Tryptophan high-yield strain as well as preparation method and application thereof

Abstract

The invention relates to a tryptophan high-yield strain as well as a preparation method and application thereof. The strain comprises overexpression of Vhb protein in an indole synthetic pathway enhanced strain. Heterologous hemoglobin Vhb is introduced into the strain to promote C-H functionalization reaction on the C3 position of indole, and an indole synthesis path is enhanced, so that the yield of tryptophan is increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a tryptophan high-yield strain, a preparation method and application thereof. BACKGROUND

[0002] L-tryptophan, namely L-alpha-amino-beta-indole propionic acid, belongs to aromatic amino acids and is one of eight essential amino acids for human body, and is widely used in food, feed, medicine and forestry industries. Adding tryptophan in food can promote protein absorption, and when used as a feed additive, it can make animals gain weight. In addition, tryptophan can also regulate the mental rhythm, improve sleep and has a certain effect on the treatment of depression. At present, the annual output of tryptophan in the world exceeds 50,000 tons, but it is still lower than the potential market demand. The traditional production methods of tryptophan mainly include chemical synthesis and enzyme catalysis. However, due to the limited production raw materials, serious pollution problems and high cost, the above methods have been gradually replaced by microbial method. With the continuous development of biotechnology and the unique advantages of microbial method such as low cost, environmental friendliness and high-density fermentation, the modification strategies related to microbial production of tryptophan have also been widely applied and researched.

[0003] Tryptophan belongs to aromatic amino acids and is one of the amino acids with the longest metabolic pathway. In Escherichia coli, the synthesis of tryptophan involves glycolysis pathway, pentose phosphate pathway and shikimic acid pathway, wherein the synthesis of tryptophan precursor by shikimic acid pathway is an important pathway affecting synthesis. The final synthesis step of tryptophan is the synthesis of indole and serine catalyzed by tryptophan synthase beta subunit, and the beta subunit of tryptophan synthase is edited by trpB gene. The subunit needs to bind L-serine in the cell through coenzyme PLP, and catalyze the combination of serine and indole to generate tryptophan. The reaction needs serine to replace H proton in C-H functional group at position 3 of indole. Therefore, it is speculated that the carbon state at position 3 of indole has a great influence on the synthesis efficiency of tryptophan, that is, the synthesis efficiency of tryptophan can be improved by introducing an enzyme capable of catalyzing the carbon at position 3 of indole. The catalytic action of hemoprotein enzyme has great potential in the field of organic synthesis, which can help to catalyze the heterocyclic or cyclic alkenes through carbene transfer reaction to obtain C-H alkylation or cyclopropanation products. Frances H. Arnold team found that cytochrome P450 can help to catalyze the selective C-H functionalization reaction of N-unprotected indole at C3 position, so that the catalytic reaction combination efficiency at this position is higher.

[0004] Currently, the use of hemoglobin in the field of biological fermentation is related to oxygen transport. Because in the late fermentation, the density of the microbial body in the fermentation broth is high, and the oxygen concentration is low, and hemoglobin has a strong oxygen transport function, enhancing the absorption and utilization of oxygen by the strain can improve the fermentation yield and conversion rate. CN115873771B describes overexpression of hemoglobin in Xanthomonas campestris to improve oxygen utilization efficiency, and CN1086203C describes the promotion effect of heterologous expression of hemoglobin gene in genetically engineered Escherichia coli on aerobic fermentation. However, there is currently no research on the promotion of hemoglobin gene to tryptophan synthesis, because exploring the promotion of hemoglobin to tryptophan synthesis has become a problem to be solved. SUMMARY

[0005] To solve the above technical problems, the present application provides a high-yield tryptophan strain, a preparation method and an application. The present application introduces heterologous hemoglobin Vhb into the strain and enhances the indole synthesis pathway to improve the yield of tryptophan.

[0006] To achieve this purpose, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a high-yield tryptophan strain, which includes overexpression of Vhb protein in a strain with enhanced indole synthesis pathway.

[0008] In the present application, heterologous hemoglobin Vhb is introduced into the strain, and the expression of heterologous protein is enhanced, so that the pathway from indole to tryptophan is enhanced, and the conversion rate from indole to tryptophan is also improved. And overexpression of related genes in the indole synthesis pathway increases the content of indole, thereby further improving the yield of tryptophan.

[0009] Preferably, the strain with enhanced indole synthesis pathway includes a strain overexpressing aroE gene and / or aroK gene.

[0010] In the present application, by overexpressing aroE gene and aroK gene, the synthesis of indole is increased while the Vhb protein is enhanced, and the synthesis of tryptophan is further promoted while the carbon state at position 3 of indole is promoted.

[0011] Preferably, the amino acid sequence of the vgb gene includes any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9.

[0012] SEQ ID No. 1:

[0013] MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE.

[0014] SEQ ID No. 2:

[0015] MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE.

[0016] SEQ ID No. 3:

[0017] MLDQQTINIIKATVPVLKEHGVTITTTFEKNLFAKHPEVRPLIDMGRQESLEPPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE.

[0018] SEQ ID No. 4:

[0019] MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVWAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE.

[0020] SEQ ID No. 5:

[0021] MLDQQTINIIKATVPVLKEHGVTITTTFEKNLFAKHPEVRPLFDMGRQESLEPPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE.

[0022] SEQ ID No. 6:

[0023] MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQES LEQPKALAMTVLAAAQNIENLPAISPAVKKIAVKHCQAGVAAAHYPIVGQELL GAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE.

[0024] SEQ ID No. 7:

[0025] MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADEFIQVEADLYAQAVE.

[0026] SEQ ID No. 8:

[0027] MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQCKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE.

[0028] SEQ ID No. 9:

[0029] MLDQQTINIIKATVPVLKEHGVTITTTMYKNLFAKHPEVRPLFDMGRQESLEPPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE.

[0030] Preferably, the nucleic acid sequence of the aroE gene comprises the sequence as set forth in SEQ ID No. 10.

[0031] SEQ ID No. 10:

[0032] ATGGAAACCTATGCTGTTTTTGGTAATCCGATAGCCCACAGCAAATCGCCATTCATTCATCAGCAATTTGCTCAGCAACTGAATATTGAACATCCCTATGGGCGCGTGTTGGCACCCATCAATGATTTCATCAACACACTGAACGCTTTCTTTAGTGCTGGTGGTAAAGGTGCGAATGTGACGGTGCCTTTTAAAGAAGAGGCTTTTGCCAGAGCGGATGAGCTTACTGAACGGGCAGCGTTGGCTGGTGCTGTTAATACCCTCATGCGGTTAGAAGATGGACGCCTGCTGGGTGACAATACCGATGGTGTAGGCTTGTTAAGCGATCTGGAACGTCTGTCTTTTATCCGCCCTGGTTTACGTATTCTGCTTATCGGCGCTGGTGGAGCATCTCGCGGCGTACTACTGCCACTCCTTTCCCTGGACTGTGCGGTGACAATAACTAATCGGACGGTATCCCGCGCGGAAGAGTTGGCTAAATTGTTTGCGCACACTGGCAGTATTCAGGCGTTGAGTATGGACGAACTGGAAGGTCATGAGTTTGATCTCATTATTAATGCAACATCCAGTGGCATCAGTGGTGATATTCCGGCGATCCCGTCATCGCTCATTCATCCAGGCATTTATTGCTATGACATGTTCTATCAGAAAGGAAAAACTCCTTTTCTGGCATGGTGTGAGCAGCGAGGCTCAAAGCGTAATGCTGATGGTTTAGGAATGCTGGTGGCACAGGCGGCTCATGCCTTTCTTCTCTGGCACGGTGTTCTGCCTGACGTAGAACCAGTTATAAAGCAATTGCAGGAGGAATTGTCCGCGTGA.

[0033] Preferably, the nucleic acid sequence of said aroK gene comprises the sequence as set forth in SEQ ID No. 11.

[0034] SEQ ID No. 11:

[0035] ATGGCAGAGAAACGCAATATCTTTCTGGTTGGGCCTATGGGTGCCGGAAAAAGCACTATTGGGCGCCAGTTAGCTCAACAACTCAATATGGAATTTTACGATTCCGATCAAGAGATTGAGAAACGAACCGGAGCTGATGTGGGCTGGGTTTTCGATTTAGAAGGCGAAGAAGGCTTCCGCGATCGCGAAGAAAAGGTCATCAATGAGTTGACCGAGAAACAGGGTATTGTGCTGGCTACTGGCGGCGGCTCTGTGAAATCCCGTGAAACGCGTAACCGTCTTTCCGCTCGTGGCGTTGTCGTTTATCTTGAAACGACCATCGAAAAGCAACTTGCACGCACGCAGCGTGATAAAAAACGCCCGTTGCTGCACGTTGAAACACCGCCGCGTGAAGTTCTGGAAGCGTTGGCCAATGAACGCAATCCGCTGTATGAAGAGATTGCCGACGTGACCATTCGTACTGATGATCAAAGCGCTAAAGTGGTTGCAAACCAGATTATTCACATGCTGGAAAGCAACTAA.

[0036] SEQ ID No. 12:

[0037] AAGCTTACAGGACGCTGGGGTTAAAAGTATTTGAGTTTTGATGTGGATTAAGTTTTAAGAGGCAATAAAGATTATAATAAGTGCTGCTACACCATACTGATGTATGGCAAAACCATAATAATGAACTTAAGGAAGACCCTCATGTTAGACCAGCAAACCATTAACATCATCAAAGCCACTGTTCCTGTATTGAAGGAGCATGGCGTTACCATTACCACGACTTTTTATAAAAACTTGTTTGCCAAACACCCTGAAGTACGTCCTTTGTTTGATATGGGTCGCCAAGAATCTTTGGAGCAGCCTAAGGCTTTGGCGATGACGGTATTGGCGGCAGCGCAAAACATTGAAAATTTGCCAGCTATTTTGCCTGCGGTCAAAAAAATTGCAGTCAAACATTGTCAAGCAGGCGTGGCAGCAGCGCATTATCCGATTGTCGGTCAAGAATTGTTGGGTGCGATTAAAGAAGTATTGGGCGATGCCGCAACCGATGACATTTTGGACGCGTGGGGCAAGGCTTATGGCGTGATTGCAGATGTGTTTATTCAAGTGGAAGCAGATTTGTACGCTCAAGCGGTTGAATAA.

[0038] Preferably, the species of the strains include Escherichia and Corynebacterium, wherein the Escherichia includes any one of Escherichia coli, Escherichia fergusonii, and the like, or a combination of at least two thereof, and the Corynebacterium includes any one of Corynebacterium glutamicum, Corynebacterium crenatum, or Corynebacterium ammoniagenes, or a combination of at least two thereof.

[0039] In a second aspect, the present application provides a preparation method of the tryptophan high-yield strain according to the first aspect, the preparation method comprising: respectively cutting and connecting the nucleic acid fragments of the Vhb protein and the blank plasmid, and then transforming a host strain.

[0040] Preferably, the preparation method comprises: respectively cutting and connecting the fragments after connecting the aroE gene, the aroK gene, and the nucleic acid fragments of the Vhb protein, and then transforming a host strain.

[0041] Preferably, the transformation method comprises any one of electrical transformation, chemical transformation, natural transformation, or conjugation transformation, or a combination of at least two thereof.

[0042] In a second aspect, the present application provides use of the tryptophan high-yield strain according to the first aspect in the production of tryptophan.

[0043] In a third aspect, the present application provides a method for producing tryptophan, which comprises culturing the tryptophan high-yield strain according to the first aspect and then performing fermentation.

[0044] In a fourth aspect, the present application provides a method for producing tryptophan in vitro, which comprises reacting the Vhb protein according to the first aspect, serine, tryptophan synthase and indole to obtain tryptophan.

[0045] Preferably, the method comprises incubating 2-8 mM serine with 0.1-1 mg / mL tryptophan synthase to obtain product 1, and then reacting 2-8 mM indole, product 1 and 1-5 μM Vhb protein to obtain the product. The 2-8 mM may be, for example, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM or 8 mM, etc. The 0.1-1 mg / mL may be, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1 mg / mL, etc. The 1-5 μM may be, for example, 1 μM, 2 μM, 3 μM, 4 μM or 5 μM, etc.

[0046] Preferably, the incubation is performed at a temperature of 35-40 °C for 0.5-2 h. The 35-40 °C may be, for example, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or 40 °C, etc. The 0.5-2 h may be, for example, 0.5 h, 1 h, 1.5 h or 2 h, etc.

[0047] Preferably, the reaction is performed at a temperature of 20-30 °C for 4-8 h at a speed of 100-300 rpm. The 20-30 °C may be, for example, 20 °C, 22 °C, 24 °C, 26 °C, 28 °C or 30 °C, etc. The 4-8 h may be, for example, 4 h, 5 h, 6 h, 7 h or 8 h, etc. The 100-300 rpm may be, for example, 100 rpm, 150 rpm, 200 rpm, 250 rpm or 300 rpm, etc.

[0048] Compared with the prior art, the present application has at least the following beneficial effects:

[0049] 1.The present application expresses hemoglobin Vhb heterologously in a strain, and verifies that hemoglobin Vhb can enhance the reaction efficiency of indole and serine in the process of tryptophan synthesis, reduce the carbon loss in the process of tryptophan synthesis, and obviously promote the synthesis of tryptophan. The present application verifies some effective mutation sites by mutating the heterologously expressed hemoglobin Vhb, and the superposition of some mutation sites can effectively improve the yield of tryptophan.

[0050] 2.The present application overexpresses aroE gene and aroK gene in a strain, enhances the shikimic acid pathway, increases the synthesis of indole, and overexpresses vgb gene at the same time, thereby promoting the synthesis of tryptophan.

[0051] 3.The present application is verified by in vitro test, and more tryptophan is produced under the catalysis of hemoglobin Vhb after enhancing the indole synthesis pathway, which further illustrates that the addition of hemoglobin Vhb accelerates the reaction, and Vhb and tryptophan synthase TrpB synergistically act to further improve the generation amount of tryptophan. DETAILED DESCRIPTION

[0052] In order to facilitate the understanding of the present application, the present application lists the following examples. It should be understood by those skilled in the art that the examples are only used to help understand the present application, and should not be regarded as a specific limitation on the present application.

[0053] The source of the materials used in the following examples is as follows:

[0054] Escherichia coli XJFF-151106S: classified as Escherichia coli, preserved in China General Microbiological Culture Collection Center, preserved on November 17, 2015, preserved under the number of CGMCC No.11674, and preserved at No.3, Beichen West Road, Chaoyang District, Beijing.

[0055] Escherichia fergusonii ATCC35469, Corynebacterium glutamicum ATCC13032, and Corynebacterium ammoniagenes ATCC6871: standard strains purchased from Beijing Baoewei Biotechnology Co., Ltd.

[0056] Corynebacterium crenatum CICC 20662: purchased from the Industrial Microbial Culture Collection Center.

[0057] Example 1

[0058] This example constructs Escherichia coli overexpressing aroE gene, aroK gene and vgb gene

[0059] (1) Amplification of vgb gene

[0060] The strain used in the embodiment is a strain XJFF-151106S with a certain yield of tryptophan, and the preservation number is CGMCC No.11674.

[0061] The vgb gene complete fragment was obtained by PCR reaction using the coding gene vgb of the transparent Aeromonas haemolyticum hemoglobin Vhb as a template, designing primers vgb-F and vgb-R, and the nucleic acid sequence of the gene is SEQ ID No.12. The primer sequences are SEQ ID No.13 and SEQ ID No.14, the PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0062] The nucleic acid sequence of the vgb gene is SEQ ID No.12.

[0063] AAGCTTACAGGACGCTGGGGTTAAAAGTATTTGAGTTTTGATGTGGATTAAGTTTTAAGAGGCAATAAAGATTATAATAAGTGCTGCTACACCATACTGATGTATGGCAAAACCATAATAATGAACTTAAGGAAGACCCTCATGTTAGACCAGCAAACCATTAACATCATCAAAGCCACTGTTCCTGTATTGAAGGAGCATGGCGTTACCATTACCACGACTTTTTATAAAAACTTGTTTGCCAAACACCCTGAAGTACGTCCTTTGTTTGATATGGGTCGCCAAGAATCTTTGGAGCAGCCTAAGGCTTTGGCGATGACGGTATTGGCGGCAGCGCAAAACATTGAAAATTTGCCAGCTATTTTGCCTGCGGTCAAAAAAATTGCAGTCAAACATTGTCAAGCAGGCGTGGCAGCAGCGCATTATCCGATTGTCGGTCAAGAATTGTTGGGTGCGATTAAAGAAGTATTGGGCGATGCCGCAACCGATGACATTTTGGACGCGTGGGGCAAGGCTTATGGCGTGATTGCAGATGTGTTTATTCAAGTGGAAGCAGATTTGTACGCTCAAGCGGTTGAATAA.

[0064] Table 1

[0065] Components Volume Forward primer (10 μM) 1 μL Reverse primer (10 μM) 1 μL Template 1 μL 10 x Buffer 5 μL dNTP 0.5 μL Exnase 0.25 μL ddH2O 16.25 μL Total 25 μL

[0066] Table 2

[0067] Procedure Temperature Time Pre-denaturation 98℃ 30 min Denaturation 98℃ 10s Annealing 55℃ 15 min Extension 72℃ 1 min / kb Final extension 72℃ 10 min

[0068] (2) Construction of overexpression plasmid

[0069] The aroE gene, the aroK gene of E. coli and the vgb gene of P. luteola were used as templates, and the primers aroE-F, aroE-R, aroK-F, aroK-R, vgb-F and vgb-R were designed. The fragments aroE, aroK and vgb were obtained by PCR amplification. The aroE and aroK were used as templates, and the primers aroE-F and aroK-R were used for PCR amplification to obtain the gene fragment aroEK. The aroEK and vgb fragments were used as templates, and the primers aroE-F and vgb-R were used for PCR amplification to obtain the fragment aroEK-vgb. The obtained aroEK, vgb, aroEK-vgb fragments and pTrp plasmid were digested with SpeI and NcoI at 37℃ for 2h 30min, and the enzyme digestion system is shown in Table 3. The linearized plasmid fragments obtained by enzyme digestion were connected with aroEK, vgb and aroEK-vgb respectively, and the connection temperature was 22℃ and the reaction time was 30min, and the connection system is shown in Table 4. Finally, the overexpression plasmids pTrp-aroEK, pTrp-vgb and pTrp-aroEK-vgb were obtained. The sequences of the above primers are shown in Table 5.

[0070] Table 3

[0071] Components Volume PCR recovery product / plasmid 6 / 10 10 x K Buffer 1.5 Spe I 1 Nco I 1 ddH2O 5.5 / 1.5 Total 15

[0072] Table 4

[0073] Components Volume Vector 2.5 μL Gene fragment 6 μL 10 x T4 Ligation Buffer 1 μL T4 DNA Ligase 0.5 μL Total volume 10 μL

[0074] Table 5

[0075]

[0076] (3) Preparation of recombinant strains

[0077] The pTrp (blank plasmid), pTrp-aroEK, pTrp-vgb and pTrp-aroEK-vgb were electroporated (electroporation parameters: 2.5Kv, 5.8ms) into the expression host strain XJFF-151106S strain, and the overexpression strains XJFF-151106S-pTRP, XJFF-151106S-pTRP-aroEK, XJFF-151106S-pTRP-vgb and XJFF-151106S-pTRP-aroEK-vgb were obtained by resistance plate screening.

[0078] Example 2

[0079] This example detects tryptophan production

[0080] The overexpression strains XJFF-151106S-pTRP, XJFF-151106S-pTRP-aroEK, XJFF-151106S-pTRP-vgb and XJFF-151106S-pTRP-aroEK-vgb constructed in Example 1 were cultured overnight in LB solid medium, with strain XJFF-151106S as a control. The grown strains were inoculated into 250 mL angle baffled flasks containing 25 mL fermentation medium and cultured at 37°C with 200 rpm shaking for 24 hours. After the culture was completed, the tryptophan content in the fermentation broth was determined by an amino acid analyzer. The fermentation medium contained the following components: glucose 60 g / L, yeast extract 2.5 g / L, ammonium sulfate [(NH4)2SO4·7H2O] 20 g / L, magnesium sulfate (MgSO4) 1 g / L, potassium dihydrogen phosphate (KH2PO4) 2 g / L, sodium citrate 5 g / L, sodium chloride (NaCl) 1 g / L, L-tyrosine 0.1 g / L, L-phenylalanine 0.15 g / L, calcium carbonate (CaCO3) 40 g / L. The final tryptophan production and conversion rate results are shown in Table 6 (the production and conversion rate results of each strain are the average of 3 results).

[0081] Table 6

[0082] Strain name Yield / (g / L) Transformation rate / % XJFF-151106S 3.40 8.75 XJFF-151106S-pTRP 3.43 8.95 XJFF-151106S-pTRP-aroEK 3.48 8.96 XJFF-151106S-pTRP-vgb 3.26 7.90 XJFF-151106S-pTRP-aroEK-vgb 4.4 11.24

[0083] According to the above fermentation results, the overexpression of aroEK or vgb alone in the tryptophan fermentation strain has little effect on the improvement of tryptophan production, but the combination of aroEK and vgb expressed in the strain has a significant improvement in production and conversion rate, with an increase of 29.4% in production and 28.5% in conversion rate compared with the starting strain.

[0084] Example 3

[0085] This example explores the effect of hemoglobin Vhb mutant on production

[0086] The coding gene of hemoglobin Vhb was subjected to site-directed mutation by error-prone PCR, with the vgb gene of Vitreoscilla as a template and vgb-F and vgb-R as primers. The mutant sequence was obtained by error-prone PCR with a kit system. The PCR reaction program was consistent with that in Example 1, and the reaction system is shown in Table 7.

[0087] Table 7

[0088] Reagent Volume H2O 31 μL Template gene fragment 1 μL Forward primer (10 μM) 1 μL Reverse primer (10 μM) 1 μL RandomMut buffer (10X) 5 μL Mutation enhancer (10X) 5 μL dNTP (2.5 mM each) 5 μL RandomMut DNA polymerase 1 μL

[0089] Finally, eight hemoglobin Vhb mutants were obtained, and the mutant sequences obtained above were ligated with the expression vector pTRP-aroEK. The ligation process was consistent with Example 1, and the successfully ligated vector pTRP-aroEK-vgbmut was electroporated into XJFF-151106S. The operation method was consistent with Example 1. Single colonies with successful construction were picked for shake flask fermentation verification, and the tryptophan content was determined. The fermentation verification and content detection steps were consistent with Example 1. The results of the mutant strains with improved tryptophan production are shown in Table 8.

[0090] Table 8

[0091]

[0092]

[0093] Sequencing analysis of the above Vhb protein found that there were at least 1 amino acid mutation and at most 3 amino acid mutations in the amino acid sequence of the Vhb protein. However, the above mutant sequence replaced the vgb gene, and the aroE gene and the aroK gene were overexpressed, and the strain could significantly improve the tryptophan production.

[0094] Example 4

[0095] This example explores the effect of using Escherichia fergusonii as a host strain

[0096] (1) Tryptophan yield detection

[0097] The complete fragment of the vgb gene in Example 1 was used, and the nucleic acid sequence of the gene was SEQ ID No. 12. The aroE gene and the aroK gene of Escherichia coli were used as templates to obtain the expression plasmids pTrp, pTrp-aroEK, pTrp-vgb, and pTrp-aroEK-vgb, which were electroporated into the expression host strain ATCC35469. The overexpression strain was selected by resistance screening, and after successful construction, shake flask fermentation verification was performed. The starting strain ATCC35469 was used as a control, and the culture and fermentation methods in Example 2 were used for preparation. The final tryptophan yield and conversion rate results are shown in Table 9 (the yield and conversion rate results of each strain are the average of 3 results).

[0098] Table 9

[0099] Strain name Yield / (g / L) Transformation rate / % ATCC35469 0 0 ATCC35469-pTRP 0.5 1.29 ATCC35469-pTRP-aroEK 0.64 1.65 ATCC35469-pTRP-vgb 0.59 1.52 ATCC35469-pTRP-aroEK-vgb 0.78 2.01

[0100] According to the results, the combination of aroEK and vgb was expressed in the strain, and the yield and conversion rate were significantly improved. Compared with the starting strain, the yield was increased by 56%, and the conversion rate was increased by 55.8%.

[0101] (2) Expression of Vhb mutants in strains

[0102] The 8 Vhb mutants in Example 3 were ligated with the expression vectors using the method in Example 1, and were introduced into the strain ATCC35469. The strains successfully introducing the Vhb mutants were screened and fermented, and the tryptophan content in the strains was detected. The results of the strains with improved tryptophan yield are shown in Table 10.

[0103] Table 10

[0104]

[0105] From the above data, it can be concluded that the mutated Vhb protein can also promote the synthesis of tryptophan, improve the yield and conversion rate of tryptophan in the Escherichia genus other than Escherichia coli.

[0106] Example 5

[0107] This example investigates the effect of using Corynebacterium glutamicum as a host strain

[0108] (1) Tryptophan yield detection

[0109] The complete fragment of vgb gene in Example 1 was used, and the nucleic acid sequence of the gene was SEQ ID No. 12. The expression plasmids pTrp, pTrp-aroEK, pTrp-vgb, and pTrp-aroEK-vgb were obtained by using the aroE gene and aroK gene of Escherichia coli as templates, and were electroporated into the expression host strain ATCC13032. The overexpression strains were screened, and after successful construction, they were inoculated into the seed culture medium for shake flask fermentation verification. The content of each component in the seed culture medium was as follows: glucose 20 g / L, (NH4)2S04 20 g / L, MgS04·7H2O 0.4 g / L, peptone 10 g / L, yeast powder 10 g / L, KH2PO4 1 g / L, K2HP041 g / L, phenylalanine 0.1 g / L, tyrosine 0.1 g / L, biotin 0.2 mg / L, pH 7.0. The content of each component in the fermentation medium was as follows: glucose 60 g / L, (NH4)2S04 20 g / L, KH2P04 1 g / L, K2HP04 1 g / L, MgS04·7H20 0.4 g / L, FeS04·7H2O 10 mg / L, MnS04·H2O 10 mg / L, ZnS04·7H2O 1 mg / L, CuS04·5H2O 0.2 mg / L, NiCl2·6H2O 0.02 mg / L, yeast powder 2 g / L, phenylalanine 0.1 g / L, tyrosine 0.1 g / L, vitamin B1 0.2 mg / L, biotin 0.2 mg / L, 2% CaC03, pH 7.0.

[0110] The final tryptophan yield and conversion rate results are shown in Table 11 (the yield and conversion rate results of each strain are the average of 3 results).

[0111] Table 11

[0112] Strain name Yield / (g / L) Transformation rate / % ATCC13032 0.21 5.23 ATCC13032-pTRP 1.33 6.17 ATCC13032-pTRP-aroEK 1.79 7.14 ATCC13032-pTRP-vgb 1.66 7.09 ATCC13032-pTRP-aroEK-vgb 2.24 7.94

[0113] According to the results, the combination of aroEK and vgb expressed in the strain increased the yield by 68.4% and the conversion rate by 28.69% compared with the starting strain.

[0114] (2) Expression of Vhb mutants in the strain

[0115] The 8 Vhb mutants in Example 3 were connected to the expression vector using the method in Example 1 and transferred into the strain ATCC13032, and the strains successfully transferred were screened for fermentation to detect the tryptophan content therein. The results of the tryptophan yield of the Vhb mutant strains are shown in Table 12.

[0116] Table 12

[0117]

[0118] From the data, it can be seen that the mutated Vhb protein also increased the yield and conversion rate of the strain ATCC13032, with the yield increased by 44.6% to 54.5% and the conversion rate increased by 24.2% to 41.8%.

[0119] Example 6

[0120] This example explores the effect of using C. crenatum as a host strain

[0121] (1) Tryptophan yield detection

[0122] The complete fragment of the vgb gene in Example 1 was used, and the nucleic acid sequence of the gene was SEQ ID No. 12. The expression plasmids pTrp, pTrp-aroEK, pTrp-vgb, and pTrp-aroEK-vgb were obtained using the aroE gene and aroK gene of E. coli as templates, and were electroporated into the expression host strain CICC 20662 using the method in Example 1. The overexpression strains were screened for resistance, and after successful construction, the shake flask fermentation was verified. The starting strain CICC 20662 was used as a control, and the culture and fermentation methods in Example 2 were used for preparation. The final tryptophan yield and conversion rate results are shown in Table 13 (the yield and conversion rate results of each strain are the average of 3 results).

[0123] Table 13

[0124] Strain name Yield / (g / L) Transformation rate / % CICC 20662 0 0 CICC 20662-pTRP 0.31 0.80 CICC 20662-pTRP-aroEK 0.44 1.13 CICC 20662-pTRP-vgb 0.39 1.0 CICC 20662-pTRP-aroEK-vgb 0.58 1.49

[0125] According to the above results, it is found that the combination of vgb and aroEK expressed in CICC 20662 can also effectively improve the yield and conversion rate of tryptophan, and the yield of CICC 20662-pTRP is increased by 87%, and the conversion rate is increased by 86.2%.

[0126] (2) Expression of Vhb mutants in strains

[0127] The eight Vhb mutants in Example 3 were connected with the expression vector in the method of Example 1, and were transferred into the strain CICC 20662, and the strains successfully transferred were screened for fermentation, and the tryptophan content therein was detected. The results of the tryptophan yield of the Vhb mutant strains are shown in Table 14.

[0128] Table 14

[0129]

[0130]

[0131] Through the above fermentation results, it is proved that the Vhb mutant protein also has the effect of improving the yield and conversion rate of tryptophan in CICC 20662, and the highest yield is increased by 65%, and the highest conversion rate is increased by 65.8%.

[0132] Example 7

[0133] This example explores the effect of using C. ammoniagenes as a host strain

[0134] The complete fragment of vgb gene in Example 1 is used, and the nucleic acid sequence of the gene is SEQ ID No. 12. The expression plasmids pTrp, pTrp-aroEK, pTrp-vgb, and pTrp-aroEK-vgb are obtained by using the aroE gene and aroK gene of E. coli as templates, and are electroporated into the expression host strain ATCC6871. The overexpression strain is screened for resistance, and after successful construction, the shake flask fermentation is verified. The starting strain ATCC6871 is used as a control, and the culture and fermentation methods in Example 2 are used for preparation. The final tryptophan yield and conversion rate results are shown in Table 15 (the yield and conversion rate results of each strain are the average of three results).

[0135] Table 15

[0136] Strain name Yield / (g / L) Transformation rate / % ATCC6871 0 0 ATCC6871-pTRP 0.41 2.13 ATCC6871-pTRP-aroEK 0.5 2.6 ATCC6871-pTRP-vgb 0.48 2.54 ATCC6871-pTRP-aroEK-vgb 0.59 2.61

[0137] According to the above results, it is found that the highest tryptophan yield is obtained when vgb and aroEK are expressed in combination, which is 44% higher than that of ATCC6871-pTRP, and the conversion rate is increased by 22.5%.

[0138] (2) Expression of Vhb mutants in strains

[0139] In this embodiment, eight Vhb mutants in Example 3 are connected with the expression vector in the method of Example 1, and are transferred into the strain ATCC6871. The strains successfully transferred are screened for fermentation, and the tryptophan content in each strain is detected. The results of the strains with improved tryptophan yield are shown in Table 16.

[0140] Table 16

[0141]

[0142] By verifying the different mutated Vhb proteins in ATCC6871-pTRP-aroEK, it is found that the mutant proteins also promote the production of L-tryptophan in ATCC6871, and the highest yield can be increased by 50%.

[0143] Example 8

[0144] This embodiment explores the effects of adding hemoglobin Vhb in vitro and enhancing the indole synthesis pathway on the yield of tryptophan

[0145] (1) Expression and purification of Vhb of Methylosinus trichosporium

[0146] The Methylosinus trichosporium was taken out from -80℃ and cultured at 37℃, 150rpm for 6h. After activation, it was inoculated into 3L resistant LB medium at 1% inoculation amount and cultured overnight. Then it was cultured at 25℃, 120rpm under anaerobic conditions for 20h. After centrifugation, it was resuspended in 1×PBS for 5min in ice bath, and was ultrasonically broken for 1h (amplitude rod Energy 40%, ultrasonic breaking 2.5s, cooling 3.5s),

[0147] 4℃, 12000rpm centrifugation for 30min, the supernatant was collected and purified using a nickel column. The desalted protein was concentrated by centrifugation for standby.

[0148] (2) In vitro experiment

[0149] In this experiment, the first group did not add Vhb reaction, the second group used chlorinated hematin for catalysis, the third group used hemoglobin Vhb for catalysis, and the fourth group used tryptophan synthase TrpB to treat serine before using Vhb for catalysis. Each group of reactions was compared, and each group of experiments was biologically repeated three times. The substances added in each group and the reaction process are as follows.

[0150] First group: dichloromethane as solvent, indole (5 mM), serine (5 mM) were added in sequence, 25 °C, 150 rpm, reaction for 6 h.

[0151] Second group: dichloromethane as solvent, indole (5 mM), serine (5 mM), chlorocyan (2.5 mM) were added, 25 °C, 150 rpm, reaction for 6 h.

[0152] Third group: dichloromethane as solvent, indole (5 mM), serine (5 mM), hemoglobin Vhb (2.5 mM) were added, 25 °C, 150 rpm, reaction for 6 h.

[0153] Fourth group: dichloromethane as solvent, 5 mM serine was incubated with 0.5 mg / mL tryptophan synthase TrpB at 37 °C for 1 h to convert serine into an intermediate, then indole (5 mM), TrpB pretreated serine, Vhb (2.5 mM) were added, 25 °C, 150 rpm, reaction for 6 h.

[0154] After the reaction was completed, it was terminated on ice, extracted with dichloromethane, and the lower layer was dried with a rotary evaporator. HPLC was used for analysis, C18 reversed-phase chromatographic column, mobile phase: acetonitrile / water (containing 0.1% formic acid), gradient elution, detection wavelength: 280 nm. Finally, the conversion number TON value (the amount of product substance mol / Vhb usage 2 times mol) was calculated.

[0155] According to HPLC analysis, the TON value of the hemoglobin Vhb catalyzed reaction was 15, and the C-H functionalization product ratio was greater than 99%, and the specific TON value is shown in Table 17.

[0156] Table 17

[0157]

[0158]

[0159] According to the results, it was found that hemoglobin Vhb could catalyze the reaction of indole and serine to generate tryptophan, but after treating serine with tryptophan synthase TrpB, more tryptophan was generated by the catalytic reaction, and the TON value increased by 87.5%, and the C-H product functionalization ratio was greater than 99%. That is, hemoglobin Vhb can also catalyze the synthesis of tryptophan in vitro, and the synergistic effect with tryptophan synthase TrpB is better. Through this result, it is proved that the introduction of hemoglobin Vhb in the tryptophan synthesis pathway and the enhancement of the indole synthesis pathway can improve the synthesis of tryptophan both in vivo and in vitro.

[0160] In summary, the present application introduces hemoglobin Vhb in tryptophan production, catalyzes the carbon at the 3-position of indole, and further enhances the indole synthesis pathway, which significantly improves the yield of tryptophan.

[0161] The applicant states that the above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A tryptophan high-producing strain, characterized by, The strain includes a strain with enhanced indole synthesis pathway overexpressing Vhb protein.

2. The tryptophan high-producing strain according to claim 1, characterized by, The strain with enhanced indole synthesis pathway includes a strain overexpressing aroE gene and / or aroK gene.

3. The tryptophan high-producing strain according to claim 1 or 2, characterized by, The amino acid sequence of the Vhb protein includes any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No.

9.

4. The tryptophan high-producing strain according to claim 2 or 3, characterized by, The nucleic acid sequence of the aroE gene includes a sequence as shown in SEQ ID No. 10; Preferably, the nucleic acid sequence of the aroK gene includes a sequence as shown in SEQ ID No.

11.

5. The tryptophan high-producing strain according to any one of claims 1 to 4, characterized in that, The species of the strain includes any one of Escherichia coli, Escherichia fergusonii, Corynebacterium glutamicum, Corynebacterium crenatum or Corynebacterium ammoniagenes or a combination of at least two thereof.

6. A method for preparing a tryptophan high-producing strain according to any one of claims 1 to 5, characterized by, The preparation method includes: respectively cleaving the nucleic acid fragments of the Vhb protein and the blank plasmid, then connecting and transforming the host strain.

7. The method for preparing a tryptophan-high-yielding strain according to claim 6, characterized by, The preparation method includes: respectively cleaving the nucleic acid fragments of the aroE gene, the aroK gene and the Vhb protein after connecting the fragments, then connecting and transforming the host strain; Preferably, the transformation method includes any one of electrotransformation, chemical transformation method, natural transformation method or conjugation transformation method or a combination of at least two thereof.

8. Use of the tryptophan high-yield strain according to any one of claims 1-5 in the production of tryptophan.

9. A method for producing tryptophan, characterized by, The method includes culturing the tryptophan high-yield strain according to any one of claims 1-5, then performing fermentation.

10. A method for the in vitro production of tryptophan, characterized in that, The method includes preparing tryptophan by reacting the Vhb protein according to any one of claims 1-5, serine, tryptophan synthase and indole; Preferably, the method includes incubating 2-8 mM serine with 0.1-1 mg / mL tryptophan synthase to obtain product 1, and then reacting 2-8 mM indole, product 1 and 1-5 μM Vhb protein to purify and prepare; Preferably, the incubation temperature is 35-40℃ and the incubation time is 0.5-2 h; Preferably, the reaction temperature is 20-30℃, the reaction time is 4-8 h and the reaction speed is 100-300 rpm.

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