Recombinant escherichia coli for improving yield of L-tryptophan and application of recombinant escherichia coli

Abstract

The invention discloses recombinant escherichia coli capable of increasing the yield of L-tryptophan and application of the recombinant escherichia coli. According to the invention, o-aminobenzoic acid synthase TrpEQ71K and 3-deoxy-D-arabinoheptanoic acid-7-phosphate synthase (DAHP synthase) AroGQ151F, which are used for solving feedback, are subjected to integrated expression on a genome of the L-tryptophan production strain TRP30, so that the constructed engineering strain TRP30 can be used for remarkably improving the yield of the L-tryptophan. After the strain is cultured in a 5L fermentation tank for 48h, the determination shows that the yield of the L-tryptophan is increased to 52.1 g / L, which is increased by 22.59% compared with the yield of the L-tryptophan of a control strain.

Description

Technical Field

[0001] The invention relates to a recombinant Escherichia coli for improving L-tryptophan production and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] L-tryptophan, one of the eight essential amino acids, cannot be synthesized in mammals and must be obtained from sources. With the continued development and application of L-tryptophan in the market, it is widely used in food, medicine, and feed. In the food industry, L-tryptophan has multiple benefits, including as a nutritional supplement, food additive, and antioxidant. As a food additive, L-tryptophan can promote fermentation during bread production and can also be added as a flavoring to foods such as pastries and biscuits. In the feed industry, L-tryptophan can be added to feed to improve protein utilization and promote cell growth. A small amount of L-tryptophan can also regulate the amino acid balance in animals, promoting normal growth and development while also enhancing their immune system. In the pharmaceutical industry, L-tryptophan promotes the maturation and differentiation of T lymphocytes, thereby enhancing immunity. It can also be metabolized in humans and animals to produce various high-value-added derivatives such as serotonin, melatonin, and pigments, resulting in antidepressant, sleep-enhancing, antihypertensive, and analgesic effects.

[0003] In Escherichia coli, the L-tryptophan biosynthesis pathway can be divided into three modules: the central metabolic pathway, the shikimate metabolic pathway, and the L-tryptophan branch pathway. The central metabolic pathway involves the production of PEP and E4P from glucose. The shikimate pathway is a common pathway for the synthesis of aromatic amino acids, primarily involving the conversion of DAHP to chorismate via six enzymatic steps. Finally, chorismate is converted to anthranilate by anthranilate synthase (encoded by the gene trpED), which then enters the L-tryptophan biosynthesis branch pathway. L-tryptophan is then catalyzed by enzymes encoded by the L-tryptophan operon. DAHP synthase (AroG) and anthranilate synthase (TrpE) are two key rate-limiting enzymes in L-tryptophan biosynthesis and are subject to feedback inhibition by L-phenylalanine and L-tryptophan, respectively. Generally speaking, introducing anti-feedback sites is an effective strategy for relieving product inhibition of rate-limiting enzymes. Therefore, identifying novel anti-feedback sites in the rate-limiting enzymes AroG and TrpE could effectively promote L-tryptophan biosynthesis. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention uses an L-tryptophan-producing Escherichia coli engineered strain TRP30 as the starting strain and uses the CRISPR / Cas9 system-mediated genome integration technology to achieve the gene trpE Q71K and aroG Q151FIntegration into the Escherichia coli genome to construct an engineered strain that can efficiently synthesize L-tryptophan.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a recombinant Escherichia coli for improving the production of L-tryptophan, wherein the recombinant Escherichia coli is an Escherichia coli host that integrates and expresses an anthranilate synthase mutant TrpE Q71K and 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F .

[0007] In one embodiment of the present invention, the anthranilate synthase mutant TrpE Q71K The nucleotide sequence is shown in SEQ ID NO.1.

[0008] In one embodiment of the present invention, the 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F The nucleotide sequence is shown in SEQ ID NO.2.

[0009] In one embodiment of the present invention, the Escherichia coli host is an engineered Escherichia coli strain TRP30.

[0010] In one embodiment of the present invention, the engineered Escherichia coli strain TRP30 is disclosed in the non-patent document “Multidimensional engineering of Escherichia coli for efficient synthesis of L-tryptophan”, and the L-tryptophan yield in a 5L fermenter is 42.5 g / L.

[0011] In one embodiment of the present invention, the anthranilate synthase mutant TrpE Q71K The protein was integrated and expressed at the ilvG pseudogene site in the Escherichia coli genome.

[0012] In one embodiment of the present invention, the 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F The protein was integrated and expressed at the yjiP pseudogene site in the Escherichia coli genome.

[0013] The second object of the present invention is to provide the use of the recombinant Escherichia coli in the fermentation production of L-tryptophan, wherein the use is to use glucose as a carbon source and utilize the recombinant Escherichia coli to ferment and produce L-tryptophan.

[0014] In one embodiment of the present invention, the fermentation conditions are a temperature of 35-38° C., a rotation speed of 600-700 rpm, and a pH of 7.0-7.2.

[0015] In one embodiment of the present invention, the fermentation medium for producing L-tryptophan comprises 15-25 g / L glucose, 2-4 g / L yeast extract, 1-3 g / L citric acid, 3-5 g / L ammonium sulfate, 0.5-2 g / L sodium chloride, 0.5-2 g / L magnesium sulfate heptahydrate, 15-25 mg / L ferrous sulfate heptahydrate, 1-1.5 mg / L manganese sulfate monohydrate, 0.4-0.6 mg / L B1 , 0.08~0.12mg / LV H and 0.8-1.2 mL / L of trace element mixture.

[0016] The third object of the present invention is to provide a method for increasing the production of L-tryptophan in Escherichia coli, wherein the method comprises integrating and expressing an anthranilate synthase mutant TrpE into an L-tryptophan-producing Escherichia coli host. Q71K and 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F , wherein the anthranilate synthase mutant TrpE Q71K The nucleotide sequence is shown in SEQ ID NO.1, and the 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F The nucleotide sequence is shown in SEQ ID NO. 2, and the Escherichia coli host is the Escherichia coli engineered strain TRP30.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides an anthranilate synthase TrpE which is integrated and expressed in the genome of L-tryptophan producing strain TRP30 to resolve feedback. Q71K and 3-deoxy-D-arabinoheptulose-7-phosphate synthase (DAHP synthase) AroG Q151F The engineered strain TRP30 was able to significantly increase L-tryptophan production. After 48 hours of culture in a 5L fermenter, the L-tryptophan production was found to have increased to 52.1 g / L, a 22.59% increase over the control strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The L-tryptophan production abilities of the engineered strains TRP30 and TRP32 were analyzed in 5 L fermenters. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific examples. These implementation cases are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims appended hereto.

[0022] The culture medium involved in the following examples is as follows:

[0023] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0024] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L.

[0025] Competent medium: peptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L.

[0026] Recovery medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0027] Seed culture medium: glucose 30 g / L, yeast extract 5 g / L, citric acid 2 g / L, ammonium sulfate 2.5 g / L, dipotassium phosphate 4 g / L, magnesium sulfate heptahydrate 1.5 g / L, ferrous sulfate heptahydrate 2.8 mg / L, manganese sulfate monohydrate 1.2 mg / L, VB1 1 mg / L, VH 1 mg / L, and trace element mixture 1 mL / L.

[0028] Fermentation medium: glucose 30 g / L, yeast extract 3 g / L, citric acid 2 g / L, ammonium sulfate 3 g / L, dipotassium hydrogen phosphate 7 g / L, sodium chloride 1 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 30 mg / L, manganese sulfate monohydrate 10 mg / L, VB1 1 mg / L, VH 1 mg / L, and trace element mixture 1 mL / L.

[0029] The engineered Escherichia coli strain TRP30 is disclosed in the non-patent document “Multidimensional engineering of Escherichia coli for efficient synthesis of L-tryptophan”.

[0030] L-Tryptophan Detection Method: L-tryptophan in the fermentation broth was determined using high-performance liquid chromatography (HPLC) (Agilent 1260 series, CA, USA). UV detection was used. The mobile phase consisted of acetonitrile / water (10:90 v / v), the flow rate was set at 1 ml / min, and the detection wavelength was set at 278 nm.

[0031] The primers involved in the following examples are shown in Table 1:

[0032] Table 1

[0033]

[0034]

[0035] The technical solutions of the present invention are described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased from commercial sources, or prepared by conventional methods, or are commonly used in the industry.

[0036] Example 1: Construction of Escherichia coli engineered strain TRP32

[0037] In order to realize the gene TrpE in the Escherichia coli engineered strain TRP30 Q71KQ151FQ71K , and the engineered strain TRP31 was constructed. Then aroG was introduced into the pseudogene site of the genome of the engineered strain TRP31. Q151F .

[0038] TrpE Q71K For integration into the pseudogene locus ilvG in the engineered strain TRP30, an sgRNA targeting the ilvG gene was designed based on the nucleotide sequence of the desired pseudogene locus. The sgRNA for ilvG, amplified using sgRNA-F / sgRNA-R, was ligated into the plasmid pGRB (Addgene #71539) via homologous recombination to construct the plasmid pGRB-ilvG-sgRNA. Using the genome of the engineered E. coli strain TRP30 as a template, the upstream and downstream homology arms (~500 bp) of the pseudogene locus ilvG were amplified using primers ilvG-arm-F1 / R1, ilvG-arm-F2 / R2, and ilvG-arm-F3 / R3. Subsequently, the homology arms were fused together using primers ilvG-arm-F1 and ilvG-arm-R3 via fusion PCR to form a complete DNA fragment. Then the plasmid pREDCas9 (Addgene #71541), pGRB-ilvG-sgRNA and homology arm DNA fragment were transformed into strain TRP30 by electroporation.

[0039] The specific operation was as follows: the strain TRP30 carrying the plasmid pREDCas9 was cultured in a competent medium containing 50 μg / mL spectinomycin at 30°C until the OD 600 When OD is about 0.1-0.2, 0.1mM IPTG is added to induce the expression of Cas9 protein. 600 When the pH value rises to 0.6-0.7, the cells are collected by centrifugation and washed three times to prepare competent cells. Then, the corresponding homologous arm DNA fragments and plasmid pGRB-ilvG-sgRNA are electroporated into the competent cells at the same time using a voltage of 1.85 kV. After electroporation, 1 mL of recovery medium is immediately added and cultured at 30 ° C for 2 hours. Finally, the transformants are spread on LB solid plates containing 50 μg / mL ampicillin and spectinomycin. After overnight culture at 30 ° C, the grown transformants are randomly selected for colony PCR verification and DNA sequencing. After successful DNA sequencing verification, the engineered strain TRP31 is constructed. Subsequently, the gene aroG Q151F It was integrated into the yjiP pseudogene site of the genome of the engineered strain TRP31, and the operation was as described above to construct the engineered strain TRP32.

[0040] Example 2: Fermentation experiment of engineered strain TRP32 in a 5 L fermenter

[0041] The strain TRP32 was activated by culturing on LB solid medium at 37°C for 12 hours. The activated strain was inoculated into a 5L fermenter containing 3L seed medium. The pH of the fermentation medium was maintained at 7.0 by automatically adding ammonia water, and the temperature was maintained at 37°C. The dissolved oxygen was maintained above 30% by changing the stirrer speed and aeration rate. When the OD 600 When it reaches 10-12, the excess fermentation broth is discharged and only 450mL is left for batch fermentation. During the batch fermentation process, the pH is also maintained at 7.0, the temperature is maintained at 37°C and the dissolved oxygen is maintained at more than 30%. After the initial sugar is consumed. The glucose concentration of the fermentation broth is controlled below 2g / L. During the subsequent fermentation process, an 80% glucose solution is added to the fermenter at an appropriate rate to maintain bacterial growth and product synthesis. Finally, the constructed engineered strain TRP32 can significantly increase the production of L-tryptophan. After culturing in a 5L fermenter for 48 hours, it was found that the L-tryptophan production increased to 52.1g / L, which was 22.59% higher than the L-tryptophan production of the control strain.

[0042] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A recombinant Escherichia coli for increasing L-tryptophan production, characterized in that: The recombinant Escherichia coli is an L-tryptophan-producing Escherichia coli host that integrates and expresses an anthranilate synthase mutant TrpE Q71K and 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F .

2. The recombinant Escherichia coli according to claim 1, characterized in that The anthranilate synthase mutant TrpE Q71K The nucleotide sequence is shown in SEQ ID NO.

1.

3. The recombinant Escherichia coli according to claim 1, characterized in that The 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F The nucleotide sequence is shown in SEQ ID NO.

2.

4. The recombinant Escherichia coli according to any one of claims 1 to 3, characterized in that The Escherichia coli host is the Escherichia coli engineered strain TRP30.

5. The recombinant Escherichia coli according to claim 1 or 2, characterized in that The anthranilate synthase mutant TrpE Q71K The protein was integrated and expressed at the ilvG pseudogene site in the E. coli genome.

6. The recombinant Escherichia coli according to claim 1 or 3, characterized in that The 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F The protein was integrated and expressed at the yjiP pseudogene site in the Escherichia coli genome.

7. Use of the recombinant Escherichia coli according to any one of claims 1 to 6 in the fermentation production of L-tryptophan, characterized in that: The application is to use glucose as a carbon source and utilize the recombinant Escherichia coli to ferment and produce L-tryptophan.

8. The use according to claim 7, characterized in that The fermentation conditions are as follows: temperature of 35-38° C., rotation speed of 600-700 rpm, and pH of 7.0-7.

2.

9. The use according to claim 7, characterized in that The fermentation medium for producing L-tryptophan comprises 15-25 g / L glucose, 2-4 g / L yeast extract, 1-3 g / L citric acid, 3-5 g / L ammonium sulfate, 0.5-2 g / L sodium chloride, 0.5-2 g / L magnesium sulfate heptahydrate, 15-25 mg / L ferrous sulfate heptahydrate, 1-1.5 mg / L manganese sulfate monohydrate, 0.4-0.6 mg / L B1 , 0.08~0.12mg / LV H and 0.8-1.2 mL / L of trace element mixture.

10. A method for increasing the L-tryptophan production in Escherichia coli, characterized in that: The method is to integrate and express an anthranilate synthase mutant TrpE in an L-tryptophan-producing Escherichia coli host. Q71K and 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F , wherein the anthranilate synthase mutant TrpE Q71K The nucleotide sequence is shown in SEQ ID NO.1, and the 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant AroG Q151F The nucleotide sequence is shown in SEQ ID NO. 2, and the Escherichia coli host is the Escherichia coli engineered strain TRP30.

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