A bacterium producing L-threonine and a method for producing L-threonine

Abstract

The invention discloses a kind of bacteria producing L-threonine and a method for producing L-threonine thereof, belonging to the field of genetic engineering technology, Escherichia coli MG1655 does not have the ability to produce L-threonine, after knocking out the PgrR gene, the acid production of threonine is increased to 0.85g / L, and the conversion rate is increased to 3.21%. Taking Escherichia coli MG1655 as the starting strain and passing through the composite mutagenesis screen, the L-threonine production of the L-threonine producing bacteria TTC is 1.05g / L, and the conversion rate is 5.73%. The strain knocks out the PgrR gene and the L-threonine production is increased from 1.05g / L to 3.62g / L, and the sugar acid conversion rate is also increased from 5.73% to 15.91%. By knocking it out, the conversion of glucose to L-threonine can be promoted, and the L-threonine production capacity of the strain can be significantly improved, especially the strain with L-threonine production capacity, and the improvement is more obvious.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a bacterium for producing L-threonine and a method for producing L-threonine. Background Art

[0002] Peptidoglycan is an important component of bacterial cell wall and is essential for maintaining cell morphology, size and survival. In the process of peptidoglycan metabolism, peptidoglycan hydrolases have an important influence on cell shape and growth. PgrR, also known as ycjZ (DNA-binding transcriptional repressor PgrR), is a transcriptional repressor. As a repressor protein, it regulates the expression of genes involved in peptidoglycan oligopeptide degradation. It can directly bind to the operon between the upstream and reverse ycjY-ymjD-ymjC-mpaA gene clusters of PgrR to regulate the expression of the ycjY-ymjD-ymjC-mpaA gene clusters (see non-patent document 1). In addition, PgrR also regulates the expression of ycjG. Since many PgrR-regulated genes are involved in the uptake and degradation of cell wall peptidoglycan, under nitrogen starvation conditions, the growth rate of PgrR-deficient strains is faster than that of wild-type strains, which means that weakening the expression of PgrR may help bacteria use peptidoglycan as a nitrogen source, thereby overcoming nitrogen limitation. At the same time, PgrR deletion can enhance the expression of the tpx gene, which acts as a lipid hydroperoxidase and helps protect the cell membrane of the strain from oxidative damage. Summary of the invention

[0003] The object of the present invention is to solve the above problems and to provide a bacterium producing L-threonine and a method for producing L-threonine.

[0004] A bacterium for producing L-threonine, wherein the bacterium is Escherichia coli and the PgrR gene is knocked out or weakened;

[0005] The nucleotide sequence of the PgrR gene is shown in the sequence listing SEQ ID No. 1;

[0006] The bacteria is Escherichia coli MG1655.

[0007] A strain of Escherichia coli TTC was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number of CGMCC 31341.

[0008] The bacteria is a strain of Escherichia coli TTC.

[0009] A method for producing L-threonine, comprising culturing the L-threonine-producing bacterium or the Escherichia coli TTC according to claim 4 to extract L-threonine.

[0010] The present invention provides a bacterium for producing L-threonine and a method for producing L-threonine. Escherichia coli MG1655 does not have the ability to produce L-threonine. After knocking out the PgrR gene, the acid production of threonine is increased to 0.85 g / L, and the conversion rate is increased to 3.21%. The L-threonine producing bacteria TTC, which uses Escherichia coli MG1655 as the starting strain and undergoes a composite mutagenesis screen, has an L-threonine acid production of 1.05 g / L and a conversion rate of 5.73%. The L-threonine production of the strain after knocking out the PgrR gene is increased from 1.05 g / L to 3.62 g / L, and the sugar acid conversion rate is also increased from 5.73% to 15.91%. It is shown that the PgrR gene has an obstructive effect in the L-threonine synthesis pathway of Escherichia coli. By knocking out the PgrR gene, the conversion of glucose to L-threonine can be promoted, and the L-threonine production capacity of the strain can be significantly improved, especially for strains with L-threonine production capacity, the improvement is more obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the PgrR gene in Escherichia coli MG1655 and TTC.

[0012] Figure 2 It is a modified sequence in which PgrR is deleted through genetic engineering technology.

[0013] Figure 3 Electrophoresis diagram of PCR products of strains (A is the recombinant fragment PgrR (HR), B is the pTargetF-PgrR fragment)

[0014] Figure 4 This is the electrophoresis diagram of the colony PCR verification of the PgrR site genome editing of the strain (A is the fragment amplified by 2×Taq PCR Mix enzyme; B is the fragment amplified by KOD-Plus-Neo enzyme of the knockout-positive bacteria)

[0015] Figure 5 The following are the verification images of strain plasmid loss (A is the verification result of pTargetF-PgrR plasmid loss, and it can be seen that colonies 2, 3, 6, 8, and 10 have successfully lost the pTargetF-PgrR plasmid; B is the verification result of pCas plasmid loss, and it can be seen that colonies 1, 3, and 4 have successfully lost the pCas plasmid) DETAILED DESCRIPTION

[0016] CRISPR-Cas9 is a gene editing technology commonly used in Escherichia coli. It can achieve gene editing by cutting DNA sequences. Its working principle is to first synthesize the sgRNA sequence according to the target gene. The sgRNA can bind to the Cas9 protein. Because the target gene and the sgRNA sequence are complementary, the sgRNA can carry the Cas9 protein to accurately find the target gene, and shear it to produce a double-stranded DNA break, and then use homologous recombination to repair the DNA break generated during the cutting, and introduce mutations at the same time, thereby achieving gene knockout, insertion or replacement. In a specific embodiment of the present invention, the CRISPR-Cas9 method is used to complete the knockout of the PgrR gene. The nucleotide sequence of the PgrR gene is shown in the sequence table SEQ ID No.1.

[0017] The Escherichia coli described in the present invention is not particularly limited, and includes Escherichia coli W3110 (ATCC 27325) and Escherichia coli MG1655 (CGMCC1.18912) derived from the prototype wild-type strain K-12, and also includes Escherichia coli inherently having the ability to produce L-threonine, and Escherichia coli that has the ability to produce L-threonine through mutagenesis and / or genetic modification.

[0018] The Escherichia coli used in the present invention is MG1655 (CGMCC1.18912), which was purchased on May 8, 2024 from the General Microbiology Center of China Culture Collection Committee, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0019] Example 1 Preparation of PgrR gene knockout strain MG1655ΔPgrR

[0020] (1) Preparation of recombinant fragments

[0021] Using PgrR-F / PgrR-R as primers (Sangong Biosynthesis), TOYOBO's KOD-Plus-Neo PCR amplification (the program and system are as shown in the table below) was used to obtain a PCR product of about 100 bp in size. Gel recovery was performed (Tiangen Ordinary Agarose Gel DNA Recovery Kit) and the recombinant fragment PgrR (HR) was finally obtained. The results are as follows Figure 3 The specific reaction conditions are shown in Table 1.

[0022] ;

[0023] (2) Introduction of pCas plasmid and preparation of MG1655 / pCas electroporation competent cells (L-ara)

[0024] Introduction of pCas plasmid: Prepare MG1655 strain into competent cells (refer to Chapter 1, page 96 of Molecular Cloning III for transformation and competent preparation methods), take 2 μL of pCas plasmid (the plasmid comes from the literature: Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) and transform it into MG1655 competent cells to obtain MG1655 / pCas single colonies.

[0025] Preparation of MG1655 / pCas electroporation competent state (L-ara): Pick a single colony of MG1655 / pCas into an LB test tube containing 50 mg / L kanamycin, and culture it at 220 r / min at 30°C overnight. The next day, transfer 1% of the bacterial solution into an LB test tube containing 50 mg / L kanamycin. One hour before the bacterial concentration OD600 reaches 0.4, add arabinose with a final concentration of 10 mM for induction. When the OD600 reaches 0.4, centrifuge at 5000 rpm / min for 5 min to collect the bacteria, resuspend the bacteria in an equal volume of pre-cooled 10% glycerol, and centrifuge at 5000 rpm / min for 10 min. Repeat twice. Finally, resuspend the bacteria in 100 μL 10% glycerol, which is the MG1655 / pCas electroporation competent state (L-ara), and freeze at -80°C for later use.

[0026] (3) Construction of pTargetF-PgrR plasmid

[0027] Using pTargetF-cadA plasmid (derived from the literature Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9System, JiangY, ChenB, etal. Appl EnvironMicrobiol, 2015) as template and PgrR-N20-F / pTargetF-R as primers, TOYOBO's KOD-Plus-NeoPCR amplification was used to obtain the pTargetF-PgrR linear PCR product with the PgrR-sgRNA recognition sequence introduced, about 2.3kb. After digestion with FastDigest DpnI (Thermo Scientific™ digestion system, see the table below), 2μL was transformed into Dh5α competent medium, spread on LB plates containing 50mg / L spectabilis, and cultured at 37℃ overnight to obtain a single colony. After liquid culture, the pTargetF-PgrR plasmid was extracted using the Tiangen plasmid extraction kit, and the primer N20-CX sequencing (Sangon Biotechnology sequencing) was used to obtain the pTargetF-PgrR plasmid. The results are as follows Figure 3 As shown in B. Specific reaction conditions are shown in Table 2 and Table 3.

[0028] ;

[0029] .

[0030] (4) Genome editing of the PgrR locus of MG1655 strain

[0031] Knockout of PgrR in MG1655 strain: pre-cool a 2mm electroporation cuvette, electroporate 400ng of PgrR(HR) fragment and 100ng of pTargetF-PgrR plasmid into MG1655 / pCas electroporation competent medium (L-ara) at 2.5kV, 200Ω, 25μF. Immediately after electroporation, add 800μL of pre-cooled LB medium, resuscitate at 30℃ 150rpm / min for 1h, centrifuge at 5000rpm / 5min, remove 800μL of supernatant, mix the remaining supernatant with the bacteria, evenly spread on an LB plate containing 50mg / L spectinomycin and 50mg / L kanamycin, and culture at 30°C overnight; grow a single colony and use Tiangen 2×Taq PCR Mix and primers PgrR-VF / PgrR-VR for colony PCR verification (see the table below for procedures and systems). Positive 549bp, negative 1.45kb, the results are as follows Figure 4 The specific reaction conditions are shown in Table 4.

[0032] ;

[0033] MG1655△ PgrR strain sequencing: PCR positive strains were taken and the genome of the positive strains was extracted using the Tiangen Bacteria Genomic DNA Extraction Kit. A 549bp PCR product was obtained by amplification using TOYOBO's KOD-Plus-Neo and primers PgrR-VF / PgrR-VR. After sequencing with primers PgrR-VF (Sangon Biotech Sequencing), the TTC△ PgrR / pCas / pTargetF-PgrR strain was obtained. The results are as follows: Figure 4 The specific reaction conditions are shown in Table 5.

[0034] .

[0035] (4) Loss of editing plasmid

[0036] pTargetF- PgrR plasmid loss: Pick a single colony that is positive for colony PCR verification, inoculate it into an LB test tube containing 50 mg / L kanamycin, add a final concentration of 0.5 mM IPTG, and culture it at 30°C overnight; the next day, streak the bacterial solution in the test tube directly on an LB plate containing 50 mg / L kanamycin, and culture it at 30°C overnight; the next day, pick a single colony and transfer it to an LB plate containing 50 mg / L spectinomycin. If it cannot grow, it indicates that the pTargetF- PgrR plasmid has been lost, and the strain TTC△ PgrR / pCas is obtained. The plate culture results are as follows: Figure 5 As shown in A.

[0037] pCas plasmid loss: strain MG1655△ PgrR / pCas was selected and inoculated into an antibiotic-free LB tube, cultured at 37°C overnight, and the next day the bacterial solution was streaked onto an LB plate and cultured at 37°C overnight; the next day a single colony was selected and transferred onto an LB plate containing 50 mg / L kanamycin. If it failed to grow, it indicated that the pCas plasmid was lost, and the MG1655Δ PgrR strain was obtained. The plate culture results were as follows: Figure 5 As shown in B.

[0038] The primer sequences used in plasmid construction are detailed in Table 6.

[0039]

[0040] Example 2 Mutation Screening of MG1655 Mutant Strain Producing L-Threonine

[0041] The purchased lyophilized strains of E. coli MG1655 were inoculated into two test tubes containing 5 ml of complete liquid culture medium, cultured overnight in a shaking incubator at 37°C, and subcultured twice to fully restore the strain activity. The rejuvenated E. coli MG1655 was then expanded into a 150 ml Erlenmeyer flask containing 20 ml of complete liquid culture medium, cultured to the logarithmic phase, centrifuged to remove the supernatant, washed 3 times with PBS, and resuspended in physiological saline to adjust the bacterial solution concentration to 1 x 10 8 CFU / mL. Then the strain was induced by the combined mutagenesis method of ultraviolet light and diethyl sulfate (DES). The specific mutagenesis conditions are as follows:

[0042] (1) UV mutagenesis

[0043] The prepared bacterial suspension was added to a sterile plate with a diameter of 9 cm, and a 15W ultraviolet lamp was used at a distance of 30 cm. A magnetic stirrer was used for stirring. The suspension was irradiated for 0, 20, 40, 60, 80, and 120 seconds respectively, and appropriately diluted with physiological saline to prevent photoreactivation after mutagenesis. The suspension was diluted under red light from a dilution of 10 -3 , 10 -4 , 10 -5 , 10 -6 Take 0.1 ml of each dilution and spread it on a solid basic culture medium plate, wrap the culture medium plate with black paper, and culture it upside down at 37°C for 12 hours. Compare the number of colonies with the same dilution for mutagenesis, measure the lethality at different mutagenesis times, and determine the best mutagenesis conditions (select the conditions with a lethality between 85-95% as the mutagenesis conditions, because positive mutations are most likely to occur when the lethality reaches 85-95%).

[0044] (2) DES mutagenesis

[0045] Take 11 groups of 1.5ml EP tubes, add 45μL of bacterial suspension, add different amounts of 5% DES (0~100μL, in increments of 10μL), and then make the volume to 1.5ml with PBS. The mutagenesis time is 15min (from the addition of 5% DES to the addition of 25% sodium thiosulfate solution). Take 1.0ml of the treated solution and add 1.0ml of 25% sodium thiosulfate solution to terminate the mutagenesis. Then take 0.1ml of each tube and spread it on the solid basic culture medium. Invert and culture at 37℃ for 12h, measure the lethality under different mutagenesis conditions, and determine the optimal mutagenesis conditions.

[0046] The strains obtained by the composite mutagenesis screening were prepared into a bacterial suspension, 0.1 ml of the bacterial suspension was spread on a methionine-supplemented medium, and inverted for 24 h at 37°C. Then 72 colonies were picked and correspondingly planted on basic medium and new methionine-supplemented medium plates. After cultivation, the strains that grew better on the methionine-supplemented medium plates than on the basic medium were picked as suspected strains. They were then numbered and streaked on methionine-supplemented medium plates and basic medium plates, respectively, and cultured continuously for 5 generations. The strain with good genetic stability was selected and cultured in the fermentation medium for shake flask culture, with shaking culture at 37°C and 220r / min for 30 hours. The L-threonine yield in the fermentation broth was used as the technical indicator for strain screening, thereby obtaining an L-threonine-producing Escherichia coli TTC (Escherichia coli). The strain was deposited in the General Microbiology Center of China Culture Collection Administration on July 17, 2024, with the address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and the strain deposit number is CGMCC31341.

[0047] The detection of L-threonine in the fermentation broth adopts paper chromatography for qualitative detection and colorimetry for quantitative detection.

[0048] The complete medium is composed of 0.5% glucose, 0.3% beef extract, 1.0% peptone, 0.5% yeast extract, 0.5% NaCl, and a pH value of 7.0-7.2. 2.0% agar is added to the solid medium.

[0049] The basic culture medium is: glucose 2.0%, (NH4)2SO4 0.4%, urea 0.1%, K2HPO4 0.05%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.02%, MnSO4·H2O 0.02%, D-biotin 50 μg / ml, vitamin B12 mg / l (sterilized separately), pH 7.0~7.2. 2.0% agar is added to the solid culture medium.

[0050] The methionine supplemented medium is: basic medium supplemented with 100 μg / ml DL-methionine.

[0051] Example 3 Preparation of PgrR gene knockout strain TTCΔPgrR

[0052] After gene sequencing, the gene PgrR in the mutant strain TTC and the PgrR in the MG1655 strain are both wild types. All primers and plasmids used for gene knockout using CRISPR are universal. Therefore, the method for knocking out the PgrR gene in TTC and MG1655 is the same, and the specific method is the same as Example 1.

[0053] Example 4 Performance Verification of L-Threonine Production by Fermentation

[0054] 1. Seed cultivation

[0055] One loopful of MG1655, MG1655△ PgrR, TTC, and TTC(Δ PgrR) slant seeds were inoculated into 250-ml Erlenmeyer flasks containing 30 ml of seed culture medium, and cultured at 37°C and 220 r / min for 12 h.

[0056] 2. Fermentation culture

[0057] Inoculate 20 ml of seed liquid into a 1000 ml Erlenmeyer flask containing 200 ml of fermentation medium and culture at 37°C and 220 r / min with shaking for 30 h.

[0058] 3. Detection of L-threonine content in fermentation broth

[0059] The volume of the threonine fermentation broth at the end of the fermentation was weighed, and then the fermentation broth was centrifuged (11000 rpm, 5 min), the supernatant was filtered with a 0.22 μm membrane, the filtered fermentation supernatant was collected, and the L-threonine content in the supernatant of the fermentation broth of the strain was detected by HPLC. The results are shown in Table 7.

[0060] The seed culture medium comprises: 10 g / L glucose, 20 g / L beet molasses, 15 g / L (NH4)2SO4, 2 g / L KH2PO4, 0.4 g / L MgSO4·7H2O, 0.6 g / L KCl, 5 mg / L FeSO4·7H2O, 5 mg / L MnSO4·4H2O, 1 g / L betaine, 10 g / L CaCO3, and pH 7.

[0061] The fermentation medium comprises: 40 g / L glucose, 2 g / L KH2PO4, 25 g / L (NH4)2SO4, 1 g / L MgSO4·7H2O, 5 mg / L FeSO4·7H2O, 5 mg / L MnSO4·4H2O, 0.15 g / L dl-methionine, 2 g / L yeast extract, 30 g / L CaCO3, 50 mg / L corn steep liquor, and pH 6.8.

[0062] The HPLC detection method is as follows:

[0063] Chromatographic conditions: flow rate 1 ml / min, column temperature 40°C, diode array detection wavelength 254 nm, injection volume 5 μL.

[0064] Solution preparation: Take 0.3 ml PITC and place it in a 25 ml volumetric flask, dilute it to the mark with acetonitrile, shake well, and store it at 4°C. Triethylamine acetonitrile solution (0.1 mol / L) Take 1.3 ml triethylamine and place it in a 10 ml volumetric flask, dilute it to the mark with acetonitrile, shake well, and store it at 4°C.

[0065] Mobile phase A: 80% acetonitrile solution (800 ml acetonitrile plus 200 ml ultrapure water), ultrasonic degassing.

[0066] Mobile phase B: 97:3 (V / V) sodium acetate acetonitrile solution (15.2 g of anhydrous sodium acetate was dissolved in 1850 ml of ultrapure water, the pH was adjusted to 6.5 with glacial acetic acid, and 140 ml of acetonitrile was added), and ultrasonic degassing was performed.

[0067] The gradient elution program is shown in the following table:

[0068] ;

[0069] ;

[0070] *Note: Sugar-acid conversion rate (%) = concentration of threonine in fermentation liquid * volume of fermentation liquid / total sugar consumption

[0071] As can be seen from Table 8, E. coli MG1655 does not have the ability to produce L-threonine. After knocking out the PgrR gene, the acid production of threonine increased to 0.85 g / L, and the conversion rate increased to 3.21%, which was significantly improved. The L-threonine production strain TTC, which was screened by composite mutagenesis using E. coli MG1655 as the starting strain, had an L-threonine acid production of 1.05 g / L and a conversion rate of 5.73%. The L-threonine production of the strain after knocking out the PgrR gene increased from 1.05 g / L to 3.62 g / L, and the sugar acid conversion rate also increased from 5.73% to 15.91%. This shows that the PgrR gene has an inhibitory effect in the L-threonine synthesis pathway of E. coli. By knocking out the PgrR gene, the conversion of glucose to L-threonine can be promoted, and the L-threonine production capacity of the strain can be significantly improved, especially for strains with L-threonine production capacity, the increase is more obvious.

Claims

1. A bacterium for producing L-threonine, characterized in that: The bacteria is Escherichia coli in which the PgrR gene is knocked out ( Escherichia coli )TTC; The Escherichia coli TTC is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31341; The nucleotide sequence of the PgrR gene is shown in the sequence listing SEQ ID No.

1.

2. A method for producing L-threonine, characterized in that: Cultivate the L-threonine-producing bacterium according to claim 1 to extract L-threonine.

3. A method for producing L-threonine, culturing Escherichia coli with knocked-out PgrR gene ( Escherichia coli ) MG1655, extracting L-threonine; The nucleotide sequence of the PgrR gene is shown in the sequence listing SEQ ID No. 1.

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