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CN115851688A - TrpCF mutant and application thereof - Google Patents

TrpCF mutant and application thereof Download PDF

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CN115851688A
CN115851688A CN202111117369.7A CN202111117369A CN115851688A CN 115851688 A CN115851688 A CN 115851688A CN 202111117369 A CN202111117369 A CN 202111117369A CN 115851688 A CN115851688 A CN 115851688A
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宫卫波
赵津津
李岩
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Abstract

The invention relates to the technical field of microorganisms, and particularly relates to a TrpCF mutant and application thereof. The invention provides a trpCF mutant, wherein the amino acid sequence of the mutant contains an amino acid sequence obtained by mutating 421 st glycine of the amino acid sequence shown as SEQ ID No. 1. Specifically, the amino acid sequence of the mutant provided by the invention contains an amino acid sequence obtained by mutating glycine to L-serine or L-lysine at the 421 st position of the amino acid sequence shown in SEQ ID No. 1. The mutant provided by the invention can reasonably weaken a tryptophan synthesis way, further improve the yield and the conversion rate of glutamic acid, and simultaneously can ensure the growth performance of the strain. The recombinant microorganism expressing the mutant has higher glutamic acid yield and saccharic acid conversion rate, and the growth performance of the strain is good.

Description

TrpCF mutant and application thereof
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to a TrpCF mutant and application thereof.
Background
Glutamic acid of formula C 5 H 9 NO 4 The molecular weight is 147.13, and the amino acid is an acidic amino acid. The molecule contains two carboxyl groups, and the chemical name of the molecule is alpha-aminoglutaric acid. Glutamic acid is a basic unit constituting proteins, and is one of 21 amino acids constituting human proteins. Glutamic acid is generally produced by corynebacterium glutamicum, and monosodium glutamate is a food additive, has the taste of delicate flavor and is one of five basic tastes of human beings. Monosodium glutamate is the monosodium glutamate of the great famous tripod. The fermentation of Corynebacterium glutamicum can be used to increase the amount of glutamic acid which is exported by opening mechanosensitive channels in the cell membrane by adding or restricting substances, including biotin restriction, tween 40 or penicillin. However, with the progress of research, the way in which substances are added or limited to control the opening of mechanically sensitive channels has been eliminated. Instead, the mechanically sensitive channel switch is controlled by temperature. Recent studies have shown that the opening of the mechanosensitive channel MscG mainly promotes the efflux of glutamate, but not of other amino acids.
At present, the most common production method of L-glutamic acid is a fermentation method, and corynebacterium is mainly used as a production bacterium for producing the L-glutamic acid by fermentation. But the problem of the fermentation of the hybrid amino acid of the prior L-glutamic acid strain is outstanding, can not be well solved, influences the fermentation performance of the L-glutamic acid and is very unfavorable for producing the high-quality L-glutamic acid.
Disclosure of Invention
The invention aims to improve the yield of glutamic acid produced by a recombinant microorganism, and provides the following technical scheme for achieving the aim of the invention.
In a first aspect, the present invention provides a trpCF mutant, the amino acid sequence of which comprises an amino acid sequence obtained by mutating the 421 st glycine of the amino acid sequence shown in SEQ ID No. 1.
The trpCF mutant provided by the invention is a bifunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase mutant. In the research and development process, the invention discovers that the tryptophan synthesis is weakened, the metabolic flux capability of a central metabolic pathway can be enhanced, more sufficient carbon flow is provided for the synthesis of the metabolic products such as glutamic acid and the like through TCA cycle, and the yield of the metabolic products is further improved, however, the excessive weakening of the tryptophan synthesis pathway can cause obvious adverse effects on the growth and metabolism of the strain, and can reduce the growth of the strain and the production performance of target products. Therefore, the production performance of the strain for the target product can be remarkably improved by weakening the tryptophan pathway to a certain extent and promoting the balance between the growth of the strain and the production of the target product. The invention discovers two isomerase mutants which can reduce the activity of isomerase to a certain extent, can reasonably weaken the metabolic flux of a tryptophan pathway, can reduce the accumulation of heteroacid such as tryptophan and the like while ensuring the growth performance of a bacterial strain, and can obviously improve the yield and the conversion rate of metabolites such as glutamic acid and the like through TCA cycle.
Specifically, the amino acid sequence of the trpCF mutant contains an amino acid sequence obtained by mutating glycine to L-serine or L-lysine at the 421 st position of the amino acid sequence shown in SEQ ID No. 1;
preferably, the trpCF mutant has an amino acid sequence as shown in SEQ ID No.2 or SEQ ID No. 3.
It will be understood by those skilled in the art that the tag protein may be added to the amino acid sequence of the trpCF mutant at the N-terminus or C-terminus or fused with other proteins to form a fusion protein, and the tag-added protein or fusion protein is also within the scope of the present invention without altering the activity of the mutant protein itself.
In a second aspect, the invention provides a DNA molecule encoding a trpCF mutant as described above. The DNA molecule has a nucleotide sequence shown as SEQ ID NO.13 or SEQ ID NO. 14. Specifically, the nucleotide sequence shown in SEQ ID NO.13 or SEQ ID NO.14 contains, in addition to the nucleic acid encoding the trpCF mutant described above, a nucleic acid comprising an upstream homology arm and an outside identifying primer portion.
In a third aspect, the invention claims a biological material comprising the above-described DNA molecule, said biological material being 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 present invention also claims the use of the trpCF mutant as described above or the DNA molecule as described above or the biological material as described above in any of the following:
(1) Improving the yield, the conversion rate or the production intensity of the glutamic acid of the recombinant microorganism;
(2) Reducing tryptophan accumulation in the recombinant microorganism.
In a fourth aspect, the present invention provides a recombinant microorganism having a reduced expression level and/or activity of bifunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase as compared to the starting strain, based on the trpCF mutant or the DNA molecule;
preferably, the recombinant microorganism expresses the trpCF mutant or the recombinant microorganism contains the DNA molecule.
Specifically, in one embodiment of the present invention, the recombinant microorganism expresses the isomerase mutant without expressing the isomerase possessed by the original strain.
In another embodiment, the present invention provides a recombinant microorganism expressing the trpCF isomerase mutant without expressing the trpCF isomerase contained in the original strain.
Preferably, the gene encoding isomerase in the recombinant microorganism is mutated into a gene encoding the trpCF mutant described above.
The recombinant microorganism provided by the invention is a corynebacterium bacterium or a brevibacterium bacterium;
preferably, the coryneform bacterium is Corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium thermogenes (Corynebacterium efficiens), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes) or Corynebacterium ammoniagenes (Corynebacterium aminogenes);
the bacterium belonging to the genus Brevibacterium is Brevibacterium flavum or 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.
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, microbiological research institute of Chinese academy of sciences, zip code 100101) at 25.12.2015.12.25.M.in China, 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.
In a fifth aspect, the present invention provides a method for constructing the above recombinant microorganism, comprising inserting one or more bases into a gene encoding bifunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase present in the starting strain;
or replacing the transcriptional and/or translational regulatory element of the gene encoding bifunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase in the strain with a regulatory element of lower activity;
preferably, a recombinant plasmid carrying the above DNA molecule is introduced into the starting strain. The recombinant plasmid is a plasmid which can carry out homologous recombination in thalli, and the coding gene of the protein mutant on the plasmid is exchanged with the homologous gene on a chromosome.
Further, the construction method comprises the following steps:
(1) Constructing a recombinant plasmid containing a gene encoding the isomerase mutant;
(2) Transforming the recombinant plasmid into an original strain, and screening a transformant by using a selection culture medium containing antibiotics;
(3) And (4) screening the positive transformants for a recombinant microorganism with the target mutation.
The invention also claims the use of the above recombinant microorganism in any one of the following, as understood by the person skilled in the art:
(1) Producing glutamic acid;
(2) The glutamic acid yield is improved;
(3) The saccharic acid conversion rate in the production of glutamic acid is improved;
(4) Reducing the accumulation of the heteropolyacid in the fermentation production of the glutamic acid.
In a sixth aspect, the present invention provides a method for increasing the production of glutamic acid, which comprises carrying out fermentation culture using the above-described recombinant microorganism.
In fermentation culture, the seed culture medium comprises the following components: corn steep liquor 5%, glucose 2%, ammonium sulfate 2%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.2%, urea 0.1%, caCO 3 0.5％，pH 7.0。
The fermentation medium comprises the following components: 2% of corn steep liquor, 12.0% of glucose, 5% of ammonium sulfate, 0.05% of magnesium sulfate, 0.2% of monopotassium phosphate and CaCO 3 4％，pH 7.0。
Specifically, the method for producing glutamic acid comprises: inoculating the recombinant microorganism to a slant culture medium for slant culture, selecting the 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 used 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 culture usedThe base is as follows: corn steep liquor 5%, glucose 2%, ammonium sulfate 2%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.2%, urea 0.1%, caCO 3 0.5％，pH 7.0。
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 used is: 2% of corn steep liquor, 12.0% of glucose, 5% of ammonium sulfate, 0.05% of magnesium sulfate, 0.2% of monopotassium phosphate and CaCO 3 4％，pH 7.0。
The fermentation is carried out at 30-32 ℃ for 12-20h by shaking culture.
The method for producing glutamic acid provided by the invention reduces the accumulation amount of the heteroacid in the fermentation production of the glutamic acid and improves the yield of the glutamic acid. Therefore, the invention equivalently provides a method for reducing the accumulation of the heteropolyacid in the fermentation production of the glutamic acid.
The invention has the beneficial effects that:
the isomerase mutant provided by the invention has reduced enzyme activity, can reasonably weaken metabolic flux of shikimic acid synthetic pathway, reduce accumulation of tryptophan, and enhance metabolic flux capability of central metabolic pathway, thereby improving the yield and conversion rate of glutamic acid, and simultaneously ensuring growth performance of strains. The invention also provides a recombinant microorganism for expressing the isomerase mutant, wherein the glutamic acid fermentation production performance of the recombinant microorganism is obviously improved, and the recombinant microorganism has higher glutamic acid yield and sugar acid conversion rate.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
Unless otherwise specified, the experimental materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art, unless otherwise specified. The names and sequences of the primers used in the examples of the present invention are shown in Table 1.
TABLE 1 primer sequences
Figure BDA0003275961130000061
Figure BDA0003275961130000071
The starting strain MHZ-0112-8 is Corynebacterium glutamicum, and the pure culture of Corynebacterium glutamicum (Corynebacterium glutamicum) MHZ-0112-8 has been preserved in the China general microbiological culture Collection center (CGMCC for short, the address: no.3 of Xilu No.1 of Beijing city facing Yang district, institute of microbiology, china academy of sciences, zip code 100101) in 2015, 12 months and 25 days, 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.
Example 1
The MHZ-0112-8 strain is firstly mutagenized by acridine yellow, thalli in a logarithmic phase are centrifuged, washed twice by phosphate buffer (0.1 mol/L and pH 7.0), the thalli are suspended in 0.1mol/L phosphate buffer (pH 7.0) containing acridine yellow with a certain concentration to enable the final concentration to be 0.2mg/mL, the mutagenized bacteria liquid is obtained after shaking treatment for 4 hours on a shaking table at 37 ℃, diluted and coated on a bromocresol purple plate, cultured for 36 hours at 31.5 ℃ to obtain 200 strains with large discoloring circles, and the strains are sieved again by a TLC method and screened by combining shaking bottle verification and HPLC to obtain high-yield glutamic acid strains MHZ-0112-Z0 and MHZ-0112-Z1.
Activating the obtained MHZ-0112-Z0 and MHZ-0112-Z1 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 on glutamic acid and heteropolyacid in MHZ-0112-Z0 fermentation liquor shows that the yield of the glutamic acid is increased to 35.9g/L from 34.2g/L, and except that the yield of the glutamic acid is obviously increased, the conversion rate is increased by 1.1%. The content of tryptophan is reduced to 0.036g/L, and liquid chromatography detection on glutamic acid and heteropolyacid in MHZ-0112-Z1 fermentation liquor shows that the yield of glutamic acid is increased to 35.6g/L from 34.2g/L, and except that the yield of glutamic acid is obviously increased, the conversion rate is increased by 1.0%. The tryptophan content was reduced to 0.038g/L (Table 2).
TABLE 2 production of glutamic acid and tryptophan by mutagenized strains
Bacterial strains OD 600 (×100) Glu(g/L) Percent conversion% 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
The mutation strains MHZ-0112-Z0, MHZ-0112-Z1 and the starting strain MHZ-0112-8 are analyzed by utilizing comparative genomics to find the tryptophan synthetic pathway genes: the difunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase gene is mutated, and the 421 st amino acid of the difunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase gene is mutated. The above mutation results in a decrease in the activity of isomerase. The mutation causes the accumulation of tryptophan to be reduced in cells, thereby enhancing the metabolic flux capability of a central metabolic pathway, providing sufficient carbon flux for the synthesis of glutamic acid and further improving the yield of the glutamic acid.
Example 2 plasmid pK18-trpCF G421S Construction of and recombination of the Strain MHZ-0112-8-trpCF G421S Construction of
This example provides a recombinant strain MHZ-0112-8-trpCF G421S The method comprises the following specific steps:
the recombinant fragment UP was prepared using Phusion ultra-fidelity polymerase (New England BioLabs), the genome of Corynebacterium glutamicum MHZ-0112-8 as a template, A1-trpCF-U/A2-trpCF-U-R as a primer, the recombinant fragment DN was prepared using A3-trpCF-D-F/A4-trpCF-D-R as a primer, the resulting fragment was purified using an agarose gel recovery kit (Tiangen), the recombinant fragment was subsequently prepared using UP and DN as templates, A1-trpCF-U/A4-trpCF-D-R as a primer, the resulting recombinant fragment was purified using an agarose gel recovery kit (Tiangen), then digested using HindIII/XbaI, pK18-mobsacB was simultaneously digested using HindIII/XbaI, the fragment was purified using T4DNA ligase (Trans Biotech), the fragment was ligated to Trans 1-sensitive cell vector (HindIII/XbaI), and the fragment was further cloned using HindIII/XbaI as a resistance-sensitive cell, and identified by cloning using a DNA ligase such as a DNA ligase, and DNA fragment DNA polymerase (DNA) was cloned into a DNA fragment 82. A1-DNA fragmentP85 primer sequencing (Invitrogen) identified the correct inserted fragment. The resulting plasmid was designated as pK18-trpCF G421S . The plasmid pK18-trpCF was prepared G421S Transfer into Corynebacterium glutamicum MHZ-0112-8, and selection of the replacement recombinants was carried out on selection medium containing 15mg/L kanamycin. The temperature of the culture was 33 ℃ and the culture was inverted. And (3) culturing the screened transformant in a common liquid brain heart infusion culture medium overnight at the culture temperature of 33 ℃ under the shaking culture of a rotary shaking table at 220 rpm. During this culture, the transformants undergo a second recombination and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted in gradient (10) -2 Continuously diluting to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10% of sucrose, and is subjected to static culture at 33 ℃ for 48 hours. Further carrying out phenotype verification on the screened strains, and selecting Kan S The recombination is verified by A7-trpCF-T-F/A4-trpCF-D-R, the recombination containing point mutation is obtained by searching annealing temperature, the obtained positive recombination is amplified and sequenced by A5-trpCF-T-F/A6-trpCF-T-R, and after verification and screening are finished, the target mutant strain is named as MHZ-0112-20.
Example 3MHZ-0112-8-trpCF G421S (MHZ-0112-20) L-glutamic acid-producing ability of mutant Strain
This example identifies the ability of the mutant strain obtained in example 2 to produce L-glutamic acid.
The seed activation medium used in this example was: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH 7.0.
The seed culture medium used was: corn steep liquor 5%, glucose 2%, ammonium sulfate 2%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.2%, urea 0.1%, caCO 3 0.5％，pH 7.0。
The fermentation medium used was: 2 percent of corn steep liquor, 12.0 percent of glucose, 5 percent of ammonium sulfate, 0.05 percent of magnesium sulfate, 0.2 percent of monopotassium phosphate, caCO 3 4％，pH 7.0。
(1) Seed culture: selecting MHZ-0112-8 and MHZ-0112-8-trpCF G421S The slant seed 1 is circularly inoculated to 20mL seed culture mediumIn a 500mL triangular flask, carrying out shaking culture at 33 ℃ and 220r/min for 16-22h;
(2) Fermentation culture: 2mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 20mL of the fermentation medium, and cultured at 33 ℃ for 72 hours with shaking at 220 r/min.
(3) 1mL of fermentation broth was centrifuged (12000rpm, 2min), and the supernatant was collected and subjected to HPLC to detect the L-glutamic acid content in the fermentation broths of the engineered and control bacteria, the concentrations of which are shown in Table 3.
TABLE 3 detection of glutamic acid and tryptophan content in mutant strains
Bacterial strains 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
The results in Table 3 show that the starting strain MHZ-0112-8 has an accumulated amount of L-glutamic acid of 34.3g/L, while the engineering bacteria MHZ-0112-8-trpCF of the invention G421S The L-glutamic acid production amount of (MHZ-0112-20) was 35.5g/L, and it was thus found that trpCF G421S The mutation is favorable for the accumulation of L-glutamic acid.
OD in Table 3 600 The culture medium diluted 100 times is at a turbidity of 600nm and represents cell mass, glu (g/L) represents the amount of accumulated L-glutamic acid, trp (g/L) represents the amount of accumulated L-tryptophan, amino acid 421 of TrpCF in the recombinant bacteria is mutated from glycine to L-serine, specifically from GGC to AGC, tryptophan is reduced from 0.42g/L to 0.016g/L, glutamic acid is increased from 34.3g/L to 35.5g/L, and the conversion rate is increased by 1.1%.
The results in Table 3 show that properly weakening the tryptophan synthesis pathway can effectively reduce the generation of the heteropolyacid in the glutamic acid production strains and improve the conversion rate of the glutamic acid.
Example 4 recombinant Strain MHZ-0112-8-trpCF G421K Construction of (MHZ-0112-21)
Because the 421 st amino acid of the bifunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase is mutated from glycine (G) to L-serine (S), the yield of L-glutamic acid is improved. In this example, amino acid 421 of bifunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase was mutated from glycine (G) to L-lysine (K) using the method of example 2. Methods of Strain construction referring to example 2, fermentation results of mutant strains are shown in Table 4:
TABLE 4 detection of glutamic acid and heteropolyacid content in mutant strains
Bacterial strains 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
OD in Table 4 600 The medium was diluted 200 times at 600nm in turbidity and cell size, glu (g/L) represents the amount of accumulated L-glutamic acid, trp (g/L) represents the amount of accumulated L-tryptophan, amino acid 421 of TrpCF was mutated from glycine to L-lysine in MHZ-0112-21, specifically, the codon was mutated from GGC to AAG, tryptophan was reduced from 0.55g/L to 0.21g/L, while glutamic acid was increased from 35.1g/L to 36.2g/L, and the conversion rate was increased by 0.9%. The results show that the proper attenuation of the tryptophan synthesis way can effectively reduce the generation of the heteropolyacid in the glutamic acid production strain and improve the conversion rate of the glutamic acid.
Fermentation results show that after the 421 st amino acid of the bifunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase is mutated from glycine (G) to L-lysine (K) or L-serine (S), the yield of glutamic acid is improved. It is obvious that the mutation can be applied to other host bacteria such as Corynebacterium glutamicum and Escherichia coli, and can be applied to the production of derivatives using glutamic acid as a precursor, such as L-arginine, L-proline, L-histidine, and L-glutamine.
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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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 (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 (Artificial Sequence)
<400> 4
cagtgccaag cttgattcgt gagcactacc agccgg 36
<210> 5
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctgcgcagcg ttgtccggag agatgcttcc cgcgagcaaa gactttgcct tcacagcggc 60
<210> 6
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gccgctgtga aggcaaagtc tttgctcgcg ggaagcatct ctccggacaa cgctgcgcag 60
<210> 7
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggtacccggg gatcctctag attgtgtgca ccgccgtgga cgagg 45
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aagcacgaag agatcgatta ct 22
<210> 9
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caccgaagta tgcaggtagc agc 23
<210> 10
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gccgctgtga aggcaaagtc tttgctcgcg ggaa 34
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ctcgtatgtt gtgtggaatt gtg 23
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cgccctgagt gcttgcggca 20
<210> 13
<211> 1662
<212> DNA
<213> Artificial Sequence (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 (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 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

Claims (10)

  1. A trpCF mutant, characterized in that the amino acid sequence of the mutant contains an amino acid sequence obtained by mutating glycine to L-lysine at position 421 of the amino acid sequence shown in SEQ ID No. 1.
  2. 2. The trpCF mutant according to claim 1, having an amino acid sequence as set forth in SEQ ID No. 3.
  3. 3. A DNA molecule encoding the trpCF mutant of any one of claims 1-2.
  4. 4. A biological material comprising the DNA molecule of claim 3, wherein said biological material is an expression cassette, a vector or a host cell.
  5. 5. Use of a trpCF mutant according to any one of claims 1 to 2, a DNA molecule according to claim 3 or a biological material according to claim 4 or an amino acid sequence as shown in SEQ ID No.2 in any one of the following:
    (1) Improving the yield, the conversion rate or the production intensity of the recombinant microorganism glutamic acid;
    (2) Reducing tryptophan accumulation in the recombinant microorganism.
  6. 6. A recombinant microorganism characterized in that it has a reduced expression level and/or activity of bifunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase as compared to the starting strain;
    preferably, the recombinant microorganism expresses the trpCF mutant according to any one of claims 1 to 2 or the recombinant microorganism expresses the amino acid sequence shown in SEQ ID No.3 or the recombinant microorganism contains the DNA molecule according to claim 3.
  7. 7. The recombinant microorganism according to claim 6, wherein the recombinant microorganism is a bacterium of the genus Corynebacterium or Brevibacterium;
    preferably, the corynebacterium genus bacterium is corynebacterium glutamicum, corynebacterium valium, corynebacterium crenatum, corynebacterium ammoniagenes thermophilum, or corynebacterium ammoniagenes.
  8. 8. The method for constructing a recombinant microorganism according to any one of claims 6 to 7, comprising inserting one or more bases into a gene encoding bifunctional indole-3-phosphoglycerate synthase/phosphoribosyl anthranilate isomerase present in the starting strain;
    or replacing the transcription and/or translation regulatory element of the coding gene of the bifunctional indole-3-phosphoglycerol synthase/phosphoribosyl anthranilate isomerase with a regulatory element with lower activity;
    preferably, a recombinant plasmid carrying the DNA molecule of claim 3 is introduced into the starting strain.
  9. 9. Use of a recombinant microorganism according to any one of claims 6 to 7 in any one of:
    (1) Producing glutamic acid;
    (2) The glutamic acid yield is improved;
    (3) The saccharic acid conversion rate in the production of glutamic acid is improved;
    (4) Reducing the accumulation of the heteropolyacid in the fermentation production of the glutamic acid.
  10. 10. A method for increasing the production of glutamic acid, which comprises carrying out fermentation culture using the recombinant microorganism according to any one of claims 6 to 7.
CN202111117369.7A 2021-09-23 2021-09-23 TrpCF mutant and application thereof Pending CN115851688A (en)

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US20190085363A1 (en) * 2011-06-22 2019-03-21 Genomatica, Inc. Microorganisms for producing 1,3-butanediol and methods related thereto
CN105695383A (en) * 2016-03-02 2016-06-22 廊坊梅花生物技术开发有限公司 Recombinant strain and application thereof
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CN116640709A (en) * 2022-02-16 2023-08-25 中国科学院天津工业生物技术研究所 Recombinant microorganism for expressing target polypeptide as well as preparation method and application thereof

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