CN113337486B - Recombinant microorganism and preparation method and application thereof - Google Patents

Recombinant microorganism and preparation method and application thereof Download PDF

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CN113337486B
CN113337486B CN202110604094.3A CN202110604094A CN113337486B CN 113337486 B CN113337486 B CN 113337486B CN 202110604094 A CN202110604094 A CN 202110604094A CN 113337486 B CN113337486 B CN 113337486B
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arabinoheptulosonate
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transketolase
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CN113337486A (en
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牛松峰
程江红
贾贝贝
李岩
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Langfang Meihua Bio Technology Development Co Ltd
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Abstract

The invention relates to the technical field of microorganisms, in particular to a recombinant microorganism and a preparation method and application thereof. The present invention provides a protein mutant which is a transketolase mutant and/or a 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant, wherein the transketolase mutant contains a mutation that the 234 th threonine is replaced by isoleucine, and the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant contains a mutation that the 171 th glycine is replaced by aspartic acid. The mutant has reduced enzyme activity, can reasonably weaken metabolic flow of shikimic acid synthetic pathway, reduce accumulation of aromatic amino acid, further improve the yield and conversion rate of glutamic acid, and simultaneously can ensure the growth performance of the strain. The invention also provides a recombinant microorganism expressing the mutant, the glutamic acid fermentation production performance of the recombinant microorganism is obviously improved, the glutamic acid yield and the saccharic acid conversion rate are higher, and the growth performance of the strain is good.

Description

Recombinant microorganism, and preparation method and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to a recombinant microorganism and a preparation method and application thereof.
Background
L-glutamic acid (L-glutamate), chemical name is alpha-aminoglutaric acid, molecular formula is C 5 H 9 NO 4 The molecular weight is 147.13076, the molecule contains two carboxyl groups, and the amino acid is an acidic amino acid. Glutamate is one of the most abundant amino acids, and plays an important role in nutrition and signal transduction in addition to being involved in protein synthesis. Glutamic acid or monosodium salt (monosodium glutamate) thereof can be used as a food additive to improve the delicate flavor of food, so that the glutamic acid or monosodium salt thereof is widely industrially produced.
At present, the most common production method of glutamic acid is a fermentation method, and compared with the process complexity of an enzymatic method and chemical synthesis and the adopted raw material toxicity, the fermentation method has the advantages of safe raw material source, low production cost, single product and the like. Although the research on the production of glutamic acid by fermentation has been developed for decades, the productivity of the strains producing glutamic acid is still to be further improved, and the production cost of the industrial large-scale production of glutamic acid is severely restricted by the fact that the strains produce more heteropolyacid and the saccharic acid conversion rate is not high enough in the production process.
Disclosure of Invention
The invention aims to provide a protein mutant, a recombinant microorganism expressing the protein mutant, and a preparation method and application thereof.
Specifically, the invention provides the following technical scheme:
the invention provides a protein mutant which is a transketolase mutant and/or a 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant, takes an amino acid sequence of wild-type transketolase of corynebacterium glutamicum as a reference sequence, the transketolase mutant contains a mutation that threonine at the 234 th position is replaced by an amino acid except threonine,
the amino acid sequence of a wild-type 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase of corynebacterium glutamicum is taken as a reference sequence, and the mutant of the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase contains a mutation of replacing the 171 th glycine with an amino acid except glycine.
Preferably, the amino acid sequence of wild-type transketolase of Corynebacterium glutamicum is used as reference sequence, and the transketolase mutant contains a mutation of threonine at position 234 replaced by isoleucine. The amino acid sequence of a wild-type 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase of corynebacterium glutamicum is taken as a reference sequence, and the mutant of the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase contains a mutation of replacing the 171 th glycine with the aspartic acid.
Specifically, the protein mutant may be a transketolase mutant, or a combination of a transketolase mutant and a 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant.
Specifically, the transketolase mutant has an amino acid sequence shown as SEQ ID NO. 1. The 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant has an amino acid sequence shown as SEQ ID NO. 2.
It will be understood by those skilled in the art that addition of a tag protein to the N-terminus or C-terminus of the mutant sequence of the above protein or fusion of the tag protein with other proteins to form a fusion protein is also within the scope of the present invention without altering the activity of the above mutant protein itself.
In the research and development process, the invention discovers that the metabolic flux of shikimic acid synthesis is weakened, the synthesis of aromatic amino acid is reduced, the metabolic flux capability of a central metabolic pathway can be enhanced, and more sufficient carbon flux is provided for the synthesis of metabolites such as glutamic acid and the like through TCA cycle, so that the yield of the metabolites is improved. Therefore, the shikimic acid pathway is weakened to a certain extent, the balance between the growth of the strain and the production of the target product is promoted, and the production performance of the strain on the target product can be obviously improved. The invention discloses a transketolase mutant and a 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthases, DS) mutant, wherein the two mutants can reduce the activities of the transketolase and the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase to a certain extent, can reasonably weaken the metabolic flux of a shikimate pathway, reduce the accumulation of heteroacid such as aromatic amino acid and the like while ensuring the growth performance of a strain, and remarkably improve the yield and the conversion rate of metabolites such as glutamic acid and the like through TCA circulation. Meanwhile, when the two mutants are used in combination, the effect is better.
The invention also provides nucleic acid encoding the protein mutant.
Based on the amino acid sequences of the protein mutants provided above, the skilled person is able to obtain the sequences of their encoding nucleic acids. Based on the degeneracy of the codon, more than one nucleic acid sequence encoding the above amino acid sequence is provided, and all nucleic acids capable of encoding the above protein mutants are within the scope of the present invention.
As an embodiment of the present invention, the nucleic acid encoding the transketolase mutant has the nucleotide sequence shown as SEQ ID NO.3, and the nucleic acid encoding the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant has the nucleotide sequence shown as SEQ ID NO. 4.
The invention also provides biological material containing the nucleic acid, wherein the biological material is an expression cassette, a vector or a host cell.
The expression cassette is a recombinant nucleic acid molecule obtained by connecting elements for driving the transcription and expression of the nucleic acid at the upstream or downstream of the nucleic acid.
The vector may be an expression vector or a cloning vector, including but not limited to a plasmid vector, a phage vector, a transposon, and the like.
Such host cells include, but are not limited to, microbial cells.
The invention provides any one of the following applications of the protein mutant or the nucleic acid or the biological material:
(1) Improving the glutamic acid yield, production intensity or conversion rate of the microorganism;
(2) Reducing the accumulation of shikimic acid or aromatic amino acids in the microorganism.
The present invention provides a recombinant microorganism having a reduced expression level and/or activity of one or both of the enzymes described in the following (1) and (2) as compared with the starting strain, based on the protein mutant described above:
(1) A transketolase;
(2) 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase.
Preferably, the recombinant microorganism expresses the protein mutant and does not express transketolase and/or 3-deoxy-D-arabinoheptulosonate 7 phosphate synthase possessed by the original strain.
Specifically, the present invention provides, as one embodiment, a recombinant microorganism which expresses the transketolase mutant and does not express the transketolase possessed by the original strain.
In another embodiment, the present invention provides a recombinant microorganism which expresses the transketolase mutant and the 3-deoxy-D-arabinoheptulosonate 7 phosphate synthase mutant without expressing the transketolase and the 3-deoxy-D-arabinoheptulosonate 7 phosphate synthase possessed by the original strain thereof.
Preferably, the gene coding for transketolase and/or 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase in the recombinant microorganism is mutated into the gene coding for the protein mutant.
Specifically, in the recombinant microorganism, a gene encoding transketolase on a chromosome is mutated into a gene encoding the transketolase mutant, or a gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase on a chromosome is mutated into a gene encoding the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant, or a gene encoding transketolase on a chromosome is mutated into a gene encoding the transketolase mutant, and a gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase on a chromosome is mutated into a gene encoding the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant.
The recombinant microorganism of the present invention may be a microorganism of the genus Corynebacterium or a bacterium of the genus Brevibacterium.
Preferably, the Corynebacterium bacterium is one selected from the group consisting of Corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium effectivum (Corynebacterium efficiens), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium ammoniagenes thermophilum (Corynebacterium thermoaminogenes), and Corynebacterium ammoniagenes (Corynebacterium ammoniagenes);
the bacterium belonging to the genus Brevibacterium is one selected from Brevibacterium flavum and Brevibacterium lactofermentum.
More preferably, the starting strain of the recombinant microorganism is a bacterium of the genus Corynebacterium which is capable of accumulating glutamic acid.
As a preferred embodiment of the present invention, the starting strain for the recombinant microorganism construction is Corynebacterium glutamicum, which is capable of accumulating glutamic acid.
Further preferably, the starting strain contains one or more of the following mutations:
(1) Introducing a point mutation a106V in yggB;
(2) Carrying out inactivation modification on the odhA gene;
(3) Overexpresses the gdh gene;
(4) The gltA gene was overexpressed.
As an embodiment of the invention, the starting strain is Corynebacterium glutamicum MHZ-0112-8 with the preservation number of CGMCC No.11941.
The pure culture MHZ-0112-8 of Corynebacterium glutamicum has been deposited in the China general microbiological culture Collection center (CGMCC for short, address: no.3 of West Lu No.1 of Beijing city Kogyo, ministry of microbiology, zip code 100101) at 25.12.2015, and the preservation number is CGMCC No.11941. Corynebacterium glutamicum MHZ-0112-8 (CGMCC No. 11941) mentioned in the invention is disclosed in Chinese patent publication No. CN 105695383A.
The invention also provides a construction method of the recombinant microorganism, which comprises the following steps: inserting, deleting or replacing one or more bases of a gene encoding transketolase and/or 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase in the starting strain to reduce the activity of transketolase, 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase;
alternatively, the transcriptional or translational regulatory elements of the genes coding for the transketolase and/or for the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase are replaced in the starting strain by regulatory elements of lower activity.
Preferably, the method comprises: introducing a recombinant plasmid carrying a gene encoding the protein mutant into an original strain, and mutating the gene encoding transketolase and/or 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase in the original strain into the gene encoding the protein mutant.
Preferably, the recombinant plasmid is a plasmid capable of homologous recombination in bacterial cells, and a gene encoding a protein mutant on the plasmid is exchanged with a homologous gene on a chromosome.
Further preferably, the construction method comprises:
(1) Constructing a recombinant plasmid containing a gene encoding the transketolase mutant and a gene encoding the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant;
(2) Transforming the recombinant plasmid into a starting strain, and screening a transformant by using a selection culture medium containing antibiotics;
(3) And screening the positive transformants for a recombinant microorganism with a target mutation.
The invention also provides application of the recombinant microorganism in glutamic acid production.
Preferably, the glutamic acid production is microbial fermentation production.
The invention provides a method for producing glutamic acid, which comprises the step of carrying out fermentation culture on the recombinant microorganism.
Preferably, the fermentation medium is: 50-60g/L glucose, 10-20g/L ammonium sulfate and KH 2 PO 4 0.5-1.5g/L，MgSO 4 ·7H 2 O 0.2-0.5g/L，FeSO 4 ·7H 2 O 0.05-0.15mg/L，MnSO 4 ·5H 2 O 0.8-1.2mg/L，VB 1 150-250 mug/L, biotin 250-350 mug/L, soybean hydrolysate 0.3-0.6g/L, pH7.2-7.5,
alternatively, the fermentation medium comprises the following components: 15-25g/L of glucose, 15-25g/L of corn steep liquor, 0.1-0.3g/L of betaine hydrochloride, and K 2 HPO 4 6-8g/L，MgSO 4 ·7H 2 O 1-2g/L，V B1 150-250μg/L，VH 550-650μg/L，FeSO 4 ·7H 2 O 25-35mg/L、MnSO 4 ·H 2 25-35mg/L of O, 0.4-0.7g/L of urea and 7.2-7.5 of pH.
Specifically, the method for producing glutamic acid comprises: inoculating the recombinant microorganism to a slant culture medium for slant culture, selecting lawn on the slant culture medium, inoculating the lawn to a seed culture medium for seed culture, and transferring the seed culture to a fermentation culture medium for fermentation.
In some embodiments, the slant medium is: 5g/L of yeast powder, 10g/L of beef extract, 10g/L of peptone, 10g/L of sodium chloride, 2.5g/L of agar powder and pH 7.0-7.2.
The slant culture is carried out at 30-32 deg.C for 20-25h.
In some embodiments, the seed medium is: glucose 25g/L, urea 3g/L, K 2 HPO 4 ·3H 2 O 2.2g/L，MgSO 4 ·7H 2 0.9g/L of O, 33mL/L of corn steep liquor, 22mL/L of bean cake hydrolysate and 7.0-7.2 of pH.
The seed culture is performed by shaking culture at 30-32 ℃ to the middle and late logarithmic growth stage, and the culture time is 10-14h.
In some embodiments, the fermentation medium is: glucose 60g/L, ammonium sulfate 15g/L, KH 2 PO 4 1.0g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 1.0mg/L，MnSO 4 ·5H 2 O 1.0mg/L，VB 1 200 mu g/L, biotin 300 mu g/L, bean hydrolysate 0.48g/L, and pH7.2-7.5.
The fermentation is performed at 30-32 deg.C for 12-20h by shaking culture.
The invention also provides a method for reducing the accumulation of the heteropolyacid in the fermentation production of the glutamic acid, which is the same as the method for producing the glutamic acid.
The invention also provides a method for improving the yield of the glutamic acid, which is the same as the method for producing the glutamic acid.
The invention has the beneficial effects that: the transketolase mutant and the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant provided by the invention have reduced enzyme activity, can reasonably weaken metabolic flux of a shikimic acid synthesis path, reduce accumulation of aromatic amino acids, and enhance metabolic flux capability of a central metabolic path, so that the yield and the conversion rate of glutamic acid are improved, and the growth performance of a strain can be ensured. The invention also provides a recombinant microorganism expressing the transketolase mutant and the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant, wherein the glutamic acid fermentation production performance of the recombinant microorganism is obviously improved, the recombinant microorganism has higher glutamic acid yield and sugar acid conversion rate, and the growth performance of the strain is good.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The names and sequences of the primers involved in the following examples are shown in Table 1.
TABLE 1 primer sequences
Figure BDA0003093795170000071
Figure BDA0003093795170000081
The strains referred to in the following examples are as follows:
the starting strain MHZ-0112-8 is Corynebacterium glutamicum, and the pure culture of Corynebacterium glutamicum MHZ-0112-8 has been preserved in 2015 12 months and 25 days in China general microbiological culture Collection center (CGMCC for short, address: beijing city rising area North Cheng Xilu No.1 institute of microbiology, china academy of sciences, postal code 100101), with the preservation number of CGMCC No.11941. The Corynebacterium glutamicum MHZ-0112-8 (CGMCC No. 11941) mentioned in the invention is disclosed in Chinese patent publication No. CN 105695383A.
Example 1 screening of mutant strains producing glutamic acid at high yield
The MHZ-0112-8 strain is firstly prepared by acridine Huang Youbian, thallus in logarithmic phase is centrifuged, phosphate buffer solution (0.1 mol/L and pH 7.0) is used for washing twice, the thallus is suspended in 0.1mol/L phosphate buffer solution (pH 7.0) containing acridine yellow with certain concentration to enable the final concentration to be 0.2mg/mL, mutagenized bacterium solution is obtained after oscillation treatment for 4 hours on a shaking table at 37 ℃, diluted and coated on a bromocresol purple plate, the bacterium solution is cultured for 36 hours at 31.5 ℃ to obtain 200 strains with large discoloring circles, high-yield glutamic acid strains MHZ-0112-9 are obtained through TLC method rescreening and shaking bottle verification combined with HPLC screening, then ultraviolet 15W,30cm and conventional mutagenesis treatment for 20 minutes are carried out on the MHZ-0112-9 strain, and a production strain MHZ-2-10 with the highest glutamic acid yield is obtained through screening by the same screening method.
Activating the obtained MHZ-0112-10 on a brain-heart infusion solid culture medium, and culturing for 16-20h at 31.5 ℃; the cells were scraped from the plate in a ring, inoculated in 30mL seed medium at 31.5 ℃ and cultured with shaking at 220rpm for 12 hours in the middle and late logarithmic growth phase. 2mL of the seed solution was transferred to 20mL of the fermentation medium at 31.5 ℃ and cultured for 16h with shaking at 220 rpm.
Liquid chromatography detection of glutamic acid and heteroacid in fermentation liquor shows that the yield of glutamic acid is increased from 34.9g/L to 36.9g/L, and the conversion rate is increased by 3.4% except that the yield of glutamic acid is obviously increased. The content of aromatic amino acids (Phe, trp, tyr) was reduced to 0.061,0.042,0.035g/L, respectively (Table 2).
TABLE 2 yield of glutamic acid and aromatic amino acids by mutagenized strains
Bacterial strains OD 600 (×100) Glu(g/L) Percent conversion% Phe(g/L) Trp(g/L) Tyr(g/L)
MHZ-0112-8 0.432±0.021 34.9±0.12 58.1±0.10 0.36±0.012 0.42±0.011 0.28±0.005
MHZ-0112-10 0.401±0.006 36.9±0.11 61.5±0.09 0.061±0.003 0.042±0.002 0.035±0.008
A mutant strain MHZ-0112-10 and an original strain MHZ-0112-8 are analyzed by utilizing comparative genomics, and two genes in a shikimic acid synthesis pathway are found out: the transketolase gene tktA and the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase gene ds are mutated, the 234 th threonine of the transketolase is mutated into isoleucine, and the 171 th glycine of the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase is mutated into aspartic acid. The above mutations resulted in reduced activities of transketolase and 3-deoxy-D-arabinoheptulosonate 7 phosphate synthase. The shikimic acid is a precursor substance for synthesizing aromatic amino acid, and the weakening of the synthesis pathway of the shikimic acid weakens the carbon flow of the aromatic amino acid, so that the accumulation of the aromatic amino acid in cells is reduced, the metabolic flux capability of a central metabolic pathway is enhanced, sufficient carbon flow is provided for the synthesis of glutamic acid, and the yield of the glutamic acid is further improved.
Example 2 construction of recombinant plasmid pK18-tktA (T234I) and introduction of tktA Point mutation in starting Strain MHZ-0112-8
Primers were designed based on the sequence of the tktA (GenBank NO: NCgl 1512) gene in the NCBI database, and the primer sequences are shown in Table 1. A recombinant fragment is prepared by using Phusion ultra-fidelity polymerase (New England BioLabs), tktA-UP-1F/tktTA-UP-1R, tktA-DN-2F/tktTA-DN-2R as a primer pair and the genome of Corynebacterium glutamicum MHZ-0112-8 as a template, wherein the PCR program is as follows: denaturation at 98 ℃ for 10s, renaturation at 50 ℃ for 20s, and extension at 72 ℃ for 15s, and the cycle is repeated for 30 times, and the extension is completed at 72 ℃ for 10min. The obtained fragment was purified by agarose gel recovery kit (Tiangen), and then recombinant fragments were prepared using tktA-UP-1F/tktA-DN-2R as primer pair and the upstream and downstream homology arms as templates, and PCR procedure was: denaturation at 98 ℃ for 10s, renaturation at 50 ℃ for 20s, and extension at 72 ℃ for 30s, and the cycle is repeated for 30 times, and the extension is completed at 72 ℃ for 10min. The resulting recombinant fragment was purified by agarose gel recovery kit (Tiangen), then digested with XbaI/HindIII, while pK18-mobsacB was digested with XbaI/HindIII and the fragment was ligated to a vector with T4DNA ligase (TransGen Biotech), transn 1T1 competent cells (TransGen Biotech) were transformed, kanamycin-resistant clones were picked up, xbaI/HindIII digestion was identified to give positive clones with the fragment inserted into pK18mobsacB, and further the inserted fragment was identified to be correct by sequencing (Invitrogen).
The resulting plasmid was designated pK18-tktAT234I. pK18-tktAT234I was transferred into Corynebacterium glutamicum MHZ-0112-8 and the recombinant was selected for replacement on selection medium containing 15mg/L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the primer pair tktA-UP-1F/P85, P82/tktA-DN-2R, the PCR procedure being: 30 cycles of 94 ℃ 30s,50 ℃ 30s,72 ℃ 45s, and complete extension at 72 ℃ for 10min. The clone of the 1252bp and 1147bp fragments amplified by the two pairs of primer pairs is a positive clone. Inoculating the selected positive clone into a non-resistance BHI culture medium for culturing for 12-14h, diluting a bacterial liquid by 100-1000 times, coating the diluted bacterial liquid on a solid BHI culture medium containing 10% of sucrose for culturing for 36h, further carrying out kanamycin resistance phenotype verification on the selected strain, selecting a recombinant of KanS, verifying a point mutation recombinant by using an identification primer pair tktttttA-F/tkttA-DN-2R, obtaining the recombinant containing point mutation by groping the annealing temperature, carrying out amplification sequencing on the obtained positive recombinant by using the tktA-ID-F/tkttA-ID-R, wherein the length of the correct recombinant is 1223bp, and the strain with correct sequencing is named as MHZ-0112-11.
Example 3 construction of recombinant plasmid pK18-ds (G171D) and introduction of the ds Point mutation in the starting MHZ-0112-8 Strain
Primers were designed based on the sequence of the ds (GenBank NO: NCgl 0950) gene in the NCBI database, and the sequences of the primers are shown in Table 1. The recombinant fragment is prepared by using Phusion ultra-fidelity polymerase (New England BioLabs), ds-UP-1F/ds-UP-1R, ds-DN-2F/ds-DN-2R as a primer pair and the genome of Corynebacterium glutamicum MHZ-0112-8 as a template, wherein the PCR program is as follows: denaturation at 98 ℃ for 10s, renaturation at 50 ℃ for 20s, and extension at 72 ℃ for 15s, and the cycle is repeated for 30 times, and the extension is completed at 72 ℃ for 10min. The resulting fragment was purified by agarose gel recovery kit (Tiangen), followed by preparation of recombinant fragments using ds-UP-1F/ds-DN-2R as primer pair and the upstream and downstream homology arms as template, PCR procedure: denaturation at 98 ℃ for 10s, renaturation at 50 ℃ for 20s, and extension at 72 ℃ for 30s, and the cycle is repeated for 30 times, and the extension is completed at 72 ℃ for 10min. The resulting recombinant fragment was purified with an agarose gel recovery kit (Tiangen), then digested with XbaI/PtsI, while pK18-mobsacB was digested with XbaI/PtsI, and the fragment was ligated to a vector with T4DNA ligase (TransGen Biotech), trans1T1 competent cells (TransGen Biotech) were transformed, kanamycin-resistant clones were picked, xbaI/PtsI digestion was performed to identify positive clones with the fragment inserted into pK18mobsacB, and the inserted fragment was further identified by sequencing (Invitrogen).
The resulting plasmid was designated pK18-dsG D. pK18-dsG D was transferred into Corynebacterium glutamicum MHZ-0112-8 and the replacement recombinants were selected on selection medium containing 15mg/L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the ds-UP-1F/P85, P82/ds-DN-2R primer pair, the PCR procedure being: 30 cycles of 30s at 94 ℃, 30s at 50 ℃ and 33s at 72 ℃, and complete extension at 72 ℃ for 10min. The clone which is amplified by the two pairs of primer pairs to obtain the fragments of 1243bp and 1124bp is a positive clone. Inoculating the selected positive clone into a non-anti BHI culture medium for culturing for 12-14h, diluting the bacterial liquid by 100-1000 times, coating the diluted bacterial liquid on a solid BHI culture medium containing 10% sucrose for culturing for 36h, further carrying out kana resistance phenotype verification on the selected strain, selecting a recombinant of KanS, verifying a point mutation recombinant by using an identification primer pair ds-F/ds-DN-2R, obtaining a recombinant containing point mutation by groping the annealing temperature, carrying out amplification sequencing on the obtained positive recombinant by using ds-ID-F/ds-ID-R, wherein the length of the correct recombinant is 1172bp, and the strain with correct sequencing is named as MHZ-0112-12.
Example 4 double mutant Strain construction
pK18-dsG D constructed in example 3 was transferred into Corynebacterium glutamicum MHZ-0112-11 and the replacement recombinants were selected on selection medium containing 15mg/L kanamycin. KanR clones were identified by colony PCR using Fast Taq DNA polymerase (TransGen Biotech) with the ds-UP-1F/P85, P82/ds-DN-2R primer pair, the PCR procedure being: 30 cycles of 94 ℃ 30s,50 ℃ 30s,72 ℃ 33s, complete extension at 72 ℃ for 10min. The clone with 1243bp and 1124bp amplified by two pairs of primers is positive clone. Inoculating the selected positive clone into a non-anti BHI culture medium for culturing for 12-14h, diluting the bacterial liquid by 100-1000 times, coating the diluted bacterial liquid on a solid BHI culture medium containing 10% sucrose for culturing for 36h, further carrying out kana resistance phenotype verification on the selected strain, selecting a recombinant of KanS, verifying a point mutation recombinant by using an identification primer pair ds-F/ds-DN-2R, obtaining a recombinant containing point mutation by groping the annealing temperature, carrying out amplification sequencing on the obtained positive recombinant by using ds-ID-F/ds-ID-R, wherein the length of the correct recombinant is 1172bp, and the strain with correct sequencing is named as MHZ-0112-13.
Example 5 verification of the ability of the mutant strains to produce glutamic acid
The recombinant corynebacterium glutamicum constructed in examples 2, 3 and 4 was fermented to verify the glutamic acid productivity, which was as follows:
inoculating the strain frozen in a glycerin pipe with the temperature of minus 80 ℃ into a slant culture medium for activation, culturing for 24 hours at the temperature of 31.5 ℃ to grow a lawn, selecting the lawn from a fresh activated slant, inoculating the lawn into the seed culture medium, performing shake culture at the temperature of 31.5 ℃ and the speed of 220rpm until the middle and later period of logarithmic growth, wherein the culture time is 12 hours, preparing a seed solution, inoculating the seed solution into a 500ml shake flask filled with 20ml of fermentation culture medium by 10 percent of inoculation amount, and performing shake culture at the speed of 220rpm at the temperature of 31.5 ℃ for 16 hours. After the sugar was completely consumed, the concentration of L-glutamic acid accumulated in the medium was measured.
The formula of the culture medium is as follows:
slant culture medium: 5g/L of yeast powder, 10g/L of beef extract, 10g/L of peptone, 10g/L of sodium chloride, 2.5g/L of agar powder, 7.0-7.2 of pH and 30min of sterilization at 121 ℃ and 0.1 MPa;
seed culture medium: glucose 25g/L, urea 3g/L, K 2 HPO 4 ·3H 2 O 2.2g/L，MgSO 4 ·7H 2 0.9g/L of O, 33mL/L of corn steep liquor, 22mL/L of soybean cake hydrolysate, 7.0-7.2 of pH and sterilization at 121 ℃ and 0.1MPa for 15min;
fermentation medium: glucose 60g/L, ammonium sulfate 15g/L, KH 2 PO 4 1g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 1.0mg/L，MnSO 4 ·5H 2 O1 mg/L, VB1 μ g/L, biotin 300 μ g/L, soybean hydrolysate 0.48g/L, adjusting pH to 7.2-7.5 with NaOH, sterilizing at 121 deg.C under 0.1MPa for 15min, and adding 1.0g of heat sterilized calcium carbonate.
TABLE 3 detection of glutamic acid and shikimic acid content in mutant strains
Bacterial strains OD 600 (×100) Glu(g/L) Conversion rate% SA(g/L) Phe(g/L) Trp(g/L) Tyr(g/L)
MHZ-0112-8 0.416±0.021 34.9±0.12 58.1±0.10 0.23±0.009 0.36±0.012 0.42±0.011 0.28±0.005
MHZ-0112-11 0.405±0.032 35.2±0.08 58.6±0.08 0.15±0.013 0.29±0.026 0.31±0.019 0.16±0.032
MHZ-0112-12 0.410±0.014 35.6±0.04 59.3±0.02 0.10±0.01 0.17±0.006 0.19±0.01 0.10±0.012
MHZ-0112-13 0.382±0.006 36.2±0.11 60.3±0.09 0.02±0.008 0.012±0.001 0.009±0.001 0.006±0.001
The results are shown in Table 3 (OD) 600 Is the turbidity of 100-fold diluted culture broth at 600nm and represents the cell mass, glu (g/L) represents the amount of accumulated L-glutamic acid, SA (g/L) represents the amount of accumulated L-shikimic acid, phe (g/L) represents the amount of accumulated L-phenylalanine, trp (g/L) represents the amount of accumulated L-tryptophan, try (g/L) represents the amount of accumulated L-tyrosine), amino acid 234 of tktA is mutated from threonine (T) to isoleucine (I) in MHZ-0112-11, i.e., after mutation of ACC to ATC, shikimic acid is reduced from 0.23g/L to 0.15g/L, while the corresponding aromatic amino acids Phe, trp, tyr are also reduced from 0.36g/L,0.42g/L,0.28g/L to 0.29g/L,0.31g/L,0.16g/L, while glutamic acid is increased from 34.9g/L to 0.35 g/L, and the conversion rate is increased from 0.5 g/L. After mutation of amino acid 171 of encoding ds gene from glycine (G) to aspartic acid (D) in MHZ-0112-12, shikimic acid was reduced from 0.23G/L to 0.10G/L, while the corresponding aromatic amino acids Phe, trp and Tyr were also reduced from 0.36G/L,0.42G/L and 0.28G/L to 0.17G/L,0.19G/L and 0.10G/L, respectively, while glutamic acid was increased from 34.9G/L to 35.6G/L, with a 1.2% increase in conversion. In MHZ-0112-13 strain with overlapped ds gene that 171 site glycine is mutated into aspartic acid, the content of shikimic acid is sharply reduced from 0.23g/L to 0.02g/L, and the content of aromatic amino acids (Phe, trp, tyr) is respectively reduced to 0.012g/L,0.009g/L and 0.006g/L. The glutamic acid yield is correspondingly improved from 35.2g/L to 36.2g/L, and compared with the starting strain, the glutamic acid conversion rate is improved by 2.2 percent. The results show that properly weakening the shikimic acid synthesis pathway can effectively reduce glutamic acid producing strainsThe production of the medium-heteroic acid improves the conversion rate of the glutamic acid.
Example 6 verification of glutamic acid-producing Properties of mutant Strain
The recombinant corynebacterium glutamicum constructed in examples 2, 3 and 4 was subjected to 50L scale-up fermentation to verify the glutamic acid productivity as follows:
inoculating the strain frozen in a glycerin pipe at the temperature of minus 80 ℃ into a slant culture medium for activation, culturing for 24 hours at the temperature of 31.5 ℃ to grow a lawn, selecting the lawn from a fresh activated slant, inoculating the lawn into a secondary seed culture medium, performing shaking culture at the temperature of 31.5 ℃ and the speed of 150rpm until the middle and later stages of logarithmic growth, culturing for 10 hours to prepare a seed solution, and inoculating 10% of the inoculation amount into the tertiary seed culture medium; culturing in 5L automatic control fermentation tank at 30 deg.C, pH of 7.0 and dissolved oxygen of 20% for 12h to logarithmic phase, inoculating 10% of inoculum size into 30L automatic control fermentation tank containing fermentation culture medium, controlling initial culture temperature to 30 deg.C, and diluting with 20 times concentration OD 600 When the value is 0.35, raising the temperature to 37 ℃ within 5 minutes, introducing proper air, adjusting proper stirring speed, and controlling dissolved oxygen by adopting a staged oxygen supply mode: 20% in 0-10h and 5% in 10-32h, controlling pH at 7.0-7.2 by automatic feeding of ammonia water, defoaming by feeding of a proper amount of foam killer, controlling residual sugar at 1.5% by feeding of a glucose solution with a concentration of 800g/L, and stopping fermentation until 32 h.
The formula of the culture medium is as follows:
secondary seed culture medium: glucose 25g/L, urea 3g/L, K 2 HPO 4 ·3H 2 O 2.2g/L，MgSO 4 ·7H 2 0.9g/L of O, 33mL/L of corn steep liquor, 22mL/L of soybean cake hydrolysate, 7.0-7.2 of pH and sterilization at 121 ℃ and 0.1MPa for 15min;
third-level seed culture medium: glucose 15g/L, corn steep liquor 20g/L, K 2 HPO 4 3g/L，MgSO 4 ·7H 2 O 1g/L，V B1 200 mu g/L, DL-methionine 50 mu g/L, feSO 4 ·7H 2 O 2mg/L、MnSO 4 ·H 2 O2 mg/L, urea 0.55g/L (separately digested), pH7.0-7.2, sterilizing at 121 deg.C for 15min;
50L fermentation mediumThe method comprises the following steps: 20g/L of glucose, 20g/L of corn steep liquor, 0.25g/L of betaine hydrochloride and K 2 HPO 4 7.5g/L，MgSO 4 ·7H 2 O 1.5g/L，V B1 200μg/L，V H 600μg/L，FeSO 4 ·7H2O 30mg/L、MnSO 4 ·H 2 30mg/L of O, 0.55g/L of urea and 7.2-7.5 of pHs.
TABLE 4 detection of glutamic acid and heteropolyacid content in mutant strains
Figure BDA0003093795170000151
The results are shown in Table 4 (OD) 600 Is the turbidity of 200-fold diluted culture broth at 600nm and represents the cell mass, glu (g/L) represents the amount of accumulated L-glutamic acid, SA (g/L) represents the amount of accumulated L-shikimic acid, phe (g/L) represents the amount of accumulated L-phenylalanine, trp (g/L) represents the amount of accumulated L-tryptophan, try (g/L) represents the amount of accumulated L-tyrosine), amino acid 234 of tktA is mutated from threonine (T) to isoleucine (I) in MHZ-0112-11, i.e., after mutation of ACC to ATC, shikimic acid is reduced from 1.15g/L to 0.76g/L, while the corresponding aromatic amino acids Phe, trp, tyr are also reduced from 1.12g/L0.83g/L,0.99g/L to 0.87g/L,0.47g/L,0.55g/L, while glutamic acid is increased from 178.4g/L to 8g/L, and the conversion rate is increased from 1.5%. After mutation of amino acid 171 of encoding ds gene from glycine (G) to aspartic acid (D) in MHZ-0112-12, shikimic acid was reduced from 1.15G/L to 0.65G/L, while the corresponding aromatic amino acids Phe, trp and Tyr were also reduced from 1.12G/L,0.83G/L and 0.99G/L to 0.74G/L,0.38G/L and 0.26G/L, respectively, while glutamic acid was increased from 178.4G/L to 185.5G/L, with a 2.3% increase in conversion.
In MHZ-0112-13 strain superimposed with ds gene obtained by mutating 171-site glycine into aspartic acid, the content of shikimic acid is sharply reduced from 1.15g/L to 0.05g/L, and the content of aromatic amino acids (Phe, trp and Tyr) is respectively reduced to 0.038g/L,0.025g/L and 0.012g/L. The glutamic acid yield is correspondingly improved from 178.4g/L to 190.6g/L, and compared with the starting strain, the glutamic acid conversion rate is improved by 4.0 percent. The results show that properly weakening the synthesis path of shikimic acid can effectively reduce the generation of the heteroacid in the glutamic acid production strain and improve the conversion rate of the glutamic acid.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Gallery plum blossom Biotechnology development Co., ltd
<120> recombinant microorganism, and preparation method and application thereof
<130> KHP211113317.0
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 697
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Thr Leu Ser Pro Glu Leu Gln Ala Leu Thr Val Arg Asn Tyr Pro
1 5 10 15
Ser Asp Trp Ser Asp Val Asp Thr Lys Ala Val Asp Thr Val Arg Val
20 25 30
Leu Ala Ala Asp Ala Val Glu Asn Cys Gly Ser Gly His Pro Gly Thr
35 40 45
Ala Met Ser Leu Ala Pro Leu Ala Tyr Thr Leu Tyr Gln Arg Val Met
50 55 60
Asn Val Asp Pro Gln Asp Thr Asp Trp Ala Gly Arg Asp Arg Phe Val
65 70 75 80
Leu Ser Cys Gly His Ser Ser Leu Thr Gln Tyr Ile Gln Leu Tyr Leu
85 90 95
Gly Gly Phe Gly Leu Glu Met Asp Asp Leu Lys Ala Leu Arg Thr Trp
100 105 110
Asp Ser Leu Thr Pro Gly His Pro Glu Tyr Arg His Thr Lys Gly Val
115 120 125
Glu Ile Thr Thr Gly Pro Leu Gly Gln Gly Leu Ala Ser Ala Val Gly
130 135 140
Met Ala Met Ala Ala Arg Arg Glu Arg Gly Leu Phe Asp Pro Thr Ala
145 150 155 160
Ala Glu Gly Glu Ser Pro Phe Asp His His Ile Tyr Val Ile Ala Ser
165 170 175
Asp Gly Asp Leu Gln Glu Gly Val Thr Ser Glu Ala Ser Ser Ile Ala
180 185 190
Gly Thr Gln Gln Leu Gly Asn Leu Ile Val Phe Trp Asp Asp Asn Arg
195 200 205
Ile Ser Ile Glu Asp Asn Thr Glu Ile Ala Phe Asn Glu Asp Val Val
210 215 220
Ala Arg Tyr Lys Ala Tyr Gly Trp Gln Ile Ile Glu Val Glu Ala Gly
225 230 235 240
Glu Asp Val Ala Ala Ile Glu Ala Ala Val Ala Glu Ala Lys Lys Asp
245 250 255
Thr Lys Arg Pro Thr Phe Ile Arg Val Arg Thr Ile Ile Gly Phe Pro
260 265 270
Ala Pro Thr Met Met Asn Thr Gly Ala Val His Gly Ala Ala Leu Gly
275 280 285
Ala Ala Glu Val Ala Ala Thr Lys Thr Glu Leu Gly Phe Asp Pro Glu
290 295 300
Ala His Phe Ala Ile Asp Asp Glu Val Ile Ala His Thr Arg Ser Leu
305 310 315 320
Ala Glu Arg Ala Ala Gln Lys Lys Ala Ala Trp Gln Val Lys Phe Asp
325 330 335
Glu Trp Ala Ala Ala Asn Pro Glu Asn Lys Ala Leu Phe Asp Arg Leu
340 345 350
Asn Ser Arg Glu Leu Pro Ala Gly Tyr Ala Asp Glu Leu Pro Thr Trp
355 360 365
Asp Ala Asp Glu Lys Gly Val Ala Thr Arg Lys Ala Ser Glu Ala Ala
370 375 380
Leu Gln Ala Leu Gly Lys Thr Leu Pro Glu Leu Trp Gly Gly Ser Ala
385 390 395 400
Asp Leu Ala Gly Ser Asn Asn Thr Val Ile Lys Gly Ser Pro Ser Phe
405 410 415
Gly Pro Glu Ser Ile Ser Thr Glu Thr Trp Ser Ala Glu Pro Tyr Gly
420 425 430
Arg Asn Leu His Phe Gly Ile Arg Glu His Ala Met Gly Ser Ile Leu
435 440 445
Asn Gly Ile Ser Leu His Gly Gly Thr Arg Pro Tyr Gly Gly Thr Phe
450 455 460
Leu Ile Phe Ser Asp Tyr Met Arg Pro Ala Val Arg Leu Ala Ala Leu
465 470 475 480
Met Glu Thr Asp Ala Tyr Tyr Val Trp Thr His Asp Ser Ile Gly Leu
485 490 495
Gly Glu Asp Gly Pro Thr His Gln Pro Val Glu Thr Leu Ala Ala Leu
500 505 510
Arg Ala Ile Pro Gly Leu Ser Val Leu Arg Pro Ala Asp Ala Asn Glu
515 520 525
Thr Ala Gln Ala Trp Ala Ala Ala Leu Glu Tyr Lys Glu Gly Pro Lys
530 535 540
Gly Leu Ala Leu Thr Arg Gln Asn Ile Pro Val Leu Glu Gly Thr Lys
545 550 555 560
Glu Lys Ala Ala Glu Gly Val Arg Arg Gly Gly Tyr Val Leu Val Glu
565 570 575
Gly Ser Lys Glu Thr Pro Asp Val Ile Leu Met Gly Ser Gly Ser Glu
580 585 590
Val Gln Leu Ala Val Asn Ala Ala Lys Ala Leu Glu Ala Glu Gly Val
595 600 605
Ala Ala Arg Val Val Ser Val Pro Cys Met Asp Trp Phe Gln Glu Gln
610 615 620
Asp Ala Glu Tyr Ile Glu Ser Val Leu Pro Ala Ala Val Thr Ala Arg
625 630 635 640
Val Ser Val Glu Ala Gly Ile Ala Met Pro Trp Tyr Arg Phe Leu Gly
645 650 655
Thr Gln Gly Arg Ala Val Ser Leu Glu His Phe Gly Ala Ser Ala Asp
660 665 670
Tyr Gln Thr Leu Phe Glu Lys Phe Gly Ile Thr Thr Asp Ala Val Val
675 680 685
Ala Ala Ala Lys Asp Ser Ile Asn Gly
690 695
<210> 2
<211> 366
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Ser Ser Pro Val Ser Leu Glu Asn Ala Ala Ser Thr Ser Asn Lys
1 5 10 15
Arg Val Val Ala Phe His Glu Leu Pro Ser Pro Thr Asp Leu Ile Ala
20 25 30
Ala Asn Pro Leu Thr Pro Lys Gln Ala Ser Lys Val Glu Gln Asp Arg
35 40 45
Gln Asp Ile Ala Asp Ile Phe Ala Gly Asp Asp Asp Arg Leu Val Val
50 55 60
Val Val Gly Pro Cys Ser Val His Asp Pro Glu Ala Ala Ile Asp Tyr
65 70 75 80
Ala Asn Arg Leu Ala Pro Leu Ala Lys Arg Leu Asp Gln Asp Leu Lys
85 90 95
Ile Val Met Arg Val Tyr Phe Glu Lys Pro Arg Thr Thr Val Gly Trp
100 105 110
Lys Gly Leu Ile Asn Asp Pro His Leu Asn Glu Thr Tyr Asp Ile Pro
115 120 125
Glu Gly Leu Arg Ile Ala Arg Lys Val Leu Ile Asp Val Val Asn Leu
130 135 140
Asp Leu Pro Val Gly Cys Glu Phe Leu Glu Pro Asn Ser Pro Gln Tyr
145 150 155 160
Tyr Ala Asp Thr Val Ala Trp Gly Ala Ile Asp Ala Arg Thr Thr Glu
165 170 175
Ser Gln Val His Arg Gln Leu Ala Ser Gly Met Ser Met Pro Ile Gly
180 185 190
Phe Lys Asn Gly Thr Asp Gly Asn Ile Gln Val Ala Val Asp Ala Val
195 200 205
Gln Ala Ala Gln Asn Pro His Phe Phe Phe Gly Thr Ser Asp Asp Gly
210 215 220
Ala Leu Ser Val Val Glu Thr Ala Gly Asn Ser Asn Ser His Ile Ile
225 230 235 240
Leu Arg Gly Gly Thr Ser Gly Pro Asn His Asp Ala Ala Ser Val Glu
245 250 255
Ala Val Val Glu Lys Leu Gly Glu Asn Ala Arg Leu Met Ile Asp Ala
260 265 270
Ser His Ala Asn Ser Gly Lys Asp His Ile Arg Gln Val Glu Val Val
275 280 285
Arg Glu Ile Ala Glu Gln Ile Ser Gly Gly Ser Glu Ala Val Ala Gly
290 295 300
Ile Met Ile Glu Ser Phe Leu Val Gly Gly Ala Gln Asn Leu Asp Pro
305 310 315 320
Ala Lys Leu Arg Ile Asn Gly Gly Glu Gly Leu Val Tyr Gly Gln Ser
325 330 335
Val Thr Asp Lys Cys Ile Asp Ile Asp Thr Thr Ile Asp Leu Leu Ala
340 345 350
Glu Leu Ala Ala Ala Val Arg Glu Arg Arg Ala Ala Ala Lys
355 360 365
<210> 3
<211> 2094
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttgacgctgt cacctgaact tcaggcgctc actgtacgca attacccctc tgattggtcc 60
gatgtggaca ccaaggctgt agacactgtt cgtgtcctcg ctgcagacgc tgtagaaaac 120
tgtggctccg gccacccagg caccgcaatg agcctggctc cccttgcata caccttgtac 180
cagcgggtta tgaacgtaga tccacaggac accgactggg caggccgtga ccgcttcgtt 240
ctttcttgtg gccactcctc tttgacccag tacatccagc tttacttggg tggattcggc 300
cttgagatgg atgacctgaa ggctctgcgc acctgggatt ccttgacccc aggacaccct 360
gagtaccgcc acaccaaggg cgtagagatc accactggcc ctcttggcca gggtcttgca 420
tctgcagttg gtatggccat ggctgctcgt cgtgagcgtg gcctattcga cccaaccgct 480
gctgagggcg aatccccatt cgaccaccac atctacgtca ttgcttctga tggtgacctg 540
caggaaggtg tcacctctga ggcatcctcc atcgctggca cccagcagct gggcaacctc 600
atcgtgttct gggatgacaa ccgcatctcc atcgaagaca acactgagat cgctttcaac 660
gaggacgttg ttgctcgtta caaggcttac ggctggcaga tcattgaggt tgaggctggc 720
gaggacgttg cagcaatcga agctgcagtg gctgaggcta agaaggacac caagcgacct 780
accttcatcc gcgttcgcac catcatcggc ttcccagctc caaccatgat gaacaccggt 840
gctgtgcacg gtgctgctct tggcgcagct gaggttgcag caaccaagac tgagcttgga 900
ttcgatcctg aggctcactt cgcgatcgac gatgaggtta tcgctcacac ccgctccctc 960
gcagagcgcg ctgcacagaa gaaggctgca tggcaggtca agttcgatga gtgggcagct 1020
gccaaccctg agaacaaggc tctgttcgat cgcctgaact cccgtgagct tccagcgggc 1080
tacgctgacg agctcccaac atgggatgca gatgagaagg gcgtcgcaac tcgtaaggct 1140
tccgaggctg cacttcaggc actgggcaag acccttcctg agctgtgggg cggttccgct 1200
gacctcgcag gttccaacaa caccgtgatc aagggctccc cttccttcgg ccctgagtcc 1260
atctccaccg agacctggtc tgctgagcct tacggccgta acctgcactt cggtatccgt 1320
gagcacgcta tgggatccat cctcaacggc atttccctcc acggtggcac ccgcccatac 1380
ggcggaacct tcctcatctt ctccgactac atgcgtcctg cagttcgtct tgcagctctc 1440
atggagaccg acgcttacta cgtctggacc cacgactcca tcggtctggg cgaagatggc 1500
ccaacccacc agcctgttga aaccttggct gcactgcgcg ccatcccagg tctgtccgtc 1560
ctgcgtcctg cagatgcgaa cgagaccgcc caggcttggg ctgcagcact tgagtacaag 1620
gaaggcccta agggtcttgc actaacccgc cagaacattc ctgttctgga aggcaccaag 1680
gagaaggctg ctgaaggcgt tcgccgcggt ggctacgtcc tggttgaggg ttccaaggaa 1740
accccagatg tgatcctcat gggctccggc tccgaggttc agcttgcagt taacgctgcg 1800
aaggctctgg aagctgaggg cgttgcagct cgcgttgttt ccgttccttg catggattgg 1860
ttccaggagc aggacgcaga gtacatcgag tccgttctgc ctgcagctgt gaccgctcgt 1920
gtgtctgttg aagctggcat cgcaatgcct tggtaccgct tcttgggcac ccagggccgt 1980
gctgtctccc ttgagcactt cggtgcttct gcggattacc agaccctgtt tgagaagttc 2040
ggcatcacca ccgatgcagt cgtggcagcg gccaaggact ccattaacgg ttaa 2094
<210> 4
<211> 1101
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgagttctc cagtctcact cgaaaacgcg gcgtcaacca gcaacaagcg cgtcgtggct 60
ttccacgagc tgcctagccc tacagatctc atcgccgcaa acccactgac accaaagcag 120
gcttccaagg tggagcagga tcgccaggac atcgctgata tcttcgctgg cgacgatgac 180
cgcctcgttg tcgttgtggg accttgctca gttcacgatc ctgaagcagc catcgattac 240
gcaaaccgcc tggctccgct ggcaaagcgc cttgaccagg acctcaagat tgtcatgcgc 300
gtgtacttcg agaagcctcg caccaccgtc ggatggaagg gattgatcaa tgatcctcac 360
ctcaacgaaa cctacgacat cccagagggc ttgcgcattg cgcgcaaagt gcttatcgac 420
gttgtgaacc ttgatctccc agtcggctgc gaattcctcg aaccaaacag ccctcagtac 480
tacgccgaca ctgtcgcatg gggagcaatc gacgctcgta ccaccgaatc tcaggtgcac 540
cgccagctgg cttctgggat gtctatgcca attggtttca agaacggaac tgacggaaac 600
atccaggttg cagtcgacgc ggtacaggct gcccagaacc cacacttctt cttcggaacc 660
tccgacgacg gcgcgctgag cgtcgtggag accgcaggca acagcaactc ccacatcatt 720
ttgcgcggcg gtacctccgg cccgaatcat gatgcagctt cggtggaggc cgtcgtcgag 780
aagcttggtg aaaacgctcg tctcatgatc gatgcttccc atgctaactc cggcaaggat 840
catatccgac aggttgaggt tgttcgtgaa atcgcagagc agatttctgg cggttctgaa 900
gctgtggctg gaatcatgat tgagtccttc ctcgttggtg gcgcacagaa ccttgatcct 960
gcgaaattgc gcatcaatgg cggtgaaggc ctggtgtacg gacagtctgt gaccgataag 1020
tgcatcgaca ttgacaccac catcgatttg ctcgctgagc tggccgcagc agtaagggaa 1080
cgccgagcag cagccaagta a 1101
<210> 5
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gctctagaca gcgggttatg aacgtagat 29
<210> 6
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gccagcctca acctcaatga tctgccagcc gtaagccttg 40
<210> 7
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
attgaggttg aggctggcga ggacgttgca gcaatcgaag 40
<210> 8
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cccaagcttg ttggaacctg cgaggtcag 29
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ggcttacggc tggcagat 18
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tcgctgcaga cgctgtag 18
<210> 11
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
acggataccg aagtgcagg 19
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gctctagaat gagttctcca gtctcactcg 30
<210> 13
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gattcggtgg tacgagcgtc gattgctccc catgcgacag 40
<210> 14
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cgctcgtacc accgaatctc aggtgcaccg ccagctggct 40
<210> 15
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tgcactgcag atcggtcaca gactgtccgt a 31
<210> 16
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cgcatgggga gcaatcga 18
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cggagtcgag cagcacctc 19
<210> 18
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ttactgctgc ggccagctc 19
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ctcgtatgtt gtgtggaatt gtg 23
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cgccctgagt gcttgcggca 20

Claims (10)

1. The protein mutant is characterized in that the protein mutant is a transketolase mutant and/or a 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant,
wherein the amino acid sequence of the transketolase mutant is shown as SEQ ID NO.1,
the amino acid sequence of the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant is shown as SEQ ID NO. 2.
2. Nucleic acid encoding the protein mutant of claim 1.
3. A biological material comprising the nucleic acid of claim 2, wherein the biological material is an expression cassette, a vector, or C.
4. Use of a protein mutant according to claim 1 or a nucleic acid according to claim 2 or a biological material according to claim 3 for the production of glutamic acid.
5. A recombinant Corynebacterium glutamicum which expresses a transketolase mutant and/or a 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant and does not express transketolase and/or 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase which are possessed by the starting strain;
the amino acid sequence of the transketolase mutant is shown as SEQ ID NO. 1;
the amino acid sequence of the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant is shown as SEQ ID NO. 2;
the starting strain of the recombinant corynebacterium glutamicum is corynebacterium glutamicum capable of accumulating glutamic acid.
6. The recombinant Corynebacterium glutamicum of claim 5, wherein the gene encoding a transketolase in the recombinant Corynebacterium glutamicum is mutated to a gene encoding the transketolase mutant, and/or the gene encoding a 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase in the recombinant Corynebacterium glutamicum is mutated to a gene encoding the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant.
7. The method for constructing recombinant Corynebacterium glutamicum of claim 5 or 6, comprising: introducing a recombinant plasmid carrying a gene encoding a transketolase mutant and/or a 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant into an initial strain, and mutating the gene encoding the transketolase in the initial strain into a gene encoding the transketolase mutant, and/or mutating the gene encoding the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase in the initial strain into a gene encoding the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase mutant.
8. Use of the recombinant corynebacterium glutamicum of claims 5 or 6, in the production of glutamic acid.
9. A method for producing glutamic acid comprising the step of subjecting the recombinant Corynebacterium glutamicum of claim 5 or 6 to fermentation culture.
10. The method of claim 9, wherein the fermentation medium comprises the following components: 50-60g/L glucose, 10-20g/L ammonium sulfate and KH 2 PO 4 0.5-1.5g/L，MgSO 4 ·7H 2 O 0.2-0.5g/L，FeSO 4 ·7H 2 O 0.05-0.15mg/L，MnSO 4 ·5H 2 O 0.8-1.2mg/L，VB 1 150-250 mug/L, biotin 250-350 mug/L, soybean hydrolysate 0.3-0.6g/L, pH7.2-7.5,
alternatively, the fermentation medium comprises the following components: 15-25g/L of glucose, 15-25g/L of corn steep liquor, 0.1-0.3g/L of betaine hydrochloride, and K 2 HPO 4 6-8g/L，MgSO 4 ·7H 2 O1-2g/L，V B1 150-250μg/L，V H 550-650μg/L，FeSO 4 ·7H 2 O 25-35mg/L、MnSO 4 ·H 2 25-35mg/L of O, 0.4-0.7g/L of urea and 7.2-7.5 of pH.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107227283A (en) * 2017-05-26 2017-10-03 廊坊梅花生物技术开发有限公司 A kind of Corynebacterium glutamicum and its construction method and application
CN112322594A (en) * 2020-11-17 2021-02-05 廊坊梅花生物技术开发有限公司 Corynebacterium glutamicum capable of producing glutamic acid in high yield and application thereof

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EP2628792A1 (en) * 2012-02-17 2013-08-21 Evonik Industries AG Cell with reduced ppGppase activity
US20150203880A1 (en) * 2013-11-06 2015-07-23 Massachusetts Institute Of Technology Co-culture based modular engineering for the biosynthesis of isoprenoids, aromatics and aromatic-derived compounds

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CN107227283A (en) * 2017-05-26 2017-10-03 廊坊梅花生物技术开发有限公司 A kind of Corynebacterium glutamicum and its construction method and application
CN112322594A (en) * 2020-11-17 2021-02-05 廊坊梅花生物技术开发有限公司 Corynebacterium glutamicum capable of producing glutamic acid in high yield and application thereof

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Mutational analysis of the catalytic and feedback sites of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli;J M Ray等;《J Bacteriol》;19881231;第170卷(第12期);第5500-5506页 *

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