An intestinal probiotic engineered bacteria and its application in a living Chinese medicine drug delivery system

Abstract

The present invention relates to the field of biotechnology, and in particular to an intestinal probiotic engineered bacterium and its application in a live Chinese medicine drug delivery system. The present invention successfully constructs an engineered bacterium for synthesizing terpenoid compounds by overexpressing key genes of the methylerythritol pathway in Escherichia coli and optimizing the metabolic pathway, thereby overexpressing a gene encoding patchouli alcohol synthase. ， The engineered Escherichia coli bacteria capable of producing patchouli alcohol are constructed. The engineered Escherichia coli bacteria of the present invention can be used for the administration of Chinese medicine in vivo, and for maintaining intestinal health and treating intestinal diseases, and have application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an intestinal probiotic engineering bacterium and its application in a living Chinese medicine drug delivery system. Background Art

[0002] As a non-pathogenic probiotic strain, Nissle 1917 (EcN) has been widely studied and applied in the intestine. It can enhance the tight junctions of intestinal epithelial cells and reduce the penetration of harmful substances, thereby improving the intestinal barrier function. It can also interact with the intestinal immune system, regulate immune responses, and reduce inflammation. It also helps maintain the integrity of the intestinal mucus layer and the balance of intestinal microbiota. Currently, EcN has shown positive effects in the treatment of inflammatory bowel disease (IBD).

[0003] Another traditional Chinese medicine prescription that can also be used to treat intestinal diseases is Huoxiang Zhengqi Shui, whose main ingredient is patchouli alcohol. Patchouli alcohol is a sesquiterpenoid compound naturally found in patchouli, which has multiple pharmacological effects, especially in intestinal regulation, such as anti-inflammatory, antibacterial, antiviral, analgesic, and immune system regulation.

[0004] The invention constructs an engineered bacterium of Escherichia coli Nissle 1917 that produces patchouli alcohol, providing a new strategy for intestinal health and disease treatment. This engineered strain can enhance intestinal barrier function, exert anti-inflammatory effects, regulate the balance of intestinal microbiota, achieve local delivery of drugs, ensure the safety of treatment and reduce systemic side effects. In addition, the production of patchouli alcohol can be controlled by gene regulation to meet different treatment needs. The application of this engineered bacterium will play an important role in the treatment of intestinal health and intestinal diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Summary of the invention

[0005] The present invention knocks out the gene encoding alpha-galactosidase in Escherichia coli agaI α-galactosidase is a hydrolase that catalyzes the hydrolysis of α-galactosidic bonds, breaking down oligosaccharides, polysaccharides or glycosides into smaller sugar molecules and releasing monosaccharides such as galactose; knocking out the gene encoding maltodextrin phosphorylase malP , an enzyme involved in maltose metabolism; knocking out the gene encoding alkyl / aryl-sulfatase yjcS , which hydrolyzes alkyl or aryl sulfates, decomposes sulfate compounds and generates alcohols and inorganic sulfates, etc. And overexpresses the following related genes, overexpresses the gene encoding isopentenyl pyrophosphate isomerase idi, which can catalyze the interconversion between isoprene diphosphate (IPP) and dimethylallyl diphosphate (DMAPP); overexpression of the gene encoding 1-deoxy-D-xylulose-5-phosphate synthase dxs , dxs Catalyzes the condensation reaction of pyruvate and D-glyceraldehyde-3-phosphate to generate 1-deoxy-D-xylulose-5-phosphate (DXP), which is a key step in the MEP pathway; Overexpresses the gene encoding farnesyl pyrophosphate synthase ispA, Involved in the biosynthesis of isoprenoids, it catalyzes a series of reactions that polymerize isoprene units to produce farnesyl pyrophosphate (FPP), a basic precursor for many biomolecules. Overexpression of a gene encoding a tandem fusion enzyme of farnesyl pyrophosphate synthase and patchouliol synthase SynSmFPS-SynPS , which catalyzes a series of reactions to obtain patchouli alcohol. Thus, an Escherichia coli engineered bacterium producing patchouli alcohol is constructed, thereby completing the present invention.

[0006] The invention provides an intestinal probiotic engineering bacterium, which is based on a starting bacterium of Escherichia coli and overexpresses a gene encoding isopentenyl pyrophosphate isomerase, a gene encoding 1-deoxy-D-xylulose-5-phosphate synthase, a gene encoding farnesyl pyrophosphate synthase, a gene encoding a fusion enzyme of farnesyl pyrophosphate synthase and patchouli alcohol synthase.

[0007] Preferably, the starting Escherichia coli strain is Nissle 1917 (EcN).

[0008] Specifically, yjcs, agaI, malP The loci overexpress a gene encoding isopentenyl pyrophosphate isomerase, a gene encoding 1-deoxy-D-xylulose-5-phosphate synthase, a gene encoding farnesyl pyrophosphate synthase, and a gene encoding a tandem fusion enzyme of farnesyl pyrophosphate synthase and patchouli alcohol synthase.

[0009] More specifically,

[0010] Said yjcs The locus is located between 4572730 and 4573890 in the EcN genome (GenBank accession number: CP058217.1), encoding an alkylsulfatase. yjcS a segment of a gene;

[0011] Said agaI The locus is the 609885 to 610545 gene interval of the EcN genome (GenBank accession number: CP058217.1). For Encoding galactosamine-6-phosphate isomerase agaI a segment of a gene;

[0012] Said malPThe locus is the gene interval from 356458 to 357181 of the EcN genome (GenBank accession number: CP058217.1), which is the gene encoding maltodextrin phosphorylase malP Encoding HTH-type transcriptional regulator malT Promoter transcription regulatory region between genes.

[0013] Specifically, the amino acid sequence of isopentenyl pyrophosphate isomerase is SEQ ID NO: 5, the amino acid sequence of 1-deoxy-D-xylulose-5-phosphate synthase is SEQ ID NO: 7, the amino acid sequence of farnesyl pyrophosphate synthase is SEQ ID NO: 3, and the amino acid sequence of the fusion enzyme of farnesyl pyrophosphate synthase and patchouli alcohol synthase is SEQ ID NO: 9.

[0014] The present invention thus provides the use of the intestinal probiotic engineering bacteria in producing patchouli alcohol.

[0015] The present invention further provides the use of the intestinal probiotic engineering bacteria in the preparation of a living Chinese medicine drug delivery system. Specifically, the Chinese medicine ingredient is patchouli alcohol.

[0016] The present invention also provides a method for preparing a living Chinese medicine drug delivery system, which utilizes the intestinal probiotic engineered bacteria to prepare a living Chinese medicine drug delivery system, colonizes the intestinal probiotic engineered bacteria in the human intestine, and causes the strain to synthesize patchouli alcohol by taking an inducer to achieve the purpose of preventing or treating intestinal diseases.

[0017] The present invention successfully constructs an engineered bacterium for synthesizing terpenoid compounds by overexpressing key genes of the methylerythritol pathway in Escherichia coli and optimizing the metabolic pathway, thereby overexpressing the gene encoding patchouli alcohol synthase. ， The engineered Escherichia coli bacteria capable of producing patchouli alcohol are constructed. The engineered Escherichia coli bacteria of the present invention can be used for the administration of Chinese medicine in vivo, and for maintaining intestinal health and treating intestinal diseases, and have application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A is the GC-MS analysis result of the strain product, EcN- Para-idi-dxs-ispA Represents EcN- Para-idi- dxs-ispA The fermentation product of the strain, EcN-PA represents the fermentation product of the EcN-PA engineered strain.

[0019] Figure 1B This is the MS graph of patchouli alcohol in the database.

[0020] Figure 1C This is the MS graph of patchouli alcohol in the product of EcN-PA strain.

[0021] Figure 2 This is a GC quantitative analysis chart. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0023] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0024] The culture medium and components used in the following examples are as follows:

[0025] LB liquid medium: 0.5% yeast extract (YEAST EXTRACT), 1% tryptone (Tryptone), 1% sodium chloride, fixed volume to 100 ml.

[0026] LB-cm liquid selection medium: 0.5% yeast extract (YEAST EXTRACT), 1% tryptone (Tryptone), 1% sodium chloride, 0.1% chloramphenicol (50 mg / ml), fixed volume to 100 ml.

[0027] LB-Kan liquid selection medium: 0.5% yeast extract (YEAST EXTRACT), 1% tryptone (Tryptone), 1% sodium chloride, 0.1% kanamycin (10 mg / ml), fixed volume to 100 ml.

[0028] LB solid medium: 0.5% yeast extract (YEAST EXTRACT), 1% tryptone (Tryptone), 1% sodium chloride, 2% agar powder, fixed to 100 ml.

[0029] LB-cm solid selection medium: 0.5% yeast extract (YEAST EXTRACT), 1% tryptone (Tryptone), 1% sodium chloride, 2% agar powder, 0.1% chloramphenicol (50 mg / ml), fixed volume to 100 ml.

[0030] LB-Kan solid selection medium: 0.5% yeast extract (YEAST EXTRACT), 1% tryptone (Tryptone), 1% sodium chloride, 2% agar powder, 0.1% kanamycin (10 mg / ml), fixed volume to 100 ml.

[0031] LB-Kan-cm solid selection medium: 0.5% yeast extract (YEAST EXTRACT), 1% tryptone (Tryptone), 1% sodium chloride, 2% agar powder, 0.1% kanamycin (10 mg / ml), 0.1% chloramphenicol (50 mg / ml), fixed volume to 100 ml.

[0032] Example 1: Construction of promoter elements, acquisition of functional genes and tool optimization

[0033] 1. Genome extraction:

[0034] The bacterial genomic DNA extraction kit (purchased from Tianjin Newsai Biotechnology Co., Ltd.) was used to extract the genomes of Escherichia coli ATCC8739 (recorded in the article: Li Z, Chen Q, Tang J, et al. Integrating balanced mevalonate pathway into chromosome for improving lycopene production in Escherichia coli[J]. Sheng wu Gong Cheng xue bao=Chinese Journal of Biotechnology, 2019, 35(3): 404-414.) and EcN (purchased from Beina Bio--Henan Industrial Microbial Strain Engineering Technology Research Center) according to the instructions.

[0035] 2. PCR amplification

[0036] Using ATCC8739 genomic DNA as a template, primers were designed to amplify the gene fragment farnesyl pyrophosphate synthase ( ispA ) 、 Isopentenyl pyrophosphate isomerase ( idi ) 、 1-deoxy-D-xylulose-5-phosphate synthase ( dxs ) 。Using pRedcas9 plasmid as template (the plasmid is recorded in the article: Zhao, D.; Yuan, S.; Xiong, B.; Sun, H.; Ye, L.; Li, J.; Zhang, X.; Bi, C. Development of a fast and easy method for Escherichia coligenome editing with CRISPR / Cas9. Microb. Cell. Fact. 2016, 15, 205.), primers were designed to amplify the promoter fragment. Para. Using the genomic DNA of Saccharomyces cerevisiae PS03 as a template (recorded in the article: Guo Shuang, Wang Dong, Yang Tingting, Li Wenhao, Li Rongsheng, Zhang Guowei, Zhang Xueli, Dai Zhubo. Creation of Saccharomyces cerevisiae cell factory for fermentation production of patchouli alcohol [J]. Chinese Journal of Traditional Chinese Medicine, 2023, 48 (09): 2316-2324.) to design primers for amplification SynSmFPS- SynPS The amplified product was recovered and stored by gel tapping.

[0037] 3. Fusion of gene fragments

[0038] With the above fragment Para, idi, dxs As a template, design primers to amplify the fragment Para-dxs-idi ;by Para, ispA As template, primers were designed to amplify Para-ispA Fragment.

[0039] The relevant sequences are listed in the sequence list and are numbered as follows:

[0040] surface

[0041] 1. Construction of recombinant plasmid

[0042] 1. Construction of CRISPR plasmid:

[0043] Construction of pCas-Rha plasmid: pCas plasmid (pCas plasmid is recorded in the article: Li Q, Sun B, Chen J, et al. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genomeediting in Escherichia coli[J]. Acta Biochimica et Biophysica Sinica, 2021, 53(5): 620-627.) was used as a template to design primers to amplify the backbone pCas, and ATCC8739 genome was used as a template to amplify the fragment rhaPBAD (SEQ ID NO: 16) 。 The amplified products were recovered and stored by gel tapping.

[0044] Use ClonExpress® MultiS one-step cloning kit (purchased from Bio-Tech [D7010M] Seamless Cloning Kit) and follow the instructions to clone the fragments. rhaPBAD Connect with the backbone pCas to obtain the recombinant plasmid pCas-Rha.

[0045] 2. pTargetF- yjcs::idi-dxs Construction of recombinant plasmid

[0046] pTargetF-g yjcS Construction of recombinant plasmid: Using pTargetF plasmid DNA as template (pTargetF plasmid is recorded in the article: Li Q, Sun B, Chen J, et al. A modified pCas / pTargetF system forCRISPR-Cas9-assisted genome editing in Escherichia coli[J]. Acta Biochimicaet Biophysica Sinica, 2021, 53(5): 620-627.), primers were designed to reversely amplify the target site. yjcS The N20 sequence of pTargetF-g yjcS The PCR product was treated with DPN1 enzyme for three hours, added to competent cells DH5α, electroporated, incubated at 37°C for 1 hour, and coated on chloramphenicol (50 mg / ml) resistant LB plates to screen plasmids. Primers were designed for sequencing verification to obtain the correct pTargetF-g yjcS Recombinant plasmid.

[0047] Linearize pTargetT-g with primersyjcS The recombinant plasmid obtained the gene fragment pT-g yjcS ; Then amplify the upstream homology arms of the target site from the EcN genome template yjcS -up and downstream homology arms yjcS -down; with fragment pT-g yjcS, yjcS -up, yjcS -down was used as a template, and fusion PCR amplification was performed to obtain a long fragment backbone pT- yjcS- HR.

[0048] ClonExpress® MultiS One-Step Cloning Kit (purchased from Bio-Tech [D7010M] Seamless Cloning Kit) was used to clone the long fragment backbone pT- yjcS- HR, Para-idi-dxs The fragments were seamlessly cloned and incubated on chloramphenicol (50 mg / ml) resistant LB plates. The plates were cultured at 37°C overnight. Single clones were streaked and amplified with primers for preliminary verification. Single clones with the same band size were selected for sequencing verification to obtain the correct pTargetT- yjcS::idi-dxs The circular connection arrangement of the above four fragments is as follows: the homology arm fragment upstream of the target site, Para Promoter idi-dxs Gene fragment, homology arm fragment downstream of target site and linearized pTargetT-g yjcS Fragment.

[0049] 3. pTargetF- agaI :: ispA Construction of recombinant plasmid

[0050] pTargetF- gagaI Construction of recombinant plasmid: Using pTargetF plasmid DNA as template, primers were designed to reversely amplify the target site. agaI The N20 sequence of pTargetF- gagaI The PCR product was treated with DPN1 enzyme for three hours, added to the competent cells DH5α, electroporated, incubated at 37℃ for 1 hour and spread on chloramphenicol (50mg / ml) resistant LB plates to screen the plasmid, and designed primers for sequencing verification to obtain the correct pTargetF- gagaI Recombinant plasmid.

[0051] Linearize pTargetT- gagaI The recombinant plasmid obtained the gene fragment pT- gagaI ; Then amplify the upstream homology arms of the target site from the EcN genome template agaI-up and downstream homology arms agaI -down; with fragment pT- agaI, agaI -up, agaI -down was used as a template, and fusion PCR amplification was performed to obtain a long fragment backbone pT- agaI- HR.

[0052] ClonExpress® MultiS One-Step Cloning Kit (purchased from Bio-Tech [D7010M] Seamless Cloning Kit) was used to clone the long fragment backbone pT- agaI- HR, Para-ispA The fragments were seamlessly cloned and incubated on chloramphenicol (50 mg / ml) resistant LB plates. The plates were cultured at 37°C overnight. Single clones were streaked and amplified with primers for preliminary verification. Single clones with the same band size were selected for sequencing verification to obtain the correct pTargetF- agaI :: ispA The circular connection arrangement of the above four fragments is as follows: the homology arm fragment upstream of the target site, Para Promoter ispA Gene fragment, homology arm fragment downstream of target site, linearized pTargetF-g agaI Fragment.

[0053] pTargetF- malP :: SynSmFPS-SynPS Construction of recombinant plasmid

[0054] pTargetF-g malP Construction of recombinant plasmid: Using pTargetF plasmid DNA as template, primers were designed to reversely amplify the target site. malP The N20 sequence of pTargetF-g malP The PCR product was treated with DPN1 enzyme for three hours, added to competent cells DH5α, electroporated, incubated at 37°C for 1 hour, and coated on chloramphenicol (50 mg / ml) resistant LB plates to screen plasmids. Primers were designed for sequencing verification to obtain the correct pTargetF-g malP Recombinant plasmid.

[0055] Linearize pTargetT- gmalP The recombinant plasmid obtained the gene fragment pT- gmalP ; Then amplify the upstream homology arms of the target site from the EcN genome template malP -up and downstream homology arms malP -down; with fragment pT- malP, malP -up, malP-down was used as a template, and fusion PCR amplification was performed to obtain a long fragment backbone pT- malP- HR.

[0056] ClonExpress® MultiS One-Step Cloning Kit (purchased from Bio-Tech [D7010M] Seamless Cloning Kit) was used to clone the long fragment backbone pT- malP- HR, Para- SynSmFPS-SynPS The fragments were seamlessly cloned and incubated on chloramphenicol (50 mg / ml) resistant LB plates. The plates were cultured at 37°C overnight. Single clones were streaked and amplified with primers for preliminary verification. Single clones with the same band size were selected for sequencing verification to obtain the correct pTargetT- malP :: SynSmFPS-SynPS The circular connection arrangement of the above four fragments is as follows: the homology arm fragment upstream of the target site, Para Promoter SynSmFPS-SynPS Gene fragment, homology arm fragment downstream of target site and linearized pTargetF-g malP Fragment.

[0057] 2. Construction of recombinant strains

[0058] 1. Construction of EcN / pCas-Rha strain:

[0059] Pick a single colony activated on the LB solid plate (source: Henan Industrial Microbial Strain Engineering Technology Research Center, No.: BNCC361741, purchased from Beina Chuanglian Biotechnology Co., Ltd.) into 3 ml LB liquid culture medium and culture overnight; take an appropriate amount of fresh bacterial liquid into 25 ml LB liquid culture medium containing 5% arabinose, and culture it in a 37 ℃ 225rpm incubator until the OD is about 0.6; place the bacterial liquid at 4 ℃ for 15-20 minutes to precool it; collect the bacteria by centrifugation at 4 ℃ 5000r / min for 5 minutes; wash once with precooled sterile water and twice with precooled 10% glycerol; use the residual liquid to resuspend the bacteria and divide them into sterilized precooled 1.5ml centrifuge tubes, which are EcN competent. Take 50ul of EcN competent cells and add about 500ng of pCas-Rha plasmid for electroporation, incubate at 30℃ 75r / min for 1h, spread on LB-Kan solid selection medium plate, and culture in a 30℃ incubator overnight. The grown single clone was confirmed as the strain EcN / pCas-Rha after PCR verification and sequencing.

[0060] 2. EcN- Para-idi-dxs / Construction of pCas-Rha strain

[0061] The competent state of EcN / pCas-Rha strain was prepared by the above method, and an appropriate amount of pTargetF- yjcs::idi- dxs The plasmid was electroporated, incubated at 30℃ 75r / min for 1h, coated on a double-antibody plate of LB-Kan-cm solid selection medium, and cultured in a 30℃ incubator overnight. Single clones were picked and streaked, and primers were designed to amplify the characteristic fragments EcN- Para-idi-dxs -1.EcN- Para-idi-dxs -2, the product was observed by gelatinization and sequenced to obtain the recombinant strain EcN- Para-idi-dxs / pCas-Rha / pTargetF- yjcs::idi-dxs (with pCas-Rha, pTargetF- yjcs::idi-dxs Double plasmid EcN- Para-idi-dxs strains).

[0062] The positive clones with correct sequencing were selected and inoculated into 2 ml LB-Kan liquid selection medium containing rhamnose (0.5 mM) for overnight culture. The above cultured bacterial liquid was streaked on a plate of LB-Kan solid selection medium for 3 zones until a single colony was cultured. The pTargetF- yjcs::idi-dxs Whether the plasmid is eliminated, that is, the normal growth on the plate of LB-Kan solid selection medium and the normal growth on the double-antibody plate of LB-Kan-cm solid selection medium indicates that pTargetF- yjcs::idi-dxs Plasmid recombinant strain EcN- Para-idi- dxs / pCas-Rha.

[0063] 3. EcN- Para-idi-dxs-ispA Construction of strains

[0064] Select EcN- Para-idi-dxs / pCas-Rha strain was prepared as described above, and pTargetT- agaI::ispA Plasmid was added to EcN- Para-idi-dxs / pCas-Rha competent medium electroporation, incubated at 30℃ 75r / min for 1h, coated on the double-antibody plate of LB-Kan-cm solid selection medium, cultured in a 30℃ incubator overnight, picked single clones and streaked, designed primers to amplify the fragments EcN- Para-ispA -1.EcN- Para-ispA -2, the product was observed by gelatinization and sequenced to obtain the correct recombinant strain EcN- Para-idi-dxs-ispA / pCas-Rha / pTargetT- agaI::ispA .

[0065] The positive clones with correct sequencing were selected and inoculated into 2 ml LB-Kan liquid selection medium containing rhamnose (0.5 mM) for overnight culture. The above cultured bacterial liquid was streaked on a plate of LB-Kan solid selection medium until a single colony was cultured. The double-antibody plate of LB-Kan-cm solid selection medium was used to test pTargetT- agaI::ispA Whether the plasmid is eliminated, that is, the plasmid grows normally on the plate of LB-Kan solid selection medium and does not grow on the double-antibody plate of LB-Kan-cm solid selection medium, which means that pTargetT- agaI::ispA Plasmid recombinant strain EcN- Para-idi-dxs-ispA / pCas-Rha.

[0066] The above recombinant strain EcN- Para-idi-dxs-ispA / pCas-Rha was inoculated into LB liquid medium, cultured overnight at 37°C, streaked on LB solid plates in three zones, cultured overnight at 37°C, and single clones were picked and streaked on LB plates and LB-Kan solid selective medium plates to check whether the pCas-Rha plasmid was eliminated. The clones that grew normally on the LB plate and did not grow on the LB-Kan solid selective medium plate were EcN- Para-idi-dxs-ispA Engineered bacterial strains.

[0067] 4. EcN- PA Engineered bacterial strains

[0068] Select EcN- Para-idi-dxs-ispA / pCas-Rha strains were prepared to be competent, and pTargetT- malp:: SynSmFPS-SynPS Plasmid was added to EcN- Para-idi-dxs-ispA / pCas-Rha, incubate at 30℃ 75r / min for 1h, spread on a double-antibody plate of LB-Kan-cm solid selection medium, culture in a 30℃ incubator overnight, pick a single clone and streak, design primers to amplify the fragments EcN- Para-PS -1.EcN- Para-PS -2, the product was observed by gelatinization and sequenced to obtain the correct recombinant strain EcN- PA / pCas-Rha / pTargetT- agaI::ispA .

[0069] The positive clones with correct sequencing were selected and inoculated into 2 ml LB-Kan liquid selection medium containing rhamnose (0.5 mM) for overnight culture. The above cultured bacterial liquid was streaked on a plate of LB-Kan solid selection medium until a single colony was cultured. The double-antibody plate of LB-Kan-cm solid selection medium was used to test pTargetT- malP::SynSmFPS-SynPSWhether the plasmid is eliminated, that is, the plasmid grows normally on the plate of LB-Kan solid selection medium and does not grow on the double-antibody plate of LB-Kan-cm solid selection medium, which means that pTargetT- malp::SynSmFPS-SynPS Plasmid recombinant strain EcN- PA / pCas-Rha.

[0070] Eliminate the above pTargetT- malP::SynSmFPS-SynPS Plasmid EcN- PA The / pCas-Rha strain was inoculated into LB liquid medium, cultured overnight at 37°C, streaked on LB solid plates at 3 zones, cultured overnight at 37°C, and single clones were picked and streaked on LB plates and LB-Kan solid selective medium plates to check whether the pCas-Rha plasmid was eliminated. The one that grew normally on the LB plate and did not grow on the LB-Kan solid selective medium plate was EcN- PA Engineered bacterial strains.

[0071] 3. Evaluation of engineered bacteria

[0072] Shake flask fermentation: Activate the EcN-PA engineered strains and EcN- Para- idi-dxs-ispA For the recombinant strain, a single clone was picked and inoculated into 10 ml LB liquid medium containing 2% glycerol and placed in a 100 ml conical flask. The bacteria were shaken (37 °C, 250 rpm) until the bacterial liquid OD was about 4. Different concentrations of arabinose were added for induction. The culture was shaken at 37 °C and 250 rpm for 3 days. 5 ml of the bacterial liquid was placed in a 15 ml centrifuge tube, 2 ml of n-hexane was added and vortexed for 30 seconds, and the upper organic phase was collected for later use. The experiment was repeated three times, and the collected organic phase was passed through an organic nylon membrane (0.22 μm) and detected by GC-MS. Detection instrument: Agilent GC-MS Agilent 7890A / 5975C.

[0073] The fermentation products were qualitatively analyzed by GC-MS and the engineered strain EcN- PA The fermentation product showed a new peak at 15.7min that was identical to the patchouli alcohol standard. Figure 1A GC-MS analysis results of the products of the indicated strains, Figure 1B The MS graph of patchouli alcohol in the database shown is Figure 1C The MS graph of patchouli alcohol in the product of the EcN-PA strain is shown. PA The activity of the fermentation product PS.

[0074] GC-MS determination conditions of the fermentation products of the strains: injection port temperature 250 ℃, injection volume 1μL, no splitting, solvent delay 3 min; chromatographic column: HP-5ms (30m*0.25 mM); chromatographic conditions: 45 ℃, 1 min, 10 ℃ / min to 300 ℃ for 5 min; MS conditions: Full Scan: 50-750 amu. Patchouli alcohol standards were used for qualitative analysis.

[0075] Different concentrations of arabinose were used as inducer gradient experiments, and GC-MS was used to quantitatively analyze the effects of the engineered bacteria EcN- PA The fermentation products were quantitatively analyzed. Figure 2 The patchouli alcohol production of the EcN-PA strain was shown in Figure 1. The results showed that the addition of the inducer arabinose affected the expression of the patchouli alcohol gene. When 0.2% arabinose was added, the patchouli alcohol production was 1.62±0.2 mg / L.

Claims

1. An intestinal probiotic engineering bacteria, characterized in that: The invention is based on the starting bacterium Escherichia coli Nissle 1917, and overexpresses a gene encoding isopentenyl pyrophosphate isomerase, a gene encoding 1-deoxy-D-xylulose-5-phosphate synthase, a gene encoding farnesyl pyrophosphate synthase, a gene encoding farnesyl pyrophosphate synthase and a gene encoding a fusion enzyme of patchouli alcohol synthase; the overexpressed genes are controlled by an arabinose-induced promoter; And, respectively, yjcs, agaI, malP The locus overexpresses a gene encoding isopentenyl pyrophosphate isomerase, a gene encoding 1-deoxy-D-xylulose-5-phosphate synthase, a gene encoding farnesyl pyrophosphate synthase, and a gene encoding a tandem fusion enzyme of farnesyl pyrophosphate synthase and patchouliol synthase; Said yjc The locus is the 4572730 to 4573890 gene interval of the EcN genome with GenBank accession number CP058217.1, which encodes an alkylsulfatase oeLh The region of the gene; Said agarI The locus is the 609885 to 610545 gene interval of the EcN genome with GenBank accession number CP058217.1, which encodes galactosamine-6-phosphate isomerase. agarI The region of the gene; Said malP The locus is the gene interval from 356458 to 357181 of the EcN genome with GenBank accession number CP058217.1, which is the gene encoding maltodextrin phosphorylase malP Encoding HTH-type transcriptional regulator t Promoter transcription regulatory regions between genes; The amino acid sequence of isopentenyl pyrophosphate isomerase is SEQ ID NO: 5, the amino acid sequence of 1-deoxy-D-xylulose-5-phosphate synthase is SEQ ID NO: 7, the amino acid sequence of farnesyl pyrophosphate synthase is SEQ ID NO: 3, and the amino acid sequence of the fusion enzyme of farnesyl pyrophosphate synthase and patchouli alcohol synthase is SEQ ID NO:

9.

2. Use of the intestinal probiotic engineering bacteria as claimed in claim 1 in producing patchouli alcohol.

3. Use of the intestinal probiotic engineered bacteria as claimed in claim 1 in the preparation of a living Chinese medicine drug delivery system; the Chinese medicine is patchouli alcohol.

4. A method for preparing a Chinese medicine live drug delivery system, characterized in that: A living Chinese medicine drug delivery system is prepared using the intestinal probiotic engineered bacteria as claimed in claim 1.

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