Engineering probiotic with high colonization capability

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a Nissle 1917 recombinant strain with high colonization ability and application of the Nissle 1917 recombinant strain. According to the invention, gene editing is carried out on xthA and ynjE genes and fragments between the xthA and ynjE genes on a wild type Nissle 1917 genome in a genome simplification manner, a long non-essential fragment on the genome is knocked out, and an engineering bacterium with high colonization ability is constructed, so that the purpose of efficient colonization in intestinal tracts of a bee animal model can be achieved. Meanwhile, the engineering strain is relatively simple to prepare, the activity of the strain is not influenced after modification, and the engineering strain has good genome stability. And for the engineering strain, functional modules can be efficiently introduced, and a new technical means is provided for treating and researching intestinal diseases of model animals by applying the engineering strain.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a Nissle 1917 recombinant strain with high colonization ability and application thereof. Background Art

[0002] The microbial communities in the bee gut occupy a unique ecological niche. They not only interact with each other, but also have complex interactions with the host bees, playing an indispensable role in key physiological processes such as nutrient absorption, disease defense, and learning and memory. Subtle changes in the gut microbiome are not only related to the health and behavior of individual bees, but may also have broad and far-reaching impacts on agricultural output and ecosystems. Therefore, restoring and enhancing the health of bees by modifying the gut microbiome has become an effective strategy.

[0003] In beekeeping, the use of probiotics to treat or prevent microbial infections in beehives is common. Most commercially available bee probiotics consist of non-indigenous microorganisms, mainly Lactobacillus Genus and Bifidobacterium The main human probiotics are from the genus A. spp. However, it is difficult for such probiotics to stably colonize in the bee intestine. Even with continuous feeding, their proportion in the bee intestinal flora is usually less than 1%, and they will quickly die after stopping feeding. Another method is to isolate the native intestinal microorganisms of bees, such as isolating intestinal homogenates from worker bees and feeding them to unhealthy bees with a lack of microbiota or disturbed microbiota. However, harmful pathogens may be mixed in the process of intestinal microbial isolation. At the same time, these intestinal probiotics are generally difficult to genetically manipulate, and there are obvious limitations in expanding the functions of intestinal bacteria, such as enhancing defense against specific diseases or establishing the ability to degrade harmful compounds. Therefore, exploring the construction methods of engineered bee intestinal bacteria and their role in improving bee health has become an area that urgently needs in-depth research, and has the potential to bring innovative strategies for the prevention and treatment of bee diseases. Transforming heterologous bacteria with rich genetic manipulation tools into engineered bacteria that can colonize the bee intestine is a possible way to achieve this goal.

[0004] As a traditional probiotic, Escherichia coli Nissle 1917 has shown great potential in the prevention and treatment of gastrointestinal dysfunction and immune diseases. Nissle 1917 has inherent antibacterial and anti-inflammatory properties and has been widely used to treat diarrhea, inflammatory bowel diseases (IBDs), and diseases associated with the accumulation of toxic metabolites. It is not only widely used in human medicine, but also has shown wide adaptability in the intestinal environment of a variety of animals including pigs, rodents, poultry, fish, etc. In current scientific research, there has been no report on the application of Nissle 1917 probiotics in the field of bees. As a non-native intestinal bacteria of bees, Nissle 1917 also faces the problem of difficulty in colonization in bees.

[0005] The present invention successfully transformed the Nissle 1917 probiotic into a chassis strain that can stably colonize in the bee's intestine through innovative transformation. This transformation not only gives the Nissle 1917 probiotic the ability to inhibit the growth of exogenous pathogenic bacteria in the bee's intestine, but also provides a new protection mechanism for the health of the bees through its probiotic function. The present invention further extends to the field of biosynthesis, and by expressing the production pathway of lycopene in Nissle 1917, the symptoms of bee intestinal inflammatory diseases can be effectively alleviated.

[0006] This invention is not only of great significance in the field of bee health, but also opens up new possibilities for the application of synthetic biology in non-traditional hosts. The engineered Nissle 1917 can be used as a chassis strain to load other functional elements to expand the application of the strain in the diagnosis and treatment of bee diseases, and can also help explore the mechanism of interaction between intestinal microorganisms and host health, and also provide new ideas for the development of new biotechnology products. Summary of the invention

[0007] The present invention uses genome simplification to edit the wild-type Nissle 1917, knocks out a long non-essential fragment on the genome, and constructs an engineered bacterium with high colonization ability, which can achieve the purpose of efficient colonization in the intestine of the bee animal model. At the same time, the preparation of the engineered strain is relatively simple, and the activity of the strain is not affected after the modification, and it has good genome stability. In addition, for the engineered strain, functional modules can be introduced efficiently, which also provides a new technical means for the treatment and research of intestinal diseases of model animals using engineered strains.

[0008] The technical route adopted by the present invention to achieve the above-mentioned purpose is as follows: One of the technical solutions provided by the present invention is a method for improving the colonization ability of Escherichia coli Nissle 1917, wherein the method is to knock out a gene on the genome of Nissle 1917. xthA andynjE Gene and xthA and ynjE This is achieved by fragments between genes; Furthermore, the knocked-out fragment is located at positions 309420-317765 on the Nissle 1917 genome, and the NCBI RefSeq of the genome is: NZ_CP022686; the knocked-out fragment contains 8346 base pairs, involving 9 genes: xthA, ydjX, ydjY, ydjZ, ynjA, ynjB, ynjC, ynjD, ynjE ; Furthermore, the Escherichia coli Nissle 1917 strain is derived from the German Collection of Microorganisms and Cell Cultures, with ID number DMS6601.

[0009] The second technical solution provided by the present invention is a recombinant strain of Escherichia coli, wherein the recombinant strain is obtained by knocking out the gene on the genome of Escherichia coli Nissle 1917. xthA and ynjE Gene and xthA and ynjE The fragment between genes is realized; the recombinant Escherichia coli strain is named Nissle 1917-△8346; Furthermore, the above xthA and ynjE Gene and xthA and ynjE The nucleotide sequence of the fragment between genes is shown in SEQ ID NO.1.

[0010] The third technical solution provided by the present invention is the application of the recombinant bacteria Nissle 1917-△8346, especially in the colonization of bee intestines or the improvement of bee intestinal function. This carefully modified strain can not only stably colonize in the bee intestines, but also effectively inhibit the growth of foreign pathogens and enhance the natural defense barrier of bees. This ability is crucial to maintaining the balance of bee intestinal microbial communities and helps prevent the invasion and spread of pathogens.

[0011] Further, it is used in alleviating intestinal inflammation in honey bees; Furthermore, the recombinant bacteria Nissle 1917-△8346 can be used as a base bacteria for improving the intestinal function of honey bees. The target compound is obtained by expressing each functional module in the recombinant bacteria Nissle 1917-△8346, and then the target compound plays a role in the intestinal tract of honey bees, thereby improving the intestinal function. Furthermore, the target compound includes, but is not limited to: specific enzymes, drug precursors or other beneficial compounds; such as aromatic amino acid transaminase, lycopene, etc.; Furthermore, the functional module is a synthetic pathway for the target compound, such as by overexpressing CrtE , CrtB , CrtI The plasmid was introduced into Nissle 1917-△8346, so that the modified Nissle 1917 strain also has the ability to produce antioxidants such as lycopene, which has significant benefits for alleviating intestinal inflammation in honey bees. As a powerful natural antioxidant, lycopene can neutralize harmful free radicals, reduce oxidative stress, and protect intestinal cells from damage. By expressing these beneficial compounds in the bee intestine, it helps to reduce inflammatory responses and promote intestinal health, which is crucial to improving the overall well-being and survival of honey bees.

[0012] Beneficial effects: Compared with the wild-type Nissle 1917, the genome modification of the engineered bacterium Nissle 1917-△8346 did not affect the growth of the strain, and at the same time significantly improved its intestinal colonization ability. In the single-bacteria colonization in the sterile bee model, the Nissle 1917-△8346 strain can effectively colonize in the bee intestine for more than 10 days; compared with the wild type, the number of colonies of the Nissle 1917-△8346 strain in each bee intestine on the 10th day of colonization reached about 5.9×10 7 The number of colonies increased significantly.

[0013] In the Smurf experiment test and the DSS-induced enteritis bee survival test, the Nissle 1917-△8346 strain was able to significantly alleviate the rupture of the bee intestine, and the proportion of Smurf-positive bees was only 53.3%, significantly lower than that of bees not fed with engineered bacteria. In addition, the strain implanted with the lycopene synthesis pathway can significantly increase the survival rate of enteritis bees. The survival rate of bees not fed with engineered bacteria was about 0 on the 15th day, and the survival rate of bees colonized with the strain was about 40%. The engineered Nissle 1917 strain can also serve as a versatile chassis strain for in situ expression of other beneficial chemicals in the bee gut. This flexibility opens up new possibilities for developing new biotechnological applications, such as producing specific enzymes, prodrugs, or other beneficial compounds. In this way, the Nissle 1917 strain could not only improve bee health, but could also potentially bring innovative solutions to the agricultural and biopharmaceutical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is the genome knockout sequencing verification diagram of the n102 strain.

[0015] Figure 2 The growth test of strain n102 in LB medium.

[0016] Figure 3 To test the enteritis relief effect of strain n102 through Smurf experiment.

[0017] Figure 4 To test the enteritis alleviating effect of the n102+pLycopene strain through a bee survival rate experiment.

[0018] Figure 5 To determine the ability of the n102 strain to overexpress aromatic amino acid transaminases. DETAILED DESCRIPTION

[0019] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0020] Example 1: Construction of Escherichia coli n102 strain with simplified genome Escherichia coli Nissle 1917 (serotype O6:K5:H1, genome NCBI sequence number: NZ_CP022686); knockout xthA and ynjE The nucleic acid sequence of the two genes and the region between them (a total of 8346 base pairs, located at positions 309420-317765 on the genome) (shown in SEQ ID NO.1).

[0021] Using the wild-type Nissle 1917 (referred to as "nwt") as the base bacteria, competent cells were prepared, homologous arms and sgRNA for homologous recombination were designed, and the determined gene fragments were knocked out from the genome by CRISPR-Cas9 gene editing. Because a single N20 can cause a double-strand break in the genome, but for a long editing region, its break region is too far away from the homologous recombination regions at both ends, and homologous recombination cannot be effectively initiated for double exchange. Therefore, the editing efficiency of ultra-long fragments using a single N20 system is low, and it is difficult to obtain a knockout strain. We used a double N20 method to knock out long fragments in the strain.

[0022] More specifically, knockout xthA and ynjEThe method for two genes and the gene fragment between them is as follows: (1) Using the Nissle 1917 genome as a template, LF / LR as primers, PCR amplification was performed to obtain the upstream homology arm L-arm, and RF / RR as primers to obtain the downstream homology arm R-arm. The total PCR reaction system was 50 μL, including 25 μL Primestar high-fidelity enzyme, 20 μL ddH2O, 2 μL LF (RF), 2 μL LR (RR) and 1 μL Nissle 1917 genome as a template. The PCR program was: 98℃ 1 min, 56℃ 15 s, 72℃ 80 s, and 30 cycles were set. The target size was about 0.6 kb.

[0023] (2) The upstream homologous arm L-arm and the downstream homologous arm R-arm are fused using the fusion PCR method to obtain the L-arm + R-arm fragment, making it a fragment of about 1.2 kb, which is the repair fragment.

[0024] (3) Design two upstream and downstream sgRNA sequences according to the deletion sequence, use pTargetF as a template, use sgRNA-F1 / sgRNA-R1 as primers, and sgRNA-F2 / sgRNA-R2 as primers, introduce two sgRNAs into the plasmid backbone, connect the backbone and L-arm+R-arm fragments by Gibson assembly to obtain plasmid pTargetF-1, and then transform the ligation product into 100 μL of commercial Escherichia coli JM109 competent cells, ice bath for 20 min, heat shock at 42 ℃ for 45 s, immediately put on ice for 2 min, and then immediately add 700 μL of SOC medium, and resuscitate in a shaker at 37 ℃ and 200 rpm for 45 min. After centrifugation at 5000 rpm for 2 min, remove 500 μL of supernatant, evenly spread the remaining bacterial solution on the corresponding resistance plate, place it at 37 ℃ overnight, pick a single clone, extract the plasmid for sequencing verification. If the corresponding N20 and homology arms in the sequencing results are successfully introduced, it means that the correct knockout plasmid pTargetF-1 is obtained.

[0025] (4) Electroporation: The pCas9 plasmid was transferred into the competent cell of Nissle 1917. The cell was revived and cultured in a shaking incubator at 37°C and 200 rpm for 2 h. 200 μL of the aliquot was spread on an LB plate containing 20 μg / mL kanamycin resistance and cultured at 30°C overnight to obtain the Nissle 1917 + pCas9 strain.

[0026] (5) Prepare competent cells of Nissle 1917 + pCas9 strain.

[0027] (6) Add pTargetF-1 to Nissle 1917 + pCas9 competent cells for electroporation, and use kanamycin / ampicillin to select Nissle 1917 + pCas9 + pTargetF-1 transformants.

[0028] (7) Inoculate Nissle 1917 + pCas9 + pTargetF-1 transformants into liquid culture medium, add IPTG and arabinose to induce editing, and plate plate to screen transformants.

[0029] (8) The transformants were verified using external primers F1 / R2 and internal primers F3 / R4. Primers F1 / R2 produced a band in PCR amplification, but primers F3 / R4 did not produce a band, indicating that the Nissle 1917 strain with the 8346 base pair (309420-317765) fragment deleted was obtained. The actual sequencing results are as follows: Figure 1 shown.

[0030] (9) The pTargretF-1 plasmid, whose replicon is thermosensitive, was then eliminated by inducing culture at 37°C, and the pCas9 plasmid was eliminated under sucrose plate culture conditions to obtain the n102 strain (also known as the Nissle 1917-△8346 strain), which was then stored in a -80°C refrigerator. The specific primer sequences are shown in the table below:

[0031] Example 2 Effect of knocking out gene fragments on the growth of strain n102 This example is used to illustrate that knocking out a large fragment of 8346 base pairs (309420-317765) in the genome has no significant effect on the growth of the n102 strain in the nutrient-rich LB medium.

[0032] LB medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl. The wild-type Nissle 1917 strain and the constructed n102 strain were streaked on LB plates and cultured at 37°C for 12 h. Three single colonies were picked from the plates and inoculated into LB liquid culture medium, and cultured at 37°C and 220 rpm for about 12 h. Then, it was transferred to 5 mL LB culture medium at a 1% inoculum and continued to be cultured at 37°C and 220 rpm for 24 h. Samples were taken every 2 h for the first 6 times, and samples were taken 12 h after the last time and stored at 4°C. A total of 7 samples were taken. After sampling, the OD600nm absorbance was measured together. Take 200 μL each time into a 96-well plate and use an ELISA reader to measure OD600nm. If the OD value is greater than 0.8, the bacterial solution needs to be diluted appropriately so that the OD600nm measured by the ELISA reader is between 0.2-0.8. At the same time, blank LB was used as a control, and the actual OD value was subtracted from the blank control to obtain the true OD value, and the growth curves of the wild-type Nissle 1917 strain and the n102 strain were drawn.

[0033] The results are as follows Figure 2 As shown in the figure, compared with the wild-type Nissle 1917 strain, the n102 strain showed a faster growth rate, with the OD600 reaching 1.8 at 24 h. This indicates that the knockout of this non-essential segment will not harm the growth of the n102 strain, but can accelerate the growth of the bacteria.

[0034] Example 3 Single-bacterial colonization experiment of strain n102 in axenic and germ-free bee models This example is used to illustrate the knockout of xthA and ynjE After encoding the two genes and a large fragment of 8364 base pairs between them, the effect of single bacterial colonization of strain n102 was tested in both the axenic and germ-free honey bee models.

[0035] Honey bees (Apis mellifera) pupae and newly emerged bees were obtained from the brood frames of the experimental beehives and kept in an incubator at 35 °C and 50% humidity. Late pupae were manually removed from the brood frames and placed in sterile plastic boxes. Pupae emerged in an incubator at 35 °C and 50% humidity. Newly emerged sterile (MF) bees (day 0) were kept in sterile cup cages with sterile sucrose (50%, wt / vol) for 24 hours. The gut tissue of the emerging bees was ground, and the ground liquid was mixed in sterile sucrose (50%, wt / vol) and fed to MF (sterile) bees for 2 days to prepare virulence (CV) bees.

[0036] (1) Single-bacteria colonization experiment on germ-free bees The experimental operation was as follows: the wild-type Nissle 1917 (abbreviated as "nwt") and n102 strains were cultured overnight in 5 mL of LB medium, then all were centrifuged and resuspended in 2 mL of sucrose water (25% mass concentration, wt / vol) to prepare a bacterial solution with an OD600 of 1. After feeding the sterile bees for 2 days, they were fed with 50% mass concentration sucrose water. On the 5th and 10th days of feeding, intestinal tissue samples were taken from 6 bees, ground, and diluted to 2×10 5 Spread the LB plates repeatedly and count the number of colonies on the plates to monitor changes.

[0037] The results are shown in Table 1. On the fifth day, the average colony count of the nwt strain in the intestine of each bee reached about 5.1×10 7 The average colony count of strain n102 in the intestine of each bee reached about 3.9×10 7 On the 10th day of colonization, the average colony number of the nwt strain in each bee gut reached about 1.8×10 6 The average colony count of strain n102 in the intestine of each bee reached about 5.9×10 7 , after T-test, P <0.05, indicating that in sterile bees, the n102 strain had a significant increase in colonization compared with nwt.

[0038] Table 1 shows the single-bacteria colonization plate counts of strain n102 in the germ-free bee model

[0039] (2) Single-bacteria colonization experiment on bees with bacteria The experimental operation was as follows: the wild-type Nissle 1917 (abbreviated as "nwt") and n102 strains were cultured overnight in 5 mL of LB medium, then all were centrifuged and resuspended in 2 mL of sucrose water (25% mass concentration, wt / vol) to prepare a bacterial solution with an OD600 of 1. After being fed to bees with bacteria for 2 days, they were fed with 50% mass concentration sucrose water. On the 5th and 10th days of feeding, intestinal tissue samples were taken from 6 bees, ground, and diluted to 2×10 5 Spread the LB plates repeatedly and count the number of colonies on the plates to monitor changes.

[0040] The results are shown in Table 2. On the fifth day, the average colony count of the nwt strain in the intestine of each bee reached about 6.5×10 7 The average colony count of strain n102 in the intestine of each bee reached about 1.7×10 8 On the 10th day of colonization, the average colony count of strain n102 in each bee’s gut reached 4.3×10 7The average colony number of the nwt strain in each bee's intestine was 1.5×10 6 , after T-test, P <0.05, indicating that in bees with bacteria, the colonization of strain n102 was significantly improved compared with that of nwt. Table 2 shows the single-bacteria colonization plate count of strain n102 in the bee model with bacteria

[0041] Example 4 Testing the effect of strain n102 on alleviating bee enteritis through Smurf experiment The Smurf assay, also known as the Smurf test, is a method for detecting intestinal damage in fruit flies. This experiment is used to study changes in the intestinal permeability of honey bees. Dextran Sulfate Sodium (DSS) is a chemically synthesized polysaccharide sulfate. In the establishment of an animal model of colitis, it can induce colitis symptoms by damaging colonic epithelial cells. The basic principle of the Smurf assay is to feed bees with a non-metabolizable blue dye (EDS) solution and judge the integrity of the intestine by observing the permeability of the dye in the bees. If bees develop enteritis due to feeding DSS, the blue dye after feeding will leak from the intestine to the whole body; otherwise, the blue dye will only exist in the digestive tract and will not spread.

[0042] The steps for performing a Smurf assay are roughly as follows: (1) Prepare the indicator sugar water: ① EDS group: Prepare sucrose (50% mass concentration, wt / vol) stock solution, dissolve EDS, prepare 1% (wt / vol) EDS solution, and feed sterile bees; ②DSS+EDS group: Prepare sucrose (50% mass concentration, wt / vol) stock solution, dissolve DSS and EDS, prepare 5% (wt / vol) DSS + 1% (wt / vol) EDS solution, and feed the sterile bees; ③n102+DSS+EDS group: Prepare sucrose (50% mass concentration, wt / vol) stock solution, dissolve DSS and EDS, and prepare 5% (wt / vol) DSS + 1% (wt / vol) EDS solution. Take 5 mL of overnight cultured n102 cells, centrifuge, resuspend with 2 mL sucrose (25% mass concentration, wt / vol) solution, and prepare n102 bacterial solution with OD600 of 1.

[0043] The sterile bees were first fed with n102 bacterial solution for 2 days, and then fed with sucrose water containing 5% (wt / vol) DSS + 1% (wt / vol) EDS solution.

[0044] ④nwt+DSS+EDS group: Prepare sucrose (50% mass concentration, wt / vol) stock solution, dissolve DSS and EDS, and prepare 5% (wt / vol) DSS + 1% (wt / vol) EDS solution. Take 5 mL of nwt overnight cultured cells, centrifuge, resuspend with sucrose (25% mass concentration, wt / vol) solution, and prepare nwt bacterial solution with OD600 of 1.

[0045] The sterile bees were first fed with nwt bacterial solution for 2 days, and then fed with sucrose water containing 5% (wt / vol) DSS + 1% (wt / vol) EDS solution.

[0046] (2) Place the bees to be tested in a feeding cup and feed them with the above-mentioned sugar water for 7 days.

[0047] (3) Observe the distribution of blue dye in the bee’s body. If the digestive tract is obviously blue, it is judged as intestinal leakage, indicating that the bee’s intestinal structure is damaged.

[0048] (4) There are 15 bees in each group, and the number of Smurf-positive bees is counted within 7 days.

[0049] like Figure 3 As shown, the exterior of each bee's digestive tract was inspected daily, and when blue coloration was observed, the bee was considered Smurf-positive and had lost barrier integrity.

[0050] The results are shown in Table 3. Among the four groups, the number of Smurf-positive bees in the EDS group reached 1, accounting for 6.7%, indicating that EDS dye has no effect on the intestinal health of bees. The number of Smurf-positive bees in the DSS+EDS group that was not fed with engineered bacteria reached 12, accounting for 80%, indicating that 5% DSS can effectively induce intestinal damage in bees. The number of Smurf-positive bees in the nwt+DSS+EDS group colonized with wild-type nwt strains reached 11, accounting for 73.3%; the number of Smurf-positive bees in the n102+ DSS+ EDS group reached 8, accounting for 53.3%. This shows that the n102 strain can significantly alleviate the rupture of the bee intestine, and is significantly lower than the bees in the DSS+EDS group that were not fed with engineered bacteria and the nwt+DSS+EDS group that was fed with wild-type nwt bacteria.

[0051] Table 3 The effect of strain n102 on alleviating bee enteritis tested by Smurf experiment

[0052] Example 5 Testing the effect of strain n102 on alleviating bee enteritis through bee survival rate experiment In order to better understand the intestinal permeability caused by DSS (Dextran Sulfate Sodium) in the bee intestine, and the alleviating effects of nwt+pLycopene (wild type + lycopene expression strain) and n102+pLycopene (mutant + lycopene expression strain) on enteritis, the experiment was designed as follows: (1) CrtE is the gene encoding geranylgeranyl pyrophosphate synthase, CrtB is the gene encoding phytoene synthase, CrtI is the gene encoding phytoene desaturase / isomerase, CrtE (Genbank ID: BCZ76196.1), CrtB (Genbank ID: BCZ76199.1), CrtI (Genbank ID: BCZ76198.1) was connected to the pBR322 plasmid backbone to obtain a recombinant plasmid that can express lycopene, and the recombinant plasmid was transformed into the nwt and n102 strains to obtain nwt+pLycopene and n102+pLycopene strains. They were cultured overnight in LB (Luria-Bertani) medium. After the culture was completed, the bacteria were separated by centrifugation.

[0053] (2) The experimental groups for colonization with n102, nwt+pLycopene, and n102+pLycopene were DSS+n102, DSS+nwt+pLycopene, and DSS+ n102+pLycopene, respectively.

[0054] DSS+n102 group: The centrifuged n102 cells were mixed with sucrose water (25% mass concentration, wt / vol) to obtain an n102 bacterial solution with an OD600 of 1. The bacterial solution was used to feed sterile bees for 2 days, and then replaced with a sucrose solution (50% mass concentration, wt / vol) containing 5% (wt / vol) DSS for feeding.

[0055] DSS+nwt+pLycopene group: The centrifuged nwt+pLycopene cells were mixed with sucrose water (25% mass concentration, wt / vol) to obtain an nwt+pLycopene bacterial solution with an OD600 of 1. The bacterial solution was used to feed sterile bees for 2 days, and then changed to a sucrose solution (50% mass concentration, wt / vol) containing 5% (wt / vol) DSS for feeding.

[0056] DSS+ n102+pLycopene group: The centrifuged n102+pLycopene cells were mixed with sucrose water (25% mass concentration, wt / vol) to obtain an n102+pLycopene bacterial solution with an OD600 of 1. The bacterial solution was used to feed sterile bees for 2 days, and then changed to a sucrose (50% mass concentration, wt / vol) solution containing 5% (wt / vol) DSS for feeding.

[0057] The control group was DSS: sterile bees were fed with a 5% (wt / vol) DSS solution in sucrose (50% mass concentration, wt / vol).

[0058] (3) Each group of bees had 15 bees. The death of the bees was observed and recorded every day for 16 days. The observation was continued until the end of the experiment. The mortality rates of bees in the DSS+n102, DSS+nwt+pLycopene and DSS+ n102+pLycopene groups were compared with those in the control group to evaluate the effects of n102, nwt+pLycopene and n102+pLycopene strains on the alleviation of DSS-induced enteritis. The differences between the experimental groups were analyzed to determine which strain had a better effect on the alleviation of enteritis.

[0059] The results are as follows Figure 4 The results showed that the survival rate of bees in the DSS group that were not fed with engineered bacteria was about 0% on the 15th day. The survival rate of bees in the DSS+n102 group was 13.3% on the 15th day. The survival rate of bees in the DSS+nwt+pLycopene group was about 20% on the 15th day. The survival rate of bees in the DSS+n102+pLycopene group was about 40%. This shows that the n102+pLycopene strain can significantly improve the survival rate of bees with DSS-induced enteritis, and the n102+pLycopene strain can effectively alleviate the death caused by enteritis in bees.

[0060] Example 6 Experiment on overexpression of aromatic amino acid transaminase in strain n102 In order to test the ability of strain n102 to express proteins as a chassis bacteria, we chose to overexpress aromatic amino acid transaminases for testing. Aromatic amino acid transaminases are a class of enzymes that catalyze the reversible ammonia reaction between aromatic amino acids and corresponding keto acids, and play a key role in the synthesis and metabolism of aromatic amino acids. In situ expression of aromatic amino acid transaminases in the intestine is helpful for studying the abnormalities of aromatic amino acid metabolism and the pathogenesis of certain neurodegenerative diseases and metabolic diseases. The experiment was designed as follows: The coding gene of aromatic amino acid transaminase AraT (NCBI Reference Sequence: :WP_000462701.1) from the background source was connected to the pBR322 plasmid backbone and transformed into the n102 strain to obtain the n102+pAraT strain.

[0061] Experimental strains: The control group was n102, and the experimental group was n102+pAraT.

[0062] The experimental strains were cultured overnight in LB (Luria-Bertani) medium for 10 h. Then, the inoculation volume was transferred to 20 mL of fresh LB at 1 / 100 and cultured in a shaking incubator at 220 rpm / min and 37°C. When OD600 reached about 0.5-0.7, a final concentration of 2 mM IPTG was added to induce expression, and the cells were cultured in a shaking incubator at 220 rpm / min and 30°C for 12 h. 20 mL of the bacterial solution was centrifuged at 4°C, the bacteria were washed with PBS, and the cells were broken using an ultrasonic cell wall disruptor to serve as the crude enzyme solution.

[0063] (1) Quantification of protein content in crude enzyme solution using the Coomassie Brilliant Blue method: Take 100μL BSA and add 2.4 mL PBS to dilute to a final concentration of 0.2 mg / mL. Prepare the staining solution. Mix the 5×G250 staining solution by inverting it 3-5 times before use. Take 10 mL 5×G250 staining solution, add 40 mL double distilled water, and mix it into 1×G250 staining solution. Add different volumes of standard protein BSA and PBS respectively to change the BSA concentration gradient. Finally, add 1×G250 staining solution to 5mL, react at room temperature for 3 minutes, and measure the absorbance value A595 to prepare the standard curve. Add the appropriately diluted crude enzyme solution and 1×G250 staining solution to 5mL, measure the absorbance value A595, react at room temperature for 3 minutes, and calculate the protein concentration of the crude enzyme solution based on the absorbance value.

[0064] (2) IPYA and enzyme activity determination Preparation of Salkowski reagent: Prepared by dissolving 10 mmol / L FeCl3 in 35% v / v H2SO4.

[0065] Preparation of indole-3-pyruvic acid (IPYA) standard solution: used to prepare a standard curve with a concentration range of 3.8 to 92 μmol / L.

[0066] Prepare reaction buffer: 0.1 mol / L phosphate buffer (pH 8.0) containing 10 mmol / L L-tryptophan and 10 μmol / L thiamine pyrophosphate.

[0067] Prepare 2-ketoglutaric acid: the final concentration is 3 mmol / L.

[0068] Preparation of standard curve: Mix IPYA standard solutions of different concentrations with Salkowski reagent, react for 10 minutes, and measure the absorbance at 530 nm. Draw a standard curve based on the relationship between IPYA concentration and absorbance for quantification of IPYA.

[0069] Add crude enzyme extract to the reaction buffer, pre-incubate for 3 minutes, and then add 2-ketoglutaric acid to start the reaction. The reaction was carried out at 35°C for 30 minutes. After the reaction was completed, Salkowski reagent was added to terminate the reaction and start the color reaction. After reacting at room temperature in the dark for 10 minutes, the absorbance at 530 nm was measured. According to the absorbance of the sample, the concentration of IPYA was calculated using the standard curve.

[0070] Enzyme activity was calculated based on the sample protein concentration: one unit of enzyme activity was defined as the production of 1 nmol of indole-3-pyruvate per mg of tissue protein per minute.

[0071] The results are as follows Figure 5 As shown, the total protein amount of the nwt control group without plasmid overexpression of AraT was 1.5 mg / mL, and the total protein amount of the n102+pAraT group was 1.8 mg / mL. The concentration of IPYA catalyzed by the nwt group without plasmid overexpression of AraT was 0.14 μmol / mL, and the concentration of IPYA catalyzed by the n102+pAraT group was 1.02 μmol / mL. The enzyme activity of the nwt control group without plasmid overexpression of AraT was 4.97 U / mg prot, and the enzyme activity of the n102+pAraT group was 28.65 U / mg prot. This shows that the n102+pAraT strain can significantly increase the expression of aromatic amino acid transaminase from the background source.

[0072] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned embodiments can also make several deformations, combinations and improvements without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A method for improving the colonization ability of Escherichia coli Nissle 1917, characterized in that: The method is to knock out the Nissle 1917 genome xA and Yj Gene and xA and Yj This is achieved by fragments between genes.

2. The method for improving the colonization ability of Escherichia coli Nissle 1917 according to claim 1, characterized in that: The knocked-out fragment is located at positions 309420-317765 on the Nissle 1917 genome, and the NCBI RefSeq of the genome is: NZ_CP022686.

3. A recombinant strain of Escherichia coli, characterized in that: The recombinant strain is obtained by knocking out the Escherichia coli Nissle1917 genome xA and Yj Gene and xA and Yj The fragment between genes was obtained; the recombinant Escherichia coli strain was named Nissle 1917-△8346.

4. The recombinant Escherichia coli strain according to claim 3, characterized in that The nucleotide sequence of the knocked-out fragment is shown in SEQ ID NO.

1.

5. Use of the recombinant Escherichia coli strain according to claim 3 in colonizing the intestinal tract of honey bees or improving the intestinal function of honey bees.

6. The use according to claim 5, characterized in that It is used to relieve intestinal inflammation in bees.

7. The use according to claim 5, characterized in that It is used as a base bacteria to improve the intestinal function of honey bees.

8. The use according to claim 7, characterized in that The target compound is obtained by expressing each functional module in the recombinant E. coli strain, and then the target compound acts in the bee intestine, thereby improving the intestinal function; The target compound includes, but is not limited to: specific enzymes, drug precursors or other beneficial compounds; such as aromatic amino acid transaminase, lycopene and the like.

9. The use according to claim 8, characterized in that Overexpression CrtE , CrtB , CrtI The plasmid is introduced into the recombinant Escherichia coli strain, so that the transformed strain has the ability to produce antioxidants such as lycopene, which is used to alleviate intestinal inflammation in honey bees.

10. A recombinant bacterium for improving the intestinal function of bees, characterized in that: The recombinant bacteria are CrtE , CrtB , CrtI The plasmid was introduced into Nissle 1917-△8346.

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