those skilled in the art.

Example 1: construction of E.coli INO4 as a genetically engineered E.coli strain

1. Blocking inosine decomposition pathway

1.1 knockout of deoD Gene:

using the E.coli MG1655 genome as a template, an upstream homology arm primer (UP-deoD-S, UP-deoD-A) and a downstream homology arm primer (DN-deoD-S, DN-deoD-A) were designed based on the upstream and downstream sequences of the deoD gene (NCBI-GeneID: 945654), and the upstream and downstream homology arm fragments were PCR amplified. The above fragments were fused by overlap PCR to obtain a knockout fragment of deoD gene (upstream homology arm-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-deoD was prepared by annealing the primers gRNA-deoD-S and gRNA-deoD-A. The recombinant fragment and plasmid pGRB-deoD are electrically transferred to competent cells of E.coli MG1655, positive strains are screened, and then the plasmid is eliminated to obtain the strain INO1-1. Construction of deoD knockout fragment and PCR verification of positive strain are shown in FIG. 1A. Wherein the length of the upstream homology arm is 458bp, the length of the downstream homology arm is 613bp, and the total length of the overlapped fragments is 1029bp. When the PCR is verified, the length of the PCR amplified fragment of the positive bacterium is 1029bp, and the length of the PCR amplified fragment of the original bacterium is 1630bp.

1.2 knockdown of ppnP Gene:

using the E.coli MG1655 genome as a template, an upstream homology arm primer (UP-ppnP-S, UP-ppnP-A) and a downstream homology arm primer (DN-ppnP-S, DN-ppnP-A) were designed based on the upstream and downstream sequences of its ppnP gene (NCBI-GeneID: 945048), and the upstream and downstream homology arm fragments thereof were PCR amplified. The above fragments were fused by overlap PCR to obtain a knocked-out fragment of the ppnP gene (upstream homology arm-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-ppnP was prepared by annealing the primers gRNA-ppnP-S and gRNA-ppnP-A. The recombinant fragment and plasmid pGRB-ppnP are electrically transferred to competent cells of INO1-1, and the plasmid is eliminated after positive strains are screened to obtain the strain INO1-2. Construction of ppnP knockout fragment and PCR-verified electrophoresis of positive strains are shown in FIG. 1B. Wherein the length of the upstream homology arm is 394bp, the length of the downstream homology arm is 690bp, and the total length of the overlapped fragment is 1040bp. When the PCR is verified, the length of the PCR amplified fragment of the positive bacterium is 1040bp, and the length of the PCR amplified fragment of the original bacterium is 1361bp.

1.3 knockout of the rilA Gene:

using the E.coli MG1655 genome as a template, an upstream homology arm primer (UP-rihA-S, UP-rihA-a) and a downstream homology arm primer (DN-rihA-S, DN-rihA-a) were designed based on the upstream and downstream sequences of its rihA gene (NCBI-GeneID: 945503), and the upstream and downstream homology arm fragments thereof were PCR amplified. The above fragments were fused by overlap PCR to obtain a knockout fragment of the rilA gene (upstream homology arm-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-rihA was prepared by annealing the primers gRNA-rihA-S and gRNA-rihA-a. The recombinant fragment and plasmid pGRB-rihA are electrotransferred to competent cells of INO1-2, and the plasmid is eliminated after screening positive strains to obtain the strain INO1-3. Construction of the rilA knockout fragment and PCR-verified electrophoresis of the positive strain are shown in FIG. 1C. Wherein the length of the upstream homology arm is 547bp, the length of the downstream homology arm is 536bp, and the total length of the overlapped fragments is 1042bp. When the PCR is verified, the length of the PCR amplified fragment of the positive bacterium is 1042bp, and the length of the PCR amplified fragment of the original bacterium is 1857bp.

1.4 knockout of the rihB gene:

using the E.coli MG1655 genome as ase:Sub>A template, an upstream homology arm primer (UP-rib-S, UP-rib-A) and ase:Sub>A downstream homology arm primer (DN-rib-S, DN-rib-A) were designed based on the upstream and downstream sequences of its rib gene (NCBI-GeneID: 946646), and the upstream and downstream homology arm fragments thereof were PCR amplified. The above fragments were fused by overlap PCR to obtain a knockout fragment of the rihB gene (upstream homology arm-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-rihB was prepared by annealing the primers gRNA-rihB-S and gRNA-rihB-A. The recombinant fragment and plasmid pGRB-rihB are electrotransferred to competent cells of INO1-3, and the plasmid is eliminated after positive strains are screened to obtain the strain INO1-4. Construction of the rib knockout fragment and PCR-verified electrophoresis of the positive strain are shown in FIG. 1D. Wherein the length of the upstream homology arm is 494bp, the length of the downstream homology arm is 459bp, and the total length of the overlapped fragment is 912bp. When the PCR is verified, the length of the PCR amplified fragment of the positive bacterium is 912bp, and the length of the PCR amplified fragment of the original bacterium is 1789bp.

1.5 knockout of the rilc gene:

using the E.coli MG1655 genome as se:Sub>A template, an upstream homology arm primer (UP-rihC-S, UP-rihC-A) and se:Sub>A downstream homology arm primer (DN-rihC-S, DN-rihC-A) were designed based on the upstream and downstream sequences of the rihC gene (NCBI-GeneID: 944796), and the upstream and downstream homology arm fragments thereof were PCR amplified. The above fragments were fused by overlap PCR to obtain a knockout fragment of the rilC gene (upstream homology arm-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-rihC was prepared by annealing the primers gRNA-rihC-S and gRNA-rihC-A. The recombinant fragment and plasmid pGRB-rihC are electrically transferred to competent cells of INO1-4, and the plasmid is eliminated after positive strains are screened to obtain the strain INO1-5. Construction of the rilc knockout fragment and PCR verification of the positive strain are shown in fig. 1E. Wherein the length of the upstream homology arm is 539bp, the length of the downstream homology arm is 475bp, and the total length of the overlapped fragments is 975bp. When the PCR is verified, the length of the PCR amplified fragment of the positive bacterium is 975bp, and the length of the PCR amplified fragment of the original bacterium is 1787bp.

2. Enhancing inosine synthesis pathway

2.1 purine operon mutant Gene purEKBCSQLF of Bacillus amyloliquefaciens ^K316Q MNCHD integration at the yghE Gene locus of E.coli INO1-5

The purine operon purEKBCSQLFMNCD (comprising purE, purK, purB, purC, purS, purQ, purL, purF, purM, purN, purH, purD twelve genes) of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens TA) is 12797bp in total, and six segments pur1, pur2, pur3, pur4, pur5, pur6 are sequentially integrated into the gene locus of E.coli yghE in this example (wherein the contained purF is replaced by a mutant gene purF) ^K316Q ) And is composed of promoter P _trc The transcription expression of the exogenous operon is started, and the strain E.coli INO2-6 is constructed. The method specifically comprises the following steps:

2.1.1P _trc integration of pur 1:

designing an upstream homology arm primer (UP-yghE-S, UP-yghE-A) and a downstream homology arm primer (DN-yghE-S1, DN-yghE-A) according to the upstream and downstream sequences of the yghE gene by taking the E.coli MG1655 genome as a template, and amplifying the upstream and downstream homology arm fragments by PCR; designing primers (UP-pur 1-S, UP-pur 1-A) according to pur1 (1 st-2113 rd position of the nucleotide sequence shown in SEQ ID NO: 3) by taking Bacillus amyloliquefaciens TA208 genome as a template, and amplifying pur1 fragment by PCR; promoter P _trc Then the design is made in the downstream primer of the upstream homology arm and the upstream primer of pur1 gene. The above fragments are fused by overlapping PCR to obtain an integrated fragment of pur1 gene (upstream homology arm-P _trc Pur 1-downstream homology arm), the DNA fragment containing the target sequence used for constructing pGRB-yghE was prepared byAnnealing of the primers gRNA-yghE-S and gRNA-yghE-A. The recombinant fragment and plasmid pGRB-yghE are electrically transferred to competent cells of INO1-5, and the plasmid is eliminated after screening positive strains to obtain the strain INO2-1.P (P) _trc During integration of the pur1 fragment, the construction of the integrated fragment and the PCR-verified electrophoresis of the positive strain are shown in FIG. 2A. Wherein the length of the upstream homology arm is 559bp, the length of pur1 gene fragment is 2236bp, the length of the downstream homology arm is 554bp, and the length of the overlapped fragment is 3244bp. During PCR verification, the length of the amplified fragment of the identification primer should be 1188bp, and the primordium should be free of bands.

2.1.2 integration of pur2:

using Bacillus amyloliquefaciens TA208 genome as template, designing upstream homology arm primer (UP-pur 2-S, UP-pur 2-A) according to pur2 (nucleotide sequence 1603-4203 shown in SEQ ID NO: 3) and upstream sequence thereof, and PCR amplifying upstream homology arm fragment; the E.coli MG1655 genome was used as a template, and downstream homology arm primers (DN-yghE-S2, DN-yghE-A) were designed based on the downstream sequence of the yghE gene, and the downstream homology arm fragments were amplified by PCR. The above fragments were fused by overlap PCR to obtain an integrated fragment of pur2 (upstream fragment of pur 2-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-pur2 was prepared by annealing the primers gRNA-pur2-S and gRNA-pur 2-A. The recombinant fragment and plasmid pGRB-pur2 are electrically transferred to competent cells of INO2-1, and the plasmid is eliminated after positive strains are screened to obtain the strain INO2-2. In the pur2 fragment integration process, the construction of the integrated fragment and the PCR verification of the positive strain are shown in the figure 2B. Wherein the total length of the upstream fragment of pur2, is 2664bp, the length of the downstream homology arm is 554bp, and the length of the overlapping fragment is 3155bp. During PCR verification, the length of the amplified fragment of the identification primer should be 1513bp, and the primordium should be free of bands.

2.1.3 integration of pur3:

using Bacillus amyloliquefaciens TA208 genome as template, designing upstream homology arm primer (UP-pur 3-S, UP-pur 3-A) according to pur3 (nucleotide sequence shown in SEQ ID NO:3 at 3494-6132) and upstream sequence thereof, and PCR amplifying upstream homology arm fragment; the E.coli MG1655 genome was used as a template, and downstream homology arm primers (DN-yghE-S1, DN-yghE-A) were designed based on the downstream sequence of the yghE gene, and the downstream homology arm fragments were amplified by PCR. The above fragments were fused by overlap PCR to obtain an integrated fragment of pur3 (upstream fragment of pur 3-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-pur3 was prepared by annealing the primers gRNA-pur3-S and gRNA-pur 3-A. The recombinant fragment and plasmid pGRB-pur3 are electrically transferred to competent cells of INO2-2, and the plasmid is eliminated after positive strains are screened to obtain the strain INO2-3. In the pur3 fragment integration process, the construction of the integrated fragment and the PCR verification of the positive strain are shown in the figure 2C. Wherein the total length of the upstream fragment of pur3, is 2702bp, the length of the downstream homology arm is 554bp, and the length of the overlapped fragment is 3193bp. During PCR verification, the length of the amplified fragment of the identification primer is 1393bp, and the primordium is free of bands.

2.1.4 integration of pur4:

based on Bacillus amyloliquefaciens TA208 genome, obtaining pur4 fragment (5543-8228 site of nucleotide sequence shown in SEQ ID NO: 3) by chemical synthesis method, designing primer (UP-pur 4-S, UP-pur 4-A) according to pur4 fragment, and amplifying pur4 fragment; the E.coli MG1655 genome was used as a template, and downstream homology arm primers (DN-yghE-S2, DN-yghE-A) were designed based on the downstream sequence of the yghE gene, and the downstream homology arm fragments were amplified by PCR. The above fragments were fused by overlap PCR to obtain an integrated fragment of pur4 (upstream fragment of pur 4-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-pur2 was prepared by annealing the primers gRNA-pur2-S and gRNA-pur 2-A. The recombinant fragment and plasmid pGRB-pur2 are electrically transferred to competent cells of INO2-3, and the plasmid is eliminated after positive strains are screened to obtain the strain INO2-4. In the pur4 fragment integration process, the construction of the integrated fragment and the PCR verification of the positive strain are shown in the figure 2D. Wherein the total length of the upstream fragment of pur4, pur3, is 2749bp, the length of the downstream homology arm is 554bp, and the length of the overlapped fragment is 3240bp. During PCR verification, the length of the amplified fragment of the identification primer should be 1328bp, and the primordium should be free of bands.

2.1.5 integration of pur5:

based on Bacillus amyloliquefaciens TA208 genome, obtaining pur5 fragment (7704-10592 site of nucleotide sequence shown in SEQ ID NO: 3) by chemical synthesis method, designing primer (UP-pur 5-S, UP-pur 5-A) according to pur5 fragment, and amplifying pur5 fragment; the E.coli MG1655 genome was used as a template, and downstream homology arm primers (DN-yghE-S2, DN-yghE-A) were designed based on the downstream sequence of the yghE gene, and the downstream homology arm fragments were amplified by PCR. The above fragments were fused by overlap PCR to obtain an integrated fragment of pur5 (upstream fragment of pur 5-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-pur3 was prepared by annealing the primers gRNA-pur3-S and gRNA-pur 3-A. The recombinant fragment and plasmid pGRB-pur3 are electrically transferred to competent cells of INO2-4, and the plasmid is eliminated after positive strains are screened to obtain the strain INO2-5. In the pur5 fragment integration process, the construction of the integrated fragment and the PCR verification of the positive strain are shown in the figure 2E. Wherein the total length of the upstream fragment of pur5, is 2952bp, the length of the downstream homology arm is 554bp, and the length of the overlapped fragment is 3443bp. During PCR verification, the length of the amplified fragment of the identification primer should be 1308bp, and the primordium should be free of bands.

2.1.6 integration of pur6:

using Bacillus amyloliquefaciens TA208 genome as template, designing upstream homology arm primer (UP-pur 6-S, UP-pur 6-A) according to pur6 (nucleotide sequence 10088-12797 shown in SEQ ID NO: 3) and upstream sequence thereof, and PCR amplifying upstream homology arm fragment; the E.coli MG1655 genome was used as a template, and downstream homology arm primers (DN-yghE-S3, DN-yghE-A) were designed based on the downstream sequence of the yghE gene, and the downstream homology arm fragments were amplified by PCR. The above fragments were fused by overlap PCR to obtain an integrated fragment of pur6 (upstream fragment of pur 6-downstream homology arm). The DNA fragment containing the target sequence used for constructing pGRB-pur2 was prepared by annealing the primers gRNA-pur2-S and gRNA-pur 2-A. The recombinant fragment and plasmid pGRB-pur2 are electrically transferred to competent cells of INO2-5, and the plasmid is eliminated after positive strains are screened to obtain the strain INO2-6. In the process of pur6 integration, the construction of the integration fragment and the PCR verification of the positive strain are shown in FIG. 2F. Wherein the total length of the upstream fragment of pur6 is 2773bp, the length of the downstream homology arm is 554bp, and the total length of the overlapped fragment is 3288bp. During PCR verification, the length of the amplified fragment of the identification primer should be 1114bp, and the primordium should be free of bands.

2.2 integration of PRPP Transamidase mutant Gene

To enhance the transcriptional expression of PRPP transamidase, purF was selected ^K326Q,P410W ，purF ^{D293V,K316Q,S400W} ，purF ^K316Q Several PRPP transamidase mutant genes from different sources are integrated and over-expressed.

Identification of mutants: amino acid residues are indicated by single letter symbols using accepted IUPAC nomenclature. "amino acid substituted at the original amino acid position" is used to refer to the mutated amino acid in the PRPP transamidase mutant. Such as purF ^K316Q The amino acid at position 316 is replaced by the parent lysine (K) with glutamine (Q), the numbering of the position corresponding to the amino acid sequence numbering of the parent PRPP transamidase. Such as purF ^K326Q,P410W Indicating that both amino acids 326 and 410 were mutated. The method comprises the following steps:

2.2.1 mutant PRPP transamidase Gene purF of E.coli MG1655 ^K326Q,P410W Integration at the yeeP gene locus of E.coli INO 2-6:

designing an upstream homology arm primer (UP-yeeP-S, UP-yeeP-A) and a downstream homology arm primer (DN-yeeP-S, DN-yeeP-A) according to the upstream and downstream sequences of the yeeP gene by taking the E.coli MG1655 genome as a template, and amplifying the upstream and downstream homology arm fragments by PCR; based on E.coli MG1655 genome, purF is obtained by chemical synthesis method ^K326Q,P410W Gene according to purF ^K326Q,P410W Gene design primer (ecoF-S, ecoF-A), amplification purF ^K326Q,P410W Gene fragment, promoter P _trc Then design of the downstream primer of the upstream homology arm and purF ^K326Q,P410W In the upstream primer of the gene, terminator T _trc Then design of the upstream primer and purF at the downstream homology arm ^K326Q,P410W In the downstream primer of the gene. Fusion of the fragments by means of overlap PCR to obtain purF ^K326Q,P410W Integration fragment of gene(upstream homology arm-P) _trc -purF ^K326Q,P410W -T _trc Downstream homology arms), the DNA fragment containing the target sequence used for constructing pGRB-yeeP was prepared by annealing the primers gRNA-yeeP-S and gRNA-yeeP-A. The recombinant fragment and plasmid pGRB-yeeP are electrically transferred to competent cells of INO2-6, and the plasmid is eliminated after positive strains are screened to obtain the strain INO2-7.purF (PurF) ^K326Q,P410W Construction of the integration fragment and PCR-verified electrophoresis of the positive strain are shown in FIG. 2G. Wherein the upstream homology arm has a length of 618 bp and purF ^K326Q,P410W The length of the gene fragment is 1641bp, the length of the downstream homology arm is 576bp, and the total length of the integration fragment is 2704bp. When the PCR is verified, the length of the PCR amplified fragment of the positive bacterium is 2704bp, and the length of the PCR amplified fragment of the original bacterium is 1396bp.

2.2.2 PRPP transamidase mutant Gene purF of B.subilis W168 ^{D293V,K316Q,S400W} Integration at the yeeP gene locus of E.coli INO 2-6:

designing an upstream homology arm primer (UP-yeeP-S, UP-yeeP-A) and a downstream homology arm primer (DN-yeeP-S, DN-yeeP-A) according to the upstream and downstream sequences of the yeeP gene by taking the E.coli MG1655 genome as a template, and amplifying the upstream and downstream homology arm fragments by PCR; based on B.subtilis W168 genome, purF is obtained by chemical synthesis method ^D293V ^,K316Q,S400W Gene according to purF ^{D293V,K316Q,S400W} Gene design primer (bsuF-S, bsuF-Sub>A), amplification of purF ^D293V ^,K316Q,S400W Gene fragment, promoter P _trc Then design of the downstream primer of the upstream homology arm and purF ^{D293V,K316Q,S400W} In the upstream primer of the gene, terminator T _trc Then design of the upstream primer and purF at the downstream homology arm ^{D293V,K316Q,S400W} In the downstream primer of the gene. Fusion of the fragments by means of overlap PCR to obtain purF ^{D293V,K316Q,S400W} Integration fragment of Gene (upstream homology arm-P _trc -purF ^{D293V,K316Q,S400W} -T _trc Downstream homology arms), the DNA fragment containing the target sequence used for constructing pGRB-yeeP was prepared by annealing the primers gRNA-yeeP-S and gRNA-yeeP-A. Electrotransformation of the recombinant fragment and plasmid pGRB-yeeP into competent cells of INO2-6, screening of positive strains and plasmid eliminationExcept that strain INO2-8 was obtained. purF (PurF) ^{D293V,K316Q,S400W} Construction of the integration fragment and PCR-verified electrophoresis of the positive strain are shown in FIG. 2H. Wherein the upstream homology arm has a length of 618 bp and purF ^{D293V,K316Q,S400W} The length of the gene fragment is 1554bp, the length of the downstream homology arm is 576bp, and the total length of the integration fragment is 2617bp. During PCR verification, the length of the PCR amplified fragment of the positive bacteria is 2617bp, and the length of the PCR amplified fragment of the original bacteria is 1396bp.

2.2.3 mutant PRPP transamidase Gene purF of Bacillus amyloliquefaciens TA208 ^K316Q Integration at the yeeP gene locus of E.coli INO 2-6:

Designing an upstream homology arm primer (UP-yeeP-S, UP-yeeP-A) and a downstream homology arm primer (DN-yeeP-S, DN-yeeP-A) according to the upstream and downstream sequences of the yeeP gene by taking the E.coli MG1655 genome as a template, and amplifying the upstream and downstream homology arm fragments by PCR; purF is obtained by chemical synthesis based on Bacillus amyloliquefaciens TA208 genome ^K316Q Gene (SEQ ID NO: 2) according to purF ^K316Q Gene design primer (bazF-S, bazF-A), amplification purF ^K316Q Gene fragment, promoter P _trc Then design of the downstream primer of the upstream homology arm and purF ^K316Q In the upstream primer of the gene, terminator T _trc Then design of the upstream primer and purF at the downstream homology arm ^K316Q In the downstream primer of the gene. Fusion of the fragments by means of overlap PCR to obtain purF ^K316Q Integration fragment of Gene (upstream homology arm-P _trc -purF ^K316Q -T _trc Downstream homology arms), the DNA fragment containing the target sequence used for constructing pGRB-yeeP was prepared by annealing the primers gRNA-yeeP-S and gRNA-yeeP-A. The recombinant fragment and plasmid pGRB-yeeP are electrically transferred to competent cells of INO2-6, and the plasmid is eliminated after positive strains are screened to obtain the strain INO2-9.purF (PurF) ^K316Q Construction of the integration fragment and PCR-verified electrophoresis of the positive strain are shown in FIG. 2I. Wherein the upstream homology arm has a length of 618 bp and purF ^K316Q The length of the gene fragment is 1552bp, the length of the downstream homology arm is 576bp, and the total length of the integration fragment is 2615bp. During PCR verification, the length of the PCR amplified fragment of the positive bacteria is 2615bp, and the length of the PCR amplified fragment of the original bacteriaThe degree should be 1396bp.

3. Modifying inosine transport systems

The pbuE gene of Bacillus amyloliquefaciens TA was integrated at the yjiT pseudogene locus of e.coli INO 2-9:

designing an upstream homology arm primer (UP-yjiT-S, UP-yjiT-A) and a downstream homology arm primer (DN-yjiT-S, DN-yjiT-A) according to the upstream and downstream sequences of the yjiT gene by taking the E.coli MG1655 genome as a template, and amplifying the upstream and downstream homology arm fragments by PCR; the genome of Bacillus amyloliquefaciens TA was used as a template, and a primer (pfuE-S, pbuE-A) was designed based on the pfuE gene (SEQ ID NO: 1) to amplify the pfuE gene fragment. Promoter P _trc Then the terminator T is designed in the downstream primer of the upstream homology arm and the upstream primer of the pfuE gene _trc Then the design is in the upstream primer of the downstream homology arm and the downstream primer of the pfue gene. The above fragments are fused by overlapping PCR to obtain an integrated fragment of the pfue gene (upstream homology arm-P) _trc -pbuE-T _trc Downstream homology arms), the DNA fragment containing the target sequence used for constructing pGRB-yjiT was prepared by annealing the primers gRNA-yjiT-S and gRNA-yjiT-A. The recombinant fragment and plasmid pGRB-yjiT are electrotransformed into competent cells of INO2-9, and the plasmid is eliminated after positive strains are screened to obtain the strain INO3. Construction of the pbuE integration fragment and PCR-verified electrophoresis patterns of positive strains are shown in fig. 3. Wherein the length of the upstream homology arm is 373bp, the length of the pbue gene fragment is 1284bp, the length of the downstream homology arm is 530bp, and the total length of the integration fragment is 2108bp. During PCR verification, the length of the PCR amplified fragment of the positive bacterium should be 2108bp, and the length of the PCR amplified fragment of the original bacterium should be 1873bp.

4. Weakening an adenosine synthesis branch

Substitution of the purA Gene of E.coli MG1655 with the mutant PurA Gene of the adenosine succinate synthase of B.subtilis W168 ^P242N . The method comprises the following steps:

designing an upstream homology arm primer (UP-purA-S, UP-purA-a) and a downstream homology arm primer (DN-purA-S, DN-purA-a) according to the upstream and downstream sequences of the purA gene by taking the E.coli MG1655 genome as a template, and amplifying the upstream and downstream homology arm fragments by PCR; based on the B.subtilis W168 genome by chemical synthesispurA is obtained by the method ^P242N Gene (SEQ ID NO: 4) according to purA ^P242N Gene design primer (bsuA-S, bsuA-a), amplification purA ^P242N A gene fragment. The fragment is fused by an overlapping PCR method to obtain purA ^P242N Integration fragment of mutant Gene (upstream homology arm-purA ^P242N Downstream homology arms), the DNA fragment containing the target sequence used for constructing pGRB-purA was prepared by annealing the primers gRNA-purA-S and gRNA-purA-a. The recombinant fragment and plasmid pGRB-purA are electrically transferred to competent cells of INO3, and the plasmid is eliminated after positive strains are screened to obtain the strain INO4.purA (PurA) ^P242N Construction of the integration fragment and PCR verification of the positive strain are shown in FIG. 4. Wherein the length of the upstream homology arm is 515bp, purA ^P242N The length of the mutant gene fragment is 1337bp, the length of the downstream homology arm is 511bp, and the total length of the integrated fragment is 2275bp. During PCR verification, the length of the amplified fragment of the identification primer should be 1879bp, and the primordium should be free of bands.

5. The primers involved in the above construction are shown in the following table:

example 2: the experimental shake flask culture method for producing inosine by shake flask fermentation of strains INO2-6, INO2-7, INO2-8 and INO2-9 is as follows:

slant activation culture: inoculating the preserved strain at-80deg.C on the activated slant, culturing at 37deg.C for 12 hr, and passaging once again;

seed culture: scraping a ring of inclined seeds by an inoculating loop, inoculating the inclined seeds into a 500mL triangular flask filled with 30mL of seed culture medium, sealing a nine-layer gauze, and culturing at 37 ℃ for 7-10h at 200 rpm;

fermentation culture: inoculating into 500mL triangular flask (final volume of 30 mL) filled with fermentation medium according to inoculum size of 10-15% of seed culture solution volume, sealing nine layers of gauze, shake culturing at 37deg.C at 200r/min, and maintaining pH at 7.0-7.2 by adding ammonia water during fermentation; adding 60% (m/v) glucose solution to maintain fermentation; the fermentation period is 24 hours.

Slant culture medium: glucose 1-5g/L, peptone 5-10g/L, yeast powder 1-5g/L, beef extract 5-10g/L, adenine 0.1-0.3g/L, naCl 1-2.5g/L, agar 15-20g/L, and water in balance, and pH 7.0-7.2.

Seed culture medium: : 15-30g/L glucose, 1-5g/L yeast powder, 1-3g/L peptone, 0.1-0.3g/L adenine and KH ₂ PO ₄ ·3H ₂ O 0.1-1.2g/L，MgSO ₄ ·7H ₂ O 0.1-0.5g/L，FeSO ₄ ·7H ₂ O 2-10mg/L，MnSO ₄ ·H ₂ O 2-10mg/L，V _B1 、V _B3 、V _B5 、V _B12 And V _H 0.1-1mg/L each, 2 drops of defoamer, and the balance of water, and the pH is 7.0-7.2.

Fermentation medium: 15-25g/L glucose, 1-4g/L yeast powder, 1-5g/L peptone, 0.1-2g/L sodium citrate, 0.1-0.3g/L adenine and KH ₂ PO ₄ ·3H ₂ O 0.1-2g/L，MgSO ₄ ·7H ₂ O 0.1-2g/L，FeSO ₄ ·7H ₂ O 5-20mg/L，MnSO ₄ ·H ₂ O 5-20mg/L，V _B1 、V _B3 、V _B5 、V _B12 And V _H 0.1-2mg/L each, 2 drops of defoamer, the balance of water, and pH 7.0-7.2.

Shaking the flask fermentation result:

respectively overexpressing purF by taking INO2-6 as an initial strain ^K326Q,P410W 、purF ^{D293V,K316Q,S400W} 、purF ^K316Q After mutation of the genes, strains INO2-7, INO2-8, INO2-9 were obtained. Shake flask fermentation results (fig. 5), OD of three strains ₆₀₀ The inosine yield is improved to different degrees without obvious change. Wherein the yield of the inosine of INO2-9 is highest and reaches 1.3g/L, and is improved by 85.7 percent compared with INO 2-6. INO2-7 increased 14.3% and 71.4% compared to INO2-8 inosine yield, respectively, for INO2-6. We speculate that Bacillus amyloliquefaciens TA208 derived PurF ^K316Q The mutant (the amino acid sequence is shown as SEQ ID NO: 6) has better feedback effect, and further brings the highest inosine yield.

Example 3: experiment for producing inosine by fermenting INO4 on 5-L tank

The fermentation tank culture method comprises the following steps:

slant activation culture: inoculating the preserved strain at-80deg.C on the activating slant, culturing at 37deg.C for 12 hr, and transferring to eggplant bottle for continuous culture for 12-16 hr;

seed culture: taking a proper amount of sterile water in an eggplant-shaped bottle, inoculating the bacterial suspension into a seed culture medium, stabilizing the pH at about 7.0, stabilizing the temperature at 37 ℃, and culturing until the dry weight of cells reaches 5-6g/L with dissolved oxygen between 25 and 35%;

Fermentation culture: inoculating fresh fermentation medium according to 15-20% inoculum size, starting fermentation, controlling pH at 7.0, maintaining temperature at 35deg.C, and dissolving oxygen at 25-35%; after the glucose in the medium was consumed, 80% (m/v) glucose solution (containing 1g/L adenine) was fed in, and the glucose concentration in the fermentation medium was maintained at 0.1-2g/L.

Fed-batch fermentation results in 5L fermentor: as shown in FIG. 6, the strain grew rapidly at the initial stage of fermentation at an OD of 16h ₆₀₀ The value reaches a maximum and then gradually decreases. The inosine keeps higher synthesis rate within 12-32 hours, the yield within 48 hours reaches 20.16g/L, and the sugar acid conversion rate and the production strength reach 0.114g/g glucose and 0.42g/L/h respectively.

Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> university of Tianjin science and technology

<120> a genetically engineered strain for producing inosine, construction method and application thereof

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 1161

<212> DNA

<213> Bacillus amyloliquefaciens TA208

<400> 1

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cttgtctgat aaaaaaacga tttttcccgg cgccgtcatc ggcattatcg gaggcggaca 540

gctcggaaaa atgatggctg tcgccgcaaa acagatgggg tataaagtcg cagtcgtcga 600

tcccgtaaaa gactcgccat gcggacagat cgccgatatt gagattaccg cccagtacaa 660

tgaccgtgaa gcgattcaaa aattggccgg agtcagtgac atcattacct acgaatttga 720

aaatatcgat tacgaggcgc tcaactggct caaagaacat gcatatcttc cccagggaag 780

cgagctgctg ctcatcaccc aaaacaggga aacggaaaaa aaggcgatcc agtcagccgg 840

atgtcaggtc gctccttatc ggatcgtaaa cagcagacgg gagcttgagg aagccgttca 900

gtcattgggt cttccggcgg tgctgaaaac atgccggggc ggatatgacg gcaaaggtca 960

atttgtcatt aaggaagaag gacagacaga tgaagcggcc gcactgttag aaaacggcgc 1020

gtgcatactt gaaagctggg tgtcattccg aatggagctt tccgttatcg tgacgagatc 1080

ggtgcacgga gagatttcaa cctttcccgc tgctgaaaat atccaccacc ataatattct 1140

gttccaaagc atcgtgcccg cgagagcgga ggaaacggtt caaaagcggg cagaggcgct 1200

tgccgttcag ctcgcggaga aactggagct cgtcgggccg cttgcggtgg aaatgttcgt 1260

cacggaagac ggagaccttt tgattaatga attggcgccg cgtcctcaca attcagggca 1320

ttatacgctc gatctttgtg aaacgagcca gttcgaacag cacatcagag cggtctgcgg 1380

acttcctctc ggcagaaccg atctgcttaa accgggaatg atggtgaatc ttctcggtga 1440

cgaagtgaag ctggcggagg agcatacgga gcttttaaag gaagccaaac tgtacctgta 1500

cggaaaacat gagattaaaa aaggccgcaa aatggggcat atgacatttt tgcgggagcc 1560

tgatgaaaaa tggattcagg acatcacgaa catatggatg aaaagagacg gaggacgagc 1620

ataatgatcg aacgttattc aagacctgaa atgtccgcga tctggacgga cgaaaacaga 1680

taccaggcat ggctggaagt cgagatttta gcctgtgaag cctgggctga acttggcgtc 1740

attccgaaag aagacgtcaa agtcatgcga gaaaacgcgt ctttcgacat taaccgcatt 1800

ttagaaatcg agcaggatac gcgccatgac gtcgtggcat tcacacgcgc ggtttcagaa 1860

tcattgggcg aagaaagaaa atgggttcac tacggcctga cgtcaacaga tgtcgtggat 1920

accgctcttt cctatctatt aaaacaggcg aatgagattt tactcaagga cattgagaga 1980

tttgttgaca ttataaaaga aaaagcgaaa gaacataaat acacggtcat gatgggccgc 2040

acacacggcg tacacgcaga accaacgaca ttcggcctga agctcgcgct gtggcacgaa 2100

gaaatgaaac gcaaccttga acgtttcaag caggcaaaag aaggaatcga agtcgggaag 2160

ctttccggag cggtcggcac atatgcaaat attgatccgt tcgtagagca gtatgtctgt 2220

gaaaaactcg gcctgaaagc ggcgccgatc tccactcaga cattacagcg cgaccgtcat 2280

gcggattaca tggcggcact tgccctgatc gcgacgagca ttgaaaaatt cgcagtggaa 2340

atccgcggtc tgcaaaagag tgaaacacgg gaagtggaag agttttttgc aaaaggacaa 2400

aaaggctcat cagctatgcc acataaacgg aatccgatcg ggtctgaaaa tatgacgggg 2460

atggcgcgcg tgatccgcgg atacatgctg acggcatacg aaaatgttcc gttatggcat 2520

gagcgtgata tttctcattc atcagctgag cgcatcattc ttcctgacgc gacaaccgcg 2580

ctgaattaca tgctgaaccg tttcagcaat atcgtcaaaa acttaacggt attcccggaa 2640

aatatgaaac gcaacatgga ccgcacactg ggtctaatct attctcagcg cgtgctgctc 2700

gctttaattg acacgggtct gcctcgtgaa gaagcatatg acacggttca gccgaaagcg 2760

atggaagcgt gggaaaaaca agtgccgttc cgtcagcttg tcgaagcgga ggaaaaaatc 2820

acgtcccgtc ttacaccgga acagattgcc gattgctttg actacaatta tcatttgaaa 2880

aacgtcgact tgatctttga tcgtttaggt ttatagaaga agccagccgg aggcggcttc 2940

ttcagccgcc atagattgaa tattcccaac attcgggtta ggaggccttc cgtgaatatt 3000

gtgaaaagta atcttcttta tgaaggaaaa gcgaaacgaa tttatcaaac tgaggacgaa 3060

cagattctcc gtgtggtcta caaggattcc gcaacagcct ttaacggcga gaaaaaagcg 3120

gagatcactg gaaagggccg tctgaacaat gaaatttcaa gcctgatctt caaacatctg 3180

cacgccaaag gaattgacaa ccattttgtg gagcgtgttt cagaatcaga gcagcttatc 3240

aaaaaagtaa gcatcgttcc gcttgaagtc gtggtcagaa acattgccgc cggaagcatg 3300

tcgaaacgcc tcggcatccc ggaaggaaca gagcttccgc agccgattat cgaattttac 3360

tataaagatg acgcactcgg tgatccgctc ataaccgaag atcatatctg gctgttaaaa 3420

gcagcttcat ccgaacaggt ggaaacgatc aaatcaatta caagacaggt gaataaagag 3480

ctgcagctta tttttgaaga ctgcggtgtc agattaatag attttaagct ggaattcggc 3540

ttagacgcag agggacgggt gcttttagcg gatgagattt ctcctgacac gtgccgtctg 3600

tgggacaaag acacgaacga aaagctcgat aaagatttgt tcagacggaa cctgggaagc 3660

ttaaccgacg catatgaaga gattttcaaa agactgggag gcatttcata atgtataaag 3720

tgaaagttta tgtcagctta aaagaaagtg tgcttgatcc tcaaggaagc gcggtgcagc 3780

atgcattgca cagcatgacc tacaatgaag tgcaggatgt gcgcatcgga aaatacatgg 3840

agctgacgct ggaaaaatca gaccgcgatc ttgatgaact cgtgaaagaa atgtgtgaga 3900

agctccttgc caatacagtc attgaagact accgatatga agttgaggag gttgtcgcac 3960

agtgaaattt gcggtgattg tgttaccagg ctctaactgc gatattgata tgtatcacgc 4020

cgtaaaggac gaactcggcg aagaagtgga gtatgtctgg cacgaggaaa caagccttga 4080

cggatttgac ggcgtgctca tccccggcgg cttttcttac ggcgactacc tgagatgcgg 4140

cgccatcgcc agattcgcca acatcatgcc ggccgtgaaa aaagcggctg ctgaaggaaa 4200

accggttctc ggcgtctgca acggattcca gattttgcag gagctcggtc tgctgcccgg 4260

cgccatgaga cgcaataaag atttaaaatt catttgccgc ccggttgaat taatcgtgca 4320

gaacggtgaa acaatgttca cttcttccta caaagaggga caatcaatta cgattcccgt 4380

tgcccacggc gaaggcaatt tctactgtga tgacgaaacg cttgaaagat taaaagaaaa 4440

caatcaaatc gctttcacat acggcggcga tattaacgga agcgtcagcg gcattgccgg 4500

cgtcgtgaat gagaaaggca acgtattagg catgatgcct cacccggagc gcgcggtcga 4560

tgaactgctc ggcagcgcag acggtcttac attgttccag tctatcgtga aaaattggag 4620

ggaaattcat gtcgctactg cttgaaccaa gtaaagaaca aataaaagaa gagaaactct 4680

atcagcaaat gggtgtcagt gatgacgagt tcgcactcat tgaatctatt atcggaagat 4740

tgccaaacta cacggaaatc gggatttttt ccgtgatgtg gtcagagcac tgcagctata 4800

aaaattctaa gccgatttta cgcaaattcc cgacaagcgg cgaacgcgtg ctgcaaggtc 4860

ccggggaagg cgcggggatc gttgacatcg gtgacaatca ggcggttgtg ttcaaaattg 4920

aatcgcataa ccacccgtca gcgcttgagc cataccaggg tgctgcgacg ggagtgggcg 4980

gcatcatccg tgacgttttc tcaatgggcg cccgtccgat agctgtatta aactctcttc 5040

gatttggtga actcacttca ccgcgtgtga agtacttgtt tgaagaagta gtggctggaa 5100

tcgcgggata cggaaactgt atcggcattc cgacggtcgg cggggaagtt cagtttgacg 5160

caagctatga gggcaatccg cttgtcaatg ccatgtgcgt cggcctaatt gatcataagg 5220

atattaaaaa aggccaggcg aaaggtgtcg gcaacacggt tatgtacgtc ggcgctaaga 5280

cgggacgcga cggcattcac ggcgctactt tcgcatcaga agaaatgtca gattcatctg 5340

aagaaaaacg ctccgcggtg caggtcggcg atcctttcat ggaaaagctt cttcttgaag 5400

cctgcctgga agtcatccag tgcgacgcgt tagtcggcat tcaggatatg ggagcggccg 5460

gtctgacaag ttcaagcgct gaaatggcaa gtaaagccgg atcaggcatt gaaatgaacc 5520

ttgatctcat tccgcagcgg gaaacgggta tgaccgctta tgaaatgatg ctttccgaat 5580

ctcaggaacg catgcttctc gttattgaac gcgggcgtga acaggaaatt gtcgatattt 5640

ttaataaata tgatcttgaa gcggtttccg tgggtcatgt cacggatgat aaaatgctcc 5700

gcctccgcca taacggagag gttgtttgcg agcttccggt tgacgcgctg gcggaagaag 5760

cccctgtata tcataagccg tcagcagaac ccgcgtacta ccgcgagttt caggaaactg 5820

aagttcccgc gcctgaagta aaagacgcga cagagacgct ttttgccctg ctgcagcagc 5880

cgacaattgc gagcaaagag tgggtgtacg atcaatatga ttacatggtg cgcacgaaca 5940

cggtggtggc tccgggctct gacgcgggag tgctcagaat ccgcggcacg aaaaaggcgc 6000

tggcaatgac gacggattgc aacgcccgct atttgtatct cgatcctgaa gaaggcggaa 6060

aaatcgccgt tgccgaagcg gcgcgcaaca tcgtttgctc cggtgccgag ccgcttgcgg 6120

tcacggataa tctgaatttc ggaaacccgg aaaaacctga aattttctgg cagatcgaaa 6180

aagcggccga cggcatcagc gaggcatgca atgttctcag cacacctgtc atcggcggaa 6240

acgtatcact ttataatgaa tcgaacggaa cggccatcta tccgacgccg gtcatcggta 6300

tggtcgggtt aatcgaagat acggctcata ttacgacaca gcatgtcaaa gcggcgggag 6360

acttgattta cgtcatcggt gaaacgaagc ctgagtatgc aggaagtgaa cttcagaaaa 6420

tgactgaagg gaaaatctat ggcaaagcgc ctgaaattga tcttgacgtt gaaaaagccc 6480

gccaaacatc gcttctgaac gccattaaac aaggtctggt ccaatctgcg catgacgtgt 6540

cagaaggcgg attgggtgtg gcgattgcag aaagcgtcat gacgacagac ggactcggcg 6600

caaacatcac ggcatttaat gaagcggctc ttttgttcag tgagtctcaa tcccgcttcg 6660

tcgtttccgt aaaggaagaa aacaaggcgg cgtttgaagc ggctgcggca gatgccgttc 6720

atatcggtga agtgacaggg gacggacagt tgacgatccg aagccaagaa ggacaacaat 6780

tggttcacgc gcaaacgaaa gaacttgagc gcgcgtggaa aggagctatt ccatgcttgc 6840

tgaaatcaaa ggcctgaatg aagaatgcgg tgtgtttggc atctgggggc atgaagaggc 6900

tccgcagatt acgtattacg gcctgcacag cctgcagcac agagggcagg agggcgccgg 6960

catcgtggcg accgacggcc aaaaactgac ggctcataaa gggcaggggc ttattaccga 7020

ggtttttcaa aacggcgaac tgagcaaagt gaaaggcaaa ggcgcgatcg gtcacgtccg 7080

ctatgcgacg gccggcggcg gcggatatga aaatgtccag ccgctcctgt tccgttcgca 7140

aaacaacggc agtctcgcgc tcgcccataa cggcaacctg gtcaatgcca cacaattgaa 7200

acagcagctt gaaaaccaag ggagcatctt tcagacttcc tccgatacgg aagtgctggc 7260

tcatctgatt aaacgcagcg gccacttttc actgaaggat cagatcaaaa attcgctatc 7320

catgctgaaa ggcgcttacg cctttttaat catgacagaa acagaaatga ttgtagcgct 7380

tgacccgaac ggactcagac cgctttcact cggcatgctc ggcgacgctt acgtcgtcgc 7440

atcagaaaca tgcgcatttg atgtggtcgg cgccacgtac cttcgtgacg tagaaccggg 7500

cgaaatgctt atcataaacg atgaaggctt gaaatcagag cgtttctcca tgaatatcaa 7560

ccgttctatc tgcagcatgg agtatatcta tttttcccgt ccggacagca atatcgacgg 7620

cattaacgtg cacagcgccc ggaagagcct cgggaaaatg cttgcccaag agtccgctgt 7680

tgaagcggat gtcgtcacag gcgtgcctga ttccagtatt tccgcggcca tcggctatgc 7740

cgaggcaacg ggcattccgt acgaactcgg tctcattcaa aaccgttacg tcggcagaac 7800

gtttatccag ccgtctcaag ctcttcgtga gcaaggagta agaatgaagc tgtccgccgt 7860

ccgcggtgtt gttgaaggaa aacgggtcgt catggttgat gattccatcg tgcgcgggac 7920

gacaagccgc cggatcgtca caatgctccg agaagcggga gcgacagagg tgcatgtaaa 7980

gatcagttcg cctccgatcg cccatccttg cttctacggc atcgacacat caacccatga 8040

ggagctgatc gcttcctcgc attcagtgga agaaatccgc cagattatcg gcgccgacac 8100

gctttctttc ttaagtgtag acggattgtt aaaaggagtc ggccgaaaat ttgaagacac 8160

caattgcgga caatgcctcg cttgttttac gggcaaatat ccgacggaaa tttatcagga 8220

tacagtgctt cctcacgtaa aagaagcagt gctgacaaaa taaagtctga aaatgatata 8280

aaggcagcgc gatttcaggc tgcctttctc tttctgcctt ttgaggagaa acgaattgac 8340

aaggagtgat agggatgtct gatgcttata aaaatgccgg agttgacatt gaagccggat 8400

atgaagccgt caaacgaatg aaaaaacatg tggagcgcac gaaaaggctc ggcgttatgg 8460

gaagcttggg cggttttggc ggaatgtttg atctgtcaga gctcccgtat caaaagcctg 8520

ttttaatttc cggaacagac ggtgtcggca ccaagctgaa actcgctttt tcaatggata 8580

agcatgacac gatcggcgta gatgcggtgg cgatgtgtgt aaatgatgtg ctcgcccaag 8640

gagcggagcc gctgtttttc cttgattatt tagcagtcgg caaggccgat ccggtaaaaa 8700

ttgagcagat cgttcagggc gtggcagatg gctgcgagca gtcaggatca gcgcttatcg 8760

gcggtgagac ggcggaaatg ccggggctct atacggctga tgaatacgat attgccggct 8820

tttcagtcgg agtcgcggaa aaagacgaaa tcgtcaccgg tgaacatatc gaagaggggc 8880

atctcttaat cggacttact tcaagcggcc ttcacagcaa cggattttcc cttgtgagaa 8940

aggtgcttct tgatgacggc ggacttgatt tggatactgt ctatgaaccc ttcgcgcggc 9000

cgctcggaga agaattgctg gaaccgacga gaatatatgt gaagccggtg cttgaagcgg 9060

tgaaaagcgg taaggtggac ggcatggcgc atgtgacagg cggaggattc atcgagaaca 9120

ttccgcggat gctgccggac ggattgagcg cggaaattga tcacgggtca tggccgatcc 9180

cgccgatttt tccgtttttg caggagcacg gcaagctgaa agaagaagaa atgttcaacg 9240

tctttaacat gggcatcggt tttgtccttg ccgttaaaga agaaaacctg acagacgtca 9300

tcgacacgct tgaagctaag ggcgagaagg cttatttgat cgggcgggtc aagcagggcg 9360

aaggcatttc tttcggcggt gcggctcttt catgaaaaaa tttgcagtat ttgcttcagg 9420

aaacggatcg aacttcgagg ccattgccaa acgcatgaga gaagagaagt gggacgcgga 9480

gctatcgctt ctcgtgacgg acaagcctca ggcgaaggcg gtggaaaggg ccgaggcttt 9540

acaaatcccg tcattcgcct ttgaaccgtc agcttttgaa aacaaggccg cctttgaacg 9600

ggccattatt gagcagcttc gccttcacgg ggttgaatta atcgttctcg ccggctatat 9660

gagactgatc ggagatacgc tgcttgaagc atacggaggc aggattatca atatacatcc 9720

gtcgcttctc ccggcgtttc cgggcattga cgctgtcgga caagcgcatc gtgccggtgt 9780

gaaagtggcg ggcattaccg ttcattacgt cgacgaaggc atggacaccg gaccgattat 9840

cgcgcaaaaa gcattcgaga tacaggaaaa cgatacgctt gaagatatgg aacatacaat 9900

acacgagctt gagcacaaat ggtatccgag cgttgtgaaa cagctgctgg gactaaataa 9960

cagaggtgaa aaggcatgac aatcaaacgc gcactaatca gtgtttctga taaaacaaat 10020

cttgtacctt tcgtaaagga actgacagag ctcggcgtcg aagtcatttc gaccggagga 10080

acaaaaaaac ttctccagga aaacggtgtg gatgtcatcg gcatttcgga agtgactgga 10140

tttcctgaaa ttatggacgg acggttaaaa acgctccatc ctaatattca cggcggcctg 10200

cttgccgtaa gagacaatga agagcatatg gcgcagatca atgaacacgg cattgcaccc 10260

attgatcttg tggtcgtcaa cctttacccg tttaaagaaa cgatttcaaa agaagacgta 10320

acatacgatg aagcgataga aaacattgat atcggcggtc ccggcatgct gcgcgccgca 10380

tcgaaaaacc atcaggatgt gacggtcatc acggacccgg ccgattacag ctccgtgctc 10440

aatgagatta aagaatacgg cggcgtttcg cttaaaagaa aacgcgagct tgcggccaaa 10500

gtattccgcc acaccgcggc atacgacgca ttaattgctg attacttaac acacgaggcc 10560

ggtgagaaag accctgagca attcaccgtt acatttgaga aaaaacaatc gctccgctac 10620

ggcgaaaacc ctcaccaaga ggctgttttc taccaaagcg cactgcctgt ctccggttcc 10680

attgcggcgg caaaacagct tcacggcaaa gagctttctt acaacaacat taaagacgcg 10740

gatgcggccg ttcaaatcgt ccgggaattt acagaacccg ccgctgttgc cgttaaacat 10800

atgaacccat gcggagtcgg tacgggagcc acaattgagg aagcgttcaa taaagcgtat 10860

gaagcggaca aaacgtccat tttcggcggc atcatcgcgc tcaaccgtga agttgatcag 10920

gcaacggccg aagcccttca cggcatcttt ttagaaatca tcatcgcccc ttctttcagt 10980

gaagaagcgc tgaatgtgct gacgtcgaag aaaaaccttc gtctgctcac gcttgacgtg 11040

aatgcagcag ggaagaaaga aaaacagctg acttccgtac agggcggcct tttgattcaa 11100

gatttagacg tgcacggatt tgatgacgca aaaatcagca ttccgacaaa aagagagccg 11160

agcgagcagg agtgggaaga tctgaagctg gcttggaaag tcgtcaaaca cgtgaaatca 11220

aacgcgatcg ttcttgcaaa agaccatatg acggtcggtg tgggtgcagg acagatgaat 11280

cgcgtcggtt cggccaaaat cgccatcgag caggccggag aaaaagcaaa aggcagcgcg 11340

ctcggatcag acgcgttttt cccgatgccg gatacagtcg aagaagccgc aaaagcgggc 11400

gtcacggcga tcatccagcc cggcggatcg gtccgcgacg aagattcaat taaaaaagcg 11460

gatgaatacg gcatcgccat ggtcttcaca ggcatcagac atttcaaaca ttaagggagg 11520

aacgaagcgt gaatgtattg attatcggta aaggcggcag agagcataca ttggcttgga 11580

aagcggcgca aagtccgctt actgacacgg tgtacgccgc gcccggaaat gacggtatgg 11640

cagactgcgc gacgctggtc agcatcgaag aaagcgatca tgccggactc attgcctttg 11700

cgaaagaaca tcatgtcggc ctgacgatta tcggtcccga ggttcctctc attgaaggga 11760

tagcggacga gtttgaaaaa gccggactcc ttgttttcgg gccgtccgaa caagcggcaa 11820

tcattgaagg aagcaaacag tttgcgaagg atttaatgaa aaaatacggt ataccgacgg 11880

cggagtatga gacgtttact tcatttgaag aggcgaaagc atatgtgcag cagaaaggcg 11940

cgccgattgt cattaaagcg gacgggcttg ccgccggaaa aggtgtgacg gtcgcgatga 12000

cggaagagga agcgattgaa tgccttcatg attttctcga ggatgagaaa ttcggcgagg 12060

cgagcgcatc cgtggtcatt gaagaatttc tcgccggtga agaattttcc ttaatggcgt 12120

ttgtaaaagg ggagaccgtc tatccgatgg tgatcgccca agaccataaa cgcgccttcg 12180

acggagacaa agggccgaat acgggcggaa tgggcgccta ctcaccggtg ccgcacattt 12240

ccgatgacat cgtcaaaagc gctgtcgaaa cgattgtgaa gccggcggca aaagctatgg 12300

tgaaagaggg acgctccttc acaggcgtgc tgtacgcggg gctgattctg acggaaaacg 12360

gatcaaaagt cattgaattc aacgcgcgct tcggtgatcc ggaaacacag gttgtggtgc 12420

cgagaatgga atcagacctc attcaggtgc ttttggatct gcttcatgag aaggatgttg 12480

acctaaggtg gaaggatacg gccgctgtca gcgttgtgct ggcttctgag ggctatcctg 12540

aaggctatgc gaaagggaca ccgatcggca gtttgacatc cgctgaagac gggatcgccg 12600

tttttcatgc cggaacgaaa aaagacggcg atcaatttgt cacaaacggc ggccgggtcg 12660

ccaatgtcac ggcatttgct gagacatttg aagaggcgag agataaagtg tacagcgccg 12720

tttccggtct gacaaaaccc ggactgtttt acagaagcga tatcggcgtc cgtgcgctga 12780

aagcatcgct gcgataa 12797

<210> 4

<211> 1293

<212> DNA

<213> artificial sequence

<400> 4

atgtcttcag tagttgtagt aggtacgcaa tggggcgatg aaggaaaagg taaaattaca 60

gatttcctat cagaaaatgc agaagtgatc gcccgttatc aaggcggaaa taacgcaggg 120

catacaatca agtttgacgg aatcacatat aagcttcact taatcccgtc tggaattttc 180

tataaggata aaacgtgtgt aatcggaaac ggaatggttg tagatccgaa agcattagtc 240

acagagcttg cgtatcttca tgagcgcaac gtgagtacag ataacctgag aatcagcaac 300

agagctcacg tcattctgcc gtatcatttg aaattggatg aagtggaaga agagcgtaaa 360

ggggctaaca agatcggcac aacgaaaaaa ggaatcggcc ctgcttacat ggataaagca 420

gcccgcatcg gaattcgcat cgcggatctg ttagaccgtg acgcgtttgc ggaaaagctt 480

gagcgcaatc ttgaagaaaa aaaccgtctt ctcgagaaaa tgtacgagac agaagggttt 540

aaacttgagg atatcttaga cgaatattat gagtacggac agcagattaa aaagtatgtt 600

tgcgatacat ctgttgtctt aaacgatgct cttgatgaag ggcgccgtgt attatttgaa 660

ggcgcacaag gggttatgct cgatatcgac caaggaacat acccgtttgt tacgtcatct 720

aacaatgttg ccggcggtgt cacgatcggt tctggtgtcg gcccgaccaa aatcaagcac 780

gttgtcggtg tatcaaaagc atatacgact cgtgtcggcg acggtccttt tccgactgag 840

ctgaaagatg aaatcggcga tcaaatccgt gaagtcggac gcgaatatgg aacaacaaca 900

ggccgcccgc gccgtgtcgg ctggtttgac agcgttgttg tccgccacgc ccgccgtgtg 960

agcggaatta cagatctttc tctgaactca attgacgtcc tagcaggaat tgaaacgttg 1020

aaaatctgtg tggcgtaccg ctacaaaggc gaaatcattg aagaattccc agcaagtctt 1080

aaggcacttg ctgaatgtga gccggtatat gaagaaatgc cgggctggac tgaggatatt 1140

acaggtgcga agagcttgag cgagcttccg gaaaatgcgc gccattatct tgagcgtgtg 1200

tctcagctga caggcattcc gctttctatt ttctctgtcg gtccagaccg ctcacaaaca 1260

aatgtccttc gcagtgtgta ccgtgcgaac taa 1293

<210> 5

<211> 74

<212> DNA

<213> artificial sequence

<400> 5

ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac 60

acaggaaaca gacc 74

<210> 6

<211> 476

<212> PRT

<213> artificial sequence

<400> 6

Met Leu Ala Glu Ile Lys Gly Leu Asn Glu Glu Cys Gly Val Phe Gly

1 5 10 15

Ile Trp Gly His Glu Glu Ala Pro Gln Ile Thr Tyr Tyr Gly Leu His

20 25 30

Ser Leu Gln His Arg Gly Gln Glu Gly Ala Gly Ile Val Ala Thr Asp

35 40 45

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

50 55 60

Phe Gln Asn Gly Glu Leu Ser Lys Val Lys Gly Lys Gly Ala Ile Gly

65 70 75 80

His Val Arg Tyr Ala Thr Ala Gly Gly Gly Gly Tyr Glu Asn Val Gln

85 90 95

Pro Leu Leu Phe Arg Ser Gln Asn Asn Gly Ser Leu Ala Leu Ala His

100 105 110

Asn Gly Asn Leu Val Asn Ala Thr Gln Leu Lys Gln Gln Leu Glu Asn

115 120 125

Gln Gly Ser Ile Phe Gln Thr Ser Ser Asp Thr Glu Val Leu Ala His

130 135 140

Leu Ile Lys Arg Ser Gly His Phe Ser Leu Lys Asp Gln Ile Lys Asn

145 150 155 160

Ser Leu Ser Met Leu Lys Gly Ala Tyr Ala Phe Leu Ile Met Thr Glu

165 170 175

Thr Glu Met Ile Val Ala Leu Asp Pro Asn Gly Leu Arg Pro Leu Ser

180 185 190

Leu Gly Met Leu Gly Asp Ala Tyr Val Val Ala Ser Glu Thr Cys Ala

195 200 205

Phe Asp Val Val Gly Ala Thr Tyr Leu Arg Asp Val Glu Pro Gly Glu

210 215 220

Met Leu Ile Ile Asn Asp Glu Gly Leu Lys Ser Glu Arg Phe Ser Met

225 230 235 240

Asn Ile Asn Arg Ser Ile Cys Ser Met Glu Tyr Ile Tyr Phe Ser Arg

245 250 255

Pro Asp Ser Asn Ile Asp Gly Ile Asn Val His Ser Ala Arg Lys Ser

260 265 270

Leu Gly Lys Met Leu Ala Gln Glu Ser Ala Val Glu Ala Asp Val Val

275 280 285

Thr Gly Val Pro Asp Ser Ser Ile Ser Ala Ala Ile Gly Tyr Ala Glu

290 295 300

Ala Thr Gly Ile Pro Tyr Glu Leu Gly Leu Ile Gln Asn Arg Tyr Val

305 310 315 320

Gly Arg Thr Phe Ile Gln Pro Ser Gln Ala Leu Arg Glu Gln Gly Val

325 330 335

Arg Met Lys Leu Ser Ala Val Arg Gly Val Val Glu Gly Lys Arg Val

340 345 350

Val Met Val Asp Asp Ser Ile Val Arg Gly Thr Thr Ser Arg Arg Ile

355 360 365

Val Thr Met Leu Arg Glu Ala Gly Ala Thr Glu Val His Val Lys Ile

370 375 380

Ser Ser Pro Pro Ile Ala His Pro Cys Phe Tyr Gly Ile Asp Thr Ser

385 390 395 400

Thr His Glu Glu Leu Ile Ala Ser Ser His Ser Val Glu Glu Ile Arg

405 410 415

Gln Ile Ile Gly Ala Asp Thr Leu Ser Phe Leu Ser Val Asp Gly Leu

420 425 430

Leu Lys Gly Val Gly Arg Lys Phe Glu Asp Thr Asn Cys Gly Gln Cys

435 440 445

Leu Ala Cys Phe Thr Gly Lys Tyr Pro Thr Glu Ile Tyr Gln Asp Thr

450 455 460

Val Leu Pro His Val Lys Glu Ala Val Leu Thr Lys

465 470 475

Claims

1. An escherichia coli genetic engineering strain, which is characterized in that:the genetically engineered strain takes E.coli MG1655 as an original strain, and SEQ ID NO:1, integrating the nucleoside transporter gene pfue shown in SEQ ID NO:2, PRPP transamidase mutant gene purF shown in the specification ^K316Q Integration at the yeeP pseudogene locus, SEQ ID NO:3, purEKBCSQLF as purine operon mutant gene ^K316Q MNHD is integrated at the yghE pseudogene locus, replacing the adenosine succinate synthase gene purA with SEQ ID NO:4, and the mutant gene purA shown in FIG. 4 ^P242N And knocks out purine nucleoside phosphorylase genes deoD, ppnP, and nucleoside hydrolase gene rihA, rihB, rihC.

2. The genetically engineered strain of claim 1, wherein: the nucleoside transporter gene pfue is connected with a promoter P _trc The method comprises the steps of carrying out a first treatment on the surface of the And/or

Purine operon mutant gene purEKBCSQLF ^K316Q MNCD is connected with promoter P _trc The method comprises the steps of carrying out a first treatment on the surface of the And/or

The PRPP transamidase mutant gene purF ^K316Q Is connected with a promoter P _trc ；

The promoter P _trc The nucleotide sequence of (2) is shown as SEQ ID NO: shown at 5.

3. The method for constructing a genetically engineered strain according to claim 1 or 2, characterized in that: the construction method comprises the following steps:

(1) Starting from the genome of the strain E.coli MG1655, knocking out purine nucleoside phosphorylase genes deoD and ppnP and knocking out nucleoside hydrolase gene rihA, rihB, rihC;

(2) The nucleotide sequence is shown as SEQ ID NO:3, the purine operon purEKBCSQLF shown in the specification ^K316Q MNCD and promoter P _trc Fusion fragment P of (C) _trc -purEKBCSQLF ^K316Q MNHD is integrated at the yghE pseudogene locus;

(3) The nucleotide sequence is shown as SEQ ID NO:2, PRPP transamidase mutant gene purF shown in the specification ^K316Q And promoter P _trc Fusion fragment P of (C) _trc -purF ^K316Q Integration at the yeeP pseudogene locus;

(4) The nucleotide sequence is shown as SEQ ID NO:1 and a promoter P _trc Fusion fragment P of (C) _trc -pbuE integration at the yjiT pseudogene locus;

(5) The nucleotide sequence of the purA gene is replaced by the nucleotide sequence shown as SEQ ID NO:4, and the mutant gene purA shown in FIG. 4 ^P242N Obtaining the genetic engineering strain.

4. Use of the genetically engineered strain of claim 1 or 2 for the fermentative production of inosine.

5. The application according to claim 4, characterized in that it comprises: culturing the genetically engineered strain under suitable conditions and collecting inosine from the culture thereof; the proper condition is that the culture temperature is 35 ℃, the pH is maintained at about 7.0, the dissolved oxygen is between 25 and 35 percent, and the culture medium comprises the following components: 15-25g/L glucose, 1-4g/L yeast powder, 1-5g/L peptone, 0.1-2g/L sodium citrate, 0.1-0.3g/L adenine and KH ₂ PO ₄ ·3H ₂ O 0.1-2g/L，MgSO ₄ ·7H ₂ O 0.1-2g/L，FeSO ₄ ·7H ₂ O 5-20mg/L，MnSO ₄ ·H ₂ O 5-20mg/L，V _B1 、V _B3 、V _B5 、V _B12 And V _H 0.1-2mg/L each, 2 drops of defoamer, the balance of water, and pH 7.0-7.2.