Method for improving L-tryptophan synthesis level in escherichia coli based on whole genome CRISPRi high-throughput screening

Abstract

The invention discloses a method for improving the synthesis level of L-tryptophan in escherichia coli based on whole genome CRISPRi high-throughput screening, and belongs to the technical field of biological engineering. According to the invention, dCas9 is integrated in a strain YB-2A, and an sgRNA plasmid library is introduced to construct a CRISPRi screening system, so that key targets spoT, rnr and the like with positive effects are obtained. When the strain is used for preparing a target inhibitor gene rnr, the L-tryptophan yield and the saccharic acid conversion rate of shake-flask fermentation of the strain reach 6.59 g.L <-1 > and 0.112 g.g <-1 > respectively and are increased by 16.1% and 8.7% respectively compared with those of a control strain, and the L-tryptophan yield and the saccharic acid conversion rate in a 3-L tank reach 32.46 g.L <-1 > and 0.171 g.g <-1 > respectively. The invention not only provides reference for high-throughput screening and transformation of the L-tryptophan production strain, but also provides reference for high-throughput screening and transformation of other amino acids.

Description

Technical Field

[0001] This invention relates to a method for improving L-tryptophan synthesis levels in Escherichia coli based on whole-genome CRISPRi high-throughput screening, belonging to the field of bioengineering technology. Background Art

[0002] L-tryptophan is one of the essential amino acids for the human body, playing an irreplaceable role in nutritional fortification and immune regulation. Currently, the modification of L-tryptophan-synthesizing strains can be mainly divided into two directions: rational modification, including classic metabolic engineering strategies such as optimizing precursor supply, regulating competitive pathways, relieving feedback inhibition of key enzymes, and optimizing transport systems; and irrational modification, including mutagenesis breeding, adaptive evolution, and directed evolution of key enzymes.

[0003] CRISPR inhibition (CRISPRi) is derived from the CRISPR-Cas system and includes clusters of regularly spaced short palindromic repeats and the Cas9 protein (dCas9), which loses its DNA cleavage activity after point mutation. Guided by sgRNA, dCas9 targets the target site and forms steric hindrance, inhibiting RNA polymerase transcription and thus downregulating the expression of the target gene. The CRISPRi system has great potential applications in high-throughput gene screening and industrial microbial modification. Currently, most studies construct the CRISPRi system by integrating the dCas9 gene into the expression strain and designing sgRNA libraries for screening and validation based on rational analysis of metabolic engineering to target gene sites directly or indirectly related to the synthesis of the target product. For example, Tang used the CRISPRi system to inhibit the genes pheA and tyrA with different intensities to weaken the competing pathways; Zhang et al. aimed to increase the D-pantothenic acid synthesis level in *E. coli*.

[69] One study selected 126 genes from four major modules—glucose metabolism, amino acid metabolism, cofactor metabolism, and cell growth morphology control—for single-gene expression inhibition validation. Li et al. selected over 30 genes related to O-acetylhomoserine synthesis in Corynebacterium glutamicum for single-gene inhibition validation. However, there is still no specific research on screening key targets for L-tryptophan synthesis in Escherichia coli based on genome-wide sgRNA plasmid libraries.

[0004] Therefore, in order to further explore key targets related to L-tryptophan synthesis, this invention will utilize an sgRNA plasmid library designed for the entire E. coli genome, and a CRISPRi library based on the entire E. coli genome, to conduct high-throughput screening of key targets related to L-tryptophan synthesis and modify them, in order to obtain strains with improved L-tryptophan synthesis performance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention utilizes CRISPRi technology to screen key targets related to L-tryptophan synthesis across the entire genome of *E. coli*, and then performs genomic modification on targets with strong promoting effects to enhance L-tryptophan synthesis levels in *E. coli*. This invention first integrates and expresses P at the yeeL-yeeJ region in the *E. coli* genome. tet -dCas9 was then introduced, followed by the L-tryptophan biosensor, and then an sgRNA plasmid library based on the whole genome of E. coli was introduced to obtain the CRISPRi strain library. The CRISPRi strain library was subjected to high-throughput screening through flow cytometry, plate screening, and shake flask screening. High-yield strains of L-tryptophan were screened and the target genes were identified. The target genes with the strongest promoting effect were validated by sgRNA expression cassette genome integration expression and knockout. The final strain was validated by 3-L tank fermentation.

[0006] The first technical solution provided by this invention is an L-tryptophan sensor plasmid, wherein the plasmid is an expression vector carrying a natural leader peptide TnaC gene and a fluorescent reporter gene, the expression vector being the pACYC plasmid, and the natural leader peptide TnaC is promoted by the P... J23107 Driven by L-tryptophan concentration signals, this type of sensor controls the ribosome arrest during TnaC translation, thereby determining the transcription of downstream fluorescent reporter genes.

[0007] In some embodiments, the nucleotide sequence of the TnaC gene is shown in SEQ ID NO.1.

[0008] In some embodiments, the fluorescent reporter gene is the eGFP gene.

[0009] Furthermore, the nucleotide sequence of the eGFP gene is shown in SEQ ID NO.2.

[0010] In some implementations, the promoter P J23107 The nucleotide sequences are shown in SEQ ID NO.3.

[0011] The second technical solution provided by this invention is a method for improving the L-tryptophan synthesis level in Escherichia coli based on the CRISPRi screening system. The method simultaneously expresses the L-tryptophan sensor plasmid and the whole-gene targeted sgRNA library plasmid described in the first technical solution in Escherichia coli that integrates dCas9. High-throughput screening is used to identify high-yield L-tryptophan-producing strains, and plasmids extracted from the high-yield L-tryptophan-producing strains are sent to Sanger sequencing to identify the targeted repressor genes.

[0012] In some embodiments, the host is strain YB-2A, and the integration location of dCas9 in the genome of strain YB-2A is gene yeeL-yeeJ to obtain strain CI-0.

[0013] In some embodiments, strain CI-0 is introduced into the L-tryptophan sensor plasmid pSensor-trp4, which is configured as pACYC-P J23107 -tnaC-egfp, then import the sgRNA plasmid library using the low-copy plasmid pSC101 as a vector to obtain the CRISPRi strain library.

[0014] In some embodiments, the screening process includes high-throughput screening procedures such as flow cytometry, primary screening using well plates, and secondary screening using shake flasks to obtain strains with improved L-tryptophan synthesis performance, and to identify target genes through sequencing and alignment.

[0015] The third technical solution provided by this invention is an Escherichia coli with increased L-tryptophan production, wherein the gene rnr in the Escherichia coli is suppressed or knocked out.

[0016] In some embodiments, the *E. coli* strain is YB-2A, in which the gene *rnr* has been knocked out using the CRISPR-Cas9 system.

[0017] In some embodiments, the *E. coli* is a gene *rnr* that integrates sgRNA via the CRISPR-Cas12a system. rnr The expression cassette was suppressed in strain CI-0.

[0018] The fourth technical solution provided by the present invention is the application of the genes spoT and / or rnr in improving the ability of Escherichia coli to synthesize L-tryptophan, wherein the application is to inhibit or knock out the genes spoT and / or rnr in Escherichia coli.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention constructs the CRISPRi screening system in strain YB-2A by integrating dCas9 and introducing it into an sgRNA plasmid library. High-throughput screening revealed previously unreported key targets with positive effects, such as spoT and rnr. When targeting and inhibiting the rnr gene, the L-tryptophan yield and sugar-acid conversion rate during shake-flask fermentation reached 6.59 g·L⁻¹. -1 and 0.112 g·g -1 Compared to the control strain, these improvements were 16.1% and 8.7%, respectively. Knockout of rnr and integration of sgRNA... rnr Expression cassettes were used to obtain strain YB-2A-Δrnr and CI-0-sgRNA. rnrVerification using shake flask and 3-L fermentation tanks showed that strain YB-2A-Δrnr exhibited superior L-tryptophan synthesis performance compared to strain CI-0-sgRNA. rnr Its shake flask yield and sugar-acid conversion rate reached 6.42 g·L⁻¹. -1 and 0.113 g·g -1 The L-tryptophan yield and sugar-acid conversion rate in the 3-L tank reached 32.46 g·L⁻¹. -1 and 0.171 g·g -1 . Attached Figure Description

[0021] Figure 1 Fermentation validation of dCas9 integrated strain

[0022] Figure 2 Validation of the response of pSensor-trp4 for L-tryptophan biosensor

[0023] Figure 3 Screening and identification of key targets related to L-tryptophan synthesis

[0024] Figure 4 For strains YB-2A-Δrnr and CI-0-sgRNA rnr Shake flask fermentation verification

[0025] Figure 5 Validation of 3-L tank fermentation of strain YB-2A-Δrnr

[0026] Figure 6 The strain CI-0-sgRNA rnr Validation of fermentation in a 3-L tank. Detailed Implementation

[0027] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0028] The methods involved in the embodiments:

[0029] Bacterial cell concentration determination: OD was measured using a spectrophotometer. 600 Characterizing cell density: After diluting the fermentation broth appropriately, the absorbance and OD were measured at a wavelength of 600 nm. 600 = Absorbance at 600nm * Dilution factor;

[0030] Glucose concentration determination: Centrifuge 1 mL of fermentation broth at 12000 rpm for 2 min, collect the supernatant, dilute to an appropriate factor, and use an M-100 biosensor analyzer (Shenzhen Xierman Technology Co., Ltd.) to detect the glucose concentration (g / L). Residual sugar value = test value * dilution factor;

[0031] L-Tryptophan concentration determination: An Agilent high-performance liquid chromatograph was used. The detection conditions were a C18 column (250 mm × 4.6 mm, 5 μm) and a mobile phase of 0.3 g·L⁻¹. -1 KH₂PO₄ aqueous solution: methanol (90:10), column temperature 39℃, flow rate 1 mL / min -1 The detection wavelength is 278nm.

[0032] Fermentation broth pretreatment: The fermentation broth was centrifuged at 12000 rpm for 2 min and the supernatant was collected. The supernatant sample was appropriately diluted and filtered through a 0.22 μm filter. The treated sample was then used for high performance liquid chromatography detection. The L-tryptophan concentration was represented by multiplying the measured value by the dilution factor.

[0033] Determination of sugar-acid conversion rate: Sugar-acid conversion rate is the ratio of L-tryptophan production to glucose consumption. Sugar-acid conversion rate = (L-tryptophan concentration × fermentation broth volume) / glucose consumption.

[0034] Fluorescence determination: Take an appropriate amount of fermentation broth, dilute and centrifuge. Wash the bacterial pellet twice with an equal volume of PBS buffer, then resuspend the bacterial cells with an equal volume of PBS buffer. For each test sample, take three replicates, adding 200 μL of each replicate to an ELISA plate. Excitation wavelength is 485 nm, emission wavelength is 535 nm, and the fluorescence gain value (Gain value 41) is fixed for each test. Subtract the fluorescence value of the control strain without eGFP expression from the test results, and then take the average to obtain the total fluorescence value of the sample. For each test sample, take three replicates, adding 200 μL of each replicate to a 96-well plate. Detect at a wavelength of 600 nm. Subtract the noise from the culture medium background from the test results, and then take the average to obtain the sample OD value. 600 Value. Total fluorescence value of sample / OD value of sample 600 The value is the unit fluorescence value.

[0035] Flow cytometry cell sorting: The bacterial growth formed by the mutagenic strain was scraped and transferred to 10 mL of M9 medium, and incubated at 200 rpm and 37°C for 12 h. OD was collected. 600 =0.9 After incubation, 1 mL of bacterial culture was centrifuged, and the bacterial cells were washed twice with PBS buffer, then resuspended in PBS buffer to a final volume of 3 mL for flow cytometry sorting. 3000-5000 strains were collected from the top 1% of fluorescent strains, incubated at 200 rpm and 37°C for 20 min, and a suitable amount of bacterial culture was spread to a concentration of 50 μg / mL. -1 Kanamycin and 25 μg·mL -1 Chloramphenicol double antibody plates were incubated at 200 rpm and 37°C for 12 h.

[0036] Initial screening in 96-well plates: Single colonies were picked from the flow cytometry-sorted and plated antibiotic-resistant plates and incubated in 96-well shallow plates at 37°C for 12 hours. Subsequently, a 1% inoculum was transferred to 96-well deep plates and incubated at 37°C for 12 hours. OD units were then measured. 600 Fluorescence value: Select 3-5 strains with high fluorescence values ​​from each plate for shake-flask fermentation and re-screening.

[0037] Secondary screening of shake-flask fermentation: Based on the results of the initial fluorescence screening of the 96-well deep-well plate, 20 μL of seed culture medium from the corresponding position of the 96-well shallow-well plate was transferred to 10 mL of a solution containing 50 μg / mL. -1 Kanamycin and 25 μg·mL -1 The chloramphenicol was activated in LB medium, and then transferred to 50 mL of medium containing 50 μg / mL chloramphenicol at an inoculation rate of 2‰. -1 In the shake flask culture medium of kanamycin, fermentation was carried out at 37°C and 200 rpm for no less than 48 h, with glucose solution added to the reaction system every 8 h.

[0038] 3-L fermentation culture: The strain was activated on LB solid medium, and then a single colony was picked and transferred to 10 ml of LB medium. After culturing at 37°C for 10 h, it was transferred to seed culture medium at an inoculation rate of 2‰ (v / v) and cultured at 37°C for approximately 10 h. 100 mL of seed culture was then transferred to a 3L fermenter containing 900 mL of fermentation medium via flame inoculation. Throughout the fermentation process, the pH was maintained at approximately 6.5, the temperature at 37°C, and the dissolved oxygen (DO) content controlled at 20% by continuously adding ammonia (50%, v / v). After a sudden increase in dissolved oxygen concentration (dissolved oxygen rebound), 80% (m / v) glucose solution was added to the 3L bioreactor to maintain the glucose concentration in the fermentation medium at 0–1 g / L. OD was monitored during fermentation. 600 Samples were taken every 3 hours to test residual sugar and L-trp concentration, and an appropriate amount of fermentation broth was retained. The L-trp concentration was measured by diluting the fermentation supernatant.

[0039] Materials used in the examples:

[0040] I. Culture media involved in the following examples: Except for yeast extract and peptone, which were purchased from Oxoid, all other reagents were purchased from Sinopharm Group.

[0041] 1. Seed culture medium: 9.6 g·L -1 KH2PO4, 24 g·L -1 K2HPO4, 10 g·L -1 Glucose, 5g·L -1 (NH4)2SO4, 15 g·L -1 Yeast powder, 2g·L -1 Citric acid, 1 g·L-1 MgSO4·7H2O. Adjust the pH to 7.2 with ammonia water.

[0042] 2. Shake flask culture medium: 9.6 g·L -1 KH2PO4, 24 g·L -1 K2HPO4, 10 g·L -1 Glucose, 5g·L -1 (NH4)2SO4, 4 g·L -1 Peptone, 2 g / L -1 Yeast powder, 1g·L -1 MgSO4·7H2O, 2g·L -1 Citric acid, 3 mL of trace element solution. Adjust the pH to 7.2 with ammonia.

[0043] 3. Trace element solution: 0.75 g·L -1 CoSO4·7H2O, 15g·L -1 ZnSO4·7H2O, 5g·L -1 CuSO4·5H2O, 2g·L -1 Al2(SO4)3·18H2O, 5g·L -1 FeSO4·7H2O, 4g·L -1 MnSO4·H2O, 3g·L -1 Na₂MoO₄·2H₂O, 2.5 g·L⁻¹ -1 NiSO4·6H2O, and 0.5 g·L -1 H3BO3.

[0044] 4. Culture medium for a 3L fermenter: 15g / L -1 K2HPO4, 7.5 g·L -1 Glucose, 2 g / L -1 MgSO4·7H2O, 2g·L -1 Yeast powder, 2g·L -1 Citric acid, 1.6 g·L -1 (NH4)2SO4, 0.0129 g·L -1 CaCl2, 0.075 g·L -1 FeSO4·7H2O and 3 mL of trace element solution. Adjust the pH to 7.2 with ammonia. Feeding medium: 800 g / L -1 glucose.

[0045] II. The strains and plasmids involved in the following examples:

[0046] 1. The genetic background of *E. coli* YB-2A is derived from *E. coli* KW3110, containing ΔtnaA, ΔtnaB, and ΔtrpR, and integrating P at panC-rpnC, ompW-yciE, and rluF-yjdD in the genome, respectively. tac -aroG S211F -P tac -trpE Q71K / S94N / C465Y -trpABCD-serA H344A / N364A Expression box.

[0047] 2. Screening and optimization of key targets related to L-tryptophan synthesis, involving plasmids: pSentrp, pTarget-dCas9 + pSensor-trp4, pSC101 unloaded (pSC101 unloaded from laboratory storage), pSC101-P J23119- sgRNA pykF pSC101-P J23119- sgRNA malM pSC101-P J23119- sgRNA spoT pSC101-P J23119- sgRNA rnr pSC101-P J23119- sgRNA rep pSC101-P J23119- sgRNA bioD pSC101-P J23119- sgRNA pheA and pSC101-P J23119- sgRNA kdpE The L-tryptophan biosensor plasmid pSentrp is referenced in the article Fang M, Wang T, Zhang C, et al. Intermediate-sensor assisted push-pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli[J]. Metabolic Engineering, 2016, 33:41-51.

[0048] 3. Plasmids involved in genomic integration and knockout of the sgRNA expression cassette targeting gene rnr: pTarget-Δrnr, pTarget-rnr + pCas9 and pCas12a (pCas9 and pCas12a are from laboratory collections).

[0049] 4. The genetic background of strains ZH-1, ZH-2, and ZH-3 is derived from Escherichia coli KW3110, containing ΔtnaA, ΔtnaB, and ΔtrpR, and integrating 1–3 copies of P at the panC-rpnC, ompW-yciE, or rluF-yjdD sites in the genome, respectively. tac -aroG S211F -P tac -trpE Q71K / S94N / C465Y -trpABCD-serA H344A / N364A Expression box. Reference article: Hou Minglei, Gao Shengqi, Wu Jing, Chen Sheng*, Zhang Kang*. Metabolic engineering of Escherichiacoli to enhance L-tryptophan biosynthesis. Systems Microbiology and Biomanufacturing, 2025. III. The primers involved in the following examples are shown in Table 1.

[0050] Table 1. Main primers involved in this invention

[0051]

[0052]

[0053] Example 1: Obtaining strain YB-2A

[0054] I. Construction and System Optimization of L-Tryptophan Biosensor

[0055] Using plasmid pSentrp as a template, the sensor expression cassette fragment tnaC-egfp was amplified using primers tnaC-egfp-F and egfp-pTrc99a-R. This fragment was then ligated to the low-copy-count plasmid vector pSC101 via a one-step cloning process to construct the sensor plasmid pSensor-trp1:pSC101-tnaC-egfp. Based on plasmid pSensor-trp1, the constitutive promoter P was introduced through two-step PCR using primers containing promoter J23109 to circularly amplify plasmid pSensor-trp1. J23107 Regulating the transcription of the leader peptide TnaC, the sensor plasmid pSensor-trp3:pSC101-P was obtained. J23107 -tnaC-egfp. Primers are shown in Table 1.

[0056] Following the above method, pSensor-trp3 was introduced into the previously obtained genome-integrated strains ZH-1, ZH-2, and ZH-3, which have different L-tryptophan synthesis capabilities, to obtain strains ZH-1-eGFP, ZH-2-eGFP, and ZH-3-eGFP. Strains ZH-1, ZH-2, and ZH-3 are referenced from the article "Metabolic engineering of Escherichia coli to enhance L-tryptophan biosynthesis." Systems Microbiology and Biomanufacturing, 2025. Further fluorescence response verification was performed on the actual L-tryptophan synthesizing strains ZH-1 / 2 / 3-eGFP. The L-tryptophan yield of strains ZH-1-eGFP, ZH-2-eGFP, and ZH-3-eGFP in shake-flask fermentation was compared with that per unit OD. 600 Fluorescence values ​​are positively correlated, R 2 A value >0.9 indicates that the sensor plasmid pSensor-trp3 exhibits good response performance in actual L-tryptophan-synthesizing strains and can be applied to high-throughput screening.

[0057] II. High-throughput screening and validation of ARTP-mutated strains

[0058] ARTP mutagenesis first requires determining the mutagenesis conditions. The mutagenic strains undergo high-throughput screening processes such as flow cytometry, primary screening using well plates, and secondary screening using shake flasks to obtain dominant mutagenic strains. These dominant mutagenic strains are then subjected to iterative mutagenesis screening to further enhance their L-tryptophan synthesis capacity.

[0059] The specific process for high-throughput screening is as follows:

[0060] Mutagenesis conditions were determined: the sensor plasmid pSensor-trp3 was introduced into strain ZH-3 to obtain the mutagenesis starting strain YB-0, which was then subjected to ARTP mutagenesis with an irradiation time of 30s.

[0061] Iterative mutagenesis: The dominant strains obtained from the previous round of mutagenesis screening were selected, and the above mutagenesis steps were repeated under the same conditions. All treated strains were plated to a concentration of 50 μg / mL. -1 ARTP mutant strains were obtained by incubating at 37°C for 12 hours on kanamycin plates.

[0062] Flow cytometry cell sorting: The bacterial growth formed by the mutagenic strain was scraped and transferred to 10 mL of M9 medium, and incubated at 200 rpm and 37°C for 12 h. OD was collected. 600=0.9 After culturing, 1 mL of bacterial culture was centrifuged, and the bacterial cells were washed twice with PBS buffer, then resuspended in PBS buffer to a final volume of 3 mL, which was used as a sample for flow cytometry sorting. 3000-5000 strains were collected from the top 0.2% or 1% of the fluorescent values, and incubated at 200 rpm and 37°C for 20 min. An appropriate amount of bacterial culture was then spread onto an antibiotic-resistant plate containing 50 μg·mL⁻¹ kanamycin and incubated at 200 rpm and 37°C for 12 h.

[0063] Flow cytometry was used to analyze the ARTP-mutated strains and sort them according to fluorescence intensity. 99.8% of the mutant strains had lower fluorescence values ​​than the control strains, and only 0.2% of the strains showed an increase in fluorescence value after mutagenesis. This may be related to the L-tryptophan synthesis capacity of the mutagenic starting strains. Therefore, 3000-5000 strains were collected from the top 0.2% of mutant strains in terms of fluorescence value for screening in 96-well plates.

[0064] Initial screening in 96-well plates: Single colonies were picked from the flow cytometry-sorted and plated antibiotic-resistant plates and incubated in 96-well shallow plates at 37°C for 12 hours. Subsequently, a 1% inoculum was transferred to 96-well deep plates and incubated at 37°C for 12 hours. OD units were then measured. 600 Fluorescence value: Select 3-5 strains with high fluorescence values ​​from each plate for shake-flask fermentation and re-screening.

[0065] Secondary screening of shake-flask fermentation: Based on the results of the initial fluorescence screening of the 96-well deep-well plate, 20 μL of seed culture medium from the corresponding position of the 96-well shallow-well plate was transferred to 10 mL of a solution containing 50 μg / mL. -1 The activator was LB medium containing kanamycin, and then transferred to 50 mL of medium containing 50 μg / mL kanamycin at a 2‰ inoculation rate. -1 In the shake flask culture medium of kanamycin, fermentation was carried out at 37°C and 200 rpm for no less than 48 h, with glucose solution added to the reaction system every 8 h.

[0066] According to the unit OD 600 Fluorescence values ​​were used to initially screen strains in 96-well plates, from which strains with specific OD values ​​were selected. 600 Strains with high fluorescence values ​​were validated through shake-flask fermentation. Based on the shake-flask fermentation results, the strain with the highest yield and unaffected growth was selected for the next round of iterative mutagenesis. After four rounds of iterative mutagenesis, one optimal strain was selected from each round, resulting in strains YB-1, YB-2, YB-3, and YB-4. These four mutagenic strains underwent shake-flask fermentation, and the L-tryptophan yields of strains YB-1, YB-2, YB-3, and YB-4 were 5.22, 5.68, 5.63, and 5.46 g·L⁻¹, respectively. -1Compared with strain YB-0, the yields of YB-3, YB-4, and YB-4 increased by 2.3%, 11.4%, 10.4%, and 7.1%, respectively. The yields of YB-3 and YB-4 decreased by 0.91% and 3.9% compared to YB-2. The sugar-acid conversion rates of strains YB-1, YB-2, YB-3, and YB-4 were 0.096, 0.103, 0.102, and 0.100 g·g⁻¹, respectively. -1 Compared with strain YB-0, the results showed increases of 3.2%, 10.7%, 9.7%, and 7.5%, respectively. These results indicate that strain YB-2 was the optimal mutagen.

[0067] III. Plasmid Elimination

[0068] Strain YB-2 was inoculated from the glycerol tube into 10 mL of LB medium at an inoculation rate of 2‰, and incubated at 200 rpm. -1 Incubate at 37℃ for 12 hours, then transfer to 10 mL of fresh LB medium at an inoculation rate of 2‰, incubating at 200 rpm. -1 Incubate at 37℃ for 12 hours, then subculture 2-3 times. Spread the culture onto LB agar plates, and pick single colonies to inoculate into untreated LB liquid and LB liquid with 50 μg / mL antibiotics. -1 In liquid LB broth containing kanamycin, growth was observed without the addition of antibiotics, but with the addition of 50 μg / mL... -1 The bacteria that do not grow in the liquid LB of kanamycin are the strains that eliminate the sensor plasmid, and are named YB-2A.

[0069] Example 2: Construction of the CRISPRi Screening System

[0070] To perform high-throughput screening based on the CRISPRi library while reducing the metabolic burden of plasmid use, expression cassette P was integrated into the intergenic locus yeeJ-yeeL of the starting strain YB-2A using the CRISPR-Cas9 system. tetR -dCas9 (SEQ ID NO.4) was first constructed using plasmid pTarget as a template. The N20 sequence of yeeJ-yeeL was replaced by a two-step PCR using primers yeeJ-N20-yeeL-F / yeeJ-N20-yeeL-R. Then, using primers yeeJ-US-F / yeeJ-US-R, yeeL-DS-F / yeeL-DS-F, and yeeJ-dCas9-F / dCas9-yeeL-R, three fragments—left and right homologous arms and the dCas9 integration expression cassette—were amplified. The plasmid pTarget-dCas9+ was then constructed using a one-step cloning process. This plasmid was introduced into competent cells of strain YB-2A containing plasmid pCas9. Colony PCR and sequencing verification confirmed successful dCas9 integration, and the resulting strain was named CI-0. Plasmid pTarget-dCas9+ Construct the required primers (Table 1).

[0071] Shake-flask fermentation was used to verify whether L-tryptophan synthesis was interfered with by the toxic effects of the dCas9 protein. The results of the shake-flask fermentation are as follows: Figure 1 As shown, strain CI-0 produced 5.60 g·L⁻¹ of L-tryptophan and achieved a sugar-acid conversion rate of 5.60 g·L⁻¹. -1 and 0.102 g·g -1 The results were largely consistent with those of shake-flask fermentation of strain YB-2A, and growth was essentially unaffected. These results indicate that integration of the dCas9 gene expression cassette has little impact on host cells and is suitable for screening key targets affecting L-tryptophan synthesis.

[0072] Based on the genomic information of *E. coli* BL21(DE3)GCF_000022665.1, 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's coding genes. 1-3 sgRNAs were designed for each coding gene, with a focus on targeting each gene as much as possible. Two sgRNAs were designed for non-template targeting, and one sgRNA was designed for template targeting. The sgRNAs were designed as close as possible to the start codon for their target sites. The designed sgRNA oligonucleotides were synthesized using a high-throughput microarray, and homologous arms of the pSC101 vector were added to both ends. The synthesized oligonucleotides were ligated into the pSC101 vector via homologous recombination to obtain *E. coli* sgRNA library plasmids.

[0073] To perform high-throughput screening of the CPISPRi strain library using flow cytometry, plasmids pACYC and pSensor-trp3 were first used as templates. The pACYC vector backbone fragment and the PJ23107-tnaC-egfp fragment were amplified using primers pACYC-F / pACYC-R and pACYC-trp4-F / pACYC-trp4-R, respectively. The low-copy-count pSC101 vector backbone of pSensor-trp3 was then replaced with the low-copy-count pACYC vector backbone via one-step cloning and ligation to obtain the sensor plasmid pSensor-trp4. The primers required for plasmid pSensor-trp4 are shown in Table 1.

[0074] L-tryptophan biosensor plasmids pSensor-trp3 and pSensor-trp4 were electroporated into E. coli JM109, respectively, and plated onto a substrate containing 25 μg·mL⁻¹ of [unclear text - likely a specific plasmid name]. -1After incubation at 37°C for 12 h on chloramphenicol plates, fluorescent response test strains were obtained by bacterial selection and culture. Fluorescent response test strains containing different sensor plasmids were inoculated into 10 mL LB solutions, incubated at 37°C for 12 h, and then transferred at a 1% inoculum to solutions containing final concentrations of 0, 20, 40, 60, 80, and 100 mg·L⁻¹. -1 After incubation of L-tryptophan in 10 mL of M9 medium at 37°C for 12 h, samples were taken to determine the OD unit. 600 Fluorescence value. Based on the fluorescence response of the test strain at different concentrations of L-tryptophan, the unit OD value is... 600 Plot a scatter plot of fluorescence values ​​and calculate R. 2 Value, in R 2 A value greater than 0.9 is used to determine whether the L-tryptophan biosensor has good response performance.

[0075] Add exogenous L-tryptophan at a concentration of 0-300 mg / L -1 Response verification was performed within the specified range, and the results are as follows: Figure 2 As shown, pSensor-trp4 at L-tryptophan concentrations of 0-100 mg·L⁻¹ -1 Within the specified range, the unit fluorescence intensity is positively correlated with the L-tryptophan concentration (200-300 mg / L). -1 Within the range, unit OD 600 The fluorescence value tended to saturate. After the sensor plasmid pSensor-trp4 was introduced into strain CI-0, it was then introduced into the sgRNA plasmid library to obtain a CRISPRi strain library based on the whole E. coli genome. This library can be used to screen and sequence genes that affect L-tryptophan synthesis across the entire genome.

[0076] The L-tryptophan biosensor plasmid pSensor-trp4 was transformed into strain CI-0 to obtain a sensing strain. The whole-gene-targeting sgRNA library plasmid was electroporated into the sensing strain to obtain the E. coli CRISPRi library.

[0077] After overnight culture of the CRISPRi library strains for 10 hours, flow cytometry samples were prepared for sorting. Flow cytometry analysis revealed that approximately 99% of the mutagenic strains had lower fluorescence values ​​than the control strains. Therefore, 3000-5000 strains were collected from the top 1% of the CRISPRi library strains in terms of fluorescence value and screened in 96-well plates.

[0078] Example 3: Screening and Optimization of Key Targets for L-Tryptophan Synthesis Based on CRISPRi Strain Library

[0079] The CRISPRi strain library obtained in Example 2 required flow cytometry sorting, 96-well plate screening, and shake-flask rescreening verification. Finally, the target genes were identified by sequencing. Sequencing verification confirmed the target genes to be pykF (Gene ID: 946179), malM (Gene ID: 948547), spoT (Gene ID: 948159), rnr (Gene ID: 948692), rep (Gene ID: 948292), bioD (Gene ID: 945387), pheA (Gene ID: 947081), and kdpE (Gene ID: 945302). Functional descriptions of the target genes and the N20 sequences of the sgRNAs are shown in Table 2.

[0080] Table 2. Functions of the target genes and N20 sequences of sgRNAs

[0081]

[0082] Using the empty vector pSC101 as a template, eight single sgRNA repressor plasmids, including pSC101-P, were reconstructed by replacing the N20 target sequence using two-step PCR with primers spoT-N20-F / spoT-N20-R and rnr-N20-F / rnr-N20-R, resulting in plasmid pSC101-P. J23119- sgRNA pykF pSC101-P J23119- sgRNA malM pSC101-P J23119- sgRNA spoT pSC101-P J23119- sgRNA rnr pSC101-P J23119- sgRNA rep pSC101-P J23119- sgRNA bioD pSC101-P J23119- sgRNA pheA and pSC101-P J23119- sgRNA kdpE The pSC101 empty vector plasmid and the eight single sgRNA repressor plasmids mentioned above were introduced into strain CI-0 to obtain strains CI-01, CI-1, CI-2, CI-3, CI-4, CI-5, CI-6, CI-7, and CI-8. These strains were then subjected to shake-flask fermentation. Examples of primers required for constructing the eight single sgRNA repressor plasmids, including pykF, are shown in Table 1.

[0083] The results of shake-flask fermentation are as follows Figure 3As shown, in addition to inhibiting malM, inhibiting seven sites including rnr still resulted in increased L-tryptophan production compared to the control strain CI-01; among them, strains CI-3 and CI-4, whose target genes are spoT and rnr, achieved L-tryptophan production of 6.28 and 6.59 g·L⁻¹, respectively. -1 Compared with the control strain CI-01, the yields were increased by 10.5% and 16.1%, respectively, with sugar-acid conversion rates reaching 0.107 and 0.112 g·g⁻¹, respectively. -1 Compared to the control strain CI-01, the yields increased by 3.9% and 8.7%, respectively; strain CI-7, whose target gene is pheA, achieved L-tryptophan production and sugar-acid conversion rate of 6.38 g·L⁻¹. -1 and 0.110 g·g -1 Compared to the control strain CI-01, it showed increases of 12.3% and 6.8%, respectively, but its OD... 600 It decreased by 17.5%.

[0084] Example 4: Validation of genomic integration and knockout of the target gene rnr using its sgRNA expression cassette.

[0085] Based on the single sgRNA plasmid validation results in Example 3, the target gene rnr, which most significantly enhances L-tryptophan synthesis, was selected. In strains YB-2A and CI-0, rnr gene knockout and sgRNA were performed using CRISPR-Cas9 and CRISPR-Cas12a systems, respectively. rnr Integration of expression cassette (SEQ ID NO.5) at melB-yjbH in the genome.

[0086] First, using plasmid pTarget as a template, the N20 or N23 sequence is replaced by a two-step PCR method using primers rnr-N20-F / rnr-N20-R or melB-N23-yjdH-F / melB-N23-yjdH-R. Then, the left and right homologous arms of rnr are amplified using primers rnr-US-F / rnr-US-R and rnr-DS-F / rnr-DS-F, and the plasmid pTarget-Δrnr is constructed by one-step cloning. Alternatively, the sgRNArnr integrated fragment and the left and right homologous arms are amplified using primers sgRNArnr-F / sgRNArnr-R, melB-US-F / melB-US-R, and yjdH-DS-F / yjdH-DS-R, and the plasmid pTarget-rnr is constructed by one-step cloning. + The plasmid pTarget-Δrnr was introduced into competent cells of strain YB-2A containing plasmid pCas9. +The plasmid pCas12a was introduced into the competent cells of strain CI-0. Colony PCR and sequencing verification confirmed successful rnr gene knockout and sgRNA removal. rnr The expression cassette was successfully integrated into the genome, and the resulting strains were named YB-2A-Δrnr and CI-0-sgRNA. rnr Plasmid pTarget-Δrnr or pTarget-rnr + The primers required for construction are shown in Table 1.

[0087] strains YB-2A-Δrnr and CI-0-sgRNA rnr The results of shake-flask fermentation are as follows Figure 4 As shown, strain YB-2A-Δrnr achieved an L-tryptophan yield and a sugar-acid conversion rate of 6.42 g·L⁻¹. -1 and 0.113 g·g -1 Compared with strain YB-2A, the levels were increased by 13.4% and 9.7%, respectively; strain CI-0-sgRNA rnr The L-tryptophan yield and sugar-acid conversion rate reached 6.26 g·L⁻¹. -1 and 0.107 g·g -1 Compared to strain CI-0, the improvements were 11.6% and 4.9%, respectively.

[0088] The L-tryptophan synthesis performance of strain YB-2A-Δrnr was further verified in a 3-L fermentation tank. The fermentation results in the 3-L tank are as follows: Figure 5 As shown, strain YB-2A-Δrnr reached its maximum concentration after 49 hours of fermentation, which was 14 hours later than strain ZH-3, with the highest OD value. 600 The L-tryptophan yield reached 68.2, a decrease of 10.4% compared to strain ZH-3; after 49 hours of fermentation, strain YB-2A-Δrnr achieved an L-tryptophan yield and sugar-acid conversion rate of 32.46 g·L⁻¹. -1 and 0.171 g·g -1 Compared with strain ZH-3, the improvements were 5.0% and 32.6%, respectively.

[0089] The L-tryptophan synthesis performance of strain CI-0-sgRNArnr was further verified in a 3-L fermentation tank. The fermentation results in the 3-L tank are as follows: Figure 6As shown, the bacterial concentration of strain CI-0-sgRNArnr tended to remain constant after about 41 hours of fermentation, with a maximum OD600 value of 67.9, which was 10.8% lower than that of strain ZH-3. After 57 hours of fermentation, the L-tryptophan yield and sugar-acid conversion rate of strain CI-0-sgRNArnr reached 27.16 g·L⁻¹ and 0.131 g·g⁻¹, respectively. Compared with strain ZH-3, the L-tryptophan yield decreased by 12.2%, while the sugar-acid conversion rate was similar. Compared with strain YB-2A-Δrnr, strain CI-0-sgRNArnr showed a longer fermentation period in a 3-L tank, and both the L-tryptophan yield and sugar-acid conversion rate decreased.

[0090] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An L-tryptophan sensor plasmid, characterized in that, The plasmid is an expression vector carrying the natural leader peptide TnaC gene and a fluorescent reporter gene. The expression vector is the pACYC plasmid, and the natural leader peptide TnaC is promoted by the P... J23107 The driver, the nucleotide sequence of the TnaC gene is shown in SEQ ID NO.1, the fluorescent reporter gene is the eGFP gene with the nucleotide sequence shown in SEQ ID NO.2, and the promoter P... J23107 The nucleotide sequence is shown in SEQ ID NO.

3.

2. A method for increasing the L-tryptophan synthesis level in Escherichia coli, characterized in that, The method simultaneously expresses the L-tryptophan sensor plasmid of claim 1 and the whole-gene-targeting sgRNA library plasmid in E. coli that integrates dCas9, and identifies high-yield L-tryptophan-producing strains through high-throughput screening. The plasmids extracted from the high-yield L-tryptophan-producing strains are sent to Sanger sequencing to identify the targeted repressor genes.

3. The method according to claim 2, characterized in that, The host was strain YB-2A, and the integration location of dCas9 in the genome of strain YB-2A was gene yeeL-yeeJ to obtain strain CI-0.

4. The method according to claim 2, characterized in that, The sgRNA library plasmid for whole-genome targeting was used as a vector by pSC101.

5. The method according to claim 2, characterized in that, The target gene is the gene rnr.

6. A method for increasing L-tryptophan production, characterized in that, The gene rnr in the E. coli was suppressed or knocked out.

7. The *Escherichia coli* according to claim 6, characterized in that, The Escherichia coli strain mentioned is YB-2A.

8. The *Escherichia coli* according to claim 6, characterized in that, The gene ID of the gene rnr is 948692.

9. The application of the genes spoT and / or rnr in enhancing the ability of Escherichia coli to synthesize L-tryptophan, characterized in that, The application involves inhibiting or knocking out the genes spoT and / or rnr in Escherichia coli.

10. The application according to claim 9, characterized in that, The gene IDs for spoT and / or rnr are 948159 and 948692, respectively.

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