A TrpCF mutant and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a TrpCF mutant and its application. The present invention provides a trpCF mutant, the amino acid sequence of the mutant contains an amino acid sequence after the 421st glycine of the amino acid sequence shown in SEQ ID No.1 is mutated. Specifically, the amino acid sequence of the mutant provided by the present invention contains an amino acid sequence after the 421st glycine of the amino acid sequence shown in SEQ ID No.1 is mutated to L-serine or L-lysine. The mutant provided by the present invention can reasonably weaken the tryptophan synthesis pathway, thereby increasing the yield and conversion rate of glutamate, while also being able to ensure the growth performance of the strain. The recombinant microorganism expressing the mutant of the present invention has a higher glutamate yield and sugar-acid conversion rate, and the growth performance of the strain is good.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a TrpCF mutant and application thereof. Background Art

[0002] Glutamic acid, with the chemical formula C5H9NO4 and a molecular weight of 147.13, is an acidic amino acid. The molecule contains two carboxyl groups and its chemical name is α-aminoglutaric acid. Glutamic acid is the basic unit of protein and one of the 21 amino acids that make up human protein. Glutamic acid is generally produced by Corynebacterium glutamicum, and monosodium glutamate monohydrate is a food additive with a "umami" taste, which is one of the five basic tastes of humans. Monosodium glutamate monohydrate is the famous MSG. By fermenting Corynebacterium glutamicum, adding or restricting some substances, and opening the mechanosensitive channels on the cell membrane, Corynebacterium glutamicum will output a large amount of glutamate. These substances include biotin restriction, addition of Tween 40 or penicillin. However, with the deepening of research, this method of controlling the opening of mechanosensitive channels by adding or restricting substances has been eliminated. Instead, the mechanosensitive channel switch is controlled by temperature. Recent studies have shown that the opening of the mechanosensitive channel MscCG mainly promotes the efflux of glutamate, rather than the efflux of other amino acids.

[0003] At present, the most commonly used method for producing L-glutamic acid is fermentation, mainly using Corynebacterium as the production bacteria to ferment L-glutamic acid. However, the problem of heteroamino acids in the fermentation of L-glutamic acid strains is prominent and cannot be well solved, which affects the performance of L-glutamic acid fermentation and is very unfavorable for the production of high-quality L-glutamic acid. Summary of the invention

[0004] The purpose of the present invention is to increase the yield of glutamic acid produced by recombinant microorganisms. In order to achieve the purpose of the present invention, the present invention provides the following technical solutions.

[0005] In a first aspect, the present invention provides a trpCF mutant, wherein the amino acid sequence of the mutant comprises an amino acid sequence in which the 421st glycine of the amino acid sequence shown in SEQ ID No. 1 is mutated.

[0006] The trpCF mutant provided by the present invention is a bifunctional indole-3-phosphoglycerol synthase / phosphoribosyl anthranilate isomerase mutant. The present invention has found in the research and development process that weakening tryptophan synthesis can enhance the metabolic flow capacity of the central metabolic pathway, provide more sufficient carbon flow for the synthesis of metabolites such as glutamate via the TCA cycle, and then increase its yield, but the excessive weakening of the synthetic pathway of tryptophan will have a significant adverse effect on the growth and metabolism of the strain, and will reduce the growth of the strain and the production performance of the target product. Therefore, weakening the tryptophan pathway to a certain extent, promoting the balance of the growth of the strain and the production of the target product, can significantly improve the production performance of the strain for the target product. The present invention has found two isomerase mutants, which can reduce the activity of isomerase to a certain extent, can reasonably weaken the metabolic flow of the tryptophan pathway, while ensuring the growth performance of the strain, reduce the accumulation of miscellaneous acids such as tryptophan, and significantly improve the yield and conversion rate of metabolites such as glutamate via the TCA cycle.

[0007] Specifically, the amino acid sequence of the trpCF mutant of the present invention comprises an amino acid sequence after the 421st position of the amino acid sequence shown in SEQ ID No. 1 is mutated from glycine to L-serine or L-lysine;

[0008] Preferably, the trpCF mutant has an amino acid sequence as shown in SEQ ID No.2 or SEQ ID No.3.

[0009] Those skilled in the art should understand that adding a tag protein to the N-terminus or C-terminus of the above-mentioned trpCF mutant amino acid sequence or fusing it with other proteins to form a fusion protein, without changing the activity of the above-mentioned mutant protein itself, the above-mentioned tagged protein or fusion protein is also within the scope of protection of the present invention.

[0010] In a second aspect, the present invention provides a DNA molecule encoding the trpCF mutant. The DNA molecule has a nucleotide sequence as shown in SEQ ID NO.13 or SEQ ID NO.14. Specifically, the nucleotide sequence as shown in SEQ ID NO.13 or SEQ ID NO.14 contains, in addition to the nucleic acid encoding the trpCF mutant, an upstream homology arm and an outer identification primer portion.

[0011] In a third aspect, the present invention seeks to protect biological materials containing the above-mentioned DNA molecules, wherein the biological materials are expression cassettes, vectors or host cells.

[0012] The expression cassette is a recombinant nucleic acid molecule obtained by connecting elements for driving the transcription and expression of the nucleic acid upstream or downstream.

[0013] 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.

[0014] The host cells include, but are not limited to, microbial cells.

[0015] According to the understanding of those skilled in the art, the present invention also seeks protection for the use of the above-mentioned trpCF mutant or the above-mentioned DNA molecule or the above-mentioned biological material in any of the following:

[0016] (1) Improving the glutamate production, conversion rate or production intensity of recombinant microorganisms;

[0017] (2) Reduce tryptophan accumulation in recombinant microorganisms.

[0018] In a fourth aspect, based on the above trpCF mutant or the above DNA molecule, the present invention provides a recombinant microorganism, wherein the expression level and / or activity of the bifunctional indole-3-phosphoglycerol synthase / phosphoribosylanthranilate isomerase of the recombinant microorganism is reduced compared with the starting strain;

[0019] Preferably, the recombinant microorganism expresses the above-mentioned trpCF mutant or the recombinant microorganism contains the above-mentioned DNA molecule.

[0020] Specifically, as one embodiment of the present invention, the recombinant microorganism expresses the isomerase mutant and does not express the isomerase possessed by its starting strain.

[0021] As another embodiment, the present invention provides a recombinant microorganism, which expresses the trpCF isomerase mutant and does not express the trpCF isomerase possessed by its starting strain.

[0022] Preferably, the gene mutation encoding isomerase in the recombinant microorganism is a gene encoding the above-mentioned trpCF mutant.

[0023] The recombinant microorganism provided by the present invention is a Corynebacterium bacterium or a Brevibacterium bacterium;

[0024] Preferably, the Corynebacterium bacterium is Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes or Corynebacteriumaminogenes;

[0025] The Brevibacterium bacterium is Brevibacterium flavum or Brevibacterium lactofermentum.

[0026] More preferably, the starting strain for constructing the recombinant microorganism is a Corynebacterium bacterium capable of accumulating glutamate.

[0027] As a preferred embodiment of the present invention, the starting strain of the recombinant microorganism construction is Corynebacterium glutamicum which can accumulate glutamate.

[0028] The starting strain is Corynebacterium glutamicum MHZ-0112-8 with a deposit number of CGMCC No.11941.

[0029] The pure culture of Corynebacterium glutamicum MHZ-0112-8 has been deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on December 25, 2015, and the deposit number is CGMCC No. 11941. Corynebacterium glutamicum MHZ-0112-8 (CGMCC No. 11941) mentioned in the present invention has been disclosed in Chinese Patent Publication No. CN 105695383 A.

[0030] In a fifth aspect, the present invention provides a method for constructing the above-mentioned recombinant microorganism, comprising inserting one or more bases into the gene encoding the bifunctional indole-3-phosphoglycerate synthase / phosphoribosylanthranilate isomerase in the starting strain;

[0031] or replacing the transcription and / or translation regulatory elements of the gene encoding the bifunctional indole-3-phosphoglycerate synthase / phosphoribosylanthranilate isomerase in the developing strain with regulatory elements with lower activity;

[0032] Preferably, a recombinant plasmid carrying the above DNA molecule is introduced into the starting strain. The recombinant plasmid is a plasmid that can undergo homologous recombination in the bacterium, exchanging the coding gene of the protein mutant on the plasmid with the homologous gene on the chromosome.

[0033] Further, the construction method comprises:

[0034] (1) constructing a recombinant plasmid containing a gene encoding the isomerase mutant;

[0035] (2) transforming the recombinant plasmid into the starting strain and screening the transformants using a selective medium containing antibiotics;

[0036] (3) Screening the recombinant microorganisms that have the target mutation among the positive transformants.

[0037] According to the understanding of those skilled in the art, the present invention also seeks to protect the use of the above-mentioned recombinant microorganism in any of the following:

[0038] (1) Glutamate production;

[0039] (2) Increase glutamate production;

[0040] (3) Improving the sugar-acid conversion rate in glutamate production;

[0041] (4) Reduce the accumulation of impurity acids in glutamate fermentation production.

[0042] In a sixth aspect, the present invention provides a method for increasing glutamate production, using the above-mentioned recombinant microorganism for fermentation culture.

[0043] In the fermentation culture, the seed culture medium used includes the following components: 5% corn steep liquor, 2% glucose, 2% ammonium sulfate, 0.05% magnesium sulfate, 0.2% potassium dihydrogen phosphate, 0.1% urea, 0.5% CaCO3, pH 7.0.

[0044] The fermentation medium used includes the following components: 2% corn steep liquor, 12.0% glucose, 5% ammonium sulfate, 0.05% magnesium sulfate, 0.2% potassium dihydrogen phosphate, 4% CaCO3, pH 7.0.

[0045] Specifically, the method for producing glutamic acid comprises: inoculating the recombinant microorganism into a slant culture medium for slant culture, picking the bacterial moss on the slant culture medium and inoculating it into a seed culture medium for seed culture, and then transferring the seed culture into a fermentation culture medium for fermentation.

[0046] In some embodiments, the slant culture medium used is: 5 g / L yeast powder, 10 g / L beef extract, 10 g / L peptone, 10 g / L sodium chloride, 2.5 g / L agar powder, pH 7.0-7.2.

[0047] The slant culture is cultured at 30-32°C for 20-25h.

[0048] In some embodiments, the seed culture medium used is: 5% corn steep liquor, 2% glucose, 2% ammonium sulfate, 0.05% magnesium sulfate, 0.2% potassium dihydrogen phosphate, 0.1% urea, 0.5% CaCO3, pH 7.0.

[0049] The seed culture is performed by shaking culture at 30-32° C. until the middle and late stages of logarithmic growth, and the culture time is 10-14 hours.

[0050] In some embodiments, the fermentation medium used is: 2% corn steep liquor, 12.0% glucose, 5% ammonium sulfate, 0.05% magnesium sulfate, 0.2% potassium dihydrogen phosphate, 4% CaCO3, pH 7.0.

[0051] The fermentation is carried out at 30-32° C. with shaking culture for 12-20 hours.

[0052] The method for producing glutamic acid provided by the present invention reduces the accumulation of miscellaneous acids in glutamic acid fermentation production and increases the yield of glutamic acid. Therefore, the present invention is equivalent to providing a method for reducing the accumulation of miscellaneous acids in glutamic acid fermentation production.

[0053] The beneficial effects of the present invention are at least:

[0054] The enzyme activity of the isomerase mutant provided by the present invention is reduced, and the metabolic flow of the shikimic acid synthesis pathway can be reasonably weakened, the accumulation of tryptophan can be reduced, and the metabolic flow capacity of the central metabolic pathway can be enhanced, thereby improving the yield and conversion rate of glutamate, while also ensuring the growth performance of the strain. The present invention also provides a recombinant microorganism expressing the isomerase mutant, and the glutamate fermentation production performance of the recombinant microorganism is significantly improved, with a higher glutamate yield and sugar-acid conversion rate. DETAILED DESCRIPTION

[0055] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of protection of the present invention.

[0056] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the examples of the present invention are all commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art. The names and sequences of the primers used in the examples of the present invention are shown in Table 1.

[0057] Table 1 Primer sequences

[0058]

[0059]

[0060] The starting strain MHZ-0112-8 is Corynebacterium glutamicum. The pure culture of Corynebacterium glutamicum MHZ-0112-8 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on December 25, 2015, with the deposit number CGMCC No.11941.

[0061] The Corynebacterium glutamicum MHZ-0112-8 (CGMCC No. 11941) mentioned in the present invention has been disclosed in Chinese Patent Publication No. CN 105695383 A.

[0062] Example 1

[0063] The MHZ-0112-8 strain was first mutated with acriflavine, the bacteria in the logarithmic phase were centrifuged, washed twice with phosphate buffer (0.1 mol / L, pH 7.0), and resuspended in 0.1 mol / L phosphate buffer (pH 7.0) containing a certain concentration of acriflavine to a final concentration of 0.2 mg / mL. The mutagenic bacterial solution obtained after shaking on a shaker at 37°C for 4 h was diluted and spread on a bromocresol purple plate, and cultured at 31.5°C for 36 h to obtain 200 strains with large color change circles. The high-yield glutamate strains MHZ-0112-Z0 and MHZ-0112-Z1 were obtained by TLC rescreening and shake flask verification combined with HPLC screening.

[0064] The obtained MHZ-0112-Z0 and MHZ-0112-Z1 were activated on brain heart infusion solid culture medium and cultured at 31.5°C for 16-20h; a loop of bacteria was scraped from the plate and inoculated into 30mL of seed culture medium at 31.5°C and 220rpm for shaking culture until the middle and late logarithmic growth period for 12h. 2mL of seed liquid was transferred to 20mL of fermentation culture medium at 31.5°C and 220rpm for shaking culture for 16h.

[0065] Liquid chromatography detection of glutamic acid and assorted acids in the fermentation broth of MHZ-0112-Z0 found that the yield of glutamic acid increased from 34.2g / L to 35.9g / L. In addition to the significant increase in the yield of glutamic acid, the conversion rate increased by 1.1%. The content of tryptophan decreased to 0.036g / L. Liquid chromatography detection of glutamic acid and assorted acids in the fermentation broth of MHZ-0112-Z1 found that the yield of glutamic acid increased from 34.2g / L to 35.6g / L. In addition to the significant increase in the yield of glutamic acid, the conversion rate increased by 1.0%. The content of tryptophan decreased to 0.038g / L (Table 2).

[0066] Table 2 The production of glutamate and tryptophan in the mutant strains

[0067] strain <![CDATA[OD 600 (×100)]]> Glu(g / L) Conversion Rate % Trp(g / L) MHZ-0112-8 0.421±0.012 34.2±0.21 58.3±0.12 0.41±0.021 MHZ-0112-Z0 0.411±0.005 35.9±0.13 59.4±0.03 0.036±0.005 MHZ-0112-Z1 0.412±0.003 35.6±0.21 59.3±0.06 0.038±0.003

[0068] Comparative genomics analysis of the mutagenic strains MHZ-0112-Z0, MHZ-0112-Z1 and the starting strain MHZ-0112-8 revealed that the genes of the tryptophan synthesis pathway: the bifunctional indole-3-phosphoglycerol synthase / phosphoribosyl anthranilate isomerase gene mutated, and the 421st amino acid of the bifunctional indole-3-phosphoglycerol synthase / phosphoribosyl anthranilate isomerase mutated. The above mutations lead to reduced activity of the isomerase. This mutation leads to reduced accumulation of tryptophan in the cell, thereby enhancing the metabolic flow capacity of the central metabolic pathway, providing sufficient carbon flow for glutamate synthesis, and thus increasing the production of glutamate.

[0069] Example 2 Plasmid pK18-trpCF G421S Construction and recombinant strain MHZ-0112-8-trpCF G421S Construction

[0070] This example provides a recombinant strain MHZ-0112-8-trpCF G421S The specific steps for building are as follows:

[0071] Phusion ultra-fidelity polymerase (New England BioLabs) was used to prepare the genome of Corynebacterium glutamicum MHZ-0112-8 as a template and A1-trpCF-UF / A2-trpCF-UR as primers to prepare the recombinant fragment UP, and A3-trpCF-DF / A4-trpCF-DR as primers to prepare the recombinant fragment DN. The obtained fragment was purified by an agarose gel recovery kit (Tian Gen). Subsequently, UP and DN were used as templates and A1-trpCF-UF / A4-trpCF-DR as primers to prepare the recombinant fragment. The obtained recombinant fragment was purified by an agarose gel recovery kit (Tian Gen), and then digested with HindIII / XbaI. At the same time, pK18-mobsacB was digested with HindIII / XbaI, and the fragment was ligated to the vector with T4 DNA ligase (TransGen Biotech) and transformed into Trans1T1 competent cells (TransGen Biotech). Biotech), picked kanamycin-resistant clones, and identified positive clones with fragments inserted into pK18mobsacB by HindIII / XbaI digestion. Further sequencing with P82 / P85 primers (Invitrogen) identified that the inserted fragments were correct. The resulting plasmid was named pK18-trpCF G421S . G421SThe transformed cells were transferred into glutamicum MHZ-0112-8 and the recombinants were selected and exchanged on a selective medium containing 15 mg / L of kanamycin. The culture temperature was 33°C and the cells were inverted. The selected transformants were cultured overnight in a common liquid brain heart infusion medium at 33°C and shaken at 220 rpm on a rotary shaker. During this culture process, the transformants underwent a second recombination and the vector sequence was removed from the genome by gene exchange. The culture was diluted in a continuous gradient (10 -2 Serial dilution up to 10 -4 ), the dilution was spread on a common solid brain heart infusion medium containing 10% sucrose and cultured at 33°C for 48 hours. S The recombinant was verified by A7-trpCF-TF / A4-trpCF-DR, and the recombinant containing point mutation was obtained by exploring the annealing temperature. The positive recombinant was amplified and sequenced by A5-trpCF-TF / A6-trpCF-TR. After the verification and screening were completed, the target mutant strain was named MHZ-0112-20.

[0072] Example 3MHZ-0112-8-trpCF G421S The performance of (MHZ-0112-20) mutant strain in producing L-glutamic acid

[0073] This example identifies the ability of the mutant strain obtained in Example 2 to produce L-glutamic acid.

[0074] The seed activation medium used in this example is: 1% yeast extract, 1% peptone, 0.5% sodium chloride, 0.5% glucose, 2% agar, pH 7.0.

[0075] The seed culture medium used was: 5% corn steep liquor, 2% glucose, 2% ammonium sulfate, 0.05% magnesium sulfate, 0.2% potassium dihydrogen phosphate, 0.1% urea, 0.5% CaCO3, pH 7.0.

[0076] The fermentation medium used was: 2% corn steep liquor, 12.0% glucose, 5% ammonium sulfate, 0.05% magnesium sulfate, 0.2% potassium dihydrogen phosphate, 4% CaCO3, pH 7.0.

[0077] (1) Seed culture: select MHZ-0112-8, MHZ-0112-8-trpCF G421S Slant seed 1 was inoculated into a 500 mL Erlenmeyer flask containing 20 mL of seed culture medium and cultured at 33°C with shaking at 220 rpm for 16-22 h.

[0078] (2) Fermentation culture: 2 mL of seed solution was inoculated into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium and cultured at 33 °C and 220 rpm for 72 h.

[0079] (3) 1 mL of the fermentation broth was centrifuged (12000 rpm, 2 min), the supernatant was collected, and the L-glutamic acid content in the fermentation broth of the engineered bacteria and the control bacteria was detected by HPLC. The concentrations are shown in Table 3.

[0080] Table 3 Detection of glutamate and tryptophan content in mutant strains

[0081] strain <![CDATA[OD 600 (×100)]]> Glu(g / L) Conversion Rate % Trp(g / L) MHZ-0112-8 0.421±0.017 34.3±0.12 58.2±0.10 0.42±0.12 MHZ-0112-20 0.411±0.022 35.5±0.08 59.3±0.08 0.016±0.012

[0082] The results in Table 3 show that the L-glutamic acid accumulation of the starting strain MHZ-0112-8 is 34.3 g / L, while the engineered strain MHZ-0112-8-trpCF G421S The L-glutamic acid yield of (MHZ-0112-20) was 35.5 g / L, which shows that trpCF G421S The mutation favors the accumulation of L-glutamate.

[0083] OD in Table 3 600 It is the turbidity of the culture solution diluted 100 times at 600nm and represents the cell amount, Glu (g / L) represents the amount of accumulated L-glutamic acid, Trp (g / L) represents the amount of accumulated L-tryptophan, and in the recombinant bacteria, the 421st amino acid of TrpCF mutates from glycine to L-serine, specifically, the codon mutates from GGC to AGC, tryptophan decreases from 0.42g / L to 0.016g / L, while glutamate increases from 34.3g / L to 35.5g / L, and the conversion rate increases by 1.1%.

[0084] The results in Table 3 show that appropriately weakening the tryptophan synthesis pathway can effectively reduce the production of miscellaneous acids in glutamate-producing strains and improve the conversion rate of glutamate.

[0085] Example 4 Recombinant strain MHZ-0112-8-trpCF G421K Construction of (MHZ-0112-21)

[0086] In view of the fact that the 421st amino acid of the bifunctional indole-3-phosphoglycerol synthase / phosphoribosyl anthranilate isomerase is mutated from glycine (G) to L-serine (S), the L-glutamate production is improved. This example adopts the method of Example 2 to mutate the 421st amino acid of the bifunctional indole-3-phosphoglycerol synthase / phosphoribosyl anthranilate isomerase from glycine (G) to L-lysine (K). The strain construction method refers to Example 2, and the fermentation results of the mutant strain are shown in Table 4:

[0087] Table 4 Detection of glutamate and miscellaneous acid contents in mutant strains

[0088] strain <![CDATA[OD 600 (×100)]]> Glu(g / L) Sugar acid conversion rate % Trp(g / L) MHZ-0112-8 0.452±0.031 35.1±0.2 58.4±0.08 0.55±0.021 MHZ-0112-21 0.441±0.023 36.2±0.3 59.3±0.11 0.21±0.018

[0089] OD in Table 4 600 It is the turbidity of the culture solution diluted 200 times at 600nm and represents the cell amount, Glu (g / L) represents the amount of accumulated L-glutamic acid, Trp (g / L) represents the amount of accumulated L-tryptophan, in MHZ-0112-21, the 421st amino acid of TrpCF is mutated from glycine to L-lysine, specifically the codon is mutated from GGC to AAG, tryptophan is reduced from 0.55g / L to 0.21g / L, while glutamic acid is increased from 35.1g / L to 36.2g / L, and the conversion rate is increased by 0.9%. The above results show that appropriately weakening the tryptophan synthesis pathway can effectively reduce the production of heteroacids in glutamic acid production strains and improve the conversion rate of glutamic acid.

[0090] The fermentation results showed that the glutamate production increased after the 421st amino acid of the bifunctional indole-3-phosphoglycerate synthase / phosphoribosyl anthranilate isomerase was mutated from glycine (G) to L-lysine (K) or L-serine (S). Obviously, this mutation can be used in other host bacteria such as Corynebacterium glutamicum and Escherichia coli, and applied to the production of derivatives with glutamate as precursor, such as L-arginine, L-proline, L-histidine, L-glutamine, etc.

[0091] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention. Sequence Listing <110> Hu Dan <120> A TrpCF mutant and its application <130> KHP211117885.4 <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 474 <212> PRT <213> Artificial Sequence <400> 1 Met Thr Ser Asn Asn Leu Pro Thr Val Leu Glu Ser Ile Val Glu Gly 1 5 10 15 Arg Arg Gly His Leu Glu Glu Ile Arg Ala Arg Ile Ala His Val Asp 20 25 30 Val Asp Ala Leu Pro Lys Ser Thr Arg Ser Leu Phe Asp Ser Leu Asn 35 40 45 Gln Gly Arg Gly Gly Ala Arg Phe Ile Met Glu Cys Lys Ser Ala Ser 50 55 60 Pro Ser Leu Gly Met Ile Arg Glu His Tyr Gln Pro Gly Glu Ile Ala 65 70 75 80 Arg Val Tyr Ser Arg Tyr Ala Ser Gly Ile Ser Val Leu Cys Glu Pro 85 90 95 Asp Arg Phe Gly Gly Asp Tyr Asp His Leu Ala Thr Val Ala Ala Thr 100 105 110 Ser His Leu Pro Val Leu Cys Lys Asp Phe Ile Ile Asp Pro Val Gln 115 120 125 Val His Ala Ala Arg Tyr Phe Gly Ala Asp Ala Ile Leu Leu Met Leu 130 135 140 Ser Val Leu Asp Asp Glu Glu Tyr Ala Ala Leu Ala Ala Glu Ala Ala 145 150 155 160 Arg Phe Asp Leu Asp Ile Leu Thr Glu Val Ile Asp Glu Glu Glu Val 165 170 175 Ala Arg Ala Ile Lys Leu Gly Ala Lys Ile Phe Gly Val Asn His Arg 180 185 190 Asn Leu His Asp Leu Ser Ile Asp Leu Asp Arg Ser Arg Arg Leu Ser 195 200 205 Lys Leu Ile Pro Ala Asp Ala Val Leu Val Ser Glu Ser Gly Val Arg 210 215 220 Asp Thr Glu Thr Val Arg Gln Leu Gly Gly His Ser Asn Ala Phe Leu 225 230 235 240 Val Gly Ser Gln Leu Thr Ser Gln Glu Asn Val Asp Leu Ala Ala Arg 245 250 255 Glu Leu Val Tyr Gly Pro Asn Lys Val Cys Gly Leu Thr Ser Pro Ser 260 265 270 Ala Ala Gln Thr Ala Arg Ala Ala Gly Ala Val Tyr Gly Gly Leu Ile 275 280 285 Phe Glu Glu Ala Ser Pro Arg Asn Val Ser Arg Glu Thr Ser Gln Lys 290 295 300 Ile Ile Ala Ala Glu Pro Asn Leu Arg Tyr Val Ala Val Ser Arg Arg 305 310 315 320 Thr Ser Gly Tyr Lys Asp Leu Leu Val Asp Gly Ile Phe Ala Val Gln 325 330 335 Ile His Ala Pro Leu Gln Gly Ser Val Glu Ala Glu Lys Ala Leu Ile 340 345 350 Ala Ala Val Arg Glu Glu Val Gly Pro Gln Val Gln Val Trp Arg Ala 355 360 365 Ile Ser Met Ser Ser Pro Leu Gly Ala Glu Val Ala Ala Ala Val Glu 370 375 380 Gly Asp Val Asp Lys Leu Ile Leu Asp Ala His Glu Gly Gly Ser Gly 385 390 395 400 Glu Val Phe Asp Trp Ala Thr Val Pro Ala Ala Val Lys Ala Lys Ser 405 410 415 Leu Leu Ala Gly Gly Ile Ser Pro Asp Asn Ala Ala Gln Ala Leu Ala 420 425 430 Val Gly Cys Ala Gly Leu Asp Ile Asn Ser Gly Val Glu Tyr Pro Ala 435 440 445 Gly Ala Gly Thr Trp Ala Gly Ala Lys Asp Ala Gly Ala Leu Leu Lys 450 455 460 Ile Phe Ala Thr Ile Ser Thr Phe His Tyr 465 470 <210> 2 <211> 474 <212> PRT <213> Artificial Sequence <400> 2 Met Thr Ser Asn Asn Leu Pro Thr Val Leu Glu Ser Ile Val Glu Gly 1 5 10 15 Arg Arg Gly His Leu Glu Glu Ile Arg Ala Arg Ile Ala His Val Asp 20 25 30 Val Asp Ala Leu Pro Lys Ser Thr Arg Ser Leu Phe Asp Ser Leu Asn 35 40 45 Gln Gly Arg Gly Gly Ala Arg Phe Ile Met Glu Cys Lys Ser Ala Ser 50 55 60 Pro Ser Leu Gly Met Ile Arg Glu His Tyr Gln Pro Gly Glu Ile Ala 65 70 75 80 Arg Val Tyr Ser Arg Tyr Ala Ser Gly Ile Ser Val Leu Cys Glu Pro 85 90 95 Asp Arg Phe Gly Gly Asp Tyr Asp His Leu Ala Thr Val Ala Ala Thr 100 105 110 Ser His Leu Pro Val Leu Cys Lys Asp Phe Ile Ile Asp Pro Val Gln 115 120 125 Val His Ala Ala Arg Tyr Phe Gly Ala Asp Ala Ile Leu Leu Met Leu 130 135 140 Ser Val Leu Asp Asp Glu Glu Tyr Ala Ala Leu Ala Ala Glu Ala Ala 145 150 155 160 Arg Phe Asp Leu Asp Ile Leu Thr Glu Val Ile Asp Glu Glu Glu Val 165 170 175 Ala Arg Ala Ile Lys Leu Gly Ala Lys Ile Phe Gly Val Asn His Arg 180 185 190 Asn Leu His Asp Leu Ser Ile Asp Leu Asp Arg Ser Arg Arg Leu Ser 195 200 205 Lys Leu Ile Pro Ala Asp Ala Val Leu Val Ser Glu Ser Gly Val Arg 210 215 220 Asp Thr Glu Thr Val Arg Gln Leu Gly Gly His Ser Asn Ala Phe Leu 225 230 235 240 Val Gly Ser Gln Leu Thr Ser Gln Glu Asn Val Asp Leu Ala Ala Arg 245 250 255 Glu Leu Val Tyr Gly Pro Asn Lys Val Cys Gly Leu Thr Ser Pro Ser 260 265 270 Ala Ala Gln Thr Ala Arg Ala Ala Gly Ala Val Tyr Gly Gly Leu Ile 275 280 285 Phe Glu Glu Ala Ser Pro Arg Asn Val Ser Arg Glu Thr Ser Gln Lys 290 295 300 Ile Ile Ala Ala Glu Pro Asn Leu Arg Tyr Val Ala Val Ser Arg Arg 305 310 315 320 Thr Ser Gly Tyr Lys Asp Leu Leu Val Asp Gly Ile Phe Ala Val Gln 325 330 335 Ile His Ala Pro Leu Gln Gly Ser Val Glu Ala Glu Lys Ala Leu Ile 340 345 350 Ala Ala Val Arg Glu Glu Val Gly Pro Gln Val Gln Val Trp Arg Ala 355 360 365 Ile Ser Met Ser Ser Pro Leu Gly Ala Glu Val Ala Ala Ala Val Glu 370 375 380 Gly Asp Val Asp Lys Leu Ile Leu Asp Ala His Glu Gly Gly Ser Gly 385 390 395 400 Glu Val Phe Asp Trp Ala Thr Val Pro Ala Ala Val Lys Ala Lys Ser 405 410 415 Leu Leu Ala Gly Ser Ile Ser Pro Asp Asn Ala Ala Gln Ala Leu Ala 420 425 430 Val Gly Cys Ala Gly Leu Asp Ile Asn Ser Gly Val Glu Tyr Pro Ala 435 440 445 Gly Ala Gly Thr Trp Ala Gly Ala Lys Asp Ala Gly Ala Leu Leu Lys 450 455 460 Ile Phe Ala Thr Ile Ser Thr Phe His Tyr 465 470 <210> 3 <211> 474 <212> PRT <213> Artificial Sequence <400> 3 Met Thr Ser Asn Asn Leu Pro Thr Val Leu Glu Ser Ile Val Glu Gly 1 5 10 15 Arg Arg Gly His Leu Glu Glu Ile Arg Ala Arg Ile Ala His Val Asp 20 25 30 Val Asp Ala Leu Pro Lys Ser Thr Arg Ser Leu Phe Asp Ser Leu Asn 35 40 45 Gln Gly Arg Gly Gly Ala Arg Phe Ile Met Glu Cys Lys Ser Ala Ser 50 55 60 Pro Ser Leu Gly Met Ile Arg Glu His Tyr Gln Pro Gly Glu Ile Ala 65 70 75 80 Arg Val Tyr Ser Arg Tyr Ala Ser Gly Ile Ser Val Leu Cys Glu Pro 85 90 95 Asp Arg Phe Gly Gly Asp Tyr Asp His Leu Ala Thr Val Ala Ala Thr 100 105 110 Ser His Leu Pro Val Leu Cys Lys Asp Phe Ile Ile Asp Pro Val Gln 115 120 125 Val His Ala Ala Arg Tyr Phe Gly Ala Asp Ala Ile Leu Leu Met Leu 130 135 140 Ser Val Leu Asp Asp Glu Glu Tyr Ala Ala Leu Ala Ala Glu Ala Ala 145 150 155 160 Arg Phe Asp Leu Asp Ile Leu Thr Glu Val Ile Asp Glu Glu Glu Val 165 170 175 Ala Arg Ala Ile Lys Leu Gly Ala Lys Ile Phe Gly Val Asn His Arg 180 185 190 Asn Leu His Asp Leu Ser Ile Asp Leu Asp Arg Ser Arg Arg Leu Ser 195 200 205 Lys Leu Ile Pro Ala Asp Ala Val Leu Val Ser Glu Ser Gly Val Arg 210 215 220 Asp Thr Glu Thr Val Arg Gln Leu Gly Gly His Ser Asn Ala Phe Leu 225 230 235 240 Val Gly Ser Gln Leu Thr Ser Gln Glu Asn Val Asp Leu Ala Ala Arg 245 250 255 Glu Leu Val Tyr Gly Pro Asn Lys Val Cys Gly Leu Thr Ser Pro Ser 260 265 270 Ala Ala Gln Thr Ala Arg Ala Ala Gly Ala Val Tyr Gly Gly Leu Ile 275 280 285 Phe Glu Glu Ala Ser Pro Arg Asn Val Ser Arg Glu Thr Ser Gln Lys 290 295 300 Ile Ile Ala Ala Glu Pro Asn Leu Arg Tyr Val Ala Val Ser Arg Arg 305 310 315 320 Thr Ser Gly Tyr Lys Asp Leu Leu Val Asp Gly Ile Phe Ala Val Gln 325 330 335 Ile His Ala Pro Leu Gln Gly Ser Val Glu Ala Glu Lys Ala Leu Ile 340 345 350 Ala Ala Val Arg Glu Glu Val Gly Pro Gln Val Gln Val Trp Arg Ala 355 360 365 Ile Ser Met Ser Ser Pro Leu Gly Ala Glu Val Ala Ala Ala Val Glu 370 375 380 Gly Asp Val Asp Lys Leu Ile Leu Asp Ala His Glu Gly Gly Ser Gly 385 390 395 400 Glu Val Phe Asp Trp Ala Thr Val Pro Ala Ala Val Lys Ala Lys Ser 405 410 415 Leu Leu Ala Gly Lys Ile Ser Pro Asp Asn Ala Ala Gln Ala Leu Ala 420 425 430 Val Gly Cys Ala Gly Leu Asp Ile Asn Ser Gly Val Glu Tyr Pro Ala 435 440 445 Gly Ala Gly Thr Trp Ala Gly Ala Lys Asp Ala Gly Ala Leu Leu Lys 450 455 460 Ile Phe Ala Thr Ile Ser Thr Phe His Tyr 465 470 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <400> 4 cagtgccaag cttgattcgt gagcactacc agccgg 36 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <400> 5 ctgcgcagcg ttgtccggag agatgcttcc cgcgagcaaa gactttgcct tcacagcggc 60 <210> 6 <211> 60 <212> DNA <213> Artificial Sequence <400> 6 gccgctgtga aggcaaagtc tttgctcgcg ggaagcatct ctccggacaa cgctgcgcag 60 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <400> 7 ggtacccggg gatcctctag attgtgtgca ccgccgtgga cgagg 45 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <400> 8 aagcacgaag agatcgatta ct 22 <210> 9 <211> twenty three <212> DNA <213> Artificial Sequence <400> 9 caccgaagta tgcaggtagc agc 23 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <400> 10 gccgctgtga aggcaaagtc tttgctcgcg ggaa 34 <210> 11 <211> twenty three <212> DNA <213> Artificial Sequence <400> 11 ctcgtatgtt gtgtggaatt gtg 23 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 cgccctgagt gcttgcggca 20 <210> 13 <211> 1662 <212> DNA <213> Artificial Sequence <400> 13 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct 60 tgattcgtga gcactaccag ccgggtgaaa tcgctcgcgt gtactctcgc tacgccagcg 120 gcatttccgt gctgtgcgag ccggatcgtt ttggtggcga ttacgatcac ctcgctaccg 180 ttgccgctac ctctcatctt ccggtgctgt gcaaagactt catcattgat cctgtccagg 240 tacacgcggc gcgttacttt ggtgctgatg ccatcctgct catgctctct gtgcttgatg 300 atgaagagta cgcagcactc gctgccgagg ctgcgcgttt tgatctggat atcctcaccg 360 aggttattga tgaggaggaa gtcgcccgcg ccatcaagct gggtgcgaag atctttggcg 420 tcaaccaccg caacctgcat gatctgtcca ttgatttgga tcgttcacgt cgcctgtcca 480 agctcattcc agcagatgcc gtgctcgtgt ctgagtctgg cgtgcgcgat accgaaaccg 540 tccgccagct aggtgggcac tccaatgcat tcctcgttgg ctcccagctg accagccagg 600 aaaacgtcga tctggcagcc cgcgaattgg tctacggccc caacaaagtc tgcggactca 660 cctcaccaag tgcagcacaa accgctcgcg cagcgggtgc ggtctacggc gggctcatct 720 tcgaagaggc atcgccacgt aatgtttcac gtgaaacatc gcaaaaaatc atcgccgcag 780 agcccaacct gcgctacgtc gcggtcagcc gtcgcacctc cgggtacaag gatttgcttg 840 tcgacggcat cttcgccgta caaatccacg ccccactgca gggcagcgtc gaagcagaaa 900 aggcattgat cgccgccgtt cgtgaagagg ttggaccgca ggtccaggtc tggcgcgcga 960 tctcgatgtc cagccccttg ggggctgaag tggcagctgc ggtggagggt gacgtcgata 1020 agctaattct tgatgcccat gaaggtggca gcggggaagt attcgactgg gctacggtgc 1080 cggccgctgt gaaggcaaag tctttgctcg cgggaagcat ctctccggac aacgctgcgc 1140 aggcactcgc tgtgggctgc gcaggtttag acatcaactc tggcgtggaa taccccgccg 1200 gtgcaggcac gtgggctggg gcgaaagatg ccggcgcgct gctgaaaatt ttcgcgacca 1260 tctccacatt ccattactaa aggtttaaat aggatcatga ctgaaaaaga aaacttgggc 1320 ggctccacgc tgctacctgc atacttcggt gaattcggcg gccagttcgt cgcggaatcc 1380 ctcctgcctg ctctcgacca gctggagaag gccttcgttg acgcgaccaa cagcccagag 1440 ttccgcgaag aactcggcgg ctacctccgc gattatctcg gccgcccaac cccgctgacc 1500 gaatgctcca acctgccact cgcaggcgaa ggcaaaggct ttgcgcggat cttcctcaag 1560 cgcgaagacc tcgtccacgg cggtgcacac aatctagagg atccccgggt accgagctcg 1620 aattcgtaat catggtcata gctgtttcct gtgtgaaatt gt 1662 <210> 14 <211> 1662 <212> DNA <213> Artificial Sequence <400> 14 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct 60 tgattcgtga gcactaccag ccgggtgaaa tcgctcgcgt gtactctcgc tacgccagcg 120 gcatttccgt gctgtgcgag ccggatcgtt ttggtggcga ttacgatcac ctcgctaccg 180 ttgccgctac ctctcatctt ccggtgctgt gcaaagactt catcattgat cctgtccagg 240 tacacgcggc gcgttacttt ggtgctgatg ccatcctgct catgctctct gtgcttgatg 300 atgaagagta cgcagcactc gctgccgagg ctgcgcgttt tgatctggat atcctcaccg 360 aggttattga tgaggaggaa gtcgcccgcg ccatcaagct gggtgcgaag atctttggcg 420 tcaaccaccg caacctgcat gatctgtcca ttgatttgga tcgttcacgt cgcctgtcca 480 agctcattcc agcagatgcc gtgctcgtgt ctgagtctgg cgtgcgcgat accgaaaccg 540 tccgccagct aggtgggcac tccaatgcat tcctcgttgg ctcccagctg accagccagg 600 aaaacgtcga tctggcagcc cgcgaattgg tctacggccc caacaaagtc tgcggactca 660 cctcaccaag tgcagcacaa accgctcgcg cagcgggtgc ggtctacggc gggctcatct 720 tcgaagaggc atcgccacgt aatgtttcac gtgaaacatc gcaaaaaatc atcgccgcag 780 agcccaacct gcgctacgtc gcggtcagcc gtcgcacctc cgggtacaag gatttgcttg 840 tcgacggcat cttcgccgta caaatccacg ccccactgca gggcagcgtc gaagcagaaa 900 aggcattgat cgccgccgtt cgtgaagagg ttggaccgca ggtccaggtc tggcgcgcga 960 tctcgatgtc cagccccttg ggggctgaag tggcagctgc ggtggagggt gacgtcgata 1020 agctaattct tgatgcccat gaaggtggca gcggggaagt attcgactgg gctacggtgc 1080 cggccgctgt gaaggcaaag tctttgctcg cgggaaagat ctctccggac aacgctgcgc 1140 aggcactcgc tgtgggctgc gcaggtttag acatcaactc tggcgtggaa taccccgccg 1200 gtgcaggcac gtgggctggg gcgaaagatg ccggcgcgct gctgaaaat ttcgcgacca 1260 tctccacatt ccattactaa aggtttaaat aggatcatga ctgaaaaaga aaacttgggc 1320 ggctccacgc tgctacctgc atacttcggt gaattcggcg gccagttcgt cgcggaatcc 1380 ctcctgcctg ctctcgacca gctggagaag gccttcgttg acgcgaccaa cagcccagag 1440 ttccgcgaag aactcggcgg ctacctccgc gattatctcg gccgcccaac cccgctgacc 1500 gaatgctcca acctgccact cgcaggcgaa ggcaaaggct ttgcgcggat cttcctcaag 1560 cgcgaagacc tcgtccacgg cggtgcacac aatctagagg atccccgggt accgagctcg 1620 aattcgtaat catggtcata gctgtttcct gtgtgaaatt gt 1662

Claims

1. trpCF mutant, characterized in that The amino acid sequence of the mutant is the amino acid sequence after the 421st position of the amino acid sequence shown in SEQ ID No. 1 is mutated from glycine to L-lysine.

2. A DNA molecule encoding the trpCF mutant according to claim 1.

3. A biological material containing the DNA molecule according to claim 2, wherein the biological material is an expression cassette, a vector or a host cell.

4. Use of the trpCF mutant according to claim 1, the DNA molecule according to claim 2, the biomaterial according to claim 3, or the amino acid sequence as shown in SEQ ID No. 2 in any of the following: (1) Improving the glutamate yield, conversion rate or production intensity of recombinant Corynebacterium glutamicum; (2) Reduce tryptophan accumulation in recombinant Corynebacterium glutamicum.

5. A recombinant Corynebacterium glutamicum, characterized in that The recombinant Corynebacterium glutamicum expresses a trpCF mutant; the amino acid sequence of the trpCF mutant is shown in SEQ ID No. 2 or SEQ ID No.

3.

6. The recombinant Corynebacterium glutamicum according to claim 5, characterized in that The recombinant Corynebacterium glutamicum expresses the trpCF mutant according to claim 1 or the recombinant Corynebacterium glutamicum contains the DNA molecule according to claim 2.

7. The method for constructing a recombinant Corynebacterium glutamicum according to any one of claims 5 to 6, characterized in that: The method comprises introducing a recombinant plasmid carrying the DNA molecule of claim 2 into a starting strain.

8. Use of the recombinant Corynebacterium glutamicum according to any one of claims 5 to 6 in any of the following: (1) Glutamate production; (2) Increase glutamate production; (3) Improving the sugar-acid conversion rate in glutamate production; (4) Reduce the accumulation of tryptophan in glutamate fermentation production.

9. A method for increasing glutamic acid production, characterized in that: The recombinant Corynebacterium glutamicum according to any one of claims 5 to 6 is used for fermentation culture.

Images