Construction and application of escherichia coli with improved l-threonine production

Abstract

The application discloses construction and application of Escherichia coli with improved L-threonine yield, and belongs to the field of genetic engineering and fermentation engineering. In the Escherichia coli TWF001, the genes nlpE, csrB and / or qseBC related to bacterial motility and biofilm formation are separately or combinedly knocked out, and the recombinant Escherichia coli with improved L-threonine yield is obtained, wherein the L-threonine yield is increased by 55.7% at most compared with that of the original strain. The research provides a new strategy for improving the L-threonine yield in the Escherichia coli.

Description

Technical Field

[0001] This invention relates to the construction and application of an Escherichia coli strain with increased L-threonine production, belonging to the fields of genetic engineering and fermentation engineering. Background Technology

[0002] L-Threonine, also known as α-amino-β-hydroxybutyric acid, has the molecular formula C4H9NO3 and a relative molecular mass of 119.12. It is a white crystalline powder with no irritating odor, readily soluble in water, and practically insoluble in organic solvents. Solutions formed are acidic, with a pH of 5.0-6.5. It is optically active. Threonine exists as four isomers, but only L-threonine is naturally occurring and physiologically active in humans. As an essential amino acid, L-threonine cannot be synthesized in the human body and must be obtained from external sources.

[0003] Biofilms are bacterial cell communities encapsulated by self-produced extracellular polymers (EPS) composed of extracellular polysaccharides and proteins. Biofilm formation is a natural growth mechanism of *E. coli*, and pathogenic *E. coli* strains utilize biofilms to protect themselves from host immune responses and antimicrobial agents. *E. coli* biofilm formation is a multi-step process. The first stage involves chemotaxis, where bacteria migrate and aggregate to find favorable fixation surfaces. Upon reaching these surfaces, *E. coli* cells lose their flagella and form aggregates, subsequently producing EPS. Therefore, inhibition of flagellated motility is a crucial step in bacterial aggregation and biofilm formation. TWF001, as an L-threonine-producing bacterium, simplifies unnecessary extracellular structures, reducing pathogenicity and is particularly important for promoting product synthesis and improving production safety. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention utilizes the individual and combined knockout of genes nlpE, csrB, and / or qseBC related to cell motility and biofilm formation in Escherichia coli TWF001. The resulting recombinant Escherichia coli exhibits a higher L-threonine yield compared to TWF001 after 36 hours of fermentation.

[0005] The first objective of this invention is to provide a recombinant Escherichia coli, using an L-threonine-producing strain as a host, by knocking out one or more of the regulatory genes nlpE, qseBC, and csrB on the genome that affect bacterial motility and biofilm formation.

[0006] In one implementation, the qseBC includes qseC and qseB.

[0007] In one implementation, the NCBI accession numbers of the nlpE, qseB, and qseC sequences are AAA86093.1, AAA69193.1, and AAA69194.1, respectively.

[0008] In one embodiment, the nucleotide sequence of the csrB is shown in SEQ ID NO.1.

[0009] In one embodiment, the L-threonine producing strain is Escherichia coli TWF001.

[0010] The second objective of this invention is to provide a method for increasing the production of L-threonine, wherein the method involves knocking out regulatory genes on the Escherichia coli genome that affect cell motility and biofilm formation.

[0011] In one embodiment, the regulatory genes affecting bacterial motility and biofilm formation include one or more of nlpE, qseBC, and csrB.

[0012] In one implementation, the qseBC includes qseC and qseB.

[0013] In one implementation, the NCBI accession numbers of the nlpE, qseB, and qseC sequences are AAA86093.1, AAA69193.1, and AAA69194.1, respectively.

[0014] In one embodiment, the nucleotide sequence of the csrB is shown in SEQ ID NO.1.

[0015] In one embodiment, the Escherichia coli is Escherichia coli TWF001.

[0016] A third objective of this invention is to provide a microbial agent for the fermentation preparation of L-threonine, wherein the microbial agent contains the recombinant Escherichia coli.

[0017] In one embodiment, the bacterial agent also contains a freeze-drying protectant.

[0018] In one embodiment, the freeze-drying protectant includes one or more of skim milk powder, trehalose, sucrose, glycine, mannitol, or lactose.

[0019] The fourth objective of this invention is to provide a method for synthesizing L-threonine, wherein the method involves fermenting L-threonine using the recombinant Escherichia coli or the bacterial agent.

[0020] In one embodiment, the method involves inoculating the seed culture of the recombinant Escherichia coli into a reaction system with glucose as the carbon source for fermentation to prepare L-threonine.

[0021] In one embodiment, the method involves activating and culturing the recombinant Escherichia coli on a plate for 24 hours, picking a single colony and inoculating it into a 5 mL test tube, and culturing it at 37°C and 200 rpm for 12 hours to obtain a primary seed culture. The initial OD... 600 The culture medium was inoculated at 0.1% with STF medium and cultured at 37°C and 200 rpm for 4 hours to obtain a secondary seed culture. Finally, the culture medium was inoculated at 0.2% with OD600 and cultured at 37°C and 200 rpm for 36 hours.

[0022] In one embodiment, the fermentation medium comprises: 1–5 g / L yeast extract, 1–5 g / L citric acid, 20–30 g / L NH4SO4, 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.

[0023] 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 NH4SO4, and 0.5–1.5 g / L MgSO4.

[0024] The present invention also provides the application of the recombinant Escherichia coli or the above-mentioned bacterial agent or the above-mentioned method in the preparation of L-threonine.

[0025] Beneficial effects

[0026] This invention screened E. coli TWF001 by individually or in combination knocking out genes nlpE, csrB, and / or qseBC, which are associated with cell motility and biofilm formation, and found that the TWF001-NQC L-threonine yield reached 17.9 g·L⁻¹ after 36 h of shake-flask fermentation. -1 The conversion rate reached 0.445 g·g -1 The production of glucose and L-threonine increased by 55.7% compared to TWF001. The L-threonine production of the remaining recombinant E. coli strains TWF001-N, TWF001-Q, TWF001-C, TWF001-NQ, TWF001-NC, and TWF001-QC reached 12.98 g·L⁻¹. -1 14.1 g·L -1 12.17 g·L -1 15.03 g·L -1 14.55 g·L -1 15.65 g·L -1The yields of L-threonine in Escherichia coli were all higher than those in the control strain TWF001. Therefore, this invention provides a novel strategy for increasing L-threonine production in Escherichia coli.

[0027] biomaterials

[0028] 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

[0029] Figure 1 Sequence comparison of TWF001, TWF001-N, TWF001-Q, TWF001-C, TWF001-NQ, TWF001-NC, TWF001-QC and TWF001-NQC.

[0030] Figure 2 : TWF001, TWF001-N, TWF001-Q, TWF001-C, TWF001-NQ, TWF001-NC, TWF001-QC and TWF001-NQC at 40g·L -1 Growth under glucose concentration, glucose consumption and L-threonine production. Detailed Implementation

[0031] (1) Culture medium:

[0032] LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl.

[0033] STF medium: 10 g / L sucrose, 20 g / L peptone, 5 g / L yeast extract, 15 g / L NH4SO4 and 1 g / L MgSO4, pH 7.3.

[0034] L-Threonine fermentation medium: 2 g / L yeast extract, 2 g / L citric acid, 25 g / L NH4SO4, 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.

[0035] (2) Determination of bacterial cell concentration

[0036] The absorbance at 600 nm was measured using a UV-1800 UV-Vis spectrophotometer.

[0037] (3) Determination of glucose content

[0038] 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 25 μL of the supernatant was taken and diluted 100 times before measurement.

[0039] (3) L-threonine concentration analysis:

[0040] The concentration of L-threonine 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.

[0041] Example 1: Construction of Escherichia coli mutant strains TWF001-N, TWF001-C, and TWF001-Q

[0042] The specific construction process is as follows:

[0043] (1) Preparation of E. coli electrotransfer competent cells containing the knockout plasmid pCas:

[0044] 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.

[0045] (2) Construction of pTargetF plasmid:

[0046] The plasmids used in this section are listed in Table 1, and the required primers are listed in Table 2. pTargetF-csrB was obtained by reverse PCR amplification using plasmid pTargetF as a template and F-csrB-f / F-csrB-r as upstream and downstream primers. Finally, Y-pTargetF-f / Y-pTargetF-r was used as sequencing primers to verify its sequence accuracy. pTargetF-qseBC was obtained by reverse PCR amplification using plasmid pTargetF as a template and F-qseBC-f / F-qseBC-r as upstream and downstream primers. Finally, Y-pTargetF-f / Y-pTargetF-r was used as sequencing primers to verify its sequence accuracy. pTargetF-nlpE was obtained by reverse PCR amplification using plasmid pTargetF as a template and F-nlpE-f / F-nlpE-r as upstream and downstream primers. Finally, Y-pTargetF-f / Y-pTargetF-r was used as sequencing primers to verify its sequence accuracy.

[0047] (3) Construction of mutant strains using CRISPR-Cas9:

[0048] The CRISPR-Cas9 system was used to knock out fimbriae synthesis genes. The strains, plasmids, and corresponding primers constructed in this section are listed in Tables 1 and 2. Strains TWF001-N, TWF001-C, and TWF001-Q knocked out nlpE, csrB, and qseBC from the TWF001 genome, respectively. For example, TWF001-N knocked out nlpE from the TWF001 genome. First, using the TWF001 genome as a template, primers nlpEf1 / nlpEf2 and nlpEr1 / nlpEr2 were used to amplify the upstream and downstream homologous arm sequences of the nlpE gene. After purification using a gel extraction kit, the upstream and downstream homologous arm sequences of the nlpE gene were overlapped by PCR using primers nlpEf1 / nlpEr2. After purification using a gel extraction kit, the overlapping homologous arm sequences of the nlpE gene were obtained. The overlapping homologous arm sequence (300 ng) and plasmid pTargetF-nlpE (200 ng) were introduced into electroporated competent TWF001 / pCas cells. The electroporated cells were then incubated at 30°C and 200 rpm for 1 hour. The electroporated cells were then plated on LB agar plates containing kanamycin and spectinomycin and cultured for 48 hours. Colonies were picked and colony PCR was performed using primers nlpEf1 / nlpEr2 to select the correct mutant strains. Finally, the mutant strains were inoculated into medium containing kanamycin and IPTG and cultured for 12 hours to remove the pTargetF-nlpE plasmid. The mutant strains without the pTargetF-nlpE plasmid were then cultured overnight at 42°C and 200 rpm to remove the pCas plasmid.

[0049] Both TWF001-C and TWF001-Q were constructed using the same method with corresponding primers and plasmids. Figure 1 ).

[0050] Table 1. Strains and plasmids

[0051]

[0052]

[0053] Table 2 Primer sequences

[0054]

[0055] Example 2: Construction of Escherichia coli mutant strains TWF001-NC, TWF001-NQ, and TWF001-QC. The specific construction process is as follows:

[0056] The CRISPR-Cas9 system was used to knock out the fimbriae synthesis gene. The strains, plasmids, and corresponding primers constructed in this section are listed in Tables 1 and 2. Strains TWF001-NC and TWF001-NQ were constructed in Example 1 by knocking out csrB and qseBC, respectively. For example, TWF001-NC knocked out csrB in the TWF001-N genome. First, primers csrBf1 / csrBf2 and csrBr1 / csrBr2 were used to amplify the upstream and downstream homologous arm sequences of the csrB gene using the TWF001 genome as a template. After purification using a gel extraction kit, the upstream and downstream homologous arm sequences of the csrB gene were then overlapped by PCR using primers csrBf1 / csrBr2. After purification using a gel extraction kit, the overlapping homologous arm sequences of the csrB gene were obtained. The overlapping homologous arm sequence (300 ng) and plasmid pTargetF-csrB (200 ng) were introduced into electroporated competent cells TWF001-N / pCas. The electroporated cells were then incubated at 30°C and 200 rpm for 1 hour. The electroporated cells were then plated on LB agar plates containing kanamycin and spectinomycin and cultured for 48 hours. Colonies were picked and colony PCR was performed using primers csrBf1 / csrBr2 to select the correct mutant strains. Finally, the mutant strains were inoculated into medium containing kanamycin and IPTG and cultured for 12 hours to remove the pTargetF-csrB plasmid. The mutant strains without the pTargetF-csrB plasmid were then cultured overnight at 42°C and 200 rpm to remove the pCas plasmid. Figure 1 Similarly, TWF001-NQ was constructed using the same method with the corresponding primers and plasmids.

[0057] The TWF001-QC strain was constructed by knocking out csrB from the TWF001-Q strain constructed in Example 1. First, using primers csrBf1 / csrBf2 and csrBr1 / csrBr2 as a template, the upstream and downstream homologous arm sequences of the csrB gene were amplified, respectively. After purification using a gel extraction kit, the upstream and downstream homologous arm sequences of the csrB gene were then subjected to overlap PCR using primers csrBf1 / csrBr2. The resulting overlapping homologous arm sequences of the csrB gene were purified using a gel extraction kit. The overlapping homologous arm sequences (300 ng) and plasmid pTargetF-csrB (200 ng) were introduced into electroporated competent cells TWF001-Q / pCas. The electroporated cells were then incubated at 30°C and 200 rpm for 1 hour. The electroporated cells were then plated on LB agar plates containing kanamycin and spectinomycin and cultured for 48 hours. Colonies were picked and colony PCR was performed using primers csrBf1 / csrBr2 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-csrB plasmid. The mutant strains without the pTargetF-csrB plasmid were then cultured overnight at 42°C and 200 rpm to remove the pCas plasmid. Figure 1 ).

[0058] Example 3: Construction of Escherichia coli mutant strain TWF001-NQC

[0059] The specific construction process is as follows:

[0060] The TWF001-NQC strain was constructed by knocking out csrB from the TWF001-NQ strain constructed in Example 2. First, using primers csrBf1 / csrBf2 and csrBr1 / csrBr2 as a template, the upstream and downstream homologous arm sequences of the csrB gene were amplified, respectively. After purification using a gel extraction kit, the upstream and downstream homologous arm sequences of the csrB gene were then subjected to overlap PCR using primers csrBf1 / csrBr2. The resulting overlapping homologous arm sequences of the csrB gene were purified using a gel extraction kit. The overlapping homologous arm sequences (300 ng) and plasmid pTargetF-csrB (200 ng) were introduced into electroporated competent cells TWF001-NQ / pCas. The electroporated cells were then incubated at 30°C and 200 rpm for 1 hour. The electroporated cells were then plated on LB agar plates containing kanamycin and spectinomycin and incubated for 48 hours. Colonies were picked and colony PCR was performed using primers csrBf1 / csrBr2 to select the correct mutant strains. Finally, the mutant strains were inoculated into medium containing kanamycin and IPTG and incubated for 12 hours to remove the pTargetF-csrB plasmid. The mutant strains without the pTargetF-csrB plasmid were then incubated overnight at 42°C and 200 rpm to remove the pCas plasmid. Figure 1 ).

[0061] Example 4: Application of Escherichia coli mutant strain TWF001-NQC in the synthesis of L-threonine

[0062] Escherichia coli TWF001 and its mutant strains TWF001-N, TWF001-Q, TWF001-C, TWF001-NQ, TWF001-NC, TWF001-QC, and TWF001-NQC were cultured on LB agar plates for 24 hours. Single colonies were picked and inoculated into 5 mL of LB medium 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 37°C for 4 hours at 200 rpm. 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, glucose concentration, and L-threonine concentration ( Figure 2 ).

[0063] The bacteria were inhibited by a combination of qseBC, nlpE, and csrB, and were present in a solution containing 40 g·L⁻¹. -1Fermentation was carried out in L-threonine fermentation medium with an initial glucose concentration for 36 h. It was found that the cell growth of mutant strains TWF001-N, TWF001-Q, TWF001-C, TWF001-NQ, TWF001-NC, TWF001-QC, and TWF001-NQC all showed varying degrees of decline. Figure 2 After 8 hours of fermentation, the growth rates of *E. coli* strains TWF001-N, TWF001-Q, and TWF001-NQC were significantly slower than other strains, with OD... 600 The values ​​were 9.9, 10.7, and 7.1, respectively, and glucose consumption was slow, with less than 50% being consumed. Figure 2 After 16 hours of fermentation, the OD values ​​of each mutant strain... 600 The gap between glucose levels and glucose consumption has narrowed. Figure 2 The TWF001-NQC strain had the lowest L-threonine production during the first 8 hours of fermentation due to its low cell biomass. However, the L-threonine production gradually increased as fermentation progressed. After 36 hours of fermentation, the L-threonine production of each mutant strain showed a gradient increase, with the TWF001-NQC strain reaching a production of 17.9 g·L⁻¹. -1 The conversion rate reached 0.445 g·g -1 The glucose yield was 11.5 g·L⁻¹, compared to the control strain TWF001. -1 The conversion rate was 0.288 g·g. -1 Glucose, TWF001-NQC L-threonine production increased by 55.7% ( Figure 2 Therefore, knocking out the genes csrB, qseBC, and nlpE, which are related to the regulation of motility and biofilm formation in *E. coli* TWF001, significantly increased the L-threonine production in *E. coli* TWF001. After 36 h of fermentation, the L-threonine production of strains TWF001-N, TWF001-Q, TWF001-C, TWF001-NQ, TWF001-NC, and TWF001-QC reached 12.98 g·L⁻¹. -1 14.1 g·L -1 12.17 g·L -1 15.03 g·L -1 14.55 g·L -1 15.65 g·L -1 All were higher than the control strain TWF001.

[0064] 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 recombinant Escherichia coli for producing L-threonine, characterized in that, Using Escherichia coli TWF001 as the host, regulatory genes affecting bacterial motility and biofilm formation were knocked out. nlpE , qseBC and csrB The qseBC include qseC and qseB .

2. The recombinant Escherichia coli according to claim 1, characterized in that, The nlpE , qseB , qseC The NCBI accession numbers for the sequences are AAA86093.1, AAA69193.1, and AAA69194.1, respectively; csrB The nucleotide sequence is shown in SEQ ID NO.

1.

3. A method for increasing the yield of L-threonine, characterized in that, The method involves knocking out regulatory genes affecting bacterial motility and biofilm formation in the genome of *E. coli* TWF001; these regulatory genes include... nlpE , qseBC and csrB ; The qseBC include qseC and qseB .

4. The method according to claim 3, characterized in that, The nlpE , qseB , qseC The NCBI accession numbers for the sequences are AAA86093.1, AAA69193.1, and AAA69194.1, respectively; csrB The nucleotide sequence is shown in SEQ ID NO.

1.

5. A microbial agent for the fermentation preparation of L-threonine, characterized in that, Contains the recombinant Escherichia coli as described in any one of claims 1 to 2.

6. A method for synthesizing L-threonine, characterized in that, The method involves fermenting L-threonine using the recombinant Escherichia coli according to any one of claims 1-2 or the bacterial agent according to claim 5.

7. The method according to claim 6, characterized in that, The method involves inoculating the seed culture of the recombinant Escherichia coli according to any one of claims 1 to 2 into a reaction system with glucose as the carbon source for fermentation to prepare L-threonine.

8. The method according to claim 7, characterized in that, The reaction system comprises: 1~5 g / L yeast powder, 1~5 g / L citric acid, 20~30 g / L NH4SO4, 5~10 g / L KH2PO4, 1~5 g / L MgSO4·7 H2O, 2~7 mg / L FeSO4·7 H2O, 2~7 mg / L MnSO4·4 H2O, 20~60 g / L glucose and 10~30 g / L CaCO3.

9. The use of the recombinant Escherichia coli according to any one of claims 1 to 2, the bacterial agent according to claim 5, or the method according to any one of claims 6 to 8 in the preparation of L-threonine.

Images