CN117467682A - Method for enhancing synthesis of L-tryptophan based on N-terminal coding sequence - Google Patents
Method for enhancing synthesis of L-tryptophan based on N-terminal coding sequence Download PDFInfo
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 title claims abstract description 69
- 229960004799 tryptophan Drugs 0.000 title claims abstract description 34
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 title claims abstract description 14
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 14
- 230000002708 enhancing effect Effects 0.000 title claims abstract description 7
- 108091026890 Coding region Proteins 0.000 title abstract description 11
- 238000000855 fermentation Methods 0.000 claims abstract description 19
- 230000004151 fermentation Effects 0.000 claims abstract description 19
- 239000002773 nucleotide Substances 0.000 claims abstract description 13
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 13
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 241000588724 Escherichia coli Species 0.000 claims description 24
- 108020001507 fusion proteins Proteins 0.000 claims description 16
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
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- 230000014509 gene expression Effects 0.000 abstract description 9
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- 238000001514 detection method Methods 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 3
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 3
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
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Abstract
The invention discloses a method for enhancing synthesis of L-tryptophan based on an N-terminal coding sequence, and belongs to the technical field of genetic engineering. According to the invention, a nucleotide sequence of 10 amino acids is randomly added before the N-terminal coding sequence of the key speed-limiting enzyme anthranilate synthase TrpE is synthesized by L-tryptophan, and is fused with green fluorescent protein to construct a TrpE-NCS random mutation library, and finally, the N-terminal coding sequence with highest expression intensity is obtained by screening and is about 6.2 times higher than that of a control strain. Finally, the optimal N-terminal coding sequence obtained by screening is inserted into the N terminal of the gene trpE, so that the expression of ANTA synthase is obviously enhanced, and the L-tryptophan yield of the constructed engineering strain TRP08 can reach 16.28g/L in a 5L fermentation tank and is improved by 18.06% compared with a control strain. In conclusion, the N-terminal coding sequences can be used as synthetic biological elements for the regulation of gene expression in metabolic pathways.
Description
Technical Field
The invention relates to a method for enhancing synthesis of L-tryptophan based on an N-terminal coding sequence, and belongs to the technical field of genetic engineering.
Background
Accurate regulation of gene expression at desired levels is of great importance for metabolic engineering and synthetic biology, in particular for fine tuning metabolic pathways and optimizing genetic circuits. Thus, a series of synthetic or engineered genetic regulatory elements for controlling gene expression at the transcriptional, translational and protein degradation levels have been established by experimental characterization or computational design, including promoters, terminators, RBS sequences, small regulatory RNAs, and proteolytic tags, among others. In addition, the N-terminal coding sequence strongly influences gene expression at the translation level by influencing ribosome binding efficiency to mRNA and ribosome elongation at the initial stage of translation, which is also an important mechanism for fine-tuning endogenous gene expression in bacteria.
L-tryptophan is an aromatic amino acid essential for humans and animals and is widely used in the production of animal feeds, food additives and pharmaceuticals. Due to the increasing concern about the demands for exhaustion of fossil resources, climate change, sustainable production of the environment, etc., methods for fermentative conversion of renewable raw materials into L-tryptophan by microbial cell factories have also become increasingly attractive. Among them, the E.coli has the advantages of fast growth, easy cultivation, strong metabolism plasticity, applicability to various genetic and genetic engineering tools, etc., and has been widely used in the production of L-tryptophan. Currently, researchers have tried various metabolic engineering strategies to improve the production of L-tryptophan and have made great progress. However, it is still difficult to achieve the desired yield by only metabolic engineering strategies. And static regulation can also lead to a number of problems. Such as difficulty in balancing cell growth with product accumulation; the imbalance between cellular metabolic flux and energy and accumulation of toxic intermediates limit the productivity to meet industrial demands. Therefore, the dynamic regulation and optimization of the synthesis of L-tryptophan by using novel synthesis biological tools has become a very promising option.
Disclosure of Invention
Technical problems:
the invention aims to further optimize the synthesis of L-tryptophan and improve the yield of the L-tryptophan.
The technical scheme is as follows:
in order to solve the technical problems, the invention constructs a TrpE-NCS random mutation library by inserting a nucleotide sequence of 10 random amino acids into the N end of the key rate-limiting enzyme TrpE for synthesizing L-tryptophan and fusing with Green Fluorescent Protein (GFP). And then high-throughput sorting is carried out by combining a flow cytometer, and after primary screening and secondary screening, the mutant with the highest fluorescence intensity is selected for sequencing identification. Finally, the identified optimal sequence is inserted into the N end of the gene trpE to realize the improvement of the yield of the L-tryptophan.
The first object of the invention is to provide a fusion protein, wherein the fusion protein is formed by connecting a nucleotide sequence shown in any one of SEQ ID NO. 1-3 to the N-terminal of a key rate-limiting enzyme TrpE for synthesizing L-tryptophan.
In one embodiment of the present invention, the nucleotide sequence of the TrpE is shown in SEQ ID NO. 5.
The second object of the invention is to provide an engineering bacterium of escherichia coli which expresses the fusion protein.
In one embodiment of the present invention, the E.coli engineering bacteria integrally express the fusion protein.
In one embodiment of the invention, the engineering bacterium of escherichia coli integrates and expresses the fusion protein at the ycgH site.
In one embodiment of the present invention, the E.coli engineering strain TRP07 is used as a host.
In one implementation method of the invention, the engineering strain TRP07 of the escherichia coli is obtained by normal pressure room temperature plasma mutagenesis and optimization of expression of an L-tryptophan synthesis operon, and the yield of L-tryptophan produced by fermentation in a 5L fermentation tank is 13.79g/L and is described in the literature: multidimensional engineering of Escherichia coli for efficient synthesis of L-trytophan, doi 10.1016/j.biortech.2023.129475.
In one implementation method of the invention, the E.coli engineering strain is ANTA synthase, and the yield of L-tryptophan produced in a 5L fermentation tank after N-terminal sequence optimization can reach 16.28g/L.
It is a third object of the present invention to provide a method for enhancing L-tryptophan synthesis by introducing the fusion protein into a host cell.
In one embodiment of the invention, the fusion protein is expressed integrally or expressed free in a host cell.
In one embodiment of the invention, the site of integrated expression is the ycgH site.
In one embodiment of the invention, the host cell comprises the engineered strain TRP07 of escherichia coli.
The invention also provides a method for producing L-tryptophan by utilizing the escherichia coli engineering bacteria through fermentation, and the escherichia coli engineering bacteria are inoculated into a reaction system containing a carbon source to carry out fermentation production of L-tryptophan.
In one embodiment of the invention, the carbon source is selected from glucose, glycerol, sucrose, starch or corn syrup.
In one embodiment of the invention, the reaction conditions are set at a temperature of 37℃at a rotational speed of 600-700rpm and a pH of 7.0-7.2.
In one embodiment of the present invention, the reaction systemComprises 20 to 40g/L glucose, 1 to 5g/L yeast extract, 1 to 5g/L citric acid, 1 to 5g/L ammonium sulfate, 5 to 10g/L dipotassium hydrogen phosphate, 1 to 2g/L sodium chloride, 1 to 2g/L magnesium sulfate heptahydrate, 20 to 40mg/L ferrous sulfate heptahydrate, 5 to 15mg/L manganese sulfate monohydrate, 1 to 5mg/L V B1 、1～5mg/L V H 1-2 mL/L of trace element mixed solution.
In one embodiment of the invention, the trace element mixed solution comprises 5-15 g/L of calcium chloride dihydrate, 0.5-1 g/L of copper sulfate dihydrate, 1-5 g/L of cobalt chloride hexahydrate and 5-10 g/L of zinc sulfate dihydrate.
The invention also provides application of the fusion protein or the escherichia coli engineering strain or the method in the production of L-tryptophan or products containing the L-tryptophan.
The beneficial effects are that:
the invention provides a method for optimizing and enhancing synthesis of L-tryptophan based on an N-terminal coding sequence, which is used for constructing a TrpE-NCS random mutation library by inserting a nucleotide sequence of 10 random amino acids into the N-terminal of a key rate-limiting enzyme TrpE for L-tryptophan synthesis and fusing the nucleotide sequence with Green Fluorescent Protein (GFP). The NCS sequence with highest expression intensity obtained after the primary screening and the re-screening of the pore plate by the combination of the flow cytometer is about 6.2 times higher than that of the control strain. Finally, inserting the NCS sequence with the nucleotide sequence shown as SEQ ID NO.1 into the N end of the gene trpE, and constructing the engineering strain TRP08 with the L-tryptophan yield reaching 16.28g/L in a 5L fermentation tank, which is improved by 18.06% compared with the control strain.
Drawings
Fig. 1: schematic construction of a TrpE-NCS random mutation library.
Fig. 2: the fluorescence intensity of the mutant was identified by re-screening in 96-well plates.
Fig. 3: the 5-L fermenter ferments and analyzes the ability of engineering strains TRP07 and TRP08 to produce L-tryptophan.
Detailed Description
Coli W3110 and JM109 and plasmids pREDCas9 and pGRB referred to in the examples below are laboratory deposited strains and plasmids; pUC19 plasmid was purchased from vast organism.
The following examples relate to media:
LB liquid medium: 10g/L peptone, 5g/L, naCl g/L yeast extract.
LB solid medium: 10g/L peptone, 5g/L, naCl g/L yeast extract and 15g/L agar.
Competent medium: 16g/L peptone, 10g/L, naCl g/L yeast extract.
Resuscitating medium: 10g/L peptone, 5g/L, naCl g/L yeast extract.
Seed culture medium: glucose 30g/L, yeast extract 5g/L, citric acid 2g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 4g/L, magnesium sulfate heptahydrate 1.5g/L, ferrous sulfate heptahydrate 2.8mg/L, manganese sulfate monohydrate 1.2mg/L, VB 1 1mg/L、V H 1mg/L and 1mL/L of trace element mixed solution; trace element mixed solution: 10g/L of calcium chloride dihydrate, 0.6g/L of copper sulfate dihydrate, 4.9g/L of cobalt chloride hexahydrate and 6.4g/L of zinc sulfate dihydrate.
Fermentation medium: glucose 30g/L, yeast extract 3g/L, citric acid 2g/L, ammonium sulfate 3g/L, dipotassium hydrogen phosphate 7g/L, sodium chloride 1g/L, magnesium sulfate heptahydrate 1g/L, ferrous sulfate heptahydrate 30mg/L, manganese sulfate monohydrate 10mg/L, VB 1 1mg/L、V H 1mg/L and 1mL/L of trace element mixed solution; trace element mixed solution: 10g/L of calcium chloride dihydrate, 0.6g/L of copper sulfate dihydrate, 4.9g/L of cobalt chloride hexahydrate and 6.4g/L of zinc sulfate dihydrate.
The L-tryptophan detection method is as follows:
the detection method of L-tryptophan in fermentation broth adopts high performance liquid chromatography HPLC (Agilent 1260series, CA, USA). The measurement was performed using an ultraviolet detector. The mobile phase was acetonitrile/water (10:90 v/v), the flow rate was set to 1ml/min, and the detection wavelength was 278nm.
The primers referred to in the following examples:
p19-NNGFP-F1：5’-CGCTCTAGAGGGTACCTTTCTCCTCTTTAATGAATTCGC-3’；
p19-NNGFP-R1：5’-CGCAAGCTTGGCGTAATCATGGTCATAGC-3’；
p19-NNGFP-F2：5’-CGCAAGCTTCTATTTGTATAGTTCATCCATGCCATG-3’；
p19-NNGFP-R2：
5’-CGCTCTAGAATGNNNNNNNNNNNNNNNNNNNNNNNNNNNCAAACACAAAAACCGACTCTCGAA CTG-3’；
p19-GFP-F1：5’-GACTGAGCTAGCCATAAAGGATCCCCGGGTACCGAG-3’；
p19-GFP-R1：5’-GATGAACTATACAAATAGAAGCTTGGCGTAATCATGGTCATAGC-3’；
p19-GFP-F2：5’-CTATTTGTATAGTTCATCCATGCCATGTGTAATCC-3’；
p19-GFP-R2：5’-ATGCACAGGAGACTTTCTGAAGTAAAGGAGAAGAACTTTTCACTGGAGTTG-3’；p19-GFP-F3：5’-AAAAGTTCTTCTCCTTTACTTCAGAAAGTCTCCTGTGCATGATGCG-3’；
p19-GFP-R3：
5’-TTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGCGAATTCATTAAAGAGGAGAAAGGTACCCAT
GCAAACACAAAAACCGACTCTCG-3’；
sgRNA-F：
5’-AGTCCTAGGTATAATACTAGTGATAATAATCCGATAAGTAAGTTTTAGAGCTAGAA-3’；
sgRNA-R：
5’-TTCTAGCTCTAAAACGTAAATCCTGACCGAATTCGACTAGTATTATACCTAGGACT-3’；
ZH-F1：5’-CACCCTCCTGGTCATCCTTATTGC-3’；
ZH-R1：
5’-CTCCTCTTTAATGAATTCCATTATACGAGCCGGATGATTAATTGTCAAAGATTAACCATCCATTCATTG GATTAACAAACATTCC-3’；
ZH-F2：
5’-TAATCATCCGGCTCGTATAATGGAATTCATTAAAGAGGAGAAAGGTACCCATGAATTATCAGAACGA CGATTTACGCATC-3’；
ZH-R2：5’-TTGAAGCCGACCGGACAGAAAAGCCCTGATGCCAGTTCG-3’；
ZH-F3：5’-GCGAACTGGCATCAGGGCTTTTCTGTCCGGTCGGCTTCAAAAATGG-3’；
ZH-R3：5’-TTTTTTAGCTAACGGCGGGATTACCCGCGACGCGCTTTTAC-3’；
ZH-F4：5’-TAAAAGCGCGTCGCGGGTAATCCCGCCGTTAGCTAAAAAACCG-3’；
ZH-R4：5’-AAGGCCGCGACGGTAAAAATG-3’。
example 1: construction of TrpE-NCS mutant library
The primer p19-NNGFP-F2/R2 is used for inserting a nucleotide sequence of 10 random amino acids before the N end of the key rate-limiting enzyme TrpE for synthesizing L-tryptophan, then the nucleotide sequence is fused with green fluorescent protein GFP, and the primer p19-NNGFP-F1/R1 is used for cloning the nucleotide sequence to a multiple cloning site of a vector pUC19 to construct a plasmid pUC19-NNtrpEgfp (figure 1). The constructed plasmid is then transformed into colibacillus JM109, a large number of transformants grow after overnight culture, the transformants grown on LB plates are washed off and then stored in glycerol, and a mutant library, i.e. a library composed of mixed mutant strains, is obtained.
GFP and TrpE were amplified by primers p19-GFP-F2/R2 and p19-GFP-F3/R3, respectively, and then TrpE and GFP were fused using primers p 19-GFP-F3/R2. And performing inverse PCR on the vector pUC19 by using the primer p19-GFP-F1/R1 to obtain the linearized vector pUC19. And then connecting the TrpE-GFP fusion fragment with a linearization vector pUC19 by a homologous recombination mode to construct a plasmid pUC19-trpEGFP, and finally transforming the constructed plasmid into Escherichia coli JM109 to obtain the recombinant strain JM109/pUC19-trpEGFP.
Example 2: sorting of high fluorescence intensity mutants
The TrpE-NCS mutant library successfully constructed in example 1 was repeatedly washed three times with PBS buffer, and the OD of the mixed mutant strain was obtained 600 Diluted to about 0.3. The recombinant JM109/pUC19-trpEGFP constructed in example 1 was used as a control strain, and the mutants with high fluorescence intensity in the mutant library were sorted by a flow cytometer, and the sorted fluorescence intensity was significantly higher than that of the mutant strain of the control strain. The strain obtained by the sorting was spread on LB plates for overnight culture. Single colonies grown on the plates were re-inoculated into 96-well plates and incubated at 37℃and 200rpm for 12h (FIG. 1). The relative fluorescence intensity of each mutant was measured to find that about 90% of the mutants had higher fluorescence values than the control strain. The mutant with the highest fluorescence intensity was increased by about 6.2-fold over the control strain (fluorescence intensity 1104) (FIG. 2). Several strains with higher fluorescence intensity than the control strain were selected for sequencing to obtain the N-terminal coding sequence (Table 2).
TABLE 2N-terminal coding sequences
| Numbering device | Sequence(s) | Fluorescence intensity (a.u.) | |
| N1 | GCCCTGATGCCCCCGCGCCGGAGTGCG | 6845 | SEQ ID NO.1 |
| N2 | CTATATGTCAGCTCACTACCATCATTA | 6675 | SEQ ID NO.2 |
| N3 | ACAGATACCCGAAGTTTTGTTAGTATG | 6521 | SEQ ID NO.3 |
| N4 | ATGCCCCATCGACCTATGCAATGCCTGCCA | 4251 | SEQ ID NO.4 |
Example 3: construction of E.coli engineering strain TRP08
The N-terminal coding sequence N1 selected in example 2 was inserted into the gene trpE S40F Is the N-terminal of (c). And designing sgRNA of the targeting gene ycgH according to the nucleotide sequence of the pseudogene locus ycgH to be inserted. The sgRNA of ycgH amplified by using sgRNA-F/sgRNA-R was ligated to plasmid pGRB (Addgene # 71539) by homologous recombination to construct plasmid pGRB-ycgH-sgRNA. Then taking the genome of the engineering strain TRP07 of the escherichia coli as a template, amplifying the upstream and downstream homology arms (-500 bp) of the pseudogene locus ycgH by using primers ZH-F1/R1, ZH-F2/R2, ZH-F3/R3 and ZH-F4/R4, and then fusing by using primers ZH-F1 and ZH-R4 to form a complete homology arm DNA fragment. Plasmids pREDCas9 (Addgene # 71541) and pGRB-ycgH-sgRNA and the homology arm DNA fragment were then transformed into strain TRP07 by means of shock transformation.
The specific operation is as follows: strain TRP07 carrying plasmid pREDCas9 was cultured at 30℃to OD in a competent medium containing 50. Mu.g/mL spectinomycin 600 About 0.1-0.2, and then 0.1mM IPTG is added to induce Cas9 protein expression. When OD is 600 Cells were collected by centrifugation and washed repeatedly three times to prepare competent cells when raised to 0.6-0.7, and then the corresponding homology arm DNA fragment and plasmid pGRB-ycgH-sgRNA were simultaneously electrotransferred into competent cells with a voltage of 1.85kV, and 1mL of resuscitation medium was added immediately after the electrotransfer was completed and cultured at 30℃for 2 hours. Finally, the transformants were plated on LB solid plates containing 50. Mu.g/mL of ampicillin and spectinomycin, cultured overnight at 30℃and randomly selected for colony PCR verification and DNA sequencing. And after successful DNA sequencing verification, the engineering strain TRP08 is constructed.
Example 4: fermentation experiment of engineering strain TRP08 in 5-L fermentation tank
The strain TRP08 constructed in example 3 was first cultured on LB solid medium at 37℃for 12 hours for activation, and the activated strain was inoculated into a 5-L fermenter containing 3-L seed medium. The pH of the seed medium was maintained at 7.0 by automatic addition of ammonia and the temperature was maintained at 37 ℃, and the dissolved oxygen was maintained above 30% by varying the stirrer speed and aeration rate. When OD is 600 When 10-12 is reached, the excess broth is drained leaving only 450mL for batch fermentation. During batch fermentation, the pH was also maintained at 7.0, the temperature was maintained at 37℃and the dissolved oxygen was maintained above 30%. After the initial sugar consumption, the glucose concentration of the fermentation liquid is controlled below 2g/L, and a glucose solution with the concentration of 80% is added into a fermentation tank in the subsequent fermentation process so as to maintain the growth of thalli and the synthesis of products. After 48 hours of fermentation culture, the yield of the engineering strain TRP08 reaches 16.28g/L, and is 18.06% higher than that of the control strain TRP07 (figure 3).
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A fusion protein is characterized in that the fusion protein is formed by connecting a nucleotide sequence shown in any one of SEQ ID NO. 1-3 with the N-terminal of TrpE which is a key rate-limiting enzyme for synthesizing L-tryptophan, and the nucleotide sequence of the TrpE is shown in SEQ ID NO. 5.
2. An engineered escherichia coli strain, wherein the fusion protein of claim 1 is expressed.
3. The engineered escherichia coli of claim 2, wherein the engineered escherichia coli is integrally expressed as the fusion protein of claim 1.
4. An engineered escherichia coli as claimed in claim 3, wherein the fusion protein of claim 1 is expressed integrally at the ycgH locus.
5. The engineered escherichia coli strain according to any one of claims 2 to 4, wherein the engineered escherichia coli strain TRP07 is used as a host.
6. A method of enhancing L-tryptophan synthesis by introducing the fusion protein of claim 1 into a host cell.
7. The method of claim 6, wherein the fusion protein is expressed integrally or expressed free in a host cell.
8. The method according to claim 6 or 7, characterized in that the host cell comprises the e.coli engineering strain TRP07.
9. A method for producing L-tryptophan by fermentation, which is characterized in that the escherichia coli engineering bacteria of any one of claims 2 to 4 are inoculated into a reaction system containing a carbon source for fermentation production of L-tryptophan.
10. Use of the fusion protein of claim 1, or the engineered strain of e.coli of any one of claims 2 to 4, or the method of claim 9, for the production of L-tryptophan or a product containing L-tryptophan.
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