CN114317386A - Gene engineering strain for producing inosine and construction method and application thereof - Google Patents
Gene engineering strain for producing inosine and construction method and application thereof Download PDFInfo
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- CN114317386A CN114317386A CN202111481366.1A CN202111481366A CN114317386A CN 114317386 A CN114317386 A CN 114317386A CN 202111481366 A CN202111481366 A CN 202111481366A CN 114317386 A CN114317386 A CN 114317386A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 72
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 title claims abstract description 61
- 229930010555 Inosine Natural products 0.000 title claims abstract description 59
- 229960003786 inosine Drugs 0.000 title claims abstract description 59
- 238000010276 construction Methods 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 claims abstract description 42
- 241000588724 Escherichia coli Species 0.000 claims abstract description 39
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims abstract description 36
- PQGCEDQWHSBAJP-TXICZTDVSA-N 5-O-phosphono-alpha-D-ribofuranosyl diphosphate Chemical compound O[C@H]1[C@@H](O)[C@@H](O[P@](O)(=O)OP(O)(O)=O)O[C@@H]1COP(O)(O)=O PQGCEDQWHSBAJP-TXICZTDVSA-N 0.000 claims abstract description 26
- 101800000628 PDH precursor-related peptide Proteins 0.000 claims abstract description 26
- 101150002764 purA gene Proteins 0.000 claims abstract description 24
- 101150013480 pbuE gene Proteins 0.000 claims abstract description 22
- 101150072043 deoD gene Proteins 0.000 claims abstract description 16
- 101150033174 ppnP gene Proteins 0.000 claims abstract description 14
- 101150093992 rihA gene Proteins 0.000 claims abstract description 14
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- 238000010353 genetic engineering Methods 0.000 claims abstract description 10
- 108010063372 N-Glycosyl Hydrolases Proteins 0.000 claims abstract description 9
- 101710101148 Probable 6-oxopurine nucleoside phosphorylase Proteins 0.000 claims abstract description 9
- 230000002018 overexpression Effects 0.000 claims abstract description 6
- OFBHPPMPBOJXRT-VWJPMABRSA-N adenylosuccinic acid Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C2=NC=NC(N[C@@H](CC(O)=O)C(O)=O)=C2N=C1 OFBHPPMPBOJXRT-VWJPMABRSA-N 0.000 claims abstract description 4
- 108091006527 nucleoside transporters Proteins 0.000 claims abstract description 3
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- 125000003729 nucleotide group Chemical group 0.000 claims description 27
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 13
- 239000008103 glucose Substances 0.000 claims description 13
- 229930024421 Adenine Natural products 0.000 claims description 11
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 11
- 229960000643 adenine Drugs 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 9
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- 102000005130 adenylosuccinate synthetase Human genes 0.000 claims description 7
- 150000001413 amino acids Chemical group 0.000 claims description 7
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 7
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 5
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
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- 239000001301 oxygen Substances 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
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- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 2
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention belongs to the technical field of genetic engineering, relates to breeding of industrial microorganisms, and particularly relates to a genetic engineering for producing inosineThe strain and its construction process and application. The gene engineering strain heterologous overexpression nucleoside transporter gene pbuE and purine operon mutant gene purEKBCSQLFK316QMNHD and PRPP transamidase mutant gene purFK316QAnd mutant gene purA with heterologous adenylosuccinate synthetaseP242NReplaces the gene purA and does not express purine nucleoside phosphorylase genes deoD, ppnP and nucleoside hydrolase genes rihA, rihB and rihC. The invention starts from the level of escherichia coli genome, and carries out comprehensive combined optimization on modules of an inosine decomposition path, an inosine synthesis path, an inosine transport system and a branch metabolic path mainly through a metabolic engineering technical means, thereby improving the inosine fermentation performance of the strain.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, relates to breeding of industrial microorganisms, and particularly relates to a genetic engineering strain for producing inosine as well as a construction method and application thereof.
Background
Inosine is a purine nucleoside, participates in metabolism of body substances and energy metabolism, and has important application value. The microbial fermentation method is the main method for industrially producing inosine at present, but because the existing strains have poor fermentation performance, the production level of inosine produced by the fermentation method is still low, and high-performance strains are urgently needed to meet the industrial demand of inosine. The breeding of the traditional inosine production strain is obtained by mutagenizing and screening structural analogue resistant strains, but the continuous improvement of the performance of the strain obtained by mutagenesis is not facilitated due to unclear genetic background and accumulation of a large number of negative mutations. With the development of system biology, the rational construction method of metabolic engineering and the like gradually becomes the first choice method for the de novo construction and continuous improvement of the inosine producing strain. The existing research results provide important reference for rational construction of inosine engineering strains, but still have a plurality of problems which are not solved. The concrete points are as follows: (1) at present, the inosine production performance of rationally constructed strains is generally low, the highest yield is only 14g/L, the production period is longer, and the length is generally as long as 72 hours or even longer. (2) The strain carries plasmid, the burden of the strain is heavy, and the plasmid is easy to lose, which can cause the instability of production. Antibiotics are required to be added for maintaining the stability of plasmids, so that the production cost is increased, and potential safety hazards such as drug resistance are increased. (3) In the aspect of construction strategy, the existing strains are mostly limited to the modification of individual nodes such as degradation pathway, head-to-head synthesis pathway, precursor synthesis pathway and branch metabolic pathway, and the system combination of all modules of the whole inosine metabolic network is lacked. (4) Most of the strains are adenine defects, adenine is required to be added in the fermentation process, the raw material cost is high, the fermentation process is complex, and the production stability is reduced.
The common inosine production strain de novo construction strategy mainly comprises four aspects of blocking degradation pathway, strengthening de novo synthesis pathway, strengthening precursor synthesis pathway and blocking branch metabolic pathway. Shimaoka M et al (Effect of amplification of expressed purF and prs on inosine accumulation in Escherichia coli. journal of bioscience and bioengineering,2007,103(3): 255-; by knocking out purR gene and overexpressing purFK326Q,P410WGenes, which enhance de novo synthetic pathways; by knocking out pgi and edd genes and over-expressing prsD128AGenes, which enhance the precursor synthesis pathway; by knocking out purA gene, the adenosine synthesis branch is blocked. After 72 hours of fermentation of the obtained strain I-9m/pMWKQ-pSTVDA, the yield of inosine reaches 7.5 g/L. Asahara T et al (Accumulation of gene-targeted Bacillus subtilis mutation and improvement of inosine production. applied Microbiol Biotechnol,2010,87(6):2195-2207.) use Bacillus subtilis W168 as an initial strain, and by knocking out deoD and punA genes, an inosine degradation pathway is blocked; 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 72h, and the yield of inosine reaches 6 g/L. Plum Hao Jian et al (De novo engineering and metabolic flux analysis of inosine biosynthesis in Bacillus subtilis, biotechnol Lett,2011,33(8): 1575) take Bacillus subtilis W168 as an initial strain, and block an inosine degradation pathway by knocking out deoD gene; by knocking out purA gene, the adenosine synthesis branch is blocked. The obtained strain BS019 is fermented for 72 hours, and the inosine yield reaches 7.6 g/L. Wenting yiyi et al (CN106906174A) use bacillus subtilis W168 as an original strain, and block the branch of adenosine synthesis by knocking out purA gene; by knocking out drmThe gene blocks the degradation pathway from ribose 1-phosphate to ribose 5-phosphate. The obtained strain IR-2 is fermented for 72 hours, and the inosine yield reaches 14g/L, which is the highest yield of inosine in rationally constructed strains.
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 produce the inosine by fermentation.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an engineered Escherichia coli strain with heterologous overexpression of the nucleotide transporter gene pbuE and the purine operon mutant gene purEKBCSQLFK316QMNHD and PRPP transamidase mutant gene purFK316QAnd mutant gene purA with heterologous adenylosuccinate synthetaseP242NReplaces the gene purA and does not express purine nucleoside phosphorylase genes deoD, ppnP and nucleoside hydrolase genes rihA, rihB and rihC.
In a second aspect, the present invention provides a method for constructing the genetically engineered escherichia coli strain, comprising: introducing a nucleotide transport protein gene pbuE and a purine operon mutant gene purEKBCSQLF into an original strain escherichia coliK316QMNHD and PRPP transamidase mutant gene purFK316QAnd mutant gene purA with heterologous adenylosuccinate synthetaseP242NThe gene purA was replaced and the purine nucleoside phosphorylase genes deoD, ppnP and the nucleoside hydrolase genes rihA, rihB, rihC were knocked out or inactivated.
In a third aspect, the present invention provides the use of the genetically engineered strain of escherichia coli as described above for high inosine production.
The invention has the following beneficial effects:
the invention utilizes a rational metabolic engineering method to obtain the engineering strain which has clear genetic background, does not contain plasmids and can efficiently produce inosine. From the production phenotype, the strain is fermented on a 5L tank for 48 hours, the inosine yield reaches 20.16g/L, the yield is improved by 42.8 percent compared with the highest yield of the currently rationally constructed strain in the prior art, the fermentation period is shortened by 1/3, and the strain does not contain plasmids. From the construction strategy, the invention carries out systematic and comprehensive combined optimization on an inosine decomposition path, an inosine synthesis path, an inosine transport system and a branch metabolic path in a sub-module manner through a metabolic engineering technical means, and the strain has clear genetic background and higher and more stable production performance. Particularly, compared with other inosine production strains, the method weakens but not directly blocks the adenosine synthesis branch, thereby ensuring the sufficient inosine branch flux and maintaining a certain growth level of the strain. The finally obtained strain can stably and efficiently produce inosine, and has good industrial application prospect.
Drawings
FIG. 1A: and (3) constructing a deoD gene knockout fragment and verifying an electrophoretogram. Wherein, M: marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacterium identification fragment 5: and (5) negative control.
FIG. 1B: and (3) constructing a ppnP gene knockout fragment and verifying an electrophoretogram. Wherein, M: marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacterium identification fragment 5: and (5) negative control.
FIG. 1C: construction of rihA knock-out fragment and confirmation of electrophoretogram. Wherein, M: marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacterium identification fragment 5: and (5) negative control.
FIG. 1D: construction of rihB gene knockout fragment and electrophoresis chart verification. Wherein, M: marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacterium identification fragment 5: and (5) negative control.
FIG. 1E: construction of a rihC gene knockout fragment and confirmation of an electrophoretogram. Wherein, M: marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacterium identification fragment 5: and (5) negative control.
FIG. 2A: pur1 integrated fragment construction and electrophoretic image verification. Wherein: m: marker; 1: an upstream homology arm; 2: pur1 fragment; 3: a downstream homology arm; 4: overlapping segments; 5: positive bacteria identification fragments; 6: and (5) negative control.
FIG. 2B: pur2 integrated fragment construction and electrophoretic image verification. Wherein: m: marker; 1: : pur2 upstream fragment-pur 2 fragment; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacteria identification fragments; 5: and (5) negative control.
FIG. 2C: pur3 integrated fragment construction and electrophoretic image verification. Wherein: m: marker; 1: : pur3 upstream fragment-pur 3 fragment; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacteria identification fragments; 5: and (5) negative control.
FIG. 2D: pur4 integrated fragment construction and electrophoretic image verification. Wherein: m: marker; 1: : pur4 upstream fragment-pur 4 fragment; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacteria identification fragments; 5: and (5) negative control.
FIG. 2E: pur5 integrated fragment construction and electrophoretic image verification. Wherein: m: marker; 1: : pur5 upstream fragment-pur 5 fragment; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacteria identification fragments; 5: and (5) negative control.
FIG. 2F: pur6 integrated fragment construction and electrophoretic image verification. Wherein: m: marker; 1: : pur6 upstream fragment-pur 6 fragment; 2: a downstream homology arm; 3: overlapping segments; 4: positive bacteria identification fragments; 5: and (5) negative control.
FIG. 2G: purFK326Q,P410WAnd (5) integrating fragment construction and verifying an electrophoretogram. Wherein: m: marker; 1: an upstream homology arm; 2: purFK326Q,P410WA gene fragment; 3: a downstream homology arm; 4: overlapping segments; 5: positive bacteria identification fragments; 6: and (5) negative control.
FIG. 2H: purFD293V,K316Q,S400WAnd (5) integrating fragment construction and verifying an electrophoretogram. Wherein: m: marker; 1: an upstream homology arm; 2: purFD293V,K316Q,S400WA gene fragment; 3: a downstream homology arm; 4: overlapping segments; 5: positive bacteria identification fragments; 6: and (5) negative control.
FIG. 2I: purFK316QIntegration of fragmentsAnd (5) constructing and verifying an electrophoretogram. Wherein: m: marker; 1: an upstream homology arm; 2: purFK316QA gene fragment; 3: a downstream homology arm; 4: overlapping segments; 5: positive bacteria identification fragments; 6: and (5) negative control.
FIG. 3: the pbuE integrated fragment was constructed and the electropherogram was verified. Wherein: m: marker; 1: an upstream homology arm; 2: a pbuE gene fragment; 3: a downstream homology arm; 4: overlapping segments; 5: positive bacteria identification fragments; 6: and (5) negative control.
FIG. 4: purAP242NAnd (5) integrating fragment construction and verifying an electrophoretogram. Wherein: m: marker; 1: an upstream homology arm; 2: purAP242NA gene fragment; 3: a downstream homology arm; 4: overlapping segments; 5: positive bacteria identification fragments; 6: and (5) negative control.
FIG. 5: effect of overexpression of different PurF mutants on inosine yield.
FIG. 6: fed batch fermentation profile of strain INO4 in a 5L fermenter.
FIG. 7: the technical scheme of the invention is schematically shown in principle.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
In a first aspect, the invention provides an engineered Escherichia coli strain with heterologous overexpression of the nucleotide transporter gene pbuE and the purine operon mutant gene purEKBCSQLFK316QMNHD and PRPP transamidase mutant gene purFK316QWherein purFK316QThe amino acid sequence of purF gene codes the amino acid sequence with the place 316 lysine replaced by glutamine, and the gene purA is mutated by the adenylosuccinic acid synthetaseP242NReplaces the gene purA, anAnd do not express purine nucleoside phosphorylase genes deoD, ppnP and nucleoside hydrolase genes rihA, rihB, rihC.
Preferably, the nucleotide sequence of the nucleoside transporter gene pbuE is as shown in SEQ ID NO: 1, its NCBI-GeneID: 12132461.
preferably, the PRPP transamidase mutant gene purFK316QThe nucleotide sequence of (a) is shown as SEQ ID NO: 2, respectively. Wherein purFK316QRepresents that lysine (K) at position 316 of the amino acid sequence encoded by purF gene is replaced by glutamine (Q). The numbering of positions is the position corresponding to the amino acid sequence of the parent PRPP transamidase.
Preferably, the purine operon mutant gene purEKBCSQLFK316QThe nucleotide sequence of MNHD is shown as SEQ ID NO: 3, respectively. purF contained thereinK316QIs a mutant gene of purF, and indicates that lysine (K) at position 316 of the amino acid sequence encoded by purF gene is replaced with glutamine (Q).
Preferably, the adenylosuccinate synthetase mutant gene purAP242NThe nucleotide sequence of (a) is shown as SEQ ID NO: 4, respectively. Wherein purAP242NRepresents that proline (P) at position 128 of the amino acid sequence encoded by purA gene is replaced by asparagine (N). The numbering of positions is the position corresponding to the amino acid sequence of the parent adenylosuccinate synthetase.
According to the present invention, the manner in which the above gene is not expressed may be by a conventional means in the art, for example, by inactivating the gene or knocking out the gene by a conventional means in the art.
According to the invention, by not expressing is meant 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%.
According to the present invention, the above-mentioned gene can be overexpressed by means conventional in the art, for example, by increasing the copy number of the gene or by linking the gene to a strong promoter.
According to the invention, said overexpression means that the amount of the expression product of the gene is significantly higher than the original level.
According to the inventionIn a preferred embodiment, the nucleotide transporter gene pbuE is linked to the promoter Ptrc(ii) a And/or the purine operon mutant gene purEKBCSQLFK316QMNHD is connected with a promoter Ptrc(ii) a And/or the PRPP transamidase mutant gene purFK316QLinked with a promoter Ptrc(ii) a Preferably, the promoter PtrcThe nucleotide sequence of (a) is shown as SEQ ID NO: 5, respectively.
The starting strain for constructing the engineered escherichia coli strain according to the present invention may be any escherichia coli, and according to a preferred embodiment of the present invention, the starting strain is e.
In a second aspect, the present invention provides a method for constructing the genetically engineered escherichia coli strain, comprising: introducing a nucleotide transport protein gene pbuE and a purine operon mutant gene purEKBCSQLF into an original strain escherichia coliK316QMNHD and PRPP transamidase mutant gene purFK316QWherein purFK316QIs the amino acid sequence with glutamine substituted by lysine at position 316, and adenylosuccinate synthetase mutant gene purAP242NThe gene purA was replaced and the purine nucleoside phosphorylase genes deoD, ppnP and the nucleoside hydrolase genes rihA, rihB, rihC were knocked out or inactivated.
The selection of each gene, the selection of a promoter, the selection of a starting strain, and the like have been described in detail in the first aspect of the present invention, and the details already described in the first aspect are not repeated herein.
According to a specific embodiment of the present invention, the method comprises:
(1) blocking inosine breakdown pathway
Knocking out purine nucleoside phosphorylase genes deoD and ppnP and knocking out nucleoside hydrolase genes rihA, rihB and rihC from a genome of a strain E.coli MG 1655; this step blocked the degradation of inosine;
(2) enhancing inosine synthesis pathway
The purine operon purEKBCSQLF of Bacillus amyloliquefaciens TA208K316QMNHD and promoter PtrcFused fragment P of (1)trc-purEKBCSQLFK316QMNHD integrates at the yghE pseudogene site; this step enhances the de novo purine anabolic flux while eliminating the feedback inhibition of PRPP amidotransferase by AMP and GMP;
the PRPP transamidase mutant gene purF of Bacillus amyloliquefaciens TA208K316QAnd the promoter PtrcFused fragment P of (1)trc-purFK316QIntegrated at the yeeP pseudogene site; the step further strengthens the transcription expression of PRPP transamidase and promotes the synthesis of inosine;
(3) modified inosine transport system
The nucleotide transport protein gene pbuE of Bacillus amyloliquefaciens TA208 and a promoter PtrcFused fragment P of (1)trc-pbuE integration at the yjiT pseudogene site; this step enhances the ability of inosine to be transported extracellularly;
(4) weakening adenosine synthesis branch
Substitution of adenylosuccinate synthetase purA Gene with mutant Gene purA of B.subtilis W168P242N(ii) a 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 seen with reference to fig. 7.
In a third aspect, the present invention provides an application of the genetically engineered strain of escherichia coli as described above in high inosine production, including: the genetically engineered strain is cultured under suitable conditions, and inosine is collected from the culture thereof.
According to a preferred embodiment of the present invention, the suitable conditions are a culture temperature of 35 ℃, a pH maintained at about 7.0, a dissolved oxygen content of 25-35%, and a 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 KH2PO4·3H2O 0.1-2g/L，MgSO4·7H2O 0.1-2g/L，FeSO4·7H2O 5-20mg/L，MnSO4·H2O 5-20mg/L，VB1、VB3、VB5、VB12And VH0.1-2mg/L of each, 2 drops of defoaming agent and the balance of water, and the pH value is 7.0-7.2.
The present invention will be described in more detail below by way of specific examples. In the following examples:
unless otherwise specified, the gene editing methods described in the examples of the present invention are performed with reference to the literature (Li Y, Lin Z, Huang C, et al. metabolic engineering of Escherichia coli using CRISPR-Cas 9 dimensional genome editing. metabolic engineering,2015,31:13-21.), and other specific manipulations related to molecular biology, genetic engineering, etc. can be performed according to technical manuals, textbooks, or literature reports readily available to those skilled in the art.
Example 1: construction of Escherichia coli genetic engineering strain E.coli INO4
1. Blocking inosine breakdown pathway
1.1 knockout of deoD Gene:
coli MG1655 genome as template, upstream homology arm primer (UP-deoD-S, UP-deoD-A) and downstream homology arm primer (DN-deoD-S, DN-deoD-A) were designed based on the upstream and downstream sequences of its deoD gene (NCBI-GeneID:945654), and its upstream and downstream homology arm fragments were PCR-amplified. The above fragments were fused by overlap PCR to obtain a knockout fragment (upstream homology arm-downstream homology arm) of deoD gene. Construction of pGRB-deoD the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-deoD-S and gRNA-deoD-A. And (3) electrically transferring the recombinant fragment and the plasmid pGRB-deoD to competent cells of E.coli MG1655, screening positive strains, and eliminating the plasmid to obtain a strain INO 1-1. The electrophoresis pattern of the construction of deoD knock-out fragment and PCR validation of positive strains is shown in FIG. 1A. Wherein, the length of the upstream homologous arm is 458bp, the length of the downstream homologous arm is 613bp, and the total length of the overlapped fragments is 1029 bp. During PCR verification, 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 1630 bp.
1.2 knockout of the ppnP Gene:
coli MG1655 genome as template, upstream homology arm primer (UP-ppnP-S, UP-ppnP-A) and 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 its upstream and downstream homology arm fragments were PCR-amplified. The above fragments were fused by an overlap PCR method to obtain a knockout fragment (upstream homology arm-downstream homology arm) of the ppnP gene. Construction of pGRB-ppnP the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-ppnP-S and gRNA-ppnP-A. The recombinant fragment and the plasmid pGRB-ppnP are electrically transferred to competent cells of INO1-1, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 1-2. The electrophoresis pattern of the construction of the ppnP knock-out fragment and the PCR verification of the positive strain is 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 fragments is 1040 bp. During PCR verification, the PCR amplified fragment length of the positive bacteria should be 1040bp, and the PCR amplified fragment length of the original bacteria should be 1361 bp.
1.3 knockout of rihA Gene:
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 its upstream and downstream homology arm fragments were PCR-amplified. The above fragments were fused by the overlap PCR method to obtain a knock-out fragment (upstream homology arm-downstream homology arm) of the rihA gene. Construction of pGRB-rihA the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-rihA-S and gRNA-rihA-A. The recombinant fragment and the plasmid pGRB-rihA are electrically transferred to competent cells of INO1-2, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 1-3. The construction of the rihA knock-out fragment and the electrophoretogram of the PCR-verified 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 1042 bp. During PCR verification, the PCR amplified fragment length of the positive bacteria should be 1042bp, and the PCR amplified fragment length of the original bacteria should be 1857 bp.
1.4 knock-out of rihB Gene:
coli MG1655 genome as template, based on its rihB gene (NCBI-GeneID:946646) upstream and downstream sequence design upstream homology arm primer (UP-rihB-S, UP-rihB-A) and downstream homology arm primer (DN-rihB-S, DN-rihB-A), and PCR amplification of its upstream and downstream homology arm fragments. The fragments are fused by an overlapping PCR method to obtain a knockout fragment (upstream homology arm-downstream homology arm) of the rihB gene. Construction of pGRB-rihB the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-rihB-S and gRNA-rihB-A. And (3) electrically transferring the recombinant fragment and the plasmid pGRB-rihB to competent cells of INO1-3, screening positive strains, and eliminating the plasmid to obtain a strain INO 1-4. The construction of the rihB knock-out fragment and the electrophoretogram of the PCR validation of the positive strain are shown in FIG. 1D. Wherein, the length of the upstream homologous arm is 494bp, the length of the downstream homologous arm is 459bp, and the total length of the overlapped fragments is 912 bp. During PCR verification, 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 1789 bp.
1.5 knockout of the rihC gene:
coli MG1655 genome as a template, an upstream homology arm primer (UP-rihC-S, UP-rihC-A) and a downstream homology arm primer (DN-rihC-S, DN-rihC-A) were designed based on the upstream and downstream sequences of its rihC gene (NCBI-GeneID:944796), and its upstream and downstream homology arm fragments were PCR-amplified. The fragment was fused by overlap PCR to obtain a knock-out fragment of the rihC gene (upstream homology arm-downstream homology arm). Construction of pGRB-rihC the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-rihC-S and gRNA-rihC-A. The recombinant fragment and the plasmid pGRB-rihC are electrically transferred to competent cells of INO1-4, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 1-5. The construction of the rihC knock-out fragment and the PCR-verified electropherogram of the positive strain are shown in figure 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 975 bp. During PCR verification, 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 1787 bp.
2. Enhancing inosine synthesis pathway
2.1 mutation of the purine operon of Bacillus amyloliquefaciens Gene purEKBCSQLFK316QMNHD is integrated at the yghE gene locus of E.coli INO1-5
The purine operon purEKBCSQLFMNHD (containing twelve genes of purE, purK, purB, purC, purS, purQ, purL, purF, purM, purN, purH and purD) of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens TA208) has 12797bp in total, and six segments of pur1, pur2, pur3, pur4, pur5 and pur6 are integrated into the yghE gene locus (containing the pur gene locus) in sequence in the embodimentrF replacement by mutant Gene purFK316Q) And from the promoter PtrcTranscription expression of the exogenous operon is started, and a strain E.coli INO2-6 is constructed. The method specifically comprises the following steps:
2.1.1Ptrcintegration of pur 1:
taking E.coli MG1655 genome as template, designing upstream homology arm primer (UP-yghE-S, UP-yghE-A) and downstream homology arm primer (DN-yghE-S1, DN-yghE-A) according to upstream and downstream sequences of yghE gene, PCR amplifying upstream and downstream homology arm fragments; using Bacillus amyloliquefaciens TA208 genome as a template, designing a primer (UP-pur1-S, UP-pur1-A) according to pur1 (1 st to 2113 th positions of a nucleotide sequence shown in SEQ ID NO: 3), and carrying out PCR amplification on a pur1 fragment; promoter PtrcThe downstream primer of the upstream homology arm and the upstream primer of pur1 gene are designed. The above fragments were fused by the overlap PCR method to obtain an integrated fragment of pur1 gene (upstream homology arm-P)trcPur 1-downstream homology arm), construction of pGRB-yghE a DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-yghE-S and gRNA-yghE-a. And (3) electrically transferring the recombinant fragment and the plasmid pGRB-yghE to competent cells of INO1-5, screening positive strains, and eliminating the plasmid to obtain a strain INO 2-1. PtrcThe electrophoresis chart of the construction of the integration fragment and the PCR verification of the positive strain during the integration of the pur1 fragment is shown in FIG. 2A. Wherein the length of the upstream homology arm is 559bp, the length of the pur1 gene fragment is 2236bp, the length of the downstream homology arm is 554bp, and the length of the overlapped fragment is 3244 bp. During PCR verification, the length of the amplified fragment of the identifying primer should be 1188bp, and the original bacterium should have no band.
2.1.2 integration of pur 2:
taking Bacillus amyloliquefaciens TA208 genome as a template, designing an upstream homology arm primer (UP-pur2-S, UP-pur2-A) according to pur2 (1603-4203 of the nucleotide sequence shown in SEQ ID NO: 3) and an upstream sequence thereof, and carrying out PCR amplification on an upstream homology arm fragment; taking E.coli MG1655 genome as a template, designing downstream homology arm primers (DN-yghE-S2 and DN-yghE-A) according to the downstream sequence of the yghE gene, and carrying out PCR amplification on the downstream homology arm fragments. The above fragments were fused by an overlap PCR method to obtain an integrated fragment of pur2 (upstream fragment-pur 2-downstream homology arm of pur 2). Construction of pGRB-pur2 the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-pur2-S and gRNA-pur 2-A. The recombinant fragment and the plasmid pGRB-pur2 are electrically transferred to competent cells of INO2-1, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 2-2. The electrophoresis chart of the construction of the integration fragment and the PCR verification of the positive strain in the integration process of the pur2 fragment is shown in FIG. 2B. Wherein the total length of the upstream fragment-pur 2 of pur2 is 2664bp, the length of the downstream homology arm is 554bp, and the length of the overlapped fragment is 3155 bp. During PCR verification, the length of the amplified fragment of the identifying primer should be 1513bp, and the original bacterium should have no band.
2.1.3 integration of pur 3:
taking Bacillus amyloliquefaciens TA208 genome as a template, designing an upstream homology arm primer (UP-pur3-S, UP-pur3-A) according to pur3 (3494-6132 site of a nucleotide sequence shown in SEQ ID NO: 3) and an upstream sequence thereof, and carrying out PCR amplification on an upstream homology arm fragment; taking E.coli MG1655 genome as a template, designing downstream homology arm primers (DN-yghE-S1 and DN-yghE-A) according to the downstream sequence of the yghE gene, and carrying out PCR amplification on the downstream homology arm fragments. The above fragments were fused by an overlap PCR method to obtain an integrated fragment of pur3 (upstream fragment-pur 3-downstream homology arm of pur 3). Construction of pGRB-pur3 the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-pur3-S and gRNA-pur 3-A. The recombinant fragment and the plasmid pGRB-pur3 are electrically transferred to competent cells of INO2-2, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 2-3. The electrophoresis chart of the construction of the integration fragment and the PCR verification of the positive strain in the integration process of the pur3 fragment is shown in FIG. 2C. Wherein the total length of the upstream fragment-pur 3 of pur3 is 2702bp, the length of the downstream homology arm is 554bp, and the length of the overlapped fragment is 3193 bp. During PCR verification, the length of the amplified fragment of the identifying primer should be 1393bp, and the original bacterium should have no band.
2.1.4 integration of pur 4:
based on Bacillus amyloliquefaciens TA208 genome, obtaining a pur4 fragment (No. 5543-8228 of a nucleotide sequence shown by SEQ ID NO: 3) by a chemical synthesis method, designing a primer (UP-pur4-S, UP-pur4-A) according to the pur4 fragment, and amplifying a pur4 fragment; taking E.coli MG1655 genome as a template, designing downstream homology arm primers (DN-yghE-S2 and DN-yghE-A) according to the downstream sequence of the yghE gene, and carrying out PCR amplification on the downstream homology arm fragments. The above fragments were fused by an overlap PCR method to obtain an integrated fragment of pur4 (upstream fragment-pur 4-downstream homology arm of pur 4). Construction of pGRB-pur2 the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-pur2-S and gRNA-pur 2-A. The recombinant fragment and the plasmid pGRB-pur2 are electrically transferred to competent cells of INO2-3, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 2-4. The electrophoresis chart of the construction of the integration fragment and the PCR verification of the positive strain in the integration process of the pur4 fragment is shown in FIG. 2D. Wherein the total length of the upstream fragment-pur 3 of pur4 is 2749bp, the length of the downstream homology arm is 554bp, and the length of the overlapped fragment is 3240 bp. During PCR verification, the length of the amplified fragment of the identifying primer should be 1328bp, and the original bacterium should have no band.
2.1.5 integration of pur 5:
based on Bacillus amyloliquefaciens TA208 genome, obtaining a pur5 fragment (7704-10592 site of a nucleotide sequence shown in SEQ ID NO: 3) by a chemical synthesis method, designing a primer (UP-pur5-S, UP-pur5-A) according to the pur5 fragment, and amplifying a pur5 fragment; taking E.coli MG1655 genome as a template, designing downstream homology arm primers (DN-yghE-S2 and DN-yghE-A) according to the downstream sequence of the yghE gene, and carrying out PCR amplification on the downstream homology arm fragments. The above fragments were fused by an overlap PCR method to obtain an integrated fragment of pur5 (upstream fragment-pur 5-downstream homology arm of pur 5). Construction of pGRB-pur3 the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-pur3-S and gRNA-pur 3-A. The recombinant fragment and the plasmid pGRB-pur3 are electrically transferred to competent cells of INO2-4, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 2-5. The electrophoresis chart of the construction of the integration fragment and the PCR verification of the positive strain in the integration process of the pur5 fragment is shown in FIG. 2E. Wherein the total length of the upstream fragment-pur 5 of pur5 is 2952bp, the length of the downstream homology arm is 554bp, and the length of the overlapped fragment is 3443 bp. During PCR verification, the length of the amplified fragment of the identifying primer should be 1308bp, and the original bacterium should have no band.
2.1.6 integration of pur 6:
taking Bacillus amyloliquefaciens TA208 genome as a template, designing an upstream homology arm primer (UP-pur6-S, UP-pur6-A) according to pur6 (10088-12797 th nucleotide sequence shown in SEQ ID NO: 3) and an upstream sequence thereof, and carrying out PCR amplification on an upstream homology arm fragment; taking E.coli MG1655 genome as a template, designing downstream homology arm primers (DN-yghE-S3 and DN-yghE-A) according to the downstream sequence of the yghE gene, and carrying out PCR amplification on the downstream homology arm fragments. The above fragments were fused by an overlap PCR method to obtain an integrated fragment of pur6 (upstream fragment-pur 6-downstream homology arm of pur 6). Construction of pGRB-pur2 the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-pur2-S and gRNA-pur 2-A. The recombinant fragment and the plasmid pGRB-pur2 are electrically transferred to competent cells of INO2-5, positive strains are screened, and then the plasmid is eliminated to obtain the strain INO 2-6. The electrophoresis chart of the construction of the integration fragment and the PCR verification of the positive strain in the integration process of pur6 is 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 3288 bp. During PCR verification, the length of the amplified fragment of the identifying primer is 1114bp, and the original bacterium has no band.
2.2 integration of PRPP transamidase mutant genes
In order to enhance the transcriptional expression of PRPP amidotransferase, purF was selectedK326Q,P410W，purFD293V,K316Q,S400W，purFK316QAnd integrating and over-expressing PRPP transamidase mutant genes from several different sources.
Identification of mutants: amino acid residues are indicated by single letter symbols using the accepted IUPAC nomenclature. The "amino acid substituted at the original amino acid position" is used to indicate the mutated amino acid in the PRPP transamidase mutant. Such as purFK316QIndicating that the amino acid at position 316 is replaced by lysine (K) of the parent to glutamine (Q), the numbering of the positions corresponding to the numbering of the amino acid sequence of the parent PRPP transglutaminase. Such as purFK326Q,P410WThis indicates that the amino acids at positions 326 and 410 are mutated. The method comprises the following specific steps:
2.2.1 PRPP transamidase mutant Gene purF of E.coli MG1655K326Q,P410WyeeP gene site integrated in e.coli INO 2-6:
taking an E.coli MG1655 genome as ｃA template, designing an upstream homology arm primer (UP-yeeP-S, UP-yeeP-A) and ｃA downstream homology arm primer (DN-yeeP-S, DN-yeeP-A) according to upstream and downstream sequences of ｃA yeeP gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; purF is obtained by a chemical synthesis method based on E.coli MG1655 genomeK326Q,P410WGene according to purFK326Q,P410WGene design primer (ecoF-S, ecoF-A), amplification purFK326Q,P410WGene fragment, promoter PtrcThen designing downstream primer and purF of upstream homology armK326Q,P410WIn the upstream primer of the gene, terminator TtrcThen upstream primers and purF of downstream homology arms are designedK326Q,P410WDownstream primer of gene. The fragments are fused by an overlapping PCR method to obtain purFK326Q,P410WIntegration fragment of Gene (upstream homology arm-P)trc-purFK326Q,P410W-TtrcDownstream homology arm) to construct pGRB-yeeP, the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-yeeP-S and gRNA-yeeP-A. The recombinant fragment and the plasmid pGRB-yeeP are electrically transferred to competent cells of INO2-6, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 2-7. purFK326Q,P410WThe electrophoresis pattern of the integrated fragment construction and PCR validation of the positive strain is shown in FIG. 2G. Wherein, the length of the upstream homology arm is 568bp, purFK326Q,P410WThe length of the gene fragment is 1641bp, the length of the downstream homology arm is 576bp, and the total length of the integrated fragment is 2704 bp. When PCR verification is carried out, 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 1396 bp.
2.2.2 mutation of B.subtilis W168 PRPP amidotransferase mutant Gene purFD293V,K316Q,S400WyeeP gene site integrated in e.coli INO 2-6:
taking E.coli MG1655 genome as template, designing upstream homologous arm primer (UP-yeeP-S, UP-yeeP-A) and downstream homologous arm primer (DN-yeeP-S, DN-yeeP-A) according to upstream and downstream sequences of yeeP gene, PCR amplifying upper and lower homologous arm primersA free homology arm segment; obtaining purF by a chemical synthesis method based on B.subtilis W168 genomeD293V ,K316Q,S400WGene according to purFD293V,K316Q,S400WGene design primers (bsuF-S, bsuF-A) to amplify purFD293V ,K316Q,S400WGene fragment, promoter PtrcThen designing downstream primer and purF of upstream homology armD293V,K316Q,S400WIn the upstream primer of the gene, terminator TtrcThen upstream primers and purF of downstream homology arms are designedD293V,K316Q,S400WDownstream primer of gene. The fragments are fused by an overlapping PCR method to obtain purFD293V,K316Q,S400WIntegration fragment of Gene (upstream homology arm-P)trc-purFD293V,K316Q,S400W-TtrcDownstream homology arm) to construct pGRB-yeeP, the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-yeeP-S and gRNA-yeeP-A. The recombinant fragment and the plasmid pGRB-yeeP are electrically transferred to competent cells of INO2-6, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 2-8. purFD293V,K316Q,S400WThe electrophoresis pattern of the integrated fragment construction and PCR validation of the positive strain is shown in FIG. 2H. Wherein, the length of the upstream homology arm is 568bp, purFD293V,K316Q,S400WThe length of the gene fragment is 1554bp, the length of the downstream homology arm is 576bp, and the total length of the integrated fragment is 2617 bp. During PCR verification, the length of the PCR amplified fragment of the positive bacterium is 2617bp, and the length of the PCR amplified fragment of the original bacterium is 1396 bp.
2.2.3 mutation of PRPP transamidase Gene purF of Bacillus amyloliquefaciens TA208K316QyeeP gene site integrated in e.coli INO 2-6:
taking an E.coli MG1655 genome as ｃA template, designing an upstream homology arm primer (UP-yeeP-S, UP-yeeP-A) and ｃA downstream homology arm primer (DN-yeeP-S, DN-yeeP-A) according to upstream and downstream sequences of ｃA yeeP gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; based on Bacillus amyloliquefaciens TA208 genome, purF is obtained by a chemical synthesis methodK316QGene (SEQ ID NO: 2), according to purFK316QGene design primer (bazF-S, bazF-A), amplification purFK316QGene fragment, promoter PtrcThen is provided withDownstream primer and purF from upstream homology armK316QIn the upstream primer of the gene, terminator TtrcThen upstream primers and purF of downstream homology arms are designedK316QDownstream primer of gene. The fragments are fused by an overlapping PCR method to obtain purFK316QIntegration fragment of Gene (upstream homology arm-P)trc-purFK316Q-TtrcDownstream homology arm) to construct pGRB-yeeP, the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-yeeP-S and gRNA-yeeP-A. The recombinant fragment and the plasmid pGRB-yeeP are electrically transferred to competent cells of INO2-6, positive strains are screened, and then the plasmid is eliminated to obtain a strain INO 2-9. purFK316QThe electrophoresis pattern of the construction of the integrated fragment and the PCR validation of the positive strain is shown in FIG. 2I. Wherein, the length of the upstream homology arm is 568bp, purFK316QThe length of the gene fragment is 1552bp, the length of the downstream homology arm is 576bp, and the total length of the integrated fragment is 2615 bp. During PCR verification, the length of the PCR amplified fragment of the positive bacterium is 2615bp, and the length of the PCR amplified fragment of the original bacterium is 1396 bp.
3. Modified inosine transport system
The pbuE gene of Bacillus amyloliquefaciens TA208 was integrated at the yjiT pseudogene site of E.coli INO 2-9:
taking an E.coli MG1655 genome as a template, 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 upstream and downstream sequences of yjiT genes of the E.coli MG1655 genome, and carrying out PCR amplification on upstream and downstream homology arm fragments of the E.coli MG1655 genome; a genome of Bacillus amyloliquefaciens TA208 is used as a template, a primer (pbuE-S, pbuE-A) is designed according to pbuE gene (SEQ ID NO: 1), and a pbuE gene fragment is amplified. Promoter PtrcThen the terminator T is designed in the downstream primer of the upstream homology arm and the upstream primer of the pbuE genetrcThe upstream primer of the downstream homology arm and the downstream primer of the pbuE gene were designed. The above fragments are fused by an overlapping PCR method to obtain an integrated fragment of the pbuE gene (upstream homology arm-P)trc-pbuE-TtrcDownstream homology arms) used for the construction of pGRB-yjiT, a DNA fragment containing the target sequence was prepared by annealing the primers gRNA-yjiT-S and gRNA-yjiT-A. The recombinant fragment and the plasmid pGRB-yjiT were electroporatedTransferring to competent cells of INO2-9, screening positive strains, and eliminating plasmids to obtain strain INO 3. The construction of the pbuE integrated fragment and the PCR-verified electropherogram of the positive strain are shown in FIG. 3. Wherein, the length of the upstream homologous arm is 373bp, the length of the pbuE gene fragment is 1284bp, the length of the downstream homologous arm is 530bp, and the total length of the integrated fragment is 2108 bp. During PCR verification, the length of the PCR amplified fragment of the positive bacterium is 2108bp, and the length of the PCR amplified fragment of the original bacterium is 1873 bp.
4. Weakening adenosine synthesis branch
Coli MG1655 purA gene was replaced with b.subtilis W168 adenylosuccinate synthetase mutant gene purAP242N. The method comprises the following specific steps:
taking an E.coli MG1655 genome as a template, 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 upstream and downstream sequences of a purA gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; obtaining purA by a chemical synthesis method based on B.subtilis W168 genomeP242NGene (SEQ ID NO: 4), according to purAP242NGene design primers (bsuA-S, bsuA-A) and amplification purAP242NA gene fragment. The purA is obtained by fusing the fragments by an overlapping PCR methodP242NIntegration fragment of mutant Gene (upstream homology arm-purA)P242NDownstream homology arms) the DNA fragment containing the target sequence used for the construction of pGRB-purA was prepared by annealing the primers gRNA-purA-S and gRNA-purA-a. And (3) electrically transferring the recombinant fragment and the plasmid pGRB-purA to competent cells of INO3, screening positive strains, and eliminating the plasmid to obtain a strain INO 4. purAP242NThe electrophoresis pattern of the construction of the integrated fragment and the PCR verification of the positive strain is shown in FIG. 4. Wherein, the length of the upstream homologous arm is 515bp, purAP242NThe 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 2275 bp. During PCR verification, the length of the amplified fragment of the identifying primer should be 1879bp, and the original bacteria should have no band.
5. The primers involved in the above construction are shown in the following table:
example 2: 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: taking a preserved strain at the temperature of minus 80 ℃, streaking and inoculating the strain on an activated inclined plane, culturing for 12h at the temperature of 37 ℃, and carrying out generation once again;
seed culture: scraping a ring of inclined plane seeds by using an inoculating ring, inoculating the seeds into a 500mL triangular flask filled with 30mL seed culture medium, sealing by nine layers of gauze, and culturing at 37 ℃ and 200rpm for 7-10 h;
fermentation culture: inoculating the seed culture solution into a 500mL triangular flask (the final volume is 30mL) filled with a fermentation culture medium according to the inoculation amount of 10-15% of the volume of the seed culture solution, sealing by nine layers of gauze, carrying out shaking culture at 37 ℃ at 200r/min, and maintaining the pH value at 7.0-7.2 by adding ammonia water in the fermentation process; adding 60% (m/v) glucose solution to maintain fermentation; the fermentation period is 24 h.
Slant culture medium: 1-5g/L glucose, 5-10g/L peptone, 1-5g/L yeast powder, 5-10g/L beef extract, 0.1-0.3g/L adenine, 1-2.5g/L NaCl, 15-20g/L agar, and the balance water, with the pH value of 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 KH2PO4·3H2O 0.1-1.2g/L，MgSO4·7H2O 0.1-0.5g/L，FeSO4·7H2O 2-10mg/L，MnSO4·H2O 2-10mg/L，VB1、VB3、VB5、VB12And VH0.1-1mg/L of each, 2 drops of defoaming agent and the balance of water, and the pH value is 7.0-7.2.
Fermentation medium: 15-25g/L of glucose, 1-4g/L of yeast powder,peptone 1-5g/L, sodium citrate 0.1-2g/L, adenine 0.1-0.3g/L, KH2PO4·3H2O 0.1-2g/L，MgSO4·7H2O 0.1-2g/L，FeSO4·7H2O 5-20mg/L，MnSO4·H2O 5-20mg/L，VB1、VB3、VB5、VB12And VH0.1-2mg/L of each, 2 drops of defoaming agent and the balance of water, and the pH value is 7.0-7.2.
And (3) shaking flask fermentation result:
InO2-6 is used as an original strain to respectively over-express purFK326Q,P410W、purFD293V,K316Q,S400W、purFK316QAfter the gene mutation, strains INO2-7, INO2-8 and INO2-9 are obtained. Shake flask fermentation results (FIG. 5), OD of three strains600The inosine yield is improved to different degrees although no obvious change exists. The inosine yield of the INO2-9 is the highest and reaches 1.3g/L, which is increased by 85.7 percent compared with that of the INO 2-6. The INO2-7 yield was improved by 14.3% and 71.4% compared with the INO2-8 inosine yield respectively in INO 2-6. We speculate that Bacillus amyloliquefaciens TA 208-derived PurFK316QThe mutant (amino acid sequence is shown as SEQ ID NO: 6) has better feedback-solving effect, thereby bringing the highest inosine yield.
Example 3: experiment for producing inosine by INO4 fermentation on 5-L tank
The fermentation tank culture method comprises the following steps:
slant activation culture: taking a preserved strain at the temperature of minus 80 ℃, streaking and inoculating the strain on an activated inclined plane, culturing for 12h at the temperature of 37 ℃, and transferring the strain to a solanaceous bottle for continuous culture for 12-16 h;
seed culture: placing 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, keeping the temperature constant at 37 ℃, and culturing until the dry weight of cells reaches 5-6g/L, wherein the dissolved oxygen is 25-35%;
fermentation culture: inoculating into fresh fermentation culture medium according to 15-20% inoculum size, starting fermentation, controlling pH to be stabilized at about 7.0, maintaining temperature at 35 deg.C, and dissolving oxygen at 25-35%; when the glucose in the medium was consumed, 80% (m/v) glucose solution (containing 1g/L adenine) was fed in to maintain the glucose concentration in the fermentation medium at 0.1-2 g/L.
Slant culture medium: 1-5g/L glucose, 5-10g/L peptone, 1-5g/L yeast powder, 5-10g/L beef extract, 0.1-0.3g/L adenine, 1-2.5g/L NaCl, 15-20g/L agar, and the balance water, with the pH value of 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 KH2PO4·3H2O 0.1-1.2g/L，MgSO4·7H2O 0.1-0.5g/L，FeSO4·7H2O 2-10mg/L，MnSO4·H2O 2-10mg/L，VB1、VB3、VB5、VB12And VH0.1-1mg/L of each, 2 drops of defoaming agent and the balance of water, and the pH value 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 KH2PO4·3H2O 0.1-2g/L，MgSO4·7H2O 0.1-2g/L，FeSO4·7H2O 5-20mg/L，MnSO4·H2O 5-20mg/L，VB1、VB3、VB5、VB12And VH0.1-2mg/L of each, 2 drops of defoaming agent and the balance of water, and the pH value is 7.0-7.2.
Fed-batch fermentation results in 5L fermenter: as shown in FIG. 6, the strain grew rapidly at the initial stage of fermentation and OD was 16h600The value reaches a maximum and then gradually decreases. 12-32 h, inosine keeps a high synthesis rate, the yield of 48h reaches 20.16g/L, and the saccharic acid conversion rate and the production intensity reach 0.114g/g glucose and 0.42g/L/h respectively.
Although the present invention has been disclosed in the form of preferred embodiments, it is not intended to limit the present invention, and those skilled in the art may make various changes, modifications, substitutions and alterations in form and detail without departing from the spirit and principle of the present invention, the scope of which is defined by the appended claims and their equivalents.
SEQUENCE LISTING
<110> Tianjin science and technology university
<120> genetic engineering 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
atgaatttca aagtatttct gcttgcagca tctactattg cagtcggatt ggttgaatta 60
attgtgggcg gtattctccc gcaaatcgct tccgacttag acatatcgat cgtcagcgcc 120
gggcagctga tcagcgtgtt cgcgctcggt tacgcggtat caggccctct gcttttggca 180
gtgacggcaa aagctgaacg aaagcggctt tatttaatcg cactttttgt tttcttcctg 240
agtaatctgg tcgcttactt cagtcccaat ttcgccgtac ttatggtgtc acgagtgctc 300
gcttccatga gcacagggct gattgtcgtc ctttctttaa cgattgctcc taaaatcgtg 360
gcgccggaat acagagcgcg ggcgatcggc atcattttca tgggcttcag ctccgcaatc 420
gctttaggcg tgcctgtcgg cattatcatc agcaatgcct tcggatggcg cgtgctgttt 480
ttgggaatcg gcgtattatc tctggtttcc atgctgatta tcagcgtctt ttttgaaaaa 540
atacctgctg aaaaaatgat cccgttccgt gagcagatta aaacgattgg gaacgccaag 600
attgccagcg cgcatcttgt taccttattt acattggcgg ggcattacac actatatgcc 660
tactttgcgc cttttttgga aacaacgctt catttgagtt ctgtttgggt cagtgtatgc 720
tactttttgt tcggcctgtc agcggtatgc ggcggcccgt tcggaggctg gctgtatgac 780
cgtttaggat catttaaaag catcatgctt gtgaccgttt ctttcgcttt gatcctgttt 840
atccttccgc tgtcaacggt ttctttaatc gttttcctgc ctgcgatggt catttgggga 900
ttgctcagct ggagccttgc gccggcgcag caaagctatt tgatcaaaat cgcgcctgag 960
tcttccgata ttcagcaaag cttcaatacg tccgctttgc aaatcggcat tgcgctcggg 1020
tcagccatcg gcggcggcgt gatcggacaa acgggttctg tcacagcaac cgcctggtgc 1080
ggcggtttga ttgtcattat cgcagtcagc ttagccgtat tctctttaac gagacccgct 1140
ttgaaaagaa aatccgcata a 1161
<210> 2
<211> 1431
<212> DNA
<213> Artificial sequence
<400> 2
atgcttgctg aaatcaaagg cctgaatgaa gaatgcggtg tgtttggcat ctgggggcat 60
gaagaggctc cgcagattac gtattacggc ctgcacagcc tgcagcacag agggcaggag 120
ggcgccggca tcgtggcgac cgacggccaa aaactgacgg ctcataaagg gcaggggctt 180
attaccgagg tttttcaaaa cggcgaactg agcaaagtga aaggcaaagg cgcgatcggt 240
cacgtccgct atgcgacggc cggcggcggc ggatatgaaa atgtccagcc gctcctgttc 300
cgttcgcaaa acaacggcag tctcgcgctc gcccataacg gcaacctggt caatgccaca 360
caattgaaac agcagcttga aaaccaaggg agcatctttc agacttcctc cgatacggaa 420
gtgctggctc atctgattaa acgcagcggc cacttttcac tgaaggatca gatcaaaaat 480
tcgctatcca tgctgaaagg cgcttacgcc tttttaatca tgacagaaac agaaatgatt 540
gtagcgcttg acccgaacgg actcagaccg ctttcactcg gcatgctcgg cgacgcttac 600
gtcgtcgcat cagaaacatg cgcatttgat gtggtcggcg ccacgtacct tcgtgacgta 660
gaaccgggcg aaatgcttat cataaacgat gaaggcttga aatcagagcg tttctccatg 720
aatatcaacc gttctatctg cagcatggag tatatctatt tttcccgtcc ggacagcaat 780
atcgacggca ttaacgtgca cagcgcccgg aagagcctcg ggaaaatgct tgcccaagag 840
tccgctgttg aagcggatgt cgtcacaggc gtgcctgatt ccagtatttc cgcggccatc 900
ggctatgccg aggcaacggg cattccgtac gaactcggtc tcattcaaaa ccgttacgtc 960
ggcagaacgt ttatccagcc gtctcaagct cttcgtgagc aaggagtaag aatgaagctg 1020
tccgccgtcc gcggtgttgt tgaaggaaaa cgggtcgtca tggttgatga ttccatcgtg 1080
cgcgggacga caagccgccg gatcgtcaca atgctccgag aagcgggagc gacagaggtg 1140
catgtaaaga tcagttcgcc tccgatcgcc catccttgct tctacggcat cgacacatca 1200
acccatgagg agctgatcgc ttcctcgcat tcagtggaag aaatccgcca gattatcggc 1260
gccgacacgc tttctttctt aagtgtagac ggattgttaa aaggagtcgg ccgaaaattt 1320
gaagacacca attgcggaca atgcctcgct tgttttacgg gcaaatatcc gacggaaatt 1380
tatcaggata cagtgcttcc tcacgtaaaa gaagcagtgc tgacaaaata a 1431
<210> 3
<211> 12797
<212> DNA
<213> Artificial sequence
<400> 3
atgcagccgc tagcaggaat catcatggga agcacctccg attgggagac aatgaaacat 60
gcatgcgaca tacttgacga acttcacatt ccttatgaaa aacaggtggt atccgcgcat 120
cggacgcctg atttgatgtt tgaatatgca gaaagcgcca ggagcagagg cttaaaagtc 180
attattgccg gagccggagg agcggcgcat ctgccgggaa tgacggcggc caaaacgaca 240
ctgccggtga tcggtgttcc ggttcagaca aaatcgctta acgggcttga ttctcttttg 300
tctatcgtac agatgcccgg cggcgtgccg gtcgcgacga cagcgatcgg aaaagcgggc 360
gcagtgaacg cgggtctgtt agccgcgcaa attttgtcgg catttgacga tgacattgcg 420
gataaactag aggcgagaag aaatgcgaca aaacaaacgg tgctggaaag cagtgatcag 480
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 (10)
1. An escherichia coli genetic engineering strain is characterized in that: the gene engineering strain heterologous overexpression nucleoside transporter gene pbuE and purine operon mutant gene purEKBCSQLFK316QMNHD and PRPP transamidase mutant gene purFK316QWherein purFK316QThe amino acid sequence of purF gene codes the amino acid sequence with the place 316 lysine replaced by glutamine, and the gene purA is mutated by heterologous adenylosuccinate synthetaseP242NReplaces the gene purA and does not express purine nucleoside phosphorylase genes deoD, ppnP and nucleoside hydrolase genes rihA, rihB and rihC.
2. The genetically engineered strain of claim 1, wherein: the nucleotide sequence of the nucleotide transporter gene pbuE is shown as SEQ ID NO: 1 is shown in the specification; and/or
The PRPP transamidase mutant gene purFK316QThe nucleotide sequence of (a) is shown as SEQ ID NO: 2 is shown in the specification; and/or
The purine operon mutant gene purEKBCSQLFK316QThe nucleotide sequence of MNHD is shown as SEQ ID NO: 3 is shown in the specification; and/or
The adenylosuccinate synthetase mutant gene purAP242NOfThe nucleotide sequence is shown as SEQ ID NO: 4, respectively.
3. The genetically engineered strain of claim 1 or 2, wherein: the nucleotide transporter gene pbuE is connected with a promoter Ptrc(ii) a And/or
The purine operon mutant gene purEKBCSQLFK316QMNHD is connected with a promoter Ptrc(ii) a And/or
The PRPP transamidase mutant gene purFK316QLinked with a promoter Ptrc；
The promoter PtrcThe nucleotide sequence of (a) is shown as SEQ ID NO: 5, respectively.
4. The genetically engineered strain of claim 1 or 2, wherein: the starting strain for constructing the escherichia coli genetic engineering strain is e.
5. The method for constructing the genetically engineered strain of any one of claims 1 to 4, wherein: the method comprises the following steps: introducing a nucleotide transport protein gene pbuE and a purine operon mutant gene purEKBCSQLF into an original strain escherichia coliK316QMNHD and PRPP transamidase mutant gene purFK316QWherein purFK316QIs the amino acid sequence with glutamine substituted by lysine at position 316 and adenylosuccinate synthetase mutant gene purAP242NThe gene purA was replaced and the purine nucleoside phosphorylase genes deoD, ppnP and the nucleoside hydrolase genes rihA, rihB, rihC were knocked out or inactivated.
6. The construction method according to claim 5, wherein: the construction method further comprises the following steps:
(1) knocking out purine nucleoside phosphorylase genes deoD and ppnP and knocking out nucleoside hydrolase genes rihA, rihB and rihC from a genome of a strain E.coli MG 1655;
(2) the nucleotide sequence is shown as SEQ ID NO: 3 of the purine operon purEKBCSQLFK316QMNHD and promoter PtrcFused fragment P of (1)trc-purEKBCSQLFK316QMNHD integrates at the yghE pseudogene site;
(3) the nucleotide sequence is shown as SEQ ID NO: 2 of PRPP transamidase mutant gene purFK316QAnd the promoter PtrcFused fragment P of (1)trc-purFK316QIntegrated at the yeeP pseudogene site;
(4) the nucleotide sequence is shown as SEQ ID NO: 1 and a promoter PtrcFused fragment P of (1)trc-pbuE integration at the yjiT pseudogene site;
(5) the nucleotide sequence of the adenylosuccinate synthetase gene purA is replaced by the nucleotide sequence shown as SEQ ID NO: 4 mutant gene purAP242NAnd obtaining the genetic engineering strain.
7. Use of the genetically engineered strain of any one of claims 1 to 4 for the fermentative production of inosine.
8. The application according to claim 7, wherein the application comprises: culturing the genetically engineered strain under suitable conditions, and collecting inosine from the culture thereof; the proper conditions are that the culture temperature is 35 ℃, the pH is maintained to be 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 KH2PO4·3H2O 0.1-2 g/L，MgSO4·7H2O 0.1-2 g/L，FeSO4·7H2O 5-20 mg/L，MnSO4·H2O 5-20 mg/L，VB1、VB3、VB5、VB12And VH0.1-2mg/L of each, 2 drops of defoaming agent and the balance of water, and the pH value is 7.0-7.2.
9. A PRPP transamidase mutant has an amino acid sequence shown as SEQ ID NO: and 6.
10. A PRPP transamidase mutant gene has a nucleotide sequence shown as SEQ ID NO: 2, respectively.
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| CN117363553A (en) * | 2023-12-07 | 2024-01-09 | 天津科技大学 | Genetically engineered bacterium for producing 2' -deoxyadenosine, construction method and application thereof |
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| CN103952422A (en) * | 2014-04-15 | 2014-07-30 | 天津大学 | Bacillus subtilis-coded PRPP (Phosphoribosyl Pyrophosphate) transamidase mutant gene pruF and application thereof |
| WO2019013696A1 (en) * | 2017-07-14 | 2019-01-17 | Biopetrolia Ab | Microbial cells for spermidine production |
| CN111201316A (en) * | 2017-07-14 | 2020-05-26 | 克里希有限公司 | Microbial cells for spermidine production |
| CN113278596A (en) * | 2021-05-24 | 2021-08-20 | 廊坊梅花生物技术开发有限公司 | Mutant capable of improving bacillus nucleoside yield and application thereof |
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| CN110656073A (en) * | 2018-06-28 | 2020-01-07 | 中国科学院青岛生物能源与过程研究所 | Recombinant bacterium for producing xanthine as well as construction method and application thereof |
| CN110656073B (en) * | 2018-06-28 | 2022-06-28 | 中国科学院青岛生物能源与过程研究所 | Recombinant bacterium for producing xanthine as well as construction method and application thereof |
| CN117363553A (en) * | 2023-12-07 | 2024-01-09 | 天津科技大学 | Genetically engineered bacterium for producing 2' -deoxyadenosine, construction method and application thereof |
| CN117363553B (en) * | 2023-12-07 | 2024-02-20 | 天津科技大学 | Genetically engineered bacterium for producing 2' -deoxyadenosine, construction method and application thereof |
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