Escherichia coli whole genome CRISPRi library construction and application

Abstract

The invention discloses a method for constructing an escherichia coli whole genome CRISPRi library and an application of the escherichia coli whole genome CRISPRi library in target product synthesis, and belongs to the technical field of gene engineering. According to the invention, an sgRNA plasmid library is designed according to escherichia coli BL21 (DE3) genome information, and a sensor plasmid and an sgRNA library plasmid are simultaneously introduced into a tetR-Ptet-dCas9 expression strain to obtain a CRISPRi library which can be used for screening strains with high yield of target products. According to the present invention, by using lactyl-N-neotetraose as a model product, eight inhibition genes, such as cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB, beneficial to the production of the lactyl-N-neotetraose are obtained; according to the invention, a screening method with feasibility is established, a key gene beneficial to synthesis of a target product is identified and obtained, and an efficient method is provided for enhancing synthesis of the target product of a strain.

Description

Technical Field

[0001] The present invention relates to the construction and application of a CRISPRi library on the scale of the entire genome of Escherichia coli, so as to screen and obtain key targets that can increase the yield of target products, and belongs to the field of biotechnology. Background Art

[0002] Escherichia coli is a model microorganism widely used in the industrial production of high-value products. Due to its clear genetic background, rapid growth, ease of genetic manipulation, and high-density fermentation, it is widely used in the production of recombinant proteins and metabolites. Traditional transformation methods, such as knocking out genes in competing and decomposition pathways, overexpressing genes in synthetic pathways, and optimizing the expression levels of key genes, are common strategies for improving target products, but these methods are highly dependent on prior knowledge. However, the close mutual constraints and dynamic connections between E. coli's internal metabolic reactions and cellular physiological processes, as well as the current lack of a systematic understanding of the mechanisms by which known and unknown genes in the genome affect target products, pose significant challenges to traditional rational design methods in further improving target product yields.

[0003] Compared to rational design methods that rely on prior knowledge of metabolic networks, irrational transformation methods (such as mutagenesis and adaptive evolution) can bypass complex metabolic mechanism analysis and directly screen for high-yielding strains, thereby more efficiently discovering potential key targets. However, these methods often suffer from high randomness and low positive mutation rates. Even if the target phenotype is achieved, key mutation sites must still be identified through methods such as genome sequencing and metabolic analysis, a process that is often time-consuming, labor-intensive, and costly.

[0004] CRISPRi (CRISPR interference) library screening can rapidly identify key genes that affect target phenotypes by constructing large-scale sgRNA libraries to target whole-genome genes and combining them with high-throughput screening methods, providing a new solution for improving the efficiency and performance of microbial cell factories.

[0005] Human milk oligosaccharides (HMOs), as important prebiotics, have many unique health effects on infants. Among them, lactoyl-N-neotetraose is the most abundant and important neutral core HMO, accounting for approximately 6% of the total HMOs. Studies have shown that lactoyl-N-neotetraose has physiological benefits such as prebiotics, immunomodulatory, anti-inflammatory, prevention of necrotizing enterocolitis, and promotion of intestinal epithelial cell maturation. Lactoyl-N-neotetraose has been approved for use in the nutritional industry, including infant formula, infant cereals, and infant nutrition products. It can also be used in dietary supplements or health functional foods for children, adults, the elderly, and lactating women, demonstrating significant commercial value and increasing interest in its efficient synthesis.

[0006] Therefore, how to apply CRISPRi library technology to identify key functional genes affecting lacto-N-neotetraose synthesis is the key to in-depth analysis of the complex regulatory network of lacto-N-neotetraose synthesis and the construction of an efficient lacto-N-neotetraose cell factory. Summary of the Invention

[0007] In response to the above-mentioned deficiencies in the prior art, the present invention provides a CRISPRi library construction and application on the whole genome scale of Escherichia coli, aiming to solve the technical problem of the lack of an efficient method for identifying key functional genes affecting lacto-N-neotetraose synthesis.

[0008] The first technical solution provided by the present invention is a method for screening key inhibitory genes for target product synthesis based on the Escherichia coli whole-genome CRISPRi library. The method is to simultaneously express a lactose sensor plasmid and a whole-gene targeted sgRNA library plasmid in Escherichia coli integrated with tetR-Ptet-dCas9, and identify high-yielding strains through high-throughput screening. The plasmids extracted from the high-yielding strains are sent for Sanger sequencing to identify the targeted inhibitory genes.

[0009] In one embodiment, the method comprises the following steps:

[0010] (1) Integrate the tetR-Ptet-dCas9 expression cassette into the ldhA site of the Escherichia coli genome to obtain a tetR-Ptet-dCas9 genome-integrated strain;

[0011] (2) Design and synthesize genome-wide encoding gene-targeting sgRNA library plasmids based on the genomic information of Escherichia coli BL21 (DE3);

[0012] (3) introducing the constructed lactose sensor plasmid into the tetR-Ptet-dCas9 genome-integrated strain of step (1) to obtain a sensing strain;

[0013] (4) introducing the sgRNA library plasmid in step (2) into the sensing strain in step (3) to obtain the E. coli CRISPRi library;

[0014] (5) Perform high-throughput screening on the E. coli CRISPRi library in step (4), and select individuals with lower green fluorescence intensity than the control group for verification.

[0015] In one embodiment, the nucleotide sequence of the tetR-Ptet-dCas9 is shown as SEQ ID NO.1.

[0016] In one embodiment, the sgRNA library plasmid construction method is as follows: the coding gene information of Escherichia coli is collected; then, sgRNA specific to the coding gene is designed using the CRISPOR (https: / / crispor.gi.ucsc.edu / crispor.py) design tool to cover 99.89% of the genome coding gene information; these oligonucleotides are synthesized by high-throughput chip and ligated to the P on the vector pSC101 plasmid by homologous recombination. J23119 The sgRNA library plasmid was then transformed into Escherichia coli competent cells.

[0017] In one embodiment, the lactose-responsive transcription factor used to construct the lactose sensor plasmid is BgaR from Bacillus megaterium or Clostridium perfringens.

[0018] In one embodiment, the plasmid used to express the BgaR gene is pACYCDuet-1 or pETDuet-1, preferably pACYCDuet-1, and the promoter used is the arabinose-inducible promoter P BAD .

[0019] In one embodiment, the promoter for expressing green fluorescent protein in the lactose sensor plasmid is the BgaR-activated promoter P BgaA / P BgaL .

[0020] In one embodiment, the amino acid sequences of the BgaR from Bacillus megaterium or Clostridium perfringens are SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0021] In one embodiment, the nucleotide sequences encoding the BgaR gene from Bacillus megaterium or Clostridium perfringens are SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

[0022] In one embodiment, the promoter P for expressing green fluorescent protein BgaA / P BgaL The sequence is shown in SEQ ID NO.6.

[0023] In one embodiment, the target product is lacto-N-neotetraose.

[0024] In one embodiment, the screening process indirectly characterizes lacto-N-neotetraose production capacity using green fluorescence intensity. Individuals with enhanced production are screened using 96-well plate culture and then screened using shake flasks to identify targeted inhibitory sites. To mitigate the effects of the sensor plasmid on lacto-N-neotetraose production, the identified sites are reintegrated into the pSC101 plasmid, further electroporated into the host bacteria, and confirmed again using shake flasks.

[0025] In one embodiment, the identification method in step (5) is Sanger sequencing.

[0026] In one embodiment, the suppressor genes include genes cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB.

[0027] The second technical solution provided by the present invention is Escherichia coli with improved lacto-N-neotetraose production, in which at least one of the following genes is inhibited: cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB.

[0028] The third technical solution provided by the present invention is the use of one or more of the genes cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB to increase the yield of lactoyl-N-neotetraose.

[0029] In one embodiment, the application is to increase the production of lacto-N-neotetraose in Escherichia coli.

[0030] In one embodiment, the NCBI accession number of the nucleotide sequence of cysW is CP053602.1 (2414505-2415380).

[0031] In one embodiment, the NCBI accession number of the nucleotide sequence of pyrG is CP053602.1 (2741916-2743553).

[0032] In one embodiment, the NCBI accession number of the nucleotide sequence of sprE is CP053602.1 (1279332-1280345).

[0033] In one embodiment, the NCBI accession number of the nucleotide sequence of pcnB is CP053602.1 (160534-161952).

[0034] In one embodiment, the NCBI accession number of the nucleotide sequence of YqjA is CP053602.1 (3115370-3116032).

[0035] In one embodiment, the NCBI accession number of the nucleotide sequence of TnaB is CP053602.1 (3780330-3781577).

[0036] In one embodiment, the NCBI accession number of the nucleotide sequence of TolB is CP053602.1 (737699-738991).

[0037] In one embodiment, the NCBI accession number of the nucleotide sequence of ihfB is CP053602.1 (970216-970500).

[0038] In one embodiment, the targeting sequences for inhibiting cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB are shown as SEQ ID NOs. 7-14, respectively.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The present invention constructs a method for constructing a whole-genome CRISPRi library of Escherichia coli and its application in improving target products.

[0041] (2) Using lacto-N-neotetraose as a model product, the genes cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB screened and obtained by the method of the present invention can effectively increase the production of lacto-N-neotetraose, resulting in an increase of lacto-N-neotetraose production by 11.33%, 18.55%, 17.07%, 30.23%, 12.42%, 25.10%, 7.32%, 16.67% and 31.25%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 To construct the CRISPRi system in Escherichia coli and verify its inhibitory effect.

[0043] Figure 2 These are the results of screening and availability verification of BgaR transcription factors.

[0044] Figure 3 The lactose-GFP response relationship was verified in a lacto-N-neotetraose producing strain.

[0045] Figure 4 A high-throughput screening process.

[0046] Figure 5 To screen the inhibitory genes for their effects on the production of lacto-N-neotetraose at the shake flask level. DETAILED DESCRIPTION

[0047] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0048] The preparation of fermentation broth samples and the detection conditions of lactoyl-N-neotetraose are as follows:

[0049] Shake flask fermentation conditions: the seed solution was transferred to a 250 mL conical flask containing 50 mL fermentation medium at a 5% inoculum volume, and the corresponding antibiotics were added to maintain the presence of the plasmid. The culture was carried out at 37°C and 200 rpm for 2.5-3 h. 600 When the enzyme expression reached 2-4, IPTG was added at a final concentration of 0.1 mM to induce enzyme expression. After culturing in an air shaker at 25°C and 200 rpm for 2 h, a final concentration of 5 g·L -1 Lactose, 25 ° C, 200 rpm continued to culture. Fermentation ended after 72 hours.

[0050] The detection method is as follows: Take 1 mL of fermentation broth sample and boil it in a metal bath at 100°C for 15 minutes, then centrifuge it at 12,000 rpm for 15 minutes in a high-speed centrifuge. The supernatant after centrifugation is filtered through a 0.22 μm aqueous needle filter into a liquid phase vial as the sample to be tested. The lactoyl-N-neotetraose in the sample is analyzed and determined by high performance liquid chromatography (HPLC) equipped with a differential detector and an Aminex HPX-87H (300×7.8 mm) column. The column temperature is 60°C, the mobile phase is 5 mM H2SO4, and the flow rate is 0.5 mL min -1 . .

[0051] Fluorescence assay:

[0052] GFP fluorescence intensity was measured using a TECAN SPARK fluorescence microplate reader. The specific method was to transfer 200 μL of the bacterial culture from the 96-well plate to a new 96-well plate, centrifuge and discard the supernatant, add an appropriate amount of PBS to resuspend the bacteria, centrifuge again, repeat the above steps, and then transfer 200 μL of the PBS-suspended bacterial solution to the 96-well fluorescence microplate reader. GFP was excited using a 488 nm semiconductor laser, and the fluorescence signal was detected through the emission channel of a 530 / 30 nm filter.

[0053] The culture medium used in the examples is:

[0054] LB medium: 5.00 g / L yeast extract, 10.00 g / L peptone, and 10.00 g / L sodium chloride.

[0055] LB solid medium: 5.00 g / L yeast extract, 10.00 g / L peptone, 10.00 g / L sodium chloride, and 20 g / L agar powder.

[0056] Fermentation medium: 30.00 g / L glycerol, 6.7.00 g / L yeast extract, 3.3 g / L corn steep liquor, 13.50 g / L potassium dihydrogen phosphate, 4.00 g / L diammonium hydrogen phosphate, 1.70 g / L citric acid, 1.40 g / L magnesium sulfate heptahydrate, and 10 mL / L trace element solution. The trace element solution included 10.00 g / L iron (III) citrate, 2.25 g / L zinc sulfate heptahydrate, 1.00 g / L copper sulfate pentahydrate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium tetraborate decahydrate, 0.11 g / L ammonium heptamolybdate, and 2.00 g / L calcium chloride.

[0057] Example 1 Construction of CRISPRi screening chassis strain in Escherichia coli

[0058] 1. E. coli genome integration of tetR-Ptet-dCas9 expression cassette

[0059] (1) Construction of tetR-Ptet-dCas9 genomic integration plasmid

[0060] 1) Replacement of N20

[0061] Using sgRNAcas9 software, we selected the predicted ldhA-N20 sequence on the ldhA gene (NCBI accession number CP053602.1 (1418711-1419700)). We designed primers (ldhA-N20-F / R) and replaced the N20 sequence on the original pTargetF plasmid via PCR. The PCR product was transformed into Escherichia coli JM109, and the plasmid was extracted and verified by sequencing. Successfully verified pTarget-ldhA was stored at -20°C until further use.

[0062] 2) Preparation of ldhA homology arms and tetR-ptet-dCas9 expression cassette

[0063] Using the genome of the strain to be knocked out as a template, the ldhA-up-F / R and ldhA-down-F / R primer pairs were used to amplify the upstream and downstream homology arms required for lhdA knockout, respectively. Both DNA fragments were purified and recovered. Using the pdCas9 plasmid as a template, the dCas9-F / R primer pair was used to amplify the tetR-Ptet-dCas9 expression cassette (SEQ ID NO. 1). The DNA fragment was then purified and recovered.

[0064] The upper and lower homology arms of ldhA and the tetR-Ptet-dCas9 expression cassette were integrated into pTarget-ldhA to construct the complete knockout plasmid pTarget-ΔldhA-tetR-Ptet-dCas9.

[0065] The upstream and downstream homology arms of ldhA and the tetR-Ptet-dCas9 expression cassette were ligated into the EcoR I and Pst I restriction sites of the pTarget-ldhA vector using a seamless cloning kit (Nanjing Novozymes Biotechnology Co., Ltd.). The ligation products were transformed into Escherichia coli JM109, and the plasmids were extracted and sent for sequencing verification. The successfully verified pTarget-ΔldhA-tetR-Ptet-dCas9 was stored at -20°C for future use.

[0066] (2) Integration of the dCas9 expression cassette into the E. coli genome Reference is made to the gene knockout method described in patent CN117343889A.

[0067] 2. P J23100 -GFP expression cassette integration into the E. coli genome

[0068] In order to verify the inhibitory ability of dCas9 protein on Escherichia coli genomic genes, the P J23100 The -gfp expression cassette (SEQ ID NO. 19) was integrated into the iclR locus of the E. coli genome, which had already been integrated with the tetR-Ptet-dCas9 expression cassette. Its NCBI accession number is CP053602.1 (4131473-4132297). (Primers and N20 sequences involved in the construction of the integration plasmid are shown in Table 1)

[0069] Table 1 Primer sequences and N20 sequences for integration plasmid construction

[0070]

[0071]

[0072] 3. Verification of CRISPRi-mediated genomic gene inhibition ability and inhibition strength screening

[0073] Four sgRNAs targeting different positions of gfp were designed and constructed into pSC101 and pACYCDuet-1 vectors respectively. The four designed targets included three sgRNAs targeting non-template chains and one sgRNA targeting template chains, and their sequences are shown in SEQ ID NO.15-18. These plasmids were electroporated into Escherichia coli respectively. After single colonies grew out, single colonies were picked and placed in LB liquid culture medium. After culturing for 8-10 hours, the bacterial solution was inoculated into the fermentation medium and cultured at 37°C and 200 rpm for 12 hours. Tetracycline was added after 2 hours of fermentation, and a tetracycline induction concentration gradient was set: 0 ng / mL, 50 ng / mL and 100 ng / mL. The fluorescence results are shown in Figure 2. Figure 1As shown, when pSC101 was used as the sgRNA expression vector, a 9.36-fold inhibitory effect was achieved without the addition of an inducer, and a maximum inhibitory effect of 36.09-fold was achieved when 50 ng / mL of tetracycline was added. When pACYCDuet-1 was used as the sgRNA expression vector, a 9.31-fold inhibitory effect was achieved without the addition of an inducer, and a maximum inhibitory effect of 30.7-fold was achieved when 50 ng / mL of tetracycline was added.

[0074] Example 2 Construction of Escherichia coli sgRNA library plasmid

[0075] (1) Escherichia coli sgRNA design

[0076] With reference to the genome information of Escherichia coli BL21(DE3)GCF_000022665.1, a total of 12,000 specific sgRNAs were designed for coding genes (4,142) using the CRISPOR (https: / / crispor.gi.ucsc.edu / crispor.py) design tool, covering 99.89% of the genome coding genes. Among them, 1-3 sgRNAs were designed for each coding gene, and as much as possible, 2 sgRNAs were designed for non-template targeting and 1 sgRNA was designed for template targeting for each coding gene. The sgRNA was designed as close to the start codon as possible for the target position.

[0077] (2) Construction of sgRNA expression plasmid

[0078] The designed sgRNA oligonucleotides were synthesized using a high-throughput chip, and homology arms of the pSC101 vector were added to both ends. The synthesized oligonucleotides were then ligated into the pSC101 vector via homologous recombination.

[0079] Example 3 Development and Validation of a Lactose Biosensor Based on the Transcription Factor BgaR

[0080] (1) Construction of recombinant plasmids of Bacillus megaterium-derived BmBgaR and Clostridium perfringens-derived CpBgaR

[0081] The genome of the Bacillus megaterium strain preserved in the laboratory was used as a template to amplify the BmBgaR (SEQ ID NO.3) expression cassette, and the CpBgaR (SEQ ID NO.4) expression cassette sequence from Clostridium perfringens was sent to Jin Weizhi for gene synthesis.

[0082] The gfp fragment was connected to the BmBgaR expression cassette and the CpBgaR expression cassette by one-step cloning homologous recombination to the corresponding positions of the pETDuet-1 vector fragment, and then the ligation product was transformed into JM109 competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 90 seconds, ice-bathed for 5 minutes, and recovered at 37°C and 200 rpm for about 1 hour. It was then spread on an LB plate containing ampicillin resistance (100 ug / mL) and cultured overnight. After a single clone grew, 1-2 were picked and inoculated into liquid LB culture medium, the plasmid was extracted, and sent for Sanger sequencing to verify whether pET-BmBgaR-gfp and pET-CpBgaR-gfp were successfully constructed.

[0083] (2) Screening and availability verification of BgaR transcription factors

[0084] The pET-BmBgaR-gfp and pET-CpBgaR-gfp plasmids constructed in step (1) were electroporated into the target Escherichia coli competent cells. After the transformants grew, they were inoculated into a vial containing liquid LB and cultured at 37°C, 200 rpm for 10-12 hours. Then, they were transferred to the fermentation medium and different lactose concentrations (0 g / L, 0.3 g / L, 0.6 g / L, 1 g / L, 1.3 g / L) were added. The relationship between the fluorescence value and the lactose concentration at different time points was detected. The results are shown in Figure 2. Figure 2 As shown, BmBgaR from Bacillus megaterium exhibits a linear relationship with GFP across a wide range of lactose concentrations, from 0 to 0.6 g / L. In contrast, CpBgaR from Clostridium perfringens reaches its critical value at 0.3 g / L lactose, resulting in a narrower response range. BmBgaR exhibits good linearity across different time points, and was therefore selected for subsequent studies.

[0085] (3) Verification of the lactose-GFP response relationship in lactoyl-N-neotetraose producing strains

[0086] The pET-BmBgaR-GFP plasmid was electroporated into the host bacteria producing lacto-N-neotetraose to verify the lactose-GFP response relationship. It was found that there was no linear relationship. The expression level of BmBgaR was optimized and the original BgaR promoter was replaced with pET-BmBgaR-GFP. BAD , the results are as follows Figure 3 As shown, there is a linear relationship between the lactose concentration of 0-0.6 g / L and the fluorescence. The relationship between the fluorescence and the lactose concentration was further detected by placing the entire expression cassette into the pACYCDuet-1 vector, as shown in FIG. Figure 3As shown in the figure, compared with expressing BmBgaR using the pETDuet-1 vector, expressing BmBgaR using the pACYCDuet-1 vector can improve the sensitivity of the sensor.

[0087] Example 4 CRISPRi library combined with sensor-mediated screening of lactoyl-N-neotetraose high-producing strains and target identification The sensor plasmid constructed in Example 3 was electroporated into the lactoyl-N-neotetraose production strain integrated with tetR-Ptet-dCas9 constructed in Example 1 to obtain transformants, and the strain was used to prepare competent cells. The whole-gene targeted sgRNA library plasmid customized in Example 2 was electroporated into competent cells containing the sensor plasmid. After electroporation, it was spread on an LB plate containing kanamycin resistance (30 μg / mL) and chloramphenicol resistance (30 μg / mL) and cultured overnight to obtain a dual-plasmid Escherichia coli CRISPRi library.

[0088] Single colonies were picked from the plates and transferred to 96-well shallow-well plates containing 130 μL LB, and kanamycin (30 μg / mL) and chloramphenicol (30 μg / mL) were added as screening markers. The seed solution was obtained by culturing at 37°C and 850 rpm for 9-12 hours. Subsequently, 30 μL of bacterial solution was pipetted into 96-well deep-well plates containing 600 μL fermentation medium for fermentation. After culturing at 37°C and 850 rpm for 3 hours, the temperature was lowered to 25°C. IPTG, lactose, and arabinose were added and cultured for 36 hours. Green fluorescence was detected using a fluorescence microplate reader. Those with lower fluorescence values ​​than the control group were selected and rescreened at the shake flask level. The strains with lacto-N-neotetraose production 10% higher than the control group obtained by shake flask level verification were extracted, and the plasmids were extracted and sent for Sanger sequencing to obtain the site of targeted inhibition. The verified inhibitory target was further reconstructed into the pSC101 vector, and the correct plasmid was re-electroporated into the lacto-N-neotetraose production strain integrated with tetR-Ptet-dCas9, and shake flask fermentation was performed again to verify the presence of the sensor plasmid to avoid interference with the detection results. Figure 4 As shown. Inhibition of cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB, ihfB (the target sequences of knockout cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB are shown in SEQ ID NO.7-14, respectively) increased lacto-N-neotetraose by 11.33%, 18.55%, 17.07%, 30.23%, 12.42%, 25.10%, 16.67% and 31.25%, respectively. These genes encode the inner membrane subunit of sulfate / thiosulfate ABC transporter, CTP synthase, regulatory factor of RpoS, poly (A) polymerase I, DedA family protein YqjA, tryptophan:H +synthase, Tol-Pal system exoplasmic protein and integration host factor subunit β, the results are as follows Figure 5 shown.

[0089] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for screening key genes for target product synthesis based on the whole-genome CRISPRi library of Escherichia coli, characterized in that: The method is to simultaneously express a lactose sensor plasmid and a full-gene targeted sgRNA library plasmid in Escherichia coli integrated with tetR-Ptet-dCas9, identify high-yielding strains through high-throughput screening, and extract plasmids from the high-yielding strains for sequencing to identify targeted inhibitory genes.

2. The method according to claim 1, characterized in that The method comprises the following steps: (1) Integrate the tetR-Ptet-dCas9 expression cassette into the ldhA site of the Escherichia coli genome to obtain a tetR-Ptet-dCas9 genome-integrated strain; (2) Design and synthesize genome-wide sgRNA library plasmids targeting coding genes based on the genomic information of Escherichia coli BL21 (DE3); (3) introducing the constructed lactose sensor plasmid into the tetR-Ptet-dCas9 genome-integrated strain of step (1) to obtain a sensing strain; (4) introducing the sgRNA library plasmid in step (2) into the sensing strain in step (3) to obtain the E. coli CRISPRi library; (5) Perform high-throughput screening on the E. coli CRISPRi library in step (4), and select individuals with lower green fluorescence intensity than the control group for verification.

3. The method according to claim 1 or 2, characterized in that The nucleotide sequence of the tetR-Ptet-dCas9 is shown in SEQ ID NO.

1.

4. The method according to claim 1 or 2, characterized in that The lactose-responsive transcription factor used to construct the lactose sensor plasmid is BgaR from Bacillus megaterium or Clostridium perfringens, and the promoter used to express green fluorescent protein in the lactose sensor plasmid is the BgaR-activated promoter P BgaA / P BgaL , the expression vector of the lactose sensor plasmid is pACYCDuet-1 or pETDuet-1; Optionally, the expression vector of the lactose sensor plasmid is pACYCDuet-1, and the promoter used is the arabinose-inducible promoter P BAD .

5. The method according to claim 1 or 2, characterized in that The target product includes but is not limited to lactoyl-N-neotetraose.

6. The method according to claim 1 or 2, characterized in that The screening process indirectly characterizes the production capacity of lactoyl-N-neotetraose by green fluorescence intensity, selects individuals with improved production through 96-well plate culture primary screening and shake flask secondary screening, and identifies target inhibition sites.

7. Escherichia coli with improved lactoyl-N-neotetraose production, characterized in that, in the Escherichia coli, at least one of the following genes is inhibited: cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB.

8. Use of one or more of the genes cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB, and ihfB for increasing the yield of lactoyl-N-neotetraose, characterized in that: The application is to inhibit or knock out one or more of the genes cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB in Escherichia coli.

9. The use according to claim 8, characterized in that The NCBI accession number of the nucleotide sequence of cysW is CP053602.1 (2414505-2415380), the NCBI accession number of the nucleotide sequence of pyrG is CP053602.1 (2741916-2743553), the NCBI accession number of the nucleotide sequence of sprE is CP053602.1 (1279332-1280345), and the NCBI accession number of the nucleotide sequence of pcnB is CP053602.1 (160534-161952). The NCBI accession number of the nucleotide sequence of the YqjA is CP053602.1 (3115370-3116032), the NCBI accession number of the nucleotide sequence of the TnaB is CP053602.1 (3780330-3781577), the NCBI accession number of the nucleotide sequence of the TolB is CP053602.1 (737699-738991), and the NCBI accession number of the nucleotide sequence of the ihfB is CP053602.1 (970216-970500).

10. The use according to claim 8, characterized in that The targeting sequences for inhibiting or knocking out cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB are shown in SEQ ID NOs. 7-14, respectively.

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