RU2806731C1 - Application of transporter carrier gene that improves l-tryptophan production efficiency in escherichia coli - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a genetically engineered bacterium for producing L-tryptophan, where the original bacterium is modified with the ywkB gene, and the resulting genetically engineered bacterium has the activity of producing a higher yield of L-tryptophan than the original bacterium; where the modification consists of modifying the pseudogenic locus yeep with the ywkB gene.

EFFECT: when the strain is fermented in a shaking flask, it can accumulate 15.2 g/l of L-tryptophan within 24 hours, which is 35% higher than that of the control strain.

13 cl, 4 dwg, 2 ex

Description

This application claims priority to Chinese Patent Application No. 201910828913.5, filed on September 3, 2019 with the National Intellectual Property Office of China, entitled “APPLICATION OF A TRANSPORT CARRIER GENE THAT IMPROVES L-TRYPTOPHAN PRODUCTION EFFICIENCY IN ESCHERICHIA COLI,” the contents of which are included in this document by reference in its entirety.

TECHNICAL FIELD

The present invention relates to metabolic engineering and biotechnology, in particular to a metabolic transporter gene derived from Bacillus subtilis, which can effectively improve the amino acid production efficiency of an engineered L-tryptophan-producing Escherichia coli strain.

BACKGROUND ART

Tryptophan, as an amino acid, which plays an important role in regulating the growth and development of organisms and has good pharmacological effects such as antidepressant effects and sleep promotion, plays an important role in food, medicine, feed and other industries. Given further research on this amino acid, its use will become more widespread, which will sooner or later ensure a sharp increase in market demand for tryptophan. Thus, the search for a more effective and cheaper way to obtain tryptophan is gradually attracting people's attention. Currently, methods for its production include a chemical synthesis method, an enzymatic reaction method and a direct microbiological fermentation method. The direct fermentation method is a method of directly producing L-tryptophan through fermentation with inexpensive raw materials such as glucose as a substrate. This method has the advantages of easy handling, low cost, high productivity, short cycle time and low environmental pollution. However, studies of the internal environment of microorganisms and the equilibrium relationships between various metabolic pathways are still not comprehensive enough, resulting in low yields of microbial fermentation methods. In addition, with regard to the synthesis of L-tryptophan, the path of its synthesis is longer and requires more precursors. Thus, in order to realize further increases in yield, it is necessary to systematically improve the microbial metabolic network through metabolic engineering.

Currently, metabolic engineering technology is widely used to improve industrial strains. Metabolic engineering primarily uses technologies such as DNA recombination and CRISPR/Cas9-mediated gene editing to optimize metabolic pathway enzymes directly involved in amino acid synthesis, key rate-limiting enzymes, and regulatory factors not directly involved in amino acid synthesis. In addition to these strategies, enhancing the target product transport ability of strains can also effectively improve amino acid production efficiency.

A number of genes encoding amino acid secreting proteins have been reported that can effectively improve the production capacity of strains. For example, increasing the expression of the lysine-secreting gene lysE can improve the production potential of lysine-producing bacteria of the genus Corynebacterium (WO9723597A2). Increasing the expression of the rhtB gene increases bacterial tolerance to L-homoserine (European patent application EP994190A2). Additional copies of the rhtC gene increase the yields of L-homoserine, L-threonine and L-leucine (European patent application EP1013765A1). Additional copies of the yahN, yeaS, yfiK and yggA genes increase the yields of L-glutamate, L-lysine, L-threonine, L-alanine and the like (European patent application EP1016710A2).

The transport protein most commonly found in aromatic amino acid-producing bacteria is encoded by E. coli's own gene, yddG. Of these, Vera Doroshenko et at. used Escherichia coli MG1655 as the parent strain and expressed the yddG gene at different intensities through plasmid or genomic integration. In fermentation experiments in test tubes, the accumulation of L-tryptophan was 0.6 μg/ml, and the accumulation of L-phenylalanine was 30,000 μg/ml. Liu Shuangping et al. introduced the yddG gene into a host strain of E. coli in the form of a plasmid through genetic engineering. Fermentation experiments showed that the ability of the strain containing the yddG gene to extracellularly transport L-phenylalanine was improved, and the final yield of L-phenylalanine reached 62 g/L. In another study, tryptophan-producing Escherichia coli strain SV164 (pGH5) was used as the parent strain, and the yddG gene was introduced into the cells under the control of a strong promoter to obtain strain SV164PL-yddG (pGH5). This strain was shaken cultured at 37°C for 48 hours in a 20 x 200 mm test tube, and the final yield of L-tryptophan was 4.17 g/L, which was approximately 12% higher than that of the parent strain.

Based on the above, it can be concluded that overexpression of the yddG gene can enhance the tolerance of engineered strains to aromatic amino acids, but the membrane proteins encoded by this gene have large differences in affinity for different aromatic amino acids, and the transport effect is not very good. This gene is derived from Escherichia coli, so it is easily self-regulated by bacteria and cannot exhibit an efficient transport function. It is difficult for this gene to meet the requirements of industrial production.

SUMMARY OF THE INVENTION

An object of the present invention is to improve the production efficiency of an L-tryptophan producing strain and to use this strain for producing L-tryptophan. In order to solve the above object, the present invention improves the production efficiency of L-tryptophan by a host strain by introducing into the host strain the ywkB gene (Gene ID: 936875) encoding a metabolite transporter derived from Bacillus subtilis subsp. ywkB is a membrane protein encoding gene that encodes a putative metabolite transporter and belongs to the auxin flux transporter family (TC 2.A.69), it is not directly involved in the synthesis of L-tryptophan, but when this gene is introduced into a strain producing L-tryptophan, it can effectively reduce the intracellular end-product concentration and alleviate end-product feedback inhibition of metabolic pathway enzymes, increase carbon flow to the target amino acid synthesis pathway, and ultimately improve product yield.

The first technical solution proposed in the present invention is a genetically engineered bacterial strain for producing L-tryptophan, where the host bacterial strain is modified with the ywkB gene, and the resulting genetically engineered bacterial strain has the activity of producing a higher yield of L-tryptophan than the bacterial strain - master.

According to the genetically engineered bacterial strain of the present invention, the ywkB gene is derived from Bacillus subtilis, and the modification is to modify the pseudogene locus ueer with the ywkB gene.

According to the genetically engineered bacterial strain of the present invention, the host bacterial strain is a bacterial host strain capable of producing L-tryptophan, and the specific bacterial species is not particularly limited as long as it has the ability to produce L-tryptophan or can gain such ability; for example, the host bacterial strain may be a prokaryotic bacterial strain capable of producing L-tryptophan, such as a strain of gram-negative bacteria capable of producing L-tryptophan; further, the host bacterial strain is an Escherichia coli strain capable of producing L-tryptophan; for example, the host bacterial strain is the L-tryptophan-producing strain E. coli TRP 03 (this strain is obtained by genetically modifying the original Escherichia coli strain W3110 and is the same strain as E. coli TRP 03 (CN 108753860 A)).

According to the genetically engineered bacterial strain of the present invention, the ywkB gene has a nucleotide sequence encoding the following polypeptide sequence:

(A) a protein containing the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;

(B) a protein that contains an amino acid sequence resulting from the deletion, substitution and/or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and still has the ability to improve the production of the L- strain tryptophan and structural analogues of L-tryptophan (such as 5-hydroxytryptophan, etc.);

(B) a protein that has at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 2, is derived from Bacillus subtilis and functions as a metabolite transporter.

According to the genetically engineered bacterial strain of the present invention, the "host bacterial strain is modified with ywkB gene" step refers to introducing a gene into the host bacterial strain.

The method of introducing a coding gene into a bacterial host strain includes, but is not limited to, means of genetic engineering known to one skilled in the art, such as introducing a target gene into a bacterial host strain through homologous recombination; or introducing the target gene into a bacterial host strain through expression nucleic acid constructs and, specifically, the expression nucleic acid construct may be a conventional plasmid which is known to one skilled in the art, and can be expressed in a prokaryotic host; or the target gene is introduced into the host chromatin through gene editing.

According to the genetically engineered bacterial strain of the present invention, where the ywkB gene is under the control of a strong promoter, the strong promoter is, for example, the BBa_j23101 promoter or the BBa_j23106 promoter.

Specifically, the modification of the present invention is to use the Escherichia coli CRISPR/Cas9 gene editing technology to introduce the transporter-encoding gene ywkB into E. coli TRP 03 and further to integrate the gene controlled by the BBa_j23106 promoter into the pseudogenic locus ueer.

The nucleotide sequence of the ywkB gene is shown in SEQ ID NO: 1.

The amino acid sequence of ywkB is shown in SEQ ID NO: 2.

The nucleotide sequence of the BBa_j23106 promoter is shown in SEQ ID NO: 3.

The second technical solution proposed in the present invention is a method for constructing a genetically engineered bacterial strain that produces L-tryptophan, where the ywkB gene is integrated into a host bacterial strain to obtain a genetically engineered bacterial strain having the activity of producing a higher yield of L-tryptophan, than the host bacterial strain.

According to the construction method of the present invention, the integration step is carried out by modifying the pseudogenic yeep locus with the ywkB gene, for example, integration is carried out by editing the CRISPR-Cas9 gene.

According to the construction method of the present invention, tryptophan-producing E. coli strain TRP 03 stored in the laboratory of Tianjin University of Science and Technology is used as the starting strain, and the ywkB gene is integrated, and the specific steps are as follows:

1) Synthesis of the integrated genetic fragment YWKB: designing fragments of the overlying and underlying homologous shoulder of the pseudogen ueer, the genetic fragment of the YWKB and the BBA_J23106 promoter sequence, where the integrated genetic fragment of the YWKB contains the ceiling shoulder of the Gene Uter, the Lower Gomological shoulder of Gena Ueer, promotional fragment of VVA_J23106 and genetic fragment ywkB;

2) expression of the integrated ywkB gene fragment in E. coli TRP 03 using CRISPR/Cas9 technology.

According to the construction method of the present invention, the ywkB gene sequence is a nucleotide sequence encoding the following polypeptide sequence:

A) a protein containing the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;

B) a protein that contains an amino acid sequence resulting from the deletion, substitution and/or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and still has the ability to improve the production of L-tryptophan by the strain and structural analogs of L-tryptophan (such as 5-hydroxytryptophan, etc.);

(B) a protein that has at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 2, which is derived from Bacillus subtilis and functions as a metabolite transporter.

The third technical solution proposed in the present invention is a genetically engineered bacterial strain obtained by the above method of constructing a genetically engineered bacterial strain that produces L-tryptophan.

The fourth technical solution proposed in the present invention is a method for producing L-tryptophan, which includes the step of fermenting a genetically engineered bacterial strain according to the first solution of the present invention or a genetically engineered bacterial strain obtained by the method according to the second solution to produce L-tryptophan.

According to the L-tryptophan production method of the present invention, it includes the following steps:

1) activation of the bacterial strain;

2) preparation of seed solution;

3) fermentation; for example, where

in step (1), a culture is prepared on the agar slant: inoculation of a bacterial strain stored at -80°C onto the activated agar slant by streaking, cultivation at 37°C for 12 hours and subculture once;

Step (2) prepares the inoculum in a shaking flask by scraping the inoculum spot on the agar slant using a microbiological loop and inoculating into a 500 ml conical flask containing 30 ml of inoculum medium, sealing the conical flask with nine layers of gauze and culturing at 37°C and 200 rpm for 8-10 hours;

In step (3), prepare the fermentation culture in a shaking flask: inoculate the seed culture at a concentration of 10-15% (v/v) into a 500 ml conical flask containing the fermentation medium (final volume: 30 ml), seal the conical flask with nine layers of gauze, cultivation at 37°C and 200 rpm on a rocking chair, adding ammonia water during fermentation to maintain pH at 7.0-7.2; adding 60% (w/v) glucose solution to maintain fermentation (with phenol red as an indicator, when the color of the fermentation broth no longer changes, it is regarded as glucose deficiency, and add 1-2 ml of 60% ( w/v) glucose solution to ensure the initial glucose concentration in the fermentation broth is 20-40 g/l); Continue fermentation for 22-26 hours.

The present invention has the following advantages and beneficial effects.

In the present invention, the ywkB gene encoding a transporter derived from Bacillus subtilis is introduced into a constructed L-tryptophan-producing strain, which significantly improves the yield of the target product. Because the gene comes from Bacillus subtilis, it will not be subject to regulation by the engineered strain and functions more simply. Thus, the present invention has wide application prospects. The engineered L-tryptophan producing strain containing the ywkB gene of the present invention can accumulate 15.2 g/L L-tryptophan after 24 hours of shaker fermentation, which is 35% more than the original strain, demonstrating the potential for industrial production of L-tryptophan.

DESCRIPTION OF GRAPHIC MATERIALS

In FIG. 1, panel (a) shows the pREDCas9 plasmid map, and panel (b) shows the pGRB plasmid map.

In FIG. Figure 2 shows an electropherogram of the construction and verification of the ywkB-BBa_j23106 gene fragment, where M: 1 kb DNA marker. (thousand pairs of nucleotides); track 1: overlying homologous arm; track 2: ywkB; track 3: underlying homologous arm; track 4: overlapping fragment; track 5: fragment of identification of positive bacteria; track 6: control strain.

In FIG. Figure 3 shows a standard curve for the determination of L-tryptophan by HPLC (high performance liquid chromatography).

In FIG. Figure 4 shows the yields of L-tryptophan during fermentation in a shaking flask.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Construction of E. coli strain TRP 05

1. Gene editing method

The gene editing method adopted in the present invention is disclosed in "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", and maps of the two plasmids used in this method are shown in FIG. 1. Namely, the pREDCas9 vector carries the pGRB plasmid elimination system for gRNA (genomic RNA) expression, the X phage Red-recombination system and the Cas9 protein expression system, spectinomycin resistance (working concentration: 100 mg/L), cultivation at 32°C ; pGRB vector uses pUC18 as a backbone and contains the J23100 promoter, gRNA-Cas9 binding domain sequence and terminator sequence, ampicillin resistance (working concentration: 100 mg/L), culture at 37°C.

Specific stages of this method.

1.1 Construction of plasmid pGRB

The objective of pGRB plasmid construction is to transcribe the corresponding gRNA to form a complex with the Cas9 protein and recognize the target site of the target gene through base pairing and PAM (adjacent protospacer motif) to achieve a double-strand break in the target DNA. Plasmid pGRB was constructed by recombining a DNA fragment containing the target sequence with a linearized vector fragment.

1.1.1 Designing a target sequence

CRISPR RGEN Tools were used to generate the target sequence (PAM: 5′-NGG-3′).

1.1.2 Obtaining a DNA fragment containing the target sequence

The primer was designed: the 5'-terminal sequence of the linearized vector (15 bp) - the restriction site, the target sequence (without the PAM sequence) - the terminal sequence of the linearized vector (15 bp) - 3' and a complementary reverse primer, and a DNA fragment, containing the target sequence was obtained by annealing single-stranded DNA. Reaction conditions: predenaturation at 95°C for 5 min; annealing at 30-50°C for 1 min. The annealing system was as follows.

Annealing system

1.1.3 Obtaining a linearized vector

To linearize the vector, the inverted PCR amplification method was used.

1.1.4 Recombination reaction

The recombination system is shown in the following table. All recombinases used were ClonExpress® II One Step Cloning Kit enzymes. Recombination conditions: 37°C, 30 min.

1.1.5 Plasmid transformation

Ten μl of the reaction solution was added to 100 ml of competent DH5α cells and mixed gently. The resulting mixture was cooled in an ice bath for 20 minutes, heat shocked at 42°C for 45-90 seconds, immediately cooled in an ice bath for 2-3 minutes, supplemented with 900 μl SOC (Super Optimal Catabolic Repressor Medium). broth with Catabolic repressor) and isolated at 37°C for 1 hour. The mixture was centrifuged at 8000 rpm for 2 min, part of the supernatant was discarded, and the remaining 200 μl of supernatant was used to resuspend the cells. The cells were then spread onto a dish containing 100 mg/L ampicillin, and the dish was placed upside down and cultured at 37°C overnight. After single colonies were grown on the plate, positive recombinants were identified and selected by molecular colony PCR.

1.1.6 Identification of clones

PCR-positive colonies were inoculated into LB medium (lysogenic medium) containing 100 mg/L ampicillin for overnight culture, and the bacteria were stored. Plasmids were extracted and identified by enzymatic digestion.

1.2 Obtaining a recombinant DNA fragment

The recombinant knockout fragment consists of the upstream and downstream homologous arms of the gene to be knocked out (upstream homologous arm-downstream homologous arm); the recombinant fragment for integronium consists of the upstream and downstream homologous arms of the integration site and the gene fragment to be integrated (upstream homologous arm - target gene - downstream homologous arm). Using primer5 primer design software, the upstream and downstream sequences of the gene to be knocked out or the site to be integrated were used as a template to design primers to amplify the upstream and downstream homologous arms (amplification product length: about 400-500 bp. ); the gene to be integrated was used as a template to design primers to amplify the integrated gene. After amplification of the upstream and downstream homologous arms and the target gene fragment by PCR, respectively, a recombinant fragment was obtained by PCR with overlapping primers. DNA polymerases used in PCR were purchased from TaKaRa, including PrimeSTAR HS High Fidelity DNA Polymerase and Ex.taq DNA Polymerase to generate PCR products with sticky ends. PCR systems and methods are shown in the following tables.

The PCR system with overlapping primers is shown in the following table.

The molecular colony PCR system is shown in the following table.

PCR reaction conditions: predenaturation at 95°C for 5 min; 30 cycles of denaturation at 98°C for 10 seconds, annealing at (Tm-3/5)°C for 15 seconds, extension at 72°C (enzyme activity: approximately 1 minute per tn); and final extension at 72°C for 10 min; stored at 4°C.

1.3 Transformation with plasmid and recombinant DNA fragment

1.3.1 Transformation of pREDCas9

The pREDCas9 plasmid was transformed by electroporation into electroporation-competent W3110 cells. Cells were isolated, cultured, and plated with LB containing spectinomycin and cultured at 32°C overnight. Single colonies grown on antibiotic plates were subjected to molecular colony PCR using identification primers to screen for positive recombinants.

1.3.2 Obtaining cells of the target strain containing pREDCas9 competent for transformation by electroporation

The strain was cultured at 32°C until the culture reached an OD600 (optical density) of 0.1 to 0.2 and then 0.1 M IPTG (isopropylthiogalactoside) was added (to a final concentration of 0.1 mM). Cultivation was continued until an OD600 value of 0.6 to 0.7 was achieved. The resulting cells were used to obtain competent cells. The purpose of adding IPTG is to induce expression of the recombinase on the pREDCas9 plasmid. The media and production method required to obtain competent cells are generally accepted standard operations.

1.3.3 Transformation of pGRB and recombinant DNA fragment

Plasmid pGRB and a donor DNA fragment were simultaneously transformed by electroporation into electroporation-competent cells containing pREDCas9. After electroporation transformation, cells were isolated, cultured, and plated with LB containing ampicillin and spectinomycin and cultured at 32°C overnight. Verification by molecular colony PCR was performed using a forward primer for the upstream homologous arm and a reverse primer for the downstream homologous arm, or using specially designed identification primers, screening for positive recombinants, and retaining the recombinant bacteria.

1.4 Plasmid elimination

1.4.1 Elimination of the pGRB plasmid

Positive recombinants were cultured overnight in LB medium containing 0.2% arabinose, and after proper dilution, they were spread onto LB plates containing spectinomycin and cultured at 32°C overnight. The recombinants were then inoculated onto LB plates containing ampicillin and spectinomycin, respectively, and single colonies did not grow on the plate containing ampicillin but grew on the plate containing spectinomycin, selected and stored.

1.4.2 Elimination of plasmid pREDCas9

Positive recombinants were transferred to liquid LB medium without antibiotics, cultured overnight at 42°C and spread onto LB plates without antibiotics after proper dilution, and cultured at 37°C overnight. The recombinants were then inoculated onto LB plates containing spectinomycin and without antibiotics, and single colonies that did not grow on the spectinomycin plate but grew on the LB plate without antibiotics were selected and stored.

2. Construction of a modified strain E. coli TRP 05, producing L-tryptophan

2.1 Synthesis of the ywkB gene

1) Using the E. coli W3110 genome as a template, PCR was performed with primers for the upstream homologous arm (upstream-S, downstream-A) and primers for the downstream homologous arm (downstream-S, downstream-S). A), designed at both ends of the ueer pseudogene to amplify the overlying and downstream homologous arms of the ueer pseudogene.

2) Primers for PCR (ywkB-S, ywkB-A) were designed in accordance with the gene sequence of the putative metabolite transporter ywkB of Bacillus subtilis (Bacillus subtilis subsp. subtilis strain 168), published in GENBANK, and the promoter sequence BBaj23106 was designed in the forward primer for the gene ywkB and its fragment were amplified with HS enzyme.

3) The amplified fragment obtained in steps (1) and (2) was used as templates to obtain the integrated gene fragment BBaj23106-ywkB by PCR with overlapping primers, and this fragment consisted of the upstream homologous arm of the yeer gene, the downstream homologous arm of the yeer gene , promoter fragment BBaj23106 and gene fragment ywkB.

2.2 ywkB gene integration

1) Construction of pGRB-yeep: plasmid pGRB containing the target sequence was prepared according to the method described in section 1.1, namely, primers, pGRB-yeep-S and pGRB-yeep-A, were constructed according to the method described in section 1.1 .2 and then a DNA fragment containing the target sequence is obtained by annealing; plasmid pGRB was linearized in accordance with the method according to section 1.1.3 and then, in accordance with the method according to section 1.1.4, plasmid pGRB-yeep was obtained containing the target sequence,

2) Obtaining competent cells E. coli TRP 03;

3) Obtaining the E. coli TRP 05 strain: in accordance with the method described in section 1.3, positive clones were selected, then E. coli TRP 03 was transformed with the pREDCas9 plasmid and then competent E. coli TRP 03 cells were obtained containing the pREDCas9 plasmid, transformed by electroporation plasmid pGRB-yeep and recombinant DNA fragments obtained in step (3) in section 2.1; after 12-16 hours of cultivation on plates, verification was carried out by PCR using the molecular colony method, positive recombinants were selected and stored; plasmid pGRB-yeep and plasmid pREDCas9, respectively, were eliminated according to the method described in section 1.4, and finally E. coli strain TRP 05 was screened by PCR identification and stored at -80°C.

An electropherogram of the construct of the integration fragment of the gene ВВа_j23106 ywkB and PCR verification of the positive strain are shown in FIG. 2, where the length of the upstream homologous arm is 512 bp, the length of the downstream homology arm is 513 bp, the length of the ywkB BBa_j23106 gene fragment is 1040 bp, and the total length of the gene/wkB BBa_j23106 integration fragment is 2065 bp. In PCR verification, the length of the PCR-amplified fragment of positive bacteria was 2065 bp, and the length of the PCR-amplified fragment of natural bacteria was 1396 bp.

3. The primers used to improve the strain are shown in the following table:

Example 2: Rocker Flask Fermentation of Genetically Engineered Escherichia coli to Produce L-Tryptophan

The specific route for using genetically engineered Escherichia coli to produce L-trypgophan through shake-flask fermentation was as follows:

agar slant culture: inoculation of a bacterial strain stored at -80°C onto an activated agar slant by streaking, culturing at 37°C for 12 hours and subculture once;

Rocker flask inoculum: scrape the inoculum spot on the agar slant using a microbiological loop and inoculate into a 500 ml conical flask containing 30 ml of inoculum medium, seal the conical flask with nine layers of gauze and culture at 37°C and 200 rpm within 8-10 hours;

shaking flask fermentation culture: inoculate the seed culture at a concentration of 10-15% (v/v) into a 500 ml conical flask containing fermentation medium (final volume: 30 ml), seal the conical flask with nine layers of gauze, culture at 37 °C and 200 rpm on a rocker, adding ammonia water during fermentation to maintain pH at 7.0-7.2; adding 60% (w/v) glucose solution to maintain fermentation (with phenol red as an indicator, when the color of the fermentation broth no longer changes, it is regarded as the absence of glucose, adding 1-2 ml of 60% (w/v) ./vol.) glucose solution to ensure the initial value of glucose concentration in the fermentation broth is 20-40 g/l); The fermentation period lasts 22-26 hours.

Determination of the concentration of L-tryptophan in the fermentation broth: 1 ml of the fermentation broth was taken, centrifuged at 13,000 rpm for 1 min and the supernatant was collected; the collected supernatant was diluted (to 0.1-0.5 g/l) with deionized water, filtered through a 0.22 μm microporous filter, and the concentration of L-tryptophan was determined by liquid chromatography; chromatography conditions were as follows: chromatographic column: Kromasil C18 column (250 mm × 460 mm, 5 μm), mobile phase: 10% acetonitrile solution, flow rate: 1.0 ml/min, column temperature: 40°C, length detection wave: 278 nm, injection volume: 20 μL, and main peak appearance time was approximately 3.5 min; the concentration of L-tryptophan in the fermentation broth was calculated based on its peak area according to the standard curve;

construction of a standard curve of L-tryptophan: solutions with L-tryptophan concentrations of 0.1 g/L, 0.3 g/L, 0.5 g/L, 0.6 g/L and 1 g/L were subjected to the above liquid chromatography for obtain peak areas corresponding to L-tryptophan concentrations, and peak areas were plotted against L-tryptophan concentration to obtain a standard curve, as shown in FIG. 3.

Components of activated agar slant medium: 1-3 g/l glucose, 5-10 g/l tryptone, 5-10 g/l beef extract, 2-5 g/l yeast extract, 2-5 g/l NaCl, 15- 30 g/l agar, the remainder is water, pH 7.0-7.2, sterilized in an autoclave at 121°C for 20 minutes.

Components of the inoculum medium: 20-40 g/l glucose, 1-5 g/l (NH ₄ ) ₂ SO ₄ , 1-5 g/l KH ₂ PO ₄ , 0.5-2 g/l MgSO ₄ ⋅7H ₂ O, 2-5 g/l yeast extract, 1-3 mg/l FeSO ₄ ⋅7H ₂ O, 1-3 mg/l MnSO ₄ ⋅H ₂ O, 0.1-0.5 mg/l _VH , 0.5-1.0 mg/l V _B1 , 1-3 ml/l mixture of trace elements, 15-30 g/l phenol red, the remainder is water, pH 7.0-7.2, sterilized in an autoclave at 115 °C for 15 min.

Components of the fermentation medium: 20-40 g/l glucose, 2-6 g/l (NH ₄ ) ₂ SO ₄ , 1-5 g/l KH ₂ PO ₄ , 0.5-2 g/l MgSO ₄ ⋅7H ₂ O, 1-5 g/l yeast extract, 30-60 mg/l FeSO ₄ ⋅7H ₂ O, 1-5 mg/l MnSO ₄ ⋅7H ₂ O, 0.1-0.5 mg/l _VH , 0.5-1.0 mg/l V _B1 , 1-3 ml/l mixture of trace elements, 15-30 g/l phenol red, the remainder is water, pH 7.0-7.2, sterilized in an autoclave at 115 °C for 15 min.

Components of a mixture of trace elements: 2.5 g/l Na ₂ MoO ₄ ⋅2H ₂ O, 2.5 g/l AlCl ₃ ⋅6H ₂ O, 2.5 g/l NiSO ₄ ⋅6H ₂ O, 1.75 g/ l CoCl ₂ ⋅6H ₂ O, 10 g/l CaCl ₂ ⋅2H ₂ O, 0.5 g/l ZnSO ₄ ⋅7H ₂ O, 0.25 g/l CuCl ₂ ⋅2H ₂ O, 0.125 g/l H ₃ VO ₃ .

The engineered L-tryptophan-producing E. coli strain TRP 05 was used for shake-flask fermentation, and the modified L-tryptophan-producing E. coli strain TRP 03 was stored at the Metabolic Engineering Laboratory of Tianjin University of Science and Technology. University of Science and Technology) was used as a control for the same fermentation in shaking flasks. The experimental results are shown in FIG. 4. The modified strain E. coli TRP 03, producing L-tryptophan, accumulated L-tryptophan up to 11.2 g/l, while E. coli TRP 05 accumulated L-tryptophan up to 15.2 g/l, which was 35% more than the control strain.

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LIST OF SEQUENCES

<110> Ningxia Eppen Biotech Co., LTD; Tianjin University of Science

& Technology

<120> USING A TRANSPORT CARRIER GENE THAT IMPROVES

EFFECTIVENESS OF L-TRYPTOPHAN PRODUCTION IN ESCHERICHIA COLI

<130> PUS1220487

<160> 11

<210> 1

<211> 993

<212> DNA

<213> Bacillus subtilis subsp. subtilis 168

<400> 1

TTGAGCATCT TAGATATCTT AATCCTCCTG GGCGCCGATCT TCTTTGTTAT CGTGCTGGGT 60

TGGTTTGCAG GACATTTTGG AAGTTATGAT GCCAAGTCGG CAAAAGGGGT AAGTACGTTA

120

GTAACGAAAT ACGCACTTCC AGCTCACTTT ATCGCTGGTA TTTTGACAAC TTCCAGAAGT

180

GAATTTTTAT CACAAGTACC TTTAATGATT TCTTTAATTA TTGGGATTGT TGGTTTCTAT

240

ATCATCATTC TTTTGGTTTG CAGATTTATA TTCAAGTATG ATTTAACGAA CTCATCTGTA

300

TTTTCTTTGA ACTCTGCACA GCCGACATTC GCATTTATGG GTATCCCGGT ATTGGGAAGC

360

TTATTCGGAG CGAATGAAGT TGCGATTCCG ATCGCGGTCA CAGGTATCGT GGTTAACGCG

420

ATTCTTGATC CGCTCGCGAT CATTATCGCT ACTGTTGGTG AGTCTTCTAA GAAAAACGAA

480

GAGAGTGGCG ACAGCTTCTG GAAGATGACA GGAAAATCAA TCCTGCATGG TCTTTGTGAG

540

CCGCTTGCAG CTGCTCCGTT AATCAGTATG ATCTTGGTGC TGGTTTTCAA TTTCACTCTT

600

CCTGAGCTGG GTGTTAAAAT GCTTGATCAG CTTGGAAGCA CAACATCTGG TGTTGCTCTC

660

TTCGCTGTTG GTGTTACCGT TGGTATTCGT AAAATTAAAC TCAGTATGCC GGCTATCGGT

720

ATTGCGTTAC TAAAAGTTGC GGTTCAGCCT GCGTTAATGT TCCTGATTGC TCTTGCTATC

780

GGACTTCCAG CTGACCAAAC AACAAAAGCA ATCCTTCTTG TTGCATTCCC TGGTTCTGCC

840

GTTGCAGCCA TGATTGCGAC TCGTTTCGAG AAACAAGAAG AAGAAACTGC AACTGCGTTT

900

GTGGTCAGTG CGATTCTGTC ATTGATTTCA CTTCCAATCA TTATCGCGCT TACTGCGTAA

960

CAAAAAAAGC TCCCTTTAAG GGAGCTTTTT GCT

993

<210> 2

<211> 330

<212>PRT

<213> Bacillus subtilis subsp. subtilis 168

<400> 2

MSILDILILL APIFFVIVLG WFAGHFGSYD AKSAKGVSTL VTKYALPAHF IAGILTTSRS 60

EFLSQVPLMI SLIIGIVGFY IIILLVCRFI FKYDLTNSSV FSLNSAQPTF AFMGIPVLGS 120

LFGANEVAIP IAVTGIVVNA ILDPLAIIIA TVGESSKKNE ESGDSFWKMT GKSILHGLCE 180

PLAAAPLISM ILVLVFNFTL PELGVKMLDQ LGSTTSGVAL FAVGVTVGIR KIKLSMPAIG 240

IALLKVAVQP ALMFLIALAI GLPADQTTKA ILLVAFPGSA VAAMIATRFE KQEEETATAF 300

VVSAILSLIS LPIIIALTA* QKKLPLRELF A 330

<210> 3

<211> 47

<212> DNA

<213> Artificial Sequence

<400> 3

TTTACGGCTA GCTCAGTCCT AGGTATAGTG CTAGCAGGAA ACAGACC 47

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 4

GGTCAGGAGG TAACTTATCA GCG 23

<210> 5

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 5

CTGCTAGCAC TATACCTAGG ACTGAGCTAG CCGTAAAATG GCAGGGCTCC GTTTT 55

<210> 6

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 6

AGGTATAGTG CTAGCAGGAA ACAGACCTTG AGCATCTTAG ATATCTTAAT CCTCC 55

<210> 7

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 7

AAATCCAGTT CAGCAAAAAG CTCCCTTAAA GGG 33

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 8

TTTTTGCTGA ACTGGATTTT CTTCTGAACC TGT 33

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 9

ACGATGTCAG CAGCCAGCA 19

<210> 10

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 10

AGTCCTAGGT ATAATACTAG TACAGAATAT TCGCGAAAAA AGTTTTAGAG CTAGAA 56

<210> 11

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 11

TTCTAGCTCT AAAACTTTTT TCGCGAATAT TCTGTACTAG TATTATACCT AGGACT 56

<---

Claims (26)

1. A genetically engineered bacterium for producing L-tryptophan, wherein the original bacterium is modified with a ywkB gene, and the resulting genetically engineered bacterium has the activity of producing a higher yield of L-tryptophan than the original bacterium; where the modification consists of modifying the pseudogenic locus yeep with the ywkB gene. 2. Genetically engineered bacterium according to claim 1, where the ywkB gene comes from Bacillus subtilis. 3. Genetically engineered bacterium according to claims. 1, 2, which is a prokaryotic bacterium, preferably a gram-negative bacterium, more preferably Escherichia coli, most preferably E. coli TRP 03. 4. A genetically engineered bacterium according to claim 1, where the ywkB gene has a nucleotide sequence encoding the following polypeptide sequence: A) a protein containing the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; B) a protein that contains an amino acid sequence obtained by deletion, substitution and/or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and still has the ability to improve the production of L- by the bacterium tryptophan and structural analogues of L-tryptophan, such as 5-hydroxytryptophan, and so on; B) a protein that has at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 2, comes from Bacillus subtilis and functions as a metabolite transporter. 5. Genetically engineered bacterium according to any one of paragraphs. 1-4, wherein the ywkB gene is introduced into the parent bacterium through homologous recombination, an expression vector or gene editing, where the expression vector is preferably a plasmid, for example, gene editing refers to the introduction of a gene of an amino acid sequence into the chromosome of the parent bacterium through the CRISPR/Cas9 method. 6. Genetically engineered bacterium according to any one of paragraphs. 1-5, where the ywkB gene is under the control of a strong promoter, for example, the strong promoter is the BBa_j23101 promoter or the BBa_j23106 promoter, for example, the amino acid sequence of the BBa_j23106 promoter is shown in SEQ ID NO: 3. 7. A method for constructing a genetically engineered bacterium that produces L-tryptophan, wherein the ywkB gene is integrated into the original L-tryptophan-producing bacterium by modifying the pseudogenic yeep locus with the ywkB gene, obtaining a genetically engineered bacterium having the activity of producing a higher yield of L-tryptophan than the original bacterium. 8. The method according to claim 7, where the starting bacterium is E. coli TRP 03. 9. The method according to claim 7, where the integration step is carried out by modifying the pseudogenic yeep locus with the ywkB gene, for example, integration is carried out by gene editing using CRISPR-Cas9. 10. The method according to claim 9, where gene editing using CRISPR-Cas9 includes the following steps: 1) synthesis of an integrated ywkB gene fragment: construction of fragments of the upstream and downstream homologous arm of the yeep pseudogene, the ywkB gene fragment and the BBa_j23106 promoter sequence, where the integrated ywkB gene fragment contains the upstream homologous arm of the yeep gene, the downstream homologous arm of the yeep gene, the promoter fragment BBa j23106 and the gene fragment ywkB; 2) expression of the integrated ywkB gene fragment in E. coli TRP 03 using CRISPR/Cas9 technology. 11. Method according to any one of paragraphs. 7-10, where the ywkB gene sequence is a nucleotide sequence encoding the following polypeptide sequence: A) a protein containing the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; B) a protein that contains an amino acid sequence obtained by deletion, substitution and/or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and still has the ability to improve the production of L- by the bacterium tryptophan and structural analogues of L-tryptophan, such as 5-hydroxytryptophan, and so on; B) a protein that has at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 2, comes from Bacillus subtilis and functions as a metabolite transporter. 12. A method for producing L-tryptophan, including the stage of fermentation of a genetically engineered bacterium according to any one of claims. 1-6 or a genetically engineered bacterium obtained by the method according to any one of paragraphs. 7-11, with the production of L-tryptophan. 13. The method for producing L-tryptophan according to claim 12, including the following steps: 1) activation of bacteria; 2) preparation of seed solution; 3) fermentation; for example, where in step (1) a culture is prepared on an agar slant: inoculation of the bacterium, stored at -80°C, onto activated agar slants using the streak method, cultured at 37°C for 12 hours and subcultured once; Step (2) prepares the inoculum in a shaking flask by scraping the inoculum spot on the agar slant using a microbiological loop and inoculating into a 500 ml conical flask containing 30 ml of inoculum medium, sealing the conical flask with nine layers of gauze and culturing at 37°C and 200 rpm for 8-10 hours; in step (3), prepare the fermentation culture in a shaking flask: inoculating the seed culture at a concentration of 10-15% (v/v) into a 500 ml conical flask containing fermentation medium with a final volume of 30 ml, sealing the conical flask with nine layers of gauze , cultivation at 37°C and 200 rpm on a shaker during fermentation, adding ammonia water to maintain the pH at 7.0-7.2; adding 60% (w/v) glucose solution to maintain fermentation with phenol red as an indicator, when the color of the fermentation broth no longer changes, it is regarded as the absence of glucose, adding 1-2 ml of 60% (w/v) glucose solution. /vol.) glucose solution to ensure the initial value of glucose concentration in the fermentation broth is 20-40 g/l; The fermentation period lasts for 22-26 hours.