Shikimic acid pathway artificial operon as well as construction method and application thereof

Abstract

The invention provides a shikimic acid pathway artificial operon and a construction method and application thereof.According to the artificial operon, shikimic acid pathway related genes are integrated and connected in different modes, a trc promoter and an RBS ribose binding site sequence are optimized, and the artificial operon formed through combination has the advantages of being stable, efficient and wide in applicability; different from a relatively extensive shikimic acid way transformation mode in the past, the operon can obviously improve the accumulation capacity of aromatic amino acid, and when the operon is applied to an L-phenylalanine production strain, the yield of L-phenylalanine reaches 105 g / L, and the saccharic acid conversion rate reaches 26.5%.

Description

Technical Field

[0001] The invention relates to the field of biotechnology and fermentation engineering technology, in particular to an artificial shikimate pathway operon and a construction method and application thereof. Background Art

[0002] L-phenylalanine is one of the eight essential amino acids for the human body. It has many applications in food, medicine, feed, and health care. The annual global demand is about 40,000 to 50,000 tons. At present, the indicators of mainstream L-phenylalanine-producing bacteria are mostly around 80g / L and 23% conversion rate. It is urgent to further improve the production efficiency of the production bacteria to further reduce production costs.

[0003] The shikimic acid pathway connects the central metabolic pathway with the aromatic amino acid synthesis pathway, and is an important intermediate component of the aromatic amino acid synthesis pathway. A total of seven enzymes participate in the reaction in the entire pathway. As an intermediate pathway connecting the upstream and downstream pathways, once the upstream carbon metabolic flow is blocked when passing through the shikimic acid pathway, it will not only affect the production of L-phenylalanine, but also accumulate toxic byproducts such as shikimic acid, affecting bacterial growth. At present, due to the complexity of enzymes related to the shikimic acid pathway, the transformation of aromatic amino acid production bacteria does not involve too many genes related to the shikimic acid pathway, but is limited to the enhanced expression and gene modification of a single gene or two genes, which undoubtedly limits the development of aromatic bio-products. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an artificial operon of shikimic acid pathway.

[0005] The technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned artificial operon of the shikimate pathway.

[0006] The technical problem to be solved by the present invention is to provide the application of the above-mentioned artificial operon of shikimate pathway.

[0007] In order to solve the above technical problems, the technical solution of the present invention is:

[0008] An artificial shikimate pathway operon, aro(GEDABK), including aroE L241I / T61W 、aroD、aroB、aroG D146N , aroA, aroK genes, the transcription of the operon is controlled by the same trc promoter and RBS09 ribosome binding site, and the transcription of the operon is controlled by the same trc terminator. The gene connection mode and the location of the gene in the operon are optimized, specifically:

[0009] (1) The operon gene sequence is trc promoter, RBS09, aroG D146N 、AroE L241I / T61W, aroD, aroA, aroK, aroB, trc terminator;

[0010] (2) trc promoter, RBS09 ribose binding site and aroG D146N The genes are directly connected end to end;

[0011] (3)aroG D146N 、aroE L241I / T61W The genes are directly connected end to end;

[0012] (4)aroE L241I / T61W , aroD gene is connected using the first spacer sequence shown in SEQ ID NO.3 in the sequence listing;

[0013] (5) aroD and aroA genes are directly linked end to end;

[0014] (6) aroA and aroK genes are linked using the second spacer sequence shown in SEQ ID NO.4 in the sequence listing;

[0015] (7) aroK and aroB genes are linked using the third spacer sequence shown in SEQ ID NO.5 in the sequence listing;

[0016] (8) The aroB and trc terminators are directly connected at the beginning and the end.

[0017] Preferably, the shikimate pathway artificial operon, the operon genes are all derived from E. coli W3110, with the accession number of ATCC 273250, wherein aroD, aroA, aroK, aroB are all wild-type genes;

[0018] AroE L241I / T61W The gene has undergone point mutations, namely, base 182 AC changed to TG, and base 721 CTG changed to ATC; aroG D146N The gene underwent a point mutation, where the G at position 436 was changed to an A.

[0019] Preferably, in the above-mentioned artificial operon of shikimate pathway, the trc promoter has the nucleotide sequence shown in SEQ ID NO.1 in the sequence list; the RBS09 ribosome binding site has the nucleotide sequence shown in SEQ ID NO.2 in the sequence list; and the aroG D146N Having the nucleotide sequence shown in SEQ ID NO.6 in the sequence list; AroE L241I / T61WIt has the nucleotide sequence shown in SEQ ID NO.7 in the sequence listing; aroD has the nucleotide sequence shown in SEQ ID NO.8 in the sequence listing; aroA has the nucleotide sequence shown in SEQ ID NO.9 in the sequence listing; aroK has the nucleotide sequence shown in SEQ ID NO.10 in the sequence listing; aroB has the nucleotide sequence shown in SEQ ID NO.11 in the sequence listing; and the trc terminator has the nucleotide sequence shown in SEQ ID NO.12 in the sequence listing.

[0020] Preferably, the above-mentioned artificial operon of the shikimate pathway, the operon aro (GEDABK) has the nucleotide sequence shown in SEQ ID NO.13 in the sequence listing.

[0021] The method for constructing the above-mentioned artificial operon of the shikimate pathway comprises the following specific steps:

[0022] ① Using the E. coli W3110 genome as a template, aroG D146N -UP-S, aroG D146N -UP-A, aroG D146N -DN-S, aroG D146N -DN-A was used as primer, and two segments of genes were obtained by HS enzyme PCR amplification. Then, aroG was obtained by HS enzyme overlapping PCR using them as templates. D146N Complete gene fragments;

[0023] ② Using the E. coli W3110 genome as a template, AroE L241I / T61W -Us, AroE L241I / T61W -Ua、AroE L241I / T61W -Ms.AroE L241I / T61W -Ma, AroE L241I / T61W -Ds, AroE L241I / T61W -Da was used as primer, and HS enzyme PCR was used to amplify three gene fragments, which were then used as templates to obtain aroE by HS enzyme overlapping PCR. L241I / T61W Complete gene fragments;

[0024] ③ Using the E. coli W3110 genome as a template and AroD-S and AroD-A as primers, HS enzyme PCR amplification was performed to obtain a gene fragment, which is the complete aroD gene fragment;

[0025] ④ Using the E. coli W3110 genome as a template and AroA-S and AroA-A as primers, HS enzyme PCR was used to obtain a gene fragment, which is the complete aroA gene fragment;

[0026] ⑤ Using the E. coli W3110 genome as a template and AroB-S and AroK-A as primers, HS enzyme PCR was used to obtain a gene fragment, which is the complete aroB and aroK gene fragment;

[0027] ⑥ With aroG D146N Complete gene fragment, aroE L241I / T61W The complete gene fragment and the complete aroD gene fragment were used as templates, and aroG D146N -UP-S and aroD-a were used as primers, and aroG was obtained by overlapping PCR with HS enzyme. D146N +aroE L241I / T61W + complete aroD gene fragment;

[0028] ⑦Using the complete aroA gene fragment, the complete aroB and aroK gene fragments as templates, and aroA-S and aroK-A as primers, the complete gene fragment of aroA+aroB+aroK was obtained by HS enzyme overlapping PCR;

[0029] ⑧ aroG D146N +aroE L241I / T61W +aroD complete gene fragment, aroA+aroB+aroK complete gene fragment as template, and aroG D146N -UP-S and aroK-a were used as primers, and the complete gene of operon aro (GEDABK) was obtained by HS enzyme overlapping PCR.

[0030] The application of the above-mentioned artificial operon of shikimate pathway in preparing L-phenylalanine producing strain.

[0031] Preferably, the application of the above-mentioned artificial operon of the shikimate pathway adopts the Escherichia coli strain F-12 as the starting bacteria, which is the Escherichia coli strain F-12 described in patent CN202211190808.1 (a phenylalanine-producing bacterium). The bacterium can also be purchased from the Metabolic Engineering Laboratory of Tianjin University of Science and Technology. The operon is applied to the strain F-12, and the beneficial effects of the operon are measured by L-phenylalanine production and sugar-acid conversion rate.

[0032] Preferably, the above-mentioned artificial operon of shikimate pathway is used to obtain the genetically engineered bacteria FG103 for producing L-phenylalanine in the form of plasmid application.

[0033] Preferably, in the application of the above-mentioned artificial operon of the shikimate pathway, the plasmid is pTrcM01, which has some characteristics of the pTrc99a plasmid vector, the LacI gene is removed from the pTrc99a plasmid vector (having the nucleotide sequence shown in SEQ ID NO.15 in the sequence listing), and the aro (GEDABK) operon is integrated into the vector, the complete gene of the operon aro (GEDABK) is connected to the modified ptrc99a plasmid vector through an enzyme ligation system to form a circular plasmid pTrcM01, the plasmid pTrcM01 has the nucleotide sequence shown in SEQ ID NO.14 in the sequence listing, and the F-12 strain having the plasmid pTrcM01 is named FG103.

[0034] Preferably, the above-mentioned artificial operon of shikimate pathway is used to obtain the genetically engineered bacteria FG105 for producing L-phenylalanine by genome integration.

[0035] Preferably, the application of the above-mentioned artificial operon of the shikimate pathway, the genome integration refers to inserting the operon sequence into the rph pseudogene site of the chromosomal genome of the starting bacteria F-12, and the F-12 L-phenylalanine producing bacteria with the aro (GEDABK) operon is named FG105.

[0036] A genetically engineered bacterium FG103 is obtained by the application method of the artificial operon of the shikimic acid pathway.

[0037] A genetically engineered bacterium FG105 is obtained by the application method of the artificial operon of the shikimic acid pathway.

[0038] The specific steps of using any one of the genetically engineered bacteria FG103 and FG105 in the fermentation production of L-phenylalanine are as follows:

[0039] (1) Inoculate any of the E. coli strains FG103 and FG105 from a 20% glycerol tube kept at -80°C for activation culture on an LB solid slant at 37°C for 12 h for two generations, and use the bacteria on the second generation solid slant as the starting bacteria for the seed tank (the slant strains are preferred if they are free of foreign bacteria and have smooth colonies);

[0040] (2) Fermentation tank seed culture: The solid slant bacteria were washed with sterile saline and then all inoculated into a 5 L fermentation tank for seed culture. The culture medium volume was 2 L. The culture conditions were 37° C., pH 7.0, and dissolved oxygen 35-55%. The culture was performed until the OD600nm reached 25.

[0041] (3) Fermentation in a fermenter with an inoculation volume of 20%, a fermentation volume of 3 L, a fermentation temperature of 36° C., a dissolved oxygen content of 30-50%, and a pH of 7.2.

[0042] Preferably, the use of any strain of the above-mentioned genetically engineered bacteria FG103 and FG105 adopts a seed culture medium comprising 30 g / L glucose, 3 g / L yeast powder, 1.2 g / L peptone, 0.5 g / L MgSO4.7H2O, 3.0 g / L KH2PO4, 2.0 g / L ammonium sulfate, 10 mg / L FeSO4.7H2O, 1 mg / L vitamin H, and 2 mg / L vitamin B1.

[0043] Preferably, the application of any strain of the genetically engineered bacteria FG103 and FG105 adopts a fermentation medium comprising: MgSO4.7H2O 2g / L, yeast powder 4g / L, peptone 2g / L, ammonium sulfate 1g / L, K2HPO4.3H2O2.3g / L, glutamic acid 2g / L, FeSO4.7H2O 30mg / L, MnSO4 10mg / L, CoCl2.6H2O 15mg / L, V B1 、V B3 、V B5 2mg / L each.

[0044] The above culture media can be prepared using standard methods.

[0045] The use of any of the genetically engineered bacteria FG103 and FG105 is used to conduct fermentation experiments in mechanically stirred fermenters to verify the L-phenylalanine production performance of strains FG103 and FG105, in order to verify the beneficial effects of the operon.

[0046] Beneficial effects:

[0047] The above-mentioned artificial operon of the shikimate pathway integrates genes related to the shikimate pathway, connects them in different ways, and optimizes the trc promoter and RBS ribose binding site sequence. The artificial operon formed by the combination has the advantages of stability, high efficiency and wide applicability. Different from the previous relatively extensive shikimate pathway modification methods, the operon can significantly improve the accumulation capacity of aromatic amino acids. When applied to L-phenylalanine production strains, the L-phenylalanine output reached 105g / L, and the sugar-acid conversion rate reached 26.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram for the operon aro(GEDABK).

[0049] Figure 2 Schematic diagram of the structure of plasmid pTrcM01. DETAILED DESCRIPTION

[0050] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0051] The percentage sign "%" involved in the embodiments, unless otherwise specified, refers to mass percentage. The percentage of the solution refers to the mass of the solute and the volume percentage of the liquid, which refers to the volume ratio of the solution at 25°C.

[0052] The starting strain F-12 used in the example, information can be found in patent CN202211190808.1.

[0053] The gene sequences involved are as follows:

[0054] The trc promoter has a nucleotide sequence shown in SEQ ID NO.1 in the sequence listing.

[0055] RBS09 has the nucleotide sequence shown in SEQ ID NO.2 in the sequence listing.

[0056] The spacer sequence 1 has a nucleotide sequence shown in SEQ ID NO.3 in the sequence listing.

[0057] The spacer sequence 2 has a nucleotide sequence shown in SEQ ID NO.4 in the sequence listing.

[0058] The spacer sequence 3 has the nucleotide sequence shown in SEQ ID NO.5 in the sequence listing.

[0059] aroG D146N It has a nucleotide sequence shown in SEQ ID NO.6 in the sequence listing.

[0060] AroE L241I / T61W It has a nucleotide sequence shown in SEQ ID NO.7 in the sequence listing.

[0061] aroD has a nucleotide sequence shown in SEQ ID NO.8 in the sequence listing.

[0062] aroA has a nucleotide sequence shown in SEQ ID NO.9 in the sequence listing.

[0063] aroK has a nucleotide sequence shown in SEQ ID NO.10 in the sequence listing.

[0064] aroB has a nucleotide sequence shown in SEQ ID NO.11 in the sequence listing.

[0065] The trc terminator has the nucleotide sequence shown in SEQ ID NO.12 in the sequence listing.

[0066] The aro (GEDABK) operon has a nucleotide sequence shown in SEQ ID NO.13 in the sequence listing.

[0067] The pTrcM01 plasmid has the nucleotide sequence shown in SEQ ID NO.14 in the sequence listing.

[0068] The pTrc99a plasmid has the nucleotide sequence shown in SEQ ID NO.15 in the sequence listing.

[0069] Example 1

[0070] 1. Methods of gene editing

[0071] The gene editing method used was referenced in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.).

[0072] The primers used in the construction of strains are listed in Table 1.

[0073] Table 1 Primers involved in strain construction

[0074]

[0075]

[0076] Example 2

[0077] This example is intended to illustrate the construction method of an operon, which has 5798 bp of bases, and is overlapped in pairs by a PCR overlap system. The specific steps are as follows:

[0078] ① Using the E. coli W3110 genome as a template, aroG D146N -UP-S, aroG D146N -UP-A, aroG D146N -DN-S, aroG D146N -DN-A was used as primer, and two segments of genes were obtained by HS enzyme PCR amplification. Then, aroG was obtained by HS enzyme overlapping PCR using them as templates. D146N Complete gene fragment.

[0079] ② Using the E. coli W3110 genome as a template, AroE L241I / T61W -Us, AroE L241I / T61W -Ua、AroE L241I / T61W -Ms.AroE L241I / T61W -Ma, AroE L241I / T61W -Ds, AroEL241I / T61W -Da was used as primer, and HS enzyme PCR was used to amplify three gene fragments, which were then used as templates to obtain aroE by HS enzyme overlapping PCR. L241I / T61W Complete gene fragment.

[0080] ③ Using the Escherichia coli W3110 genome as a template and AroD-S and AroD-A as primers, a gene fragment was obtained by HS enzyme PCR amplification, which is the complete aroD gene fragment.

[0081] ④ Using the Escherichia coli W3110 genome as a template and AroA-S and AroA-A as primers, a gene fragment was obtained by HS enzyme PCR amplification, which is the complete aroA gene fragment.

[0082] ⑤ Using the Escherichia coli W3110 genome as a template and AroB-S and AroK-A as primers, HS enzyme PCR amplification was performed to obtain a gene fragment, namely the complete aroB and aroK gene fragments.

[0083] ⑥ With aroG D146N Complete gene fragment, aroE L241I / T61W The complete gene fragment and the complete aroD gene fragment were used as templates, and aroG D146N -UP-S and aroD-a were used as primers, and aroG was obtained by overlapping PCR with HS enzyme. D146N +aroE L241I / T61W + complete aroD gene fragment.

[0084] ⑦ Using the complete aroA gene fragment, complete aroB and aroK gene fragments as templates, and aroA-S and aroK-A as primers, HS enzyme overlapping PCR was used to obtain the complete gene fragment of aroA+aroB+aroK.

[0085] ⑧ aroG D146N +aroE L241I / T61W +aroD complete gene fragment, aroA+aroB+aroK complete gene fragment as template, and aroG D146N -UP-S and aroK-a were used as primers, and the complete gene of operon aro (GEDABK) was obtained by HS enzyme overlapping PCR.

[0086] The PCR amplification system used is shown in Table 2-1, and the PCR overlap system is shown in Table 2-2:

[0087] Table 2-1 PCR amplification system

[0088]

[0089] Table 2-2PCR overlapping system

[0090]

[0091] Example 3

[0092] This example is intended to illustrate the construction method of plasmid pTrcM01, and the specific steps are as follows:

[0093] The complete gene of the operon aro (GEDABK) obtained in Example 2 was connected to the ptrc99a plasmid vector through an enzyme ligation system to form a circular plasmid.

[0094] The enzyme-linked system is shown in Table 3:

[0095] Table 3 Enzyme-linked system

[0096]

[0097] Example 4

[0098] This example aims to illustrate the method of integrating the operon aro (GEDABK) at the rph pseudogene site, that is, the method of obtaining strain FG105, and the specific steps are as follows:

[0099] ① Using pGRB-rph-S and pGRB-rph-A as primers, construct a DNA fragment containing the target sequence used in pGRB-mbhA through a PCR annealing program, and transform it into DH5α competent cells, screen to obtain positive transformants, and extract plasmid pGRB-rph.

[0100] ② The pGRB-rph plasmid obtained in step ① and the operon gene obtained in Example 2 were electroporated into the F-12 strain to obtain positive transformants as genetically engineered bacteria for producing L-phenylalanine, and named FG105.

[0101] Example 5

[0102] This example is intended to illustrate the method for obtaining strain FG103, and the specific steps are as follows:

[0103] The plasmid pTrcM01 obtained in Example 3 was electroporated into the F-12 strain to obtain a positive transformant as a genetically engineered bacterium producing L-phenylalanine, which was named FG103.

[0104] Example 6

[0105] This example is intended to illustrate the application method of strain FG103, that is, the production performance verification of strain FG103, and the steps are as follows:

[0106] ① Seed culture: Use a 5L mechanical stirring fermenter, the culture temperature is 37℃, and the culture pH is maintained at 7.0±0.2 by automatically adding 25% ammonia solution. The culture dissolved oxygen value is maintained at 45% by adjusting the stirring speed or ventilation volume. When OD 600nm The inoculation requirement is met when the PH value is 25; the seed culture medium used is: glucose 30g / L, yeast powder 3g / L, peptone 1.2g / L, MgSO4.7H2O 0.5g / L, KH2PO4 3.0g / L, ammonium sulfate 2.0g / L, FeSO4.7H2O 10mg / L, vitamin H1mg / L, vitamin B1 2mg / L, ampicillin 100mg / L.

[0107] ② Fermentation culture: A 5L mechanical stirring fermenter was used, the fermentation inoculation volume was 20%, the culture temperature was 36°C, the culture pH was maintained at 7.0±0.2 by automatically adding 25% ammonia solution, the culture dissolved oxygen value was maintained at 40% by adjusting the stirring speed or ventilation volume, and the glucose concentration in the tank was controlled at ≤0.5g / L by adding 80% glucose solution, and the fermentation cycle was 52h; the fermentation medium used was: MgSO4.7H2O 2g / L, yeast powder 4g / L, peptone 2g / L, ammonium sulfate 1g / L, K2HPO4.3H2O 2.3g / L, glutamic acid 2g / L, FeSO4.7H2O30mg / L, MnSO4 10mg / L, CoCl2.6H2O 15mg / L, V B1 、V B3 、V B5 2mg / L each, ampicillin 100mg / L.

[0108] After fermentation verification, strain FG103 was able to produce 103g / L phenylalanine, an increase of 14.2% over strain F-12, and the sugar-acid conversion rate was 26.3%, which was the same as strain F-12.

[0109] Example 7

[0110] This example is intended to illustrate the application method of strain FG105, namely, the production performance verification of strain FG105. The steps are consistent with those of Example 6, except that strain FG105 does not require the addition of any antibiotics (ampicillin).

[0111] After fermentation verification, strain FG103 was able to produce 105g / L phenylalanine, which was 16.4% higher than strain F-12, and the sugar-acid conversion rate was 26.5%, which was slightly higher than strain F-12.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. The construction steps of the strain of the present invention are not in any particular order. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. An artificial shikimate pathway operon, characterized in that: It is aro (GEDABK) and has the nucleotide sequence shown in SEQ ID NO.13 in the sequence listing.

2. The method for constructing the artificial operon of the shikimate pathway according to claim 1, characterized in that: The specific steps are as follows: ① Using the E. coli W3110 genome as a template, aroG D146N -UP-S, aroG D146N -UP-A, aroG D146N -DN-S, aroG D146N -DN-A was used as primer, and two segments of genes were obtained by HS enzyme PCR amplification. Then, aroG was obtained by HS enzyme overlapping PCR using them as templates. D146N Complete gene fragments; ② Using the E. coli W3110 genome as a template, AroE L241I / T61W -Us, AroE L241I / T61W -Ua、AroE L241I / T61W -Ms.AroE L241I / T61W -Ma, AroE L241I / T61W -Ds, AroE L241I / T61W -Da was used as primer, and HS enzyme PCR was used to amplify three gene fragments, which were then used as templates to obtain aroE by HS enzyme overlapping PCR. L241I / T61W Complete gene fragments; ③ Using the E. coli W3110 genome as a template and AroD-S and AroD-A as primers, HS enzyme PCR amplification was performed to obtain a gene fragment, which is the complete aroD gene fragment; ④ Using the E. coli W3110 genome as a template and AroA-S and AroA-A as primers, HS enzyme PCR was used to obtain a gene fragment, which is the complete aroA gene fragment; ⑤ Using the E. coli W3110 genome as a template and AroB-S and AroK-A as primers, HS enzyme PCR was used to obtain a gene fragment, which is the complete aroB and aroK gene fragment; ⑥ With aroG D146N Complete gene fragment, aroE L241I / T61W The complete gene fragment and the complete aroD gene fragment were used as templates, and aroG D146N -UP-S and aroD-a were used as primers, and aroG was obtained by overlapping PCR with HS enzyme. D146N +aroE L241I / T61W + complete aroD gene fragment; ⑦Using the complete aroA gene fragment, the complete aroB and aroK gene fragments as templates, and aroA-S and aroK-A as primers, the complete gene fragment of aroA+aroB+aroK was obtained by HS enzyme overlapping PCR; ⑧ aroG D146N +aroE L241I / T61W +aroD complete gene fragment, aroA+aroB+aroK complete gene fragment as template, and aroG D146N -UP-S and aroK-a were used as primers, and the complete gene of operon aro (GEDABK) was obtained by HS enzyme overlapping PCR.

3. Use of the artificial shikimate pathway operon according to claim 1 in preparing an L-phenylalanine producing strain.

4. The use of the artificial shikimate pathway operon according to claim 3, characterized in that: The starting bacteria used were the Escherichia coli strain F-12 described in patent CN202211190808.1, and the operon was applied to the strain F-12.

5. The use of the artificial shikimate pathway operon according to claim 3 or 4, characterized in that: The genetically engineered bacteria FG103 for producing L-phenylalanine was obtained by using plasmids.

6. The use of the artificial shikimate pathway operon according to claim 5, characterized in that: The plasmid is pTrcM01, having the nucleotide sequence shown in SEQ ID NO.14 in the sequence table. The F-12 strain having the plasmid pTrcM01 is named FG103.

7. The use of the artificial shikimate pathway operon according to claim 3 or 4, characterized in that: The genetically engineered bacterium FG105 for producing L-phenylalanine was obtained by genome integration application.

8. The use of the artificial shikimate pathway operon according to claim 7, characterized in that: The genome integration refers to inserting an operon sequence into the rph pseudogene site of the chromosome genome of the starting bacteria F-12, and the F-12 L-phenylalanine producing bacteria having the aro (GEDABK) operon is named FG105.

9. Any one of the genetically engineered bacteria FG103 and FG105, characterized in that: The genetically engineered bacterium FG103 is obtained by the application method of the artificial operon of the shikimate pathway described in claim 6; the genetically engineered bacterium FG105 is obtained by the application method of the artificial operon of the shikimate pathway described in claim 8.

10. Use of any one of the genetically engineered bacteria FG103 and FG105 according to claim 9 in the fermentation production of L-phenylalanine, characterized in that: The specific steps are as follows: (1) Inoculate any of the E. coli strains FG103 and FG105 from a 20% glycerol tube kept at -80°C on an LB solid slant for activation culture at 37°C for 12 h for two generations, and use the bacteria on the second generation solid slant as the starting bacteria for the seed tank; (2) Fermentation tank seed culture: The solid slant bacteria were washed with sterile saline and then all inoculated into a 5 L fermentation tank for seed culture. The culture medium volume was 2 L. The culture conditions were 37° C., pH 7.0, and dissolved oxygen 35-55%. The culture was performed until the OD600nm reached 25. (3) Fermentation in a fermenter with an inoculation volume of 20%, a fermentation volume of 3 L, a fermentation temperature of 36° C., a dissolved oxygen content of 30-50%, and a pH of 7.2.

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