Genetically engineered probiotic escherichia coli producing indole-3-acetic acid and application thereof

Abstract

This invention belongs to the fields of genetic engineering and microbial technology, and discloses a genetically engineered probiotic *E. coli* that produces indole-3-acetic acid and its applications. This invention uses wild-type *E. coli* Nissel 1917 as the host, and achieves this by knocking out its endogenous tryptophanase gene. tnaA The invention involves introducing a recombinant expression vector containing the tryptophan monooxygenase gene and the indoleacetamide hydrolase gene to obtain genetically engineered probiotic *E. coli*. The invention also describes the application of this genetically engineered probiotic in the microbial synthesis of indole-3-acetic acid (IAA) using tryptophan as a substrate; and its application in the preparation of drugs, probiotic preparations, or functional products for alleviating intestinal inflammation and / or maintaining intestinal barrier function. The genetically engineered probiotic *E. coli* constructed in this invention can stably and efficiently produce IAA and can be used to prepare drugs or products for alleviating intestinal inflammation and maintaining intestinal barrier function.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial technology, specifically relating to a genetically engineered probiotic Escherichia coli that produces indole-3-acetic acid and its applications. Background Technology

[0002] Indole-3-acetic acid (IAA) is an important endogenous aryl hydrocarbon receptor (AhR) ligand produced from tryptophan by gut microbiota. IAA plays a crucial role in maintaining intestinal barrier integrity, regulating immune homeostasis, and inhibiting intestinal inflammatory responses by activating the AhR signaling pathway, making it one of the core molecules in microbiota-host interactions. For example, Chinese patent CN121154706A discloses that loganic acid can significantly regulate the gut microbiota structure of arthritic mice, increase the abundance of *Lactobacillus murinus* in the gut, and promote the production of its tryptophan metabolite, indole-3-acetic acid, thereby exerting an anti-rheumatoid arthritis effect through a gut microbiota-dependent mechanism.

[0003] Currently, there is a lack of live bacterial preparations capable of efficiently and safely producing IAA in situ within the intestine. *Escherichia coli* (EcN) Escherichia coli Nissle 1917 is a non-pathogenic probiotic strain certified by the FDA as GRAS (Generally Recognized As Safe). EcN can treat intestinal diseases such as diarrhea and inflammatory bowel disease (especially ulcerative colitis) through mechanisms such as regulating gut microbiota, strengthening the intestinal barrier, and modulating immunity. Clinical studies have shown that its efficacy is comparable to mesalazine. For example, Chinese patent CN119193456A discloses an engineered intestinal probiotic strain, constructing an Escherichia coli strain capable of producing patchouli alcohol. This engineered E. coli strain can be used for live administration of traditional Chinese medicine, and it has application value for maintaining intestinal health and treating intestinal diseases.

[0004] Modifying EcN to synthesize AhR ligands such as IAA enables sustained and controllable local IAA delivery in the intestine, which can further enhance the alleviating effect of EcN on intestinal inflammation. However, natural EcN lacks an IAA synthesis pathway, and its own tryptophan metabolism mainly occurs via... tnaA The gene-encoded tryptophanase catalyzes the production of indole, not IAA; at the same time, the accumulation of indole competes for tryptophan substrates and may have adverse effects on the intestinal barrier.

[0005] Therefore, constructing an engineered probiotic strain that can efficiently convert tryptophan into IAA and is suitable for the intestinal microenvironment has important clinical translational value. Summary of the Invention

[0006] The purpose of this invention is to provide a genetically engineered probiotic Escherichia coli that produces indole-3-acetic acid and its application. The constructed genetically engineered probiotic Escherichia coli can stably and efficiently produce IAA, and can be used to prepare drugs or products that alleviate intestinal inflammation and maintain intestinal barrier function.

[0007] To achieve the above-mentioned objectives, the technical solution of this invention is as follows: A genetically engineered probiotic Escherichia coli that produces indole-3-acetic acid, using wild-type Escherichia coli Escherichia coli Using Nissle 1917 as the host, the endogenous tryptophanase gene was knocked out. tnaA (NCBI-Gene ID:948221), and a recombinant expression vector containing tryptophan monooxygenase gene and indoleacetamide hydrolase gene was introduced to obtain genetically engineered probiotic Escherichia coli.

[0008] This invention uses probiotic Escherichia coli Escherichia coli Nissle 1917 (EcN) was used as the host, and the virus was knocked out. tnaA The gene blocks the endogenous indole synthesis pathway, reducing substrate competition. Simultaneously, it expresses tryptophan monooxygenase (TMO) and indole-3-acetamide hydrolase (IaaH) with a strong promoter, enabling tryptophan to be converted to indole-3-acetic acid via the indole-3-acetamide pathway, and then converting tryptophan into indole-3-acetamide (IAM) and IAA in sequence.

[0009] The tryptophan monooxygenase gene is Bptmo The Bptmo The nucleotide sequence is shown in SEQ ID NO.1; the indoleacetamide hydrolase gene is... Bpiaah The Bpiaah The nucleotide sequence is shown in SEQ ID NO.2.

[0010] The above Bptmo and Bpiaah The gene originates from Burkholderia pyrrole ( Burkholderia pyrrocinia Tryptophan monooxygenase gene Bptmo With indoleacetamide hydrolase gene Bpiaah The nucleotide sequence is a codon-optimized sequence based on the codon preference of *E. coli*. The tryptophan monooxygenase gene and the indoleacetamide hydrolase gene encode the corresponding tryptophan monooxygenase. Bp TMO and indoleacetamide hydrolase Bp The amino acid sequences of IaaH are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0011] The recombinant expression vector uses P T7lacO ParaBAD or P trclacO Startup driver Bptmo and Bpiaah Expression. Among them, P T7lacO P araBAD or P trclacO The nucleotide sequences of the promoters are shown in SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, respectively.

[0012] The recombinant expression vector sequentially includes a promoter, a ribosome binding site (RBS), and... Bptmo Gene, ribosome binding site RBS and Bpiaah Gene expression cassette.

[0013] The present invention also provides a method for constructing the above-mentioned genetically engineered probiotic Escherichia coli, comprising the following steps: (1) Knockout of host bacteria using homologous recombination technology Escherichia coli Nissle in 1917 tnaA Gene; (2) Constructing a system containing Bptmo Genes and Bpiaa Recombinant expression vector of the h gene; (3) Introduce the recombinant expression vector tnaA The genetically engineered probiotic Escherichia coli was obtained from a host bacterium with a gene deletion.

[0014] Specifically, step (2) is as follows: (2-1) The vector was reverse amplified using PCR technology and specific primers containing homologous recombination sequences, and the recovered product was a linearized vector fragment; (2-2) PCR amplification of the promoter-tryptophan monooxygenase gene and the RBS-indoleacetamide hydrolase gene was performed using specific primers, and the target gene fragments were recovered. (2-3) The target gene fragment and the linearized vector fragment were subjected to in vitro homologous recombination reaction using the seamless cloning method, transformed into Escherichia coli TOP10 competent cells, and after positive clone screening and sequencing verification, positive clones were amplified and plasmids were extracted to obtain recombinant expression vectors.

[0015] The present invention also provides an application of the above-mentioned genetically engineered probiotic Escherichia coli in the microbial synthesis of indole-3-acetic acid using tryptophan as a substrate.

[0016] The method for microbial synthesis of indole-3-acetic acid using tryptophan as a substrate includes the following steps: (1) Genetically engineered probiotic Escherichia coli was streaked and activated into a solid culture medium containing antibiotics. Single clones were picked and inoculated into a liquid culture medium containing antibiotics to obtain seed culture. (2) The seed culture is inoculated into the fermentation medium for initial fermentation culture; (3) After fermentation for a certain period of time, add an inducing agent to induce fermentation culture; (4) During induction, tryptophan is added to the substrate for biotransformation to synthesize indole-3-acetic acid.

[0017] Preferably, the inducing agent is isopropyl-β-D-thiogalactoside or arabinose, the final concentration of tryptophan is 5 mM, the induction culture temperature is 16℃, and the induction culture time is 20 h.

[0018] The antibiotics mentioned in step (1) are chloramphenicol and ampicillin; the solid culture medium for activating the engineered bacteria mentioned in step (1) is tryptone (10 g / L), yeast extract (5 g / L) and sodium chloride (10 g / L); the culture conditions for the seed culture mentioned in step (1) are 37℃, 200 rpm, 16 h; the proportion of the seed culture medium inoculated into the fermentation medium mentioned in step (1) is 2%.

[0019] The fermentation medium in step (2) consists of tryptone (16 g / L), yeast extract (10 g / L) and sodium chloride (5 g / L); according to the above scheme, the initial fermentation conditions of the fermentation medium in step (2) are 37℃, 220 rpm, and 3h.

[0020] The inducer mentioned in step (3) is isopropyl-β-d-thiogalactoside (IPTG); the final concentration of the inducer IPTG mentioned in step (3) is 0.5 mM; the induction fermentation culture conditions of the fermentation medium mentioned in step (3) are 20℃, 220 rpm, 20 h; the final concentration of the inducer IPTG mentioned in step (3) is 0.5 mM; The final concentration of the substrate tryptophan in step (4) is 5 mM, and the addition amount is 5% v / v; This invention also provides a method for detecting indole-3-acetic acid by liquid chromatography, comprising: S1. Take 500 μL of induced fermentation product, add 1 mL of methanol, mix and then perform ultrasonic disruption. S2. Centrifuge the ultrasonically disrupted product at 12000 rpm for 10 min and collect the supernatant. S3. The supernatant is filtered through a 0.22 μm filter membrane into a 2 mL liquid chromatography vial to obtain the sample to be tested; S4. The chromatographic conditions for high performance liquid chromatography are as follows: the chromatographic column is TC-C18(2) (5 μm, 4.6×250 mm) or an equivalent column, the mobile phase A is ultrapure water containing 0.1% v / v formic acid, the mobile phase B is pure acetonitrile containing 0.1% v / v formic acid, the gradient conditions are: 0-10 min 10%B→80%B, 10-12 min 80%B, 12-15 min 10%B, 15-20 min 10%B, the flow rate is 1 mL / min, the injection volume is 10 μL, the column temperature is 35℃, and the detection wavelength is 280 nm. S5. Based on the peak area conversion of the indole-3-acetic acid standard, calculate the standard curve and finally calculate the content of indole-3-acetic acid in the sample.

[0021] The present invention also provides the application of the above-mentioned genetically engineered probiotic Escherichia coli in the preparation of drugs, probiotic preparations or functional products for relieving intestinal inflammation and / or maintaining intestinal barrier function.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention relates to genetically engineered probiotics for producing indole-3-acetic acid (IAA), which can stably and efficiently produce IAA using tryptophan as a substrate, with a maximum conversion rate of 32.86%. Using 1.02 g / L tryptophan (approximately 5 mM) as a substrate, the engineered probiotic EcN-tnaAPtrc08 constructed in this invention can convert up to 0.22 g / L IAA (approximately 1.27 mM). The engineered probiotics constructed in this invention can be used to prepare probiotic agents that alleviate intestinal inflammation and maintain intestinal barrier function. Attached Figure Description

[0023] Figure 1 A strategy for the overall metabolic remodeling of engineered probiotics that synthesize indole-3-acetic acid using tryptophan as a substrate; Figure 2 Escherichia coli chassis strain EcNΔ tnaA Electrophoresis image of positive clone PCR verification; Figure 3 Image of the recombinant plasmid vector; Figure 4 The results of SDS-PAGE detection of tryptophan monooxygenase and indoleacetamide hydrolase in this invention are shown below. M represents the standard molecular weight of the protein, Cell represents the whole cell sample after induction by the recombinant expression strain EcN-tnaAPtrc08, Sp represents the supernatant after centrifugation of the cell lysate after induction, and Sd represents the precipitate after centrifugation of the cell lysate after induction. Figure 5 This is a chromatogram of the liquid chromatography detection results for indole-3-acetic acid; Figure 6The efficiency of engineered probiotics in converting tryptophan into indole-3-acetic acid. Detailed Implementation

[0024] Example 1 Step 1, Knockout tnaA E. coli chassis strain EcNΔ tnaA Construction (1) Using pBSKLcmL plasmid as a template, PCR amplification was performed with primers dtnaA-F and dtnaA-R to obtain the knockout plasmid. tnaA The required complement fragment for the gene is 1319 bp in size. The complement fragment contains 50-60 bp upstream and downstream homologous arms at both ends, and the complement fragment LoxP-CmlR-LoxP in the middle of the homologous arms consists of a chloramphenicol resistance gene with a loxP site at each end; (2) The purified replenishment fragment was electroporated into competent cells of Escherichia coli EcN / pSim6 strain (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and after being recovered in a shaker at 30°C for 2 h, it was spread on a solid LB plate containing 20 μg / mL chloramphenicol. (3) Select positive clones and perform PCR amplification using primer pair dtnaA-yz-F / dtnaA-yz-R. Select successful knockout clones. tnaA The target chassis strain of the gene EcNΔ tnaA The electrophoresis diagram of the PCR verification of the positive strain during the process is shown in [image missing]. Figure 2 .

[0025] Step 2: Construction of engineered bacteria expressing tryptophan monooxygenase and indoleacetamide hydrolase (1) Artificially synthesized Bptmo Using the template, the nucleotide sequence is shown in SEQ ID NO.1, and primer pairs P are used respectively. T7 -TMO-F / TMO-R, P araBAD -TMO-F / TMO-R and P trclacO -TMO-F / TMO-R (P T7lacO P araBAD and P trclacO The promoter (nucleotide sequences are shown in SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7, respectively) was subjected to PCR amplification. The amplification products were purified by agarose gel electrophoresis and gel recovery to obtain P. T7 -TMO, P araBAD -TMO and P trclacO -TMO target fragments, with fragment sizes of 1728 bp, 1746 bp, and 1718 bp, respectively; (2) Artificially synthesized BpiaaHUsing the gene as a template, the nucleotide sequence is shown in SEQ ID NO.2. PCR amplification was performed using primer pair RBS-IaaH-F / IaaH-R. The amplification product was purified by agarose gel electrophoresis and gel recovery to obtain the RBS-IaaH target fragment, which is 1497 bp in size. (3) Using pETDuet-1 as a template, PCR amplification was performed with primer pair T7-pF / T7-pR to obtain the linearized vector fragment pETDuet, which is 5180 bp in size; (4) Using pSB3K3araBAD as a template, PCR amplification was performed with primer pair ParaBAD-pF / ParaBAD-pR to obtain the linearized vector fragment pSB3K3araBAD, which is 3924 bp in size; (5) Using pGEX-6P-1 as a template, PCR amplification was performed with primer pair PtarlacO-pFarlacO-pR to obtain the linearized vector fragment pGEX, which is 4330 bp in size; The primers used in steps (1) to (5) are shown in Table 1.

[0026] Table 1 Primer names and sequences

[0027] The PCR amplification program in the above steps is as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles, 72℃ final extension for 5 min, and 16℃ hold. (6) The above-mentioned target fragment P T7 -TMO and RBS-IaaH are linked to the linearized vector pETDuet, targeting fragment P araBAD -TMO and RBS-IaaH are linked to the linearized vector pSB3K3araBAD, targeting fragment P trclacO -TMO and RBS-IaaH were ligated with the linearized vector pGEX and transformed into E. coli TOP10 competent cells, respectively. After positive clones were selected and verified by sequencing, recombinant plasmids were extracted and amplified, and named pT7lacO08, paraBAD08 and pTrclacO08, respectively. (7) The pT708, paraBAD08 and pTrclacO08 plasmids were transformed into Escherichia coli EcNΔ using electroporation. tnaAThe competent cells were spread on LB solid medium containing 100 μg / mL ampicillin and incubated at 37°C for 12-16 hours until positive clones appeared. Positive clones were picked to obtain the expected engineered bacteria EcN-tnaAPT7l08, EcN-tnaAParaBAD08 and EcN-tnaAPtrc08.

[0028] Application Example 1: Microbial synthesis of indole-3-acetic acid using tryptophan as a substrate (1) The engineered bacteria EcN-tnaAPT7l08, EcN-tnaAParaBAD08 and EcN-tnaAPtrc08 were streaked on LB solid plates containing 100 μg / mL ampicillin or 50 μg / mL kanamycin and incubated at 37°C upside down overnight. (2) Select single clones and inoculate them into seed culture medium and culture overnight to obtain seed liquid. The seed culture medium contains 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride. The culture conditions for seed liquid are 37℃ and 220 rpm. (3) The above seed liquid was inoculated into the fermentation medium at an inoculation rate of 2% for fermentation culture. The fermentation medium contained 16 g / L tryptone, 10 g / L yeast extract and 5 g / L chloride. The fermentation culture conditions were 37℃ and 220 rpm. (4) After fermentation culture for 3 h, 0.5 mM IPTG or 0.5 mg / mL arabinose was added to induce expression, and tryptophan with a final concentration of 5 mM was added to induce fermentation of indole-3-acetic acid. The temperature of induction fermentation culture was 20℃ and the induction time was 20 hours. After fermentation, the yield of indole-3-acetic acid was detected by high performance liquid chromatography (HPLC).

[0029] (5) Determination of indole-3-acetic acid yield by high performance liquid chromatography ① Take 500 μL of fermentation product, add 1 mL of methanol, mix thoroughly, and then sonicate for 10 min to obtain fermentation product extract. ②The fermentation product extract was centrifuged at 12000 rpm for 10 min, and the supernatant was collected; ③ The supernatant is filtered through a 0.22 μm filter membrane into a brown liquid phase bottle to obtain the sample to be tested; ④ The chromatographic conditions for high performance liquid chromatography are as follows: the chromatographic column is TC-C18(2) (5 μm, 4.6×250 mm) or an equivalent column, the mobile phase A is ultrapure water containing 0.1% v / v formic acid, the mobile phase B is pure acetonitrile containing 0.1% v / v formic acid, the gradient conditions are: 0-10 min 10%B→80%B, 10-12 min 80%B, 12-15 min 10%B, 15-20 min 10%B, the flow rate is 1 mL / min, the injection volume is 10 μL, the column temperature is 35℃, and the detection wavelength is 280 nm. ⑤ Based on the peak area of ​​the indole-3-acetic acid standard, calculate the content of indole-3-acetic acid in the sample. The results are shown in [Figure number missing]. Figure 6 .

[0030] The above results indicate that the method provided by this invention, which uses probiotic Escherichia coli Nissle 1917 as a chassis host and knocks out the tryptophanase gene, is effective. tnaA Blocking the tryptophan-to-indole conversion pathway and using the IPTG-inducible promoter P T7lacO and P trclacO arabinose-inducible promoter P araBAD By inducing the expression of tryptophan monooxygenase TMO and indoleacetamide hydrolase IaaH, respectively, engineered probiotics capable of stably and efficiently producing IAA using tryptophan as a substrate were successfully constructed, with IAA production rates of 21.66%, 32.86%, and 9.97%, respectively. Using 1.02 g / L tryptophan (approximately 5 mM) as a substrate, the engineered probiotic EcN-tnaAPtrc08 constructed in this invention can generate up to 0.22 g / L IAA (approximately 1.27 mM). This engineered probiotic can be used to prepare probiotic agents that alleviate intestinal inflammation and maintain intestinal barrier function.

Claims

1. A genetically engineered probiotic Escherichia coli that produces indole-3-acetic acid, characterized in that, wild-type Escherichia coli Escherichia coli Using Nissle 1917 as the host, the endogenous tryptophanase gene was knocked out. tnaA The recombinant expression vector containing tryptophan monooxygenase gene and indoleacetamide hydrolase gene was introduced to obtain genetically engineered probiotic Escherichia coli. Among them, the tnaA As shown in NCBI-Gene ID:948221, the tryptophan monooxygenase gene is Bptmo The Bptmo The nucleotide sequence is shown in SEQ ID NO.1, and the indoleacetamide hydrolase gene is... Bpiaah The Bpiaah The nucleotide sequence is shown in SEQ ID NO.

2. Bptmo and Bpiaah Derived from Burkholderia pyrrole Burkholderia pyrrocinia ; The recombinant expression vector uses P T7lacO P araBAD or P trclacO Startup driver Bptmo and Bpiaah The recombinant expression vector sequentially comprises a promoter, a ribosome binding site (RBS), and... Bptmo Gene, ribosome binding site RBS and Bpiaah Gene expression cassette.

2. A method for constructing genetically engineered probiotic Escherichia coli as described in claim 1, characterized in that, Includes the following steps: (1) Knockout of host bacteria using homologous recombination technology Escherichia coli Nissle in 1917 tnaA Gene; (2) Constructing a system containing Bptmo Genes and Bpiaah Recombinant gene expression vectors; (3) Introduce the recombinant expression vector tnaA The genetically engineered probiotic Escherichia coli was obtained from a host bacterium with a gene deletion.

3. The application of the genetically engineered probiotic Escherichia coli as described in claim 1 in the microbial synthesis of indole-3-acetic acid using tryptophan as a substrate.

4. The application according to claim 3, characterized in that, The method for microbial synthesis of indole-3-acetic acid using tryptophan as a substrate includes the following steps: (1) Genetically engineered probiotic Escherichia coli was streaked and activated into a solid culture medium containing antibiotics. Single clones were picked and inoculated into a liquid culture medium containing antibiotics to obtain seed culture. (2) The seed culture is inoculated into the fermentation medium for initial fermentation culture; (3) After fermentation, add an inducing agent to induce fermentation culture; (4) During induction, tryptophan is added to the substrate for biotransformation to synthesize indole-3-acetic acid.

5. The application according to claim 4, characterized in that, The inducer is isopropyl-β-D-thiogalactoside or arabinose, the final concentration of tryptophan is 5 mM, the induced fermentation culture temperature is 20℃, and the induced fermentation culture time is 20 h.

6. The application according to claim 4, characterized in that, The indole-3-acetic acid was detected by liquid chromatography, including: S1. Take 500 μL of induced fermentation product, add 1 mL of methanol, mix and then perform ultrasonic disruption. S2. Centrifuge the ultrasonically disrupted product at 12000 rpm for 10 min and collect the supernatant. S3. The supernatant is filtered through a 0.22 μm filter membrane into a 2 mL liquid chromatography vial to obtain the sample to be tested; S4. The chromatographic conditions for high performance liquid chromatography are as follows: the chromatographic column is TC-C18(2) (5 μm, 4.6×250 mm) or an equivalent column, the mobile phase A is ultrapure water containing 0.1% v / v formic acid, the mobile phase B is pure acetonitrile containing 0.1% v / v formic acid, the gradient conditions are: 0-10 min 10%B→80%B, 10-12 min 80%B, 12-15 min 10%B, 15-20 min 10%B, the flow rate is 1 mL / min, the injection volume is 10 μL, the column temperature is 35℃, and the detection wavelength is 280 nm. S5. Based on the peak area conversion of the indole-3-acetic acid standard, calculate the standard curve and finally calculate the content of indole-3-acetic acid in the sample.

7. The use of the genetically engineered probiotic Escherichia coli according to claim 1 or the genetically engineered probiotic Escherichia coli according to any one of claims 3-6 in the preparation of drugs, probiotic preparations or functional products for relieving intestinal inflammation and / or maintaining intestinal barrier function.

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