Construction and application of escherichia coli with high yield of l-threonine

Abstract

The application discloses construction and application of Escherichia coli with high L-threonine yield, and belongs to the field of genetic engineering and fermentation engineering. In the genome of Escherichia coli TWF001, the pilus synthesis gene clusters Ycb, Yad, Yde, Yeh, Yqi, Yra, Yfc, Type1, Yhc and Sfm are knocked out to obtain mutant TWK021. The L-threonine yield of the TWK021 strain in a fermentation medium is 15.75 g / L, which is increased by 32.4% compared to the TWF001 yield, and the L-threonine yield of 62.7 g / L is obtained in a fed-batch fermentation. The application constructs a stable Escherichia coli with high L-threonine yield, and the plasmid does not need to be maintained by antibiotics in the whole fermentation process, and a new strategy for increasing the L-threonine yield of Escherichia coli is provided.

Description

Technical Field

[0001] This invention relates to the construction and application of a high-yield L-threonine-producing Escherichia coli, belonging to the fields of genetic engineering and fermentation engineering. Background Technology

[0002] L-Threonine, with the chemical formula C4H9NO3 and a molecular weight of 119.1, is an essential amino acid that must be obtained from the diet of animals. It is also an important bioactive molecule that plays a crucial regulatory role in protein synthesis, energy metabolism, and nutrient absorption. Studies have shown that threonine has a beneficial effect on gut microbiota, thus making it valuable in food, medicine, and animal feed. However, threonine synthesis competes with the TCA pathway; therefore, improving the supply of microbial matter and energy is key to high threonine production.

[0003] Pili are thin, long cell appendages that mediate bacterial adsorption, infection, DNA transfer across the cell membrane, and enhance bacterial resistance. They are also important components of biofilms. Therefore, the presence of pili structures can lead to product spoilage or contamination, increasing the risk of fermentation contamination and shortening the lifespan of fermentation equipment. Furthermore, as non-essential extracellular structures, pili consume significant amounts of energy and resources from the cells. The most widely studied pili system in *E. coli* is the guide chaperone system (CU). CU-based pili development requires a periplasmic chaperone and an outer membrane assembly platform called an intropospheric organ.

[0004] The chaperone prevents the folding and polymerization of the pili subunits and guides them to the intropospheric site, where the assembly of the pili structure is completed. There are a total of 12 CU operators in *E. coli* TWF001. Figure 1 Taking type I pili as an example, fimA, fimF, fimG, and fimH encode structural proteins, while fimC and fimD encode chaperone-intropospheric structures. Other CU pili structures are homologous to type I pili structures. Qiao J et al. constructed a pili-deficient mutant from wild-type *E. coli* MG1655 and examined the intracellular ATP / ADP ratio. Due to the absence of pili, the intracellular ATP / ADP ratio of *E. coli* increased by 2.6-fold. This demonstrates that the absence of pili affects the growth and metabolism of the strain. Summary of the Invention

[0005] This invention knocks out 10 gene clusters that synthesize CU fimbriae in the genome of threonine-producing Escherichia coli TWF001 to obtain mutant strain TWK021. Escherichia coli TWK021 can efficiently synthesize threonine in fermentation medium.

[0006] The first objective of this invention is to provide a method for increasing L-threonine production, wherein the method knocks out the fimbrial synthesis gene cluster on the genome of Escherichia coli TWF001, the fimbrial synthesis gene cluster being Ycb, Yad, Yde, Yeh, Yqi, Yra, Yfc, Type 1, Yhc, Sfm, Mat, and Ybg.

[0007] In one embodiment, the method involves knocking out the fimbrial synthesis gene cluster on the genome of Escherichia coli TWF001, wherein the fimbrial synthesis gene cluster is Ycb, Yad, Yde, Yeh, Yqi, Yra, Yfc, Type 1, Yhc, and Sfm.

[0008] The Ycb is ycbQ, ycbR, ycbS, ycbT, ycbU, ycbV, ycbF;

[0009] The Yad is yadC, yadK, yadL, yadM, htrE, ecpD, yadN;

[0010] The Yde is ydeQ, ydeR, ydeS, ydeT;

[0011] The Yeh is yehA, yehB, yehC, yehD;

[0012] The Yqi are ygiL, yqiG, yqiH, and yqiI;

[0013] The Yra is yraH, yraI, yraJ, yraK;

[0014] The Yfc is yfcO, yfcP, yfcQ, yfcR, yfcS, yfcT, yfcU, yfcV;

[0015] Type 1 is fimA, fimI, fimC, fimD, fimF, fimG, fimH;

[0016] The Yhc is yhcA, yhcD, yhcE, yhcF, gltF

[0017] The Sfm is sfmA, sfmC, sfmD, sfmH, sfmF;

[0018] The Mat is yagV, yagW, yagX, yagY, yagZ;

[0019] The Ybg is ybgO, ybgP, ybgQ, ybgD.

[0020] In one embodiment, the genes ycbQ, ycbR, ycbS, ycbT, ycbU, ycbV, ycbF, yadC, yadK, yadL, yadM, htrE, ecpD, yadN, ydeQ, ydeR, ydeS, ydeT, yehA, yehB, yehC, yehD, ygiL, yqiG, yqiH, yqiI, yraH, yraI, yraJ, yraK, yfcO, yfcP, yfcQ, yfcR, yfcS, yfcT, yfcU, yfcV, fimA, fimI, fimC, fimD, fimF, fimG, fimH, gltF, yhcA The NCBI accession numbers for the sequences yhcD, yhcE, yhcF, sfmA, sfmC, sfmD, sfmH, sfmF, yagV, yagW, yagX, yagY, yagZ, ybgO, ybgP, ybgQ, and ybgD are “AAC74024.2”, “AAC74025.1”, “AAC74026.1”, “AAC74027.1”, “AAC74028.1”, “AAC74029.2”, “AAC74030.2”, “AAC73246.1”, “AAC73247.1”, “AAC73248.1”, “AAC73249.2”, and “AAC73248.1”, “AAC73249.2”, and “AAC73248.1”, respectively. 250.1","AAC73251.1","AAC73252.1","AAC74575.1","AAC74576.1","AAC74577.1","AYC08213.1","AAC75169.1","AAC75170.1","AAC75171.1", "AAC75172.1", "AAC76079.1", "AAC76082.1", "AAC76083.2", "AAC76084.1", "AAC76176.1", "AAC76177.1", "AAC76178.1", "AAC76179.1", "AAC753 92.2","AAC75393.1","AAC75394.1","AAC75395.1","AAC75396.1","AAC7397.1","AAC7398.1","AAC75399.1","AAC77270.1","AAC77271.2","AA C77272.1", "AAC77273.1", "AAC77274.1", "AAC77275.1", "AAC77276.1", "AAC76246.1", "AAC76247.1", "AAC76248.1", "AAC76249.1", "AAC76251.1","AAC73632.2","AAC73633.1","AAC73634.1","AAC73635.2","AAC73636.1","AAC73392.2","AAC73393.1" , "AAC73394.1", "AAC73395.1", "AAC73396.1", "AAC73810.2", "AAC73811.1", "AAC73812.2", "AAC73813.1".

[0021] A second objective of this invention is to provide recombinant Escherichia coli obtained by the above-described method.

[0022] The third objective of this invention is the application of the recombinant Escherichia coli in the preparation of L-threonine, wherein the application involves inoculating the recombinant Escherichia coli into a fermentation system to prepare L-threonine.

[0023] In one embodiment, the application involves inoculating the seed culture of the recombinant Escherichia coli into a fermentation medium, setting the initial OD600 to 0.1-0.5, and culturing at 35-38°C and 150-250 rpm for 30-40 hours.

[0024] In one embodiment, the seed culture is prepared by activating and culturing recombinant Escherichia coli on a plate for 15-25 hours, inoculating it into SOB medium, and culturing it at 35-38°C and 150-250 rpm for 10-15 hours to obtain a primary seed culture; the primary seed culture is then inoculated into STF medium with an initial OD600 of 0.1 and cultured at 35-38°C and 150-250 rpm for 3-5 hours to obtain a secondary seed culture.

[0025] In one embodiment, the fermentation medium comprises 1–5 g / L yeast extract, 1–5 g / L citric acid, 20–30 g / L (NH4)2SO4, 5–10 g / L KH2PO4, 1–5 g / L MgSO4·7H2O, 2–7 mg / L FeSO4·7H2O, 2–7 mg / L MnSO4·4H2O, 20–60 g / L glucose, and 10–30 g / L CaCO3.

[0026] In one embodiment, the STF culture medium comprises: 5–15 g / L sucrose, 10–30 g / L peptone, 1–10 g / L yeast extract, 10–20 g / L (NH4)2SO4 and 0.5–1.5 g / L MgSO4.

[0027] The fourth objective of this experiment is to provide a method for preparing L-threonine, wherein the seed culture of the above-mentioned recombinant Escherichia coli is inoculated into a fed-batch fermentation medium, the aeration rate is set to 1.5 vvm, and the dissolved oxygen coupling speed is set to maintain the DO value at 25%-30%.

[0028] In one implementation, when the glucose concentration in the culture medium is below 5 g·L⁻¹ -1 The process was carried out in stages, with glucose added continuously to maintain a concentration of 15 g / L. -1 -20g·L -1 .

[0029] In one embodiment, the rotational speed does not exceed 900 rpm.

[0030] In one embodiment, the method involves inoculating the seed culture of the recombinant Escherichia coli into a fed-batch fermentation medium at a volume ratio of 5-15%.

[0031] In one embodiment, the seed culture is prepared by inoculating the recombinant Escherichia coli into SOB medium and culturing overnight to obtain an initial OD value. 600 Inoculate 0.1 g of the culture medium into STF medium and incubate at 35-38°C and 150-250 rpm for 3-5 hours; then inoculate with the initial OD value. 600 Inoculate 0.2 g into the fermentation medium and incubate at 35-38℃ and 150-250 rpm for 3-5 hours.

[0032] In one embodiment, the fed-batch fermentation medium comprises 1–5 g·L⁻¹ -1 Yeast extract, 1–10 g·L -1 (NH4)2SO4, 1–5 g·L -1 KH2PO4, 1-5 g·L -1 MgSO4·7H2O, 1–10 mg·L -1 FeSO4·7H2O, 1~10 mg·L -1 MnSO4·4H2O, 20~60g·L -1 glucose.

[0033] The present invention also provides the application of the recombinant Escherichia coli or the above-described method in the fields of fermentation, drug preparation, materials or environmental protection.

[0034] Beneficial effects

[0035] This invention knocks out the fimbrial synthesis gene clusters Ycb, Yad, Yde, Yeh, Yqi, Yra, Yfc, Type 1, Yhc, and Sfm in the genome of *E. coli* TWF001, obtaining the mutant strain TWK021. The TWK021 strain, when cultured in shake flasks, produced 15.75 g / L of L-threonine, a 32.4% increase compared to TWF001. Furthermore, in a fed-batch fermentation in a 3L fermenter, 62.7 g / L of L-threonine was obtained, with a sugar-acid conversion rate of 0.427 g / g. This invention constructs a stable, high-threonine-producing recombinant *E. coli* strain that does not require antibiotics to maintain plasmid presence throughout the fermentation process, providing a novel strategy for increasing L-threonine production in *E. coli*.

[0036] biomaterials

[0037] Escherichia coli TWF001 was published in the 2018 paper "Increasing L-threonine production in Escherichia coli by engineering the glyoxylate shunt and the L-threonine biosynthesis pathway". Attached Figure Description

[0038] Figure 1 : 12 gene clusters in Escherichia coli that synthesize CU fimbriae.

[0039] Figure 2 (A) Deletion gene cluster, (B) growth status, and (C) L-threonine production status of TWK001-TWK012, a fimbriae-deficient strain of TWF001.

[0040] Figure 3 (A) Deletion gene cluster, (B) growth status, and (C) L-threonine production status of TWF001 fimbriae-deficient strain TWK013-TWK023.

[0041] Figure 4 Feed-batch fermentation of TWK021 in a 2-L bioreactor. Detailed Implementation

[0042] (1) Culture medium:

[0043] LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl.

[0044] STF medium: 10 g / L sucrose, 20 g / L peptone, 5 g / L yeast extract, 15 g / L (NH4)2SO4 and 1 g / L MgSO4, pH 7.3.

[0045] L-Threonine fermentation medium: 2 g / L yeast extract, 2 g / L citric acid, 25 g / L (NH4)2SO4, 7.46 g / L KH2PO4, 2 g / L MgSO4·7H2O, 5 mg / L FeSO4·7H2O, 5 mg / L MnSO4·4H2O, 40 g / L glucose and 20 g / L CaCO3, pH 7.1.

[0046] SOB medium: 20 g / L peptone, 5 g / L yeast extract, 2.47 g / L MgSO4·7H2O, 0.5 g / L NaCl, 0.186 g / L KCl, and pH 7.0 ± 0.2.

[0047] Fed-batch fermentation medium: 3 g·L -1 Yeast extract, 5g·L -1 (NH4)2SO4, 2 g·L -1 KH2PO4, 2 g·L -1 MgSO4·7H2O, 5 mg·L -1 FeSO4·7H2O, 5 mg·L -1 MnSO4·4H2O, 40g·L -1 Add glucose and adjust the pH to 7.0.

[0048] (2) Determination of bacterial cell concentration

[0049] The absorbance at 600 nm was measured using a UV-1800 UV-Vis spectrophotometer.

[0050] (3) Determination of glucose content

[0051] The glucose content in the fermentation broth was detected using an SBA-40 bioanalyzer. First, 25 μL of SBA standard solution was used for calibration. Then, the fermentation broth was centrifuged at 12,000 rpm for 15 min, and 10 μL of the supernatant was taken and diluted 100 times before measurement.

[0052] (4) L-threonine concentration analysis:

[0053] L-threonine concentration was quantitatively determined using the o-phthalaldehyde pre-column derivatization method (Koros, A., Varga, Z., Molnar-Perl, I. 2008. Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives incheese by high-performance liquid chromatography. J Chromatogr A, 1203(2), 146-52.). An Agilent 1200 or 1260 series high-performance liquid chromatography system was used, equipped with a Thermo 250mm × 4.0mm ODS-2HYPERSIL C18 column.

[0054] Example 1: Construction of engineered bacteria lacking fimbriae

[0055] (1) Preparation of E. coli electrotransfer competent cells containing the knockout plasmid pCas:

[0056] E. coli cells containing the pCas knockout plasmid were seeded in 5 mL of LB medium containing kanamycin and incubated overnight at 30°C and 200 rpm. Initial OD was used as the starting point for the culture. 600 =0.05% was inoculated into 50 mL of LB medium containing kanamycin and incubated at 30°C and 200 rpm until OD reached. 600 When the concentration of L-arabinose (30 mmol / L) was 0.1, L-arabinose was added to induce recombinase expression, and the mixture was cultured until the OD value reached 0.1. 600 =0.6, incubate on ice for 30 minutes. Pour the culture into a 50 mL sterile centrifuge tube, centrifuge at 4000 rpm for 10 minutes at 4°C to collect the bacterial cells, and wash three times with 10% glycerol. Finally, resuspend the cells in 1 mL of 10% glycerol and aliquot into 10 sterile 1 mL centrifuge tubes for later use.

[0057] (2) Construction of pTargetF plasmid:

[0058] The plasmids used are listed in Table 1, and the primers required are listed in Table 2. pTargetF-01 was obtained by reverse PCR amplification using plasmid pTargetF as a template and F-ycb-f / F-ycb-r as upstream and downstream primers. Finally, Y-pTargetF-f / Y-pTargetF-r was used as sequencing primers to verify its sequence accuracy. pTargetF-02, pTargetF-03, pTargetF-04, pTargetF-05, pTargetF-06, pTargetF-07, pTargetF-08, pTargetF-09, pTargetF-10, pTargetF-11, and pTargetF-12 were constructed using the same method (Qiao, J., Tan, X., Ren, H., Wu, Z., Hu, X., Wang, X. 2021. Construction of an Escherichia coli Strain Lacking Fimbriae by Deleting 64 Genes and Its Application for Efficient Production of Poly(3-Hydroxybutyrate) and l-Threonine. Appl Environ Microbiol, 87(12), e0038121.), and the corresponding primers were used.

[0059] (3) Construction of mutant strains using CRISPR-Cas9:

[0060] The CRISPR-Cas9 system was used to knock out fimbriae synthesis genes. The constructed strains, plasmids, and corresponding primers are listed in Tables 1 and 2. Strains TWK001, TWK002, TWK003, TWK004, TWK005, TWK006, TWK007, TWK008, TWK009, TWK010, TWK011, and TWK012 are fimbriae synthesis gene clusters that were knocked out from the TWF001 genome, namely Ycb, Yad, Yde, Yeh, Yqi, Yra, Yfc, Type 1, Yhc, Sfm, Mat, and Ybg, respectively.

[0061] For example, TWK001 was obtained by knocking out the Ycb fimbriae gene cluster in the TWF001 genome. First, using primers ycbf1 / ycbf2 and ycbr1 / ycbr2 as a template, the upstream and downstream homologous arm sequences of the Ycb gene cluster were amplified, respectively. After purification using a gel extraction kit, overlap PCR was performed using ycbf1 / ycbr2, and the overlapping homologous arm sequences of the Ycb gene cluster were obtained after purification using a gel extraction kit. The overlapping homologous arm sequences (300 ng) and plasmid pTargetF-01 (200 ng) were introduced into electroporated competent TWF001 / pCas cells. The electroporated cells were incubated at 30°C and 200 rpm for 1 hour. Then, the electroporated cells were plated on LB agar plates containing kanamycin and spectinomycin and cultured for 48 hours. Colonies were picked and colony PCR was performed using primers ycbf1 / ycbr2 to select the correct mutant strains. Finally, the mutant strains were inoculated into a medium containing kanamycin and IPTG and cultured for 12 hours to remove the pTargetF-01 plasmid. The mutant strains without the pTargetF-01 plasmid were then cultured overnight at 42°C and 200 rpm to remove the pCas plasmid. TWK002, TWK003, TWK004, TWK005, TWK006, TWK007, TWK008, TWK009, TWK010, TWK011, and TWK012 were all constructed using the same method with the corresponding primers and plasmids. TWK013, TWK014, TWK015, TWK016, TWK017, TWK018, TWK019, TWK020, TWK021, TWK022, and TWK023 are multi-knockout strains constructed by knocking out the remaining 11 gene clusters one by one based on TWK001.

[0062] Table 1. Strains and plasmids

[0063]

[0064]

[0065] Table 2 Primer sequences

[0066]

[0067]

[0068]

[0069] Example 2: Knocking out a single fimbriae gene cluster

[0070] The single knockout mutant strains TWK001, TWK002, TWK003, TWK004, TWK005, TWK006, TWK007, TWK008, TWK009, TWK010, TWK011, and TWK012, constructed in Example 1, were activated on SOB plates at 37°C for 24 hours. Single colonies were picked and inoculated into 5 mL LB tubes and incubated at 37°C for 12 hours at 200 rpm. The initial OD was used as the starting point for the culture. 600 =0.1 was inoculated into 30 mL of STF medium and incubated at 200 rpm and 37 °C for 4 hours. Finally, the initial OD was used as the criterion. 600 =0.2 mg / L was inoculated into 30 mL of L-threonine fermentation medium and incubated at 37 °C for 36 hours at 200 rpm. OD was measured every 8 hours. 600 Values ​​and amino acid concentrations ( Figure 2 ).

[0071] The deletion of the fimbrial synthesis gene cluster alone does not improve bacterial growth. Figure 2 B), but it has a beneficial effect on the production of L-threonine. Figure 2 C) Compared to the wild-type strain TWF001, strains TWK001, TWK002, TWK003, TWK004, TWK005, TWK006, and TWK008 reached their peak L-threonine production earlier. TWK012, however, reached its peak L-threonine production of 12.4 g / L at 36 hours. Therefore, this result indicates that knocking out a single fimbriae gene cluster is beneficial for L-threonine production.

[0072] Example 3: Knocking out multiple fimbrial gene clusters

[0073] The results of Example 2 show that knocking out a single fimbrial gene cluster is beneficial for L-threonine production. Therefore, 11 additional multi-knockout mutant strains of fimbrial gene clusters, TWK013, TWK014, TWK015, TWK016, TWK017, TWK018, TWK019, TWK020, TWK021, TWK022, and TWK023, were constructed. The construction method is detailed in Example 1. The mutant strains TWK013, TWK014, TWK015, TWK016, TWK017, TWK018, TWK019, TWK020, TWK021, TWK022, and TWK023 were activated on SOB plates at 37°C for 24 hours. Single colonies were picked and inoculated into 5 mL LB tubes and cultured at 37°C for 12 hours at 200 rpm. The initial OD was used as the starting point for the culture. 600 =0.1 was inoculated into 30 mL of STF medium and incubated at 200 rpm and 37 °C for 4 hours. Finally, the initial OD was used as the criterion. 600=0.2 mg / L was inoculated into 30 mL of L-threonine fermentation medium and incubated at 37 °C for 36 hours at 200 rpm. OD was measured every 8 hours. 600 Values ​​and amino acid concentrations ( Figure 3 ).

[0074] Among them, mutant strains TWK013, TWK014, TWK015, TWK016, and TWK017 grew relatively quickly with high cell density and relatively high L-threonine production in the early stage of growth. However, from 24 hours onwards, the L-threonine production of mutant strains TWK018, TWK019, TWK020, TWK021, TWK022, and TWK023 increased significantly, and at 36 hours, the L-threonine production showed a gradient increase. The results showed that after 36 hours of fermentation, the L-threonine yields of the mutant strains TWK013, TWK014, TWK015, TWK016, TWK017, TWK018, TWK019, TWK020, TWK021, TWK022, and TWK023 were 11.96 g / L, 10.58 g / L, 12.8 g / L, 13.18 g / L, 10.59 g / L, 9.95 g / L, 10.26 g / L, 13.63 g / L, 15.1 g / L, 15.75 g / L, 14.76 g / L, and 11.01 g / L, respectively. This indicates that the yield of the mutant strain TWK021 was increased by 32.4% compared to the wild-type TWF001 (11.9 g / L).

[0075] Example 4: Preparation of L-threonine by fed-batch fermentation

[0076] Recombinant Escherichia coli TWK021 was inoculated into 5 mL of SOB medium and cultured overnight to determine the initial OD value. 600 0.1 mg was inoculated into a 250 mL shake flask containing 30 mL of STF medium and incubated at 37 °C and 200 rpm for 4 h. Then, the initial OD was used as the starting point. 600 0.2 μL of the culture was inoculated into a 500 mL shake flask containing 100 mL of fermentation medium and incubated at 37 °C and 200 rpm for 4 h. The entire culture was then transferred to a 900 mL fed-batch fermentation medium. During fermentation, the aeration rate was maintained at 1.5 vvm, and the dissolved oxygen coupling speed (maximum 900 rpm) was kept to maintain the dissolved oxygen (DO) value at 25%-30%. The culture was then fed with 800 g·L⁻¹ feedstock. -1 Glucose was used to maintain the glucose concentration at 15 g / L during the fermentation process. -1 -20g·L -1 The first feeding should be done when the glucose concentration in the culture medium is below 5 g / L. -1The fermentation process was carried out in stages. During fermentation, concentrated ammonia was added continuously to maintain the pH at 7.0. If the pH rose rapidly in the middle of fermentation, feeding was used to prevent the pH from rising due to glucose depletion. Samples were taken every 4 hours in the early stage of fermentation, every 2-3 hours in the middle stage, and every 4 hours in the later stage. Each sample was 1 mL in volume, and the fermentation lasted for a total of 48 hours.

[0077] The results showed that TWK021 consumed a total of 30 g·L in the first 8 hours of fermentation. -1 Glucose was added, followed by supplemental feeding. After 48 hours of fermentation, TWK021 produced 62.7 g / L of L-threonine. -1 A total of 147 g / L of glucose was consumed. -1 The sugar-to-acid conversion rate is 0.427 g / g. Therefore, TWK021 exhibits excellent L-threonine production performance. Figure 4 ).

[0078] 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. A method for increasing L-threonine production, characterized in that, The method involves knocking out the fimbriae synthesis gene cluster on the genome of Escherichia coli TWF001, wherein the fimbriae synthesis gene cluster is Ycb, Yad, Yde, Yeh, Yqi, Yra, Yfc, Type 1, Yhc and Sfm; The Ycb is ycbQ , ycbR , ycbS , ycbT , ycbU , ycbV , ycbF ; The Yad is yadC , yadK , yadL , yadM , htrE , ecpD , yadN ; The Yde is ydeQ , ydeR , ydeS , ydeT ; The Yeh is yehA , yehB , yehC , yehD ; The Yqi is ygiL , yqiG , yqiH , yqiI ; The Yra is yraH , yraI , yraJ , yraK ; The Yfc is yfcO , yfcP , yfcQ , yfcR , yfcS , yfcT , yfcU , yfcV ; The Type 1 is fimA , fimI , fimC , fimD , fimF , fimG , fimH ; The Yhc is yhcA , yhcD , yhcE , yhcF , gltF ; The Sfm is sfmA , sfmC , sfmD , sfmH , sfmF .

2. The recombinant Escherichia coli obtained by the method described in claim 1.

3. The application of the recombinant *Escherichia coli* according to claim 2 in the preparation of L-threonine, characterized in that, The application is to inoculate the recombinant Escherichia coli of claim 2 into a fermentation system to prepare L-threonine.

4. The application according to claim 3, characterized in that, The seed culture of the recombinant Escherichia coli described in claim 2 was inoculated into a fermentation medium, and cultured for 30-40 h at an initial OD600 of 0.1-0.5, at 35-38°C and 150-250 rpm.

5. A method for preparing L-threonine, characterized in that, The method involves inoculating the seed culture of the recombinant Escherichia coli described in claim 2 into a fed-batch fermentation medium, setting the aeration rate to 1.5 vvm, and the dissolved oxygen coupling speed to maintain the DO value at 25%-30%.

6. The method according to claim 5, characterized in that, When the glucose concentration in the culture medium is below 5 g·L -1 At that time, glucose was added continuously to maintain the concentration at 15 g·L⁻¹. -1 -20 g·L -1 .

7. The method according to claim 5, characterized in that, The fed-batch fermentation medium contains 1~5 g·L -1 Yeast extract, 1~10 g·L -1 (NH4)2SO4, 1~5 g·L -1 KH2PO4, 1~5 g·L -1 MgSO4·7H2O, 1~10 mg·L -1 FeSO4·7 H2O, 1~10 mg·L -1 MnSO4·4H2O, 20~60 g·L -1 glucose.

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