Anthranilic acid synthase subunit mutant as well as coding gene, vector, recombinant bacterium and application of anthranilic acid synthase subunit mutant

Abstract

The invention discloses an anthranilic acid synthase subunit mutant as well as a coding gene, a vector, recombinant bacteria and application thereof, and belongs to the technical field of enzyme engineering. According to the invention, the o-aminobenzoic acid synthase subunit from Escherichia coli K12MG1655 is mutated, and methionine (Met) at the 293 site is mutated into threonine (Thr), so that the feedback inhibition of L-tryptophan on the o-aminobenzoic acid synthase subunit can be effectively relieved, and the catalytic activity of o-aminobenzoic acid is improved, thereby promoting the synthesis of L-tryptophan. According to the present invention, the 293rd site of the anthranilic acid synthase subunit from Escherichia coli K12MG1655 is subjected to saturation mutation, such that the mutant capable of effectively relieving the synergistic feedback inhibition effect of L-tryptophan on the anthranilic acid synthase subunit is obtained, and the new idea is provided for the construction of the high-yield L-tryptophan recombinant strain.

Description

Technical Field

[0001] The present invention relates to an anthranilate synthase subunit mutant and its encoding gene, vector, recombinant bacteria and application, belonging to the technical field of enzyme engineering. Background Art

[0002] In Escherichia coli, anthranilate synthase, encoded by the trpE and trpD genes, is a key enzyme in the synthesis of the essential amino acid tryptophan. This enzyme catalyzes the formation of anthranilate, an intermediate metabolite in the tryptophan biosynthesis pathway. trpE and trpD encode two subunits of the enzyme, located downstream of the tryptophan operon and tightly regulated by intracellular tryptophan concentration. In addition to regulation by the tryptophan operon, the activity of the subunit TrpE encoded by the trpE gene itself is directly regulated by tryptophan. When intracellular tryptophan concentration is low, TrpE maintains normal catalytic activity, working with TrpD to catalyze the conversion of chorismate to anthranilate, thereby shifting metabolic flux toward tryptophan synthesis. However, when tryptophan concentration is excessive, TrpE activity is inhibited by tryptophan binding to its allosteric site, preventing the accumulation of excessive tryptophan, which can lead to metabolic burden and cytotoxicity. This feedback inhibition mechanism is important for regulating tryptophan metabolism in nature, but it can become a limiting factor in the industrial production of L-tryptophan. Industrially, it is desirable to develop E. coli that can overcome this inhibition and continuously accumulate tryptophan. Therefore, identifying mutants that are not susceptible to feedback inhibition is of great significance and could improve the efficiency and stability of L-tryptophan production. These mutants could be achieved by modifying the structure or functional sites of TrpE, thereby enabling it to maintain catalytic activity even at high tryptophan concentrations. Such engineering modifications would help optimize industrial production processes and increase the efficiency of L-tryptophan production, meeting the needs and goals of modern industrial production. Summary of the Invention

[0003] To address the above technical problems, the present invention first mutated the anthranilate synthase subunit from Escherichia coli K12MG1655, converting methionine (Met, M) at position 293 to threonine (Thr, T). This mutated subunit effectively alleviates the feedback inhibition of L-tryptophan on the anthranilate synthase subunit, enhancing the catalytic activity of anthranilate, thereby promoting the synthesis of L-tryptophan. The results showed that the mutant enzyme, TrpEM293T, effectively alleviates the synergistic feedback inhibition of L-tryptophan on TrpE, promoting the synthesis of L-tryptophan.

[0004] The first object of the present invention is to provide an anthranilate synthase subunit mutant, wherein the anthranilate synthase subunit mutant is a mutant in which the methionine at position 293 of the parent amino acid sequence shown in SEQ ID NO.1 is mutated to threonine.

[0005] TrpE (SEQ ID NO. 1):

[0006] MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLESADIDSKDDLKSLLLVDSALRITALGDTVTIQALSGNGEALLALLDNALPAGVESEQSPNCRVLRFPPVSPLLDEDARLCSLSVFDAFRLL QNLLNVPKEEREAMFFGGLFSYDLVAGFEDLPQLSAENNCPDFCFYLAETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNELRQQLTEAAPPLPVVSVPHMRCECNQSDEEFGGVVRLLQKAIRAGE IFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDNDFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGSLDRDLDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVADLTKV DRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGTLSGAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIVIRSALVENGIATVQAGAGVVLDSVPQSEADETRNKARAVLRAIATAHHAQETF

[0007] The nucleotide sequence encoding the wild-type parent anthranilate synthase subunit is shown in SEQ ID NO.3.

[0008]

[0009] The amino acid sequence of mutant M293T is as follows (SEQ ID NO.8):

[0010] MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLESADIDSKDDLKSLLLVDSALRITALGDTVTIQALSGNGEALLALLDNALPAGVESEQSPNCRVLRFPPVSPLLDEDARLCSLSVFDAFRLL QNLLNVPKEEREAMFFGGLFSYDLVAGFEDLPQLSAENNCPDFCFYLAETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNELRQQLTEAAPPLPVVSVPHMRCECNQSDEEFGGVVRLLQKAIRAGE IFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPYTFFMQDNDFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGSLDRDLDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVADLTKV DRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGTLSGAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIVIRSALVENGIATVQAGAGVVLDSVPQSEADETRNKARAVLRAIATAHHAQETF

[0011] The second object of the present invention is to provide a gene encoding the anthranilate synthase subunit mutant.

[0012] The third object of the present invention is to provide an expression vector carrying the encoding gene.

[0013] Furthermore, the vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

[0014] The fourth object of the present invention is to provide a recombinant bacterium that expresses the anthranilate synthase subunit mutant.

[0015] Furthermore, the recombinant bacteria use bacteria, fungi, plants, insects or animal cells as host cells.

[0016] The fifth object of the present invention is to provide the use of the anthranilate synthase subunit mutant in constructing a high-yield L-tryptophan recombinant strain.

[0017] The sixth object of the present invention is to provide an enzyme preparation comprising the anthranilate synthase subunit mutant.

[0018] Furthermore, the enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

[0019] The seventh object of the present invention is to provide a recombinant enzyme catalyst containing the anthranilate synthase subunit mutant, wherein the recombinant enzyme catalyst is any one of the following forms:

[0020] (1) culturing recombinant expression transformants and isolating transformant cells containing the recombinase;

[0021] (2) culturing the recombinant expression transformant, isolating the transformant cells containing the recombinase, and disrupting the transformant cells containing the recombinase to obtain a cell disrupted liquid;

[0022] (3) Cultivating the recombinant expression transformant, isolating the transformant cells containing the recombinant enzyme, disrupting the transformant cells containing the recombinant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant enzyme to obtain a freeze-dried enzyme powder.

[0023] The eighth object of the present invention is to provide a method for improving the catalytic activity and / or enzymatic activity and / or tolerance to the product L-tryptophan of an anthranilate synthase subunit, the method comprising mutating the methionine at position 293 of the anthranilate synthase subunit whose amino acid sequence is shown in SEQ ID NO.1 to threonine.

[0024] The ninth object of the present invention is to provide an anthranilate synthase subunit mutant for use in increasing the yield of L-tryptophan produced by Escherichia coli or in constructing a high-yield L-tryptophan recombinant strain.

[0025] The tenth object of the present invention is to provide a recombinant Escherichia coli, wherein the recombinant Escherichia coli expresses the anthranilate synthase subunit mutant according to claim 1;

[0026] In one embodiment of the present invention, the chassis cells of the recombinant Escherichia coli are: trpR, trpL, tyrR, tnaB genes on the genome of Escherichiacoli K12MG1655 are knocked out, and serA is overexpressed at the sdaB site on the genome. fbr Gene, overexpression of BsglnA at the rpnd locus fbr Gene, overexpression of yddG gene at the mtr site, overexpression of aroG at the yeeP site fbr Gene.

[0027] In one embodiment of the present invention, the trpR gene, trpL gene, tyrR gene, and tnaB gene have NCBI Gene ID numbers: 948917, 945856, 945879, and 948220.

[0028] In one embodiment of the present invention, the sdaB site, rpnd site, mtr site, and yeeP site are numbered on NCBI: 947262, 38094982, 947675, and 946524.

[0029] In one embodiment of the present invention, the Gene ID of the yddG gene is 945942; the serA fbr gene, BsglnA fbr gene, aroG fbr The gene sequences are as follows:

[0030] serA fbr (T372D) (SEQ ID NO. 5):

[0031]

[0032] BsglnA fbr (L159I,E304A)(SEQ ID NO.6):

[0033]

[0034] aroG fbr (S180F)(SEQ ID NO.7):

[0035]

[0036] The present invention also provides a method for preparing L-tryptophan, wherein the method comprises the following steps: using the above-mentioned recombinant Escherichia coli to ferment and prepare L-tryptophan.

[0037] In one embodiment of the present invention, the method comprises inoculating a recombinant Escherichia coli seed solution into a fermentation medium at a 10% inoculation rate, and fermenting at 36° C. to prepare L-tryptophan;

[0038] In one embodiment of the present invention, the fermentation medium comprises: 40 g / L anhydrous glucose, 3.0 g / L yeast extract, 1.6 g / L anhydrous ammonium sulfate, 2.0 g / L citric acid, 5.6 g / L anhydrous dipotassium hydrogen phosphate, 2.0 g / L anhydrous magnesium sulfate, 1 mL / L trace element solution, and 80 mg / L phenol red. The pH of the medium is adjusted to 7 using aqueous ammonia. Sterilization conditions are 115°C for 10 minutes. The formula of the trace element solution is: 7.6 g / L ferrous sulfate heptahydrate, 0.4 g / L cobalt chloride hexahydrate, 0.06 g / L copper sulfate pentahydrate, 0.64 g / L zinc sulfate, 2.0 g / L sodium sulfate, and 0.45 g / L manganese sulfate monohydrate.

[0039] The present invention also provides the use of the above-mentioned anthranilate synthase subunit mutant, or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned method or the above-mentioned recombinant Escherichia coli in L-tryptophan or products containing L-tryptophan.

[0040] Beneficial effects

[0041] The present invention mutates the anthranilate synthase subunit from Escherichia coli K12MG1655, converting methionine (Met, M) at position 293 to threonine (Thr, T). This effectively alleviates the feedback inhibition of L-tryptophan on the anthranilate synthase subunit, improves the catalytic activity of anthranilate, and thus promotes the synthesis of L-tryptophan. This provides a new approach for constructing a high-yield L-tryptophan recombinant strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The amino acid sequence alignment results of TrpE enzymes in wild-type Escherichia coli K12 and JW-5 strains are shown.

[0043] Figure 2 The graph shows tryptophan production in shake flask fermentation of strains carrying different TrpE mutants.

[0044] Figure 3 The relative enzyme activities of TrpE and TrpEM293T mutants in the presence of different concentrations of L-tryptophan.

[0045] Figure 4 The results are for a 5 L fermentation tank of the T-8 strain carrying the TrpEM293T mutant. DETAILED DESCRIPTION

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0047] In the present invention, the starting bacterium Escherichia coli K12MG1655 is a model strain.

[0048] The vector pTrcmob involved is a commercially available common expression vector for Escherichia coli.

[0049] The culture medium involved in the following examples is as follows:

[0050] Shake flask fermentation medium formula: 40 g / L anhydrous glucose, 3.0 g / L yeast extract, 1.6 g / L anhydrous ammonium sulfate, 2.0 g / L citric acid, 5.6 g / L anhydrous dipotassium hydrogen phosphate, 2.0 g / L anhydrous magnesium sulfate, 1 mL / L trace element solution, 80 mg / L phenol red. Adjust the pH of the medium to 7 with ammonia. Sterilize at 115°C for 10 min. The trace element solution formula is: 7.6 g / L ferrous sulfate heptahydrate, 0.4 g / L cobalt chloride hexahydrate, 0.06 g / L copper sulfate pentahydrate, 0.64 g / L zinc sulfate, 2.0 g / L sodium sulfate, and 0.45 g / L manganese sulfate monohydrate.

[0051] TB medium (protein expression): 24 g / L yeast extract, 12 g / L tryptone, 6.42 g / L dipotassium hydrogen phosphate, 2.31 g / L potassium dihydrogen phosphate, 4 g / L glycerol. Sterilize at 121°C for 10 min.

[0052] Seed culture medium (fermenter): yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L, potassium dihydrogen phosphate 2g / L, glucose 10g / L, casein hydrolyzate 1g / L.

[0053] Fermentation medium (fermenter): Glucose 10g / L, yeast extract 12.5g / L, potassium dihydrogen phosphate 7.5g / L, anhydrous magnesium sulfate 2.5g / L, anhydrous ammonium sulfate 5.6g / L, citric acid 2g / L, ferrous sulfate heptahydrate 0.46g / L, sodium glutamate 2g / L, L-methionine 0.5g / L, L-isoleucine 0.1g / L, complex amino acid powder 1g / L, thiamine 0.001g / L, biotin 0.005g / L, trace element solution 2mL / L. The trace element solution formula is: ammonium molybdate tetrahydrate 0.25g / L, boric acid 5g / L, cobalt chloride hexahydrate 1.4g / L, copper sulfate pentahydrate 0.5g / L, manganese chloride tetrahydrate 3.2g / L, zinc sulfate heptahydrate 0.6g / L. Adjust the prepared fermentation medium to pH 7 with ammonia. Sterilization conditions are 115°C, 10 min.

[0054] The detection methods involved in the following embodiments are as follows:

[0055] Qualitative and quantitative analysis of substrates and products and monitoring of bacterial growth:

[0056] (1) Glucose determination: A biosensor analyzer (SBA-40E, Institute of Biology, Shandong Academy of Sciences) was used.

[0057] (2) L-Tryptophan Assay: L-tryptophan in the fermentation broth was quantified by high-performance liquid chromatography using a Waters C18 column (5 μL, 4.6 × 250 mm). The mobile phase consisted of 90:10 (V (0.03% KH2PO4)):V (pure methanol) at a flow rate of 1 mL / min and a detection wavelength of 278 nm. (Agilent-1260, Agilent Technologies, Inc.)

[0058] (3) Bacterial growth determination: Take the sample bacterial solution, dilute it with distilled water to a certain multiple, use distilled water as a blank control, and use a spectrophotometer to measure the OD at a 1 cm optical path. 600 .

[0059] Enzyme activity detection method:

[0060] Reaction system: Tris-HCl buffer (100 mM), glutamine (5 mM), chorismate (1 mM), magnesium chloride (5 mM), crude enzyme solution (100 μL). Reaction temperature: 37°C, reaction time: 20 min. After completion, add 0.1 M hydrochloric acid solution to terminate the reaction. Add 4 mL of ethyl acetate to the system to extract anthranilic acid. The absorbance of anthranilic acid was measured at 336 nm, and the anthranilic acid content was calculated using an extinction coefficient of 4900. The enzyme activity unit measured is the amount of enzyme required to generate 1 μmol of anthranilic acid in 1 minute.

[0061] Example 1: Construction of expression plasmid pTrcmob-trpED

[0062] The anthranilate synthase encoding gene trpED from Escherichia coli K12MG1655 was amplified by PCR and purified to obtain the trpED fragment;

[0063] The amino acid sequence of trpED is as follows:

[0064] TrpE (SEQ ID NO. 1):

[0065] MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLESADIDSKDDLKSLLLVDSALRITALGDTVTIQALSGNGEALLALLDNALPAGVESEQSPNCRVLRFPPVSPLLDEDARLCSLSVFDAFRLL QNLLNVPKEEREAMFFGGLFSYDLVAGFEDLPQLSAENNCPDFCFYLAETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNELRQQLTEAAPPLPVVSVPHMRCECNQSDEEFGGVVRLLQKAIRAGE IFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDNDFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGSLDRDLDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVADLTKV DRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGTLSGAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIVIRSALVENGIATVQAGAGVVLDSVPQSEADETRNKARAVLRAIATAHHAQETF

[0066] TrpD (SEQ ID NO. 2):

[0067] MADILLLDNIDSFTYNLADQLRSNGHNVVIYRNHIPAQTLIERLATMSNPVLMLSPGPGVPSEAGCMPELLTRLRGKLPIIGICLGHQAIVEAYGGYVGQAGEILHGKASSIEHDGQAMFAGLTNPLPVARYHSLVGSNIPAGLTINAHFNGMVMAVRHDADRVCGFQFHPESILTTQGARLLEQTLAWAQQKLEPANTLQPILEKLYQAQTLSQQESHQLFSAVVRGELKPEQLAAALVSMKIRGEHPNEIAGAATALLENAAPFPRPDYLFADIVGTGGDGSNSINISTASAFVAAACGLKVAKHGNRSVSSKSGSSDLLAAFGINLDMNADKSRQALDELGVCFLFAPKYHTGFRHAMPVRQQLKTRTLFNVLGPLINPAHPPLALIGVYSPELVLPIAETLRVLGYQRAAVVHSGGMDEVSLHAPTIVAELHDGEIKSYQLTAEDFGLTPYHQEQLAGGTPEENRDILTRLLQGKGDAAHEAAVAANVAMLMRLHGHEDLQANAQTVLEVLRSGSAYDRVTALAARG

[0068] The nucleotide sequence of trpED is as follows:<000017ό>

[0069] trpE (SEQ ID NO.3):

[0070] It should be noted that there seems to be an incorrect character "ό" in the original text at line . If this is an error, it should be corrected for a more accurate translation.

[0071] trpD(SEQ ID NO.4):

[0072]

[0073] It was constructed between the EcoRI and BamHI restriction sites of empty pTrcmob using a one-step cloning kit from Nanjing Novozyme Biotechnology Co., Ltd. The primers used to construct the plasmid are shown in Table 1 below.

[0074] Table 1: Primers used for expression plasmid construction

[0075]

[0076] The recombinant vector pTrcmob-trpED was prepared.

[0077] Example 2: Saturation mutagenesis of amino acid 293 of trpE in vector pTrcmob-trpED

[0078] After computer simulation and sequence alignment, it was determined that amino acid 293 of trpE is an important site affecting enzyme activity; therefore, saturation mutagenesis was performed on this site. The specific steps are as follows:

[0079] (1) Construction of recombinant vectors containing different mutants

[0080] Using the expression plasmid pTrcmob-trpED as a template, PCR amplification was performed using the primers listed in Table 2, and the plasmid template was removed using DpnI digestion enzyme.

[0081] Table 2: Primers used for saturation mutagenesis

[0082]

[0083]

[0084] Note: "NNN" represents the amino acid codons TTT (L-phenylalanine), CTT (L-leucine), ATT (L-isoleucine), GTT (L-valine), TCT (L-serine), CCT (L-proline), ACT (L-threonine), TAT (L-tyrosine), CAT (L-histidine), CAA (L-glutamine), AAT (L-asparagine), AAA (L-lysine), GAT (L-aspartic acid), GAA (L-glutamic acid), TGT (L-cysteine), UGG (L-tryptophan), CGT (L-arginine), GGT (glycine), and ATG (L-methionine).

[0085] (2) The PCR product was purified and transformed into competent E. coli JM109 cells for plating culture. A single colony was picked for shake flask culture and plasmid was extracted using a plasmid rapid extraction kit from Nanjing Novozymes Biotechnology Co., Ltd.

[0086] Sequencing was performed using the sequencing primers shown in Table 2 to screen out the correct expression vector containing the mutant.

[0087] Example 3: Transformation of the mutant plasmid into L-tryptophan engineering bacteria T-8 to test its ability to produce L-tryptophan

[0088] The specific steps are as follows:

[0089] (1) L-tryptophan-producing strain T-8 (Escherichia coli K12MG1655ΔtrpR,ΔtrpL,ΔtyrR,ΔtnaB,ΔsdaB::serA) fbr ,Δrpnd::BsglnA fbr ,Δmtr::yddG,ΔyeeP::aroG fbr )

[0090] The T-8 construction method can be found in the paper Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system, specifically:

[0091] Knockout of trpR (Gene ID: 948917), trpL (Gene ID: 945856), tyrR (Gene ID: 945879), tnaB (Gene ID: 948220) on the Escherichia coli K12MG1655 genome, and overexpression of serA based on the knockout of sdaB (Gene ID: 947262) fbr (SEQ ID NO.5), overexpressed BsglnA based on knockout of rpnd (Gene ID: 38094982). fbr (SEQ ID NO.6), overexpressed yddG (Gene ID: 945942) based on the knockout of mtr (Gene ID: 947675), and overexpressed aroG based on the knockout of yeeP (Gene ID: 946524). fbr (SEQ ID NO.7).

[0092] The L-tryptophan-producing strain T-8 (Escherichia coli K12MG1655ΔtrpR,ΔtrpL,ΔtyrR,ΔtnaB,ΔsdaB::serA) was prepared. fbr ,Δrpnd::BsglnA fbr,Δmtr::yddG,ΔyeeP::aroG fbr ).

[0093] (2) Construction of recombinant strains carrying different mutants

[0094] The expression vectors carrying the mutants prepared in Example 2 were transformed into T-8 (Escherichia coli K12MG1655ΔtrpR,ΔtrpL,ΔtyrR,ΔtnaB,ΔsdaB::serA fbr ,Δrpnd::BsglnA fbr ,Δmtr::yddG,ΔyeeP::aroG fbr ) in the culture medium; respectively, recombinant strains were prepared.

[0095] (3) Preparation of L-tryptophan

[0096] In a clean bench, use a sterile inoculating loop to scoop up the recombinant strain from the glycerol tube and streak onto an LB plate containing 50 mg / L kanamycin. Incubate the plate upside down in a 37°C incubator for 10–12 hours. Use a sterile inoculating loop to scrape one loopful of cells and inoculate it into 50 mL of liquid LB medium containing 50 mg / L kanamycin. Incubate at 37°C, 200 rpm, for 10 hours to prepare the seed solution.

[0097] The obtained recombinant bacteria carrying different mutants were seeded into shake flask fermentation medium at a 10% (v / v) inoculation rate and fermented at 36°C and 200 rpm for 40 hours. After the fermentation, the fermentation broth was diluted and the L-tryptophan content in the fermentation broth was measured. The fermentation results are shown in Figure 2. Figure 2 As shown in Table 3.

[0098] Table 3: Effects of different mutants on L-tryptophan content

[0099] anthranilate synthase L-tryptophan production anthranilate synthase L-tryptophan production WT 1.810g / L M293S 1.800g / L M293A 1.795g / L M293T 2.825g / L M293G 1.660g / L M293C 1.760g / L M293V 1.785g / L M293N 1.755g / L M293L 1.320g / L M293Q 1.694g / L M293I 1.445g / L M293D 1.355g / L M293P 1.690g / L M293E 1.733g / L M293F 1.750g / L M293K 1.705g / L M293Y 1.230g / L M293R 1.785g / L M293W 1.710g / L M293H 1.700g / L

[0100] The results showed that the M293T mutant had the best effect, and the enzymatic properties of M293T were studied in the future.

[0101] Example 4: TrpE M293T In vitro enzyme activity assay of mutants

[0102] The specific steps are as follows:

[0103] 1. Detection of enzyme activity

[0104] (1) The wild-type trpED and trpE were separated by enzyme digestion and ligation. M293TThe two genes were constructed between the EcoRI and XhoI sites of the expression vector pET28a to obtain two expression plasmids, namely pET28a-trpED and pET28a-trpE M293T D.

[0105] (2) The constructed plasmids pET28a-trpED and pET28a-trpE M293T D was transformed into Escherichia coli BL21 (DE3) (this expression chassis is a model bacterium). Single colonies that were successfully transformed were picked and inoculated into 10 mL of LB liquid medium containing 50 mg / L kanamycin resistance and cultured at 37°C for 10-12 hours to prepare seed solutions.

[0106] (3) Inoculate the cultured seed solution into TB medium at a 1% (v / v) inoculation volume. First, culture at 37°C until the OD 600 To a pH of 0.5-0.6, IPTG was added to the bacterial solution in a clean hood to a final concentration of 0.1 mmol / L. The solution was allowed to stand at room temperature for 30 minutes, then incubated on a shaker at 16°C (200 rpm) for 16 hours to prepare a bacterial solution. The cultured solution was centrifuged to collect the cells, which were then washed three times with PBS buffer and disrupted. After centrifugation, the supernatant was collected to obtain a crude enzyme solution. Enzyme activity was assayed. The results are shown in Table 4 below.

[0107] Table 4: Enzyme activity assay results

[0108]

[0109] 2. Tolerance of TrpE and its mutants to L-tryptophan products

[0110] At the same time, in order to observe the effect of L-tryptophan on TrpE and TrpE M293T To determine the effect of L-tryptophan on enzyme activity, 0.5 M, 1 M, 2 M, and 3 M L-tryptophan were added to the initial reaction system for enzyme activity determination. The enzyme activity without L-tryptophan was taken as 100% for comparison. The relative enzyme activity was calculated based on the enzyme activity results. The results are shown in the figure. Figure 3 shown.

[0111] The results showed that mutant TrpE M293T Tolerance to L-tryptophan products was significantly increased.

[0112] Example 5: Overexpression of TrpE M293T Effects on bacterial growth and L-tryptophan synthesis

[0113] The specific steps are as follows:

[0114] (1) Preparation of recombinant strains:

[0115] The expression plasmid pTrcmob-trpE carrying the mutant prepared in Example 3 was M293T The recombinant strain T-8.1 was obtained by transforming the gene into the T-8 strain.

[0116] (2) T-8 and T-8.1 were inoculated into seed culture medium (fermenter) respectively, and cultured at 37°C for 8 h to prepare seed solutions;

[0117] Then, the prepared seed solution was transferred into a 5L fermentation tank containing fermentation medium (fermenter) at a 10% (v / v) inoculation rate, and the temperature was controlled at 36°C, the pH was maintained at 6.9, the rotation speed was not higher than 800 rpm, the ventilation volume was maintained within 4 vvm, the tank pressure was controlled within 0.06 MPa, the dissolved oxygen was controlled between 20 and 40, the residual sugar was controlled at about 1 g / L, and the fermentation was carried out for 44 hours. Figure 4 shown.

[0118] The results showed that at the end of fermentation, the L-tryptophan production of T-8 and T-8.1 were 11.13 g / L and 19.96 g / L, respectively; and the sugar-acid conversion rates were 10.11% and 12.03%, respectively.

[0119] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An anthranilate synthase subunit mutant, characterized in that: The mutant is obtained by mutating the 293rd methionine of the anthranilate synthase subunit as shown in the amino acid sequence of SEQ ID NO.1 to threonine.

2. A gene encoding the mutant according to claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant according to claim 1 or carrying the gene or recombinant vector according to claim 2.

4. The recombinant cell according to claim 3, characterized in that The recombinant cell uses bacteria or fungi as expression hosts.

5. A recombinant enzyme catalyst comprising the anthranilate synthase subunit mutant according to claim 1, characterized in that: The recombinase catalyst is any one of the following forms: (1) culturing recombinant expression transformants and isolating transformant cells containing the recombinase; (2) culturing the recombinant expression transformant, isolating the transformant cells containing the recombinase, and disrupting the transformant cells containing the recombinase to obtain a cell disrupted liquid; (3) Cultivating the recombinant expression transformant, isolating the transformant cells containing the recombinant enzyme, disrupting the transformant cells containing the recombinant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant enzyme to obtain a freeze-dried enzyme powder.

6. A method for improving the catalytic activity and / or enzymatic activity and / or tolerance of anthranilate synthase subunits to the product L-tryptophan, characterized in that: The method comprises the following steps: mutating the methionine at position 293 of the anthranilate synthase subunit of the amino acid sequence as shown in SEQ ID NO. 1 to threonine.

7. Use of the anthranilate synthase subunit mutant according to claim 1 in increasing the yield of L-tryptophan produced by Escherichia coli or in constructing a high-yield L-tryptophan recombinant strain.

8. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli expresses the anthranilate synthase subunit mutant according to claim 1; Preferably, the recombinant Escherichia coli chassis cells are: knocked out the trpR, trpL, tyrR, tnaB genes on the Escherichia coli K12 MG1655 genome, and overexpressed serA at the sdaB site on the genome fbr Gene, overexpression of BsglnA at the rpnd locus fbr Gene, overexpression of yddG gene at the mtr site, overexpression of aroG at the yeeP site fbr Gene.

9. A method for preparing L-tryptophan, characterized in that: The method comprises the following steps: using the recombinant Escherichia coli according to claim 8 to ferment and prepare L-tryptophan; Preferably, the method comprises inoculating the recombinant Escherichia coli seed solution into a fermentation medium at an inoculum rate of 10%, and fermenting under the conditions to prepare L-tryptophan; Preferably, the fermentation medium is: 40 g / L anhydrous glucose, 3.0 g / L yeast extract, 1.6 g / L anhydrous ammonium sulfate, 2.0 g / L citric acid, 5.6 g / L anhydrous dipotassium hydrogen phosphate, 2.0 g / L anhydrous magnesium sulfate, 1 mL / L trace element solution, 80 mg / L phenol red, wherein the formula of the trace element solution is: 7.6 g / L ferrous sulfate heptahydrate, 0.4 g / L cobalt chloride hexahydrate, 0.06 g / L copper sulfate pentahydrate, 0.64 g / L zinc sulfate, 2.0 g / L sodium sulfate, and 0.45 g / L manganese sulfate monohydrate.

10. Use of the anthranilate synthase subunit mutant according to claim 1, or the gene or recombinant vector according to claim 2, or the recombinant cell according to claim 3 or 4, or the recombinant enzyme catalyst according to claim 5, or the method according to claim 6 or 9, or the recombinant Escherichia coli according to claim 8 in L-tryptophan or a product containing L-tryptophan.

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