Method for enhancing Escherichia coli electronic respiration transfer and energy transfer efficiency and improving L-threonine yield

Abstract

The invention discloses a method for improving the yield of L-threonine by enhancing Escherichia coli electronic respiration transfer and energy transfer efficiency, and belongs to the technical field of genetic engineering. According to the invention, a cytochrome bo oxidase subunit CyoA mutant derived from escherichia coli, a cytochrome bo oxidase subunit CyoB mutant derived from pseudomonas aeruginosa, a cytochrome bd oxidase cydAB gene cluster derived from escherichia coli and a polyphosphate kinase PPK mutant derived from radiation-resistant coccus are introduced, amn genes are knocked out, and a cytochrome bo oxidase gene is obtained. The electron transfer efficiency in escherichia coli and the production efficiency of energy ATP (adenosine triphosphate) are enhanced, and the production efficiency of L-threonine and the carbon yield are further improved. Finally, the obtained engineering strain ECTHRS-5 is subjected to fed-batch fermentation in a 5L fermentation tank to accumulate 160.8 g / L of L-threonine, and the sugar-acid conversion rate is 0.62 g / g.

Description

Technical Field

[0001] The invention relates to a method for enhancing the electron respiration transfer and energy transfer efficiency of Escherichia coli and improving the L-threonine yield, belonging to the technical field of genetic engineering. Background Art

[0002] L-Threonine, an essential amino acid, has important applications in the feed, pharmaceutical, and food industries. Its global annual production exceeds 100,000 tons, of which approximately 70% is produced through Escherichia coli fermentation. Although E. coli's metabolic pathways are well-defined and easily genetically modified, industrial production still faces multiple bottlenecks, particularly metabolic flux allocation issues caused by inefficient electron transport chain (ETC) and imbalanced energy supply. Enhancing the expression of genes in the Cyo gene cluster, a crucial component of the oxidative electron transport chain, is expected to improve the efficiency of the electron respiratory chain. The CydAB gene cluster encodes genes for cytochrome bd oxidase, which efficiently catalyzes the reduction of oxygen at low oxygen concentrations while simultaneously pumping protons to maintain the transmembrane proton gradient. Furthermore, increasing the production of ATP, the intracellular energy currency, is crucial for the physiological activities and industrial applications of microbial cells. Summary of the Invention

[0003] The present invention aims to provide a method for enhancing electron respiratory transfer and energy transfer efficiency in Escherichia coli to increase L-threonine production. This method utilizes genetic engineering techniques to simultaneously enhance electron respiratory chain transfer and promote ATP production. The resulting engineered strain ECTHRS-5 accumulated 160.8 g / L of L-threonine in a 5L fermentor using fed-batch fermentation, with a sugar-to-acid conversion rate of 0.62 g / g.

[0004] The complete technical means and methods of the present invention:

[0005] By overexpressing the E. coli-derived cytochrome bo oxidase subunit CyoA mutant, the Pseudomonas aeruginosa-derived cytochrome bo oxidase subunit CyoB mutant, the E. coli-derived cytochrome bd oxidase cydAB gene cluster, and the Radiodurans radiodurans polyphosphate kinase PPK mutant in E. coli, the amn gene was knocked out to enhance the electron transfer efficiency in E. coli and increase the intracellular energy supply.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant nucleic acid of Escherichia coli, wherein the recombinant nucleic acid comprises a gene encoding the following protein:

[0008] Cytochrome bo oxidase subunit CyoA mutant from Escherichia coli, cytochrome bo oxidase subunit CyoB mutant from Pseudomonas aeruginosa, cytochrome bd oxidase cydAB gene cluster from Escherichia coli, polyphosphate kinase PPK mutant from Radiodurans deinococcus.

[0009] In one embodiment, the cytochrome bo oxidase subunit CyoA mutant derived from Escherichia coli, the cytochrome bo oxidase subunit CyoB mutant derived from Pseudomonas aeruginosa, and the cytochrome bd oxidase cydAB gene cluster derived from Escherichia coli are all expressed by the Trc promoter.

[0010] In one embodiment, the gene encoding the cytochrome bo oxidase subunit CyoA mutant is derived from Escherichia coli; the amino acid at position 161 of the cytochrome bo oxidase subunit CyoA mutant is mutated from valine to leucine; and the cytochrome bo oxidase subunit CyoA mutant is integrated into the E. coli pseudogene yeep site. The nucleotide sequence encoding the CyoA protein mutant is shown in SEQ ID NO. 1; the original nucleotide sequence encoding the CyoA protein is shown in SEQ ID NO. 2.

[0011] In one embodiment, the gene encoding the cytochrome bo oxidase subunit CyoB mutant is derived from Pseudomonas aeruginosa; the amino acid at position 126 of the cytochrome bo oxidase subunit CyoB mutant is mutated from alanine to aspartic acid; and the cytochrome bo oxidase subunit CyoB mutant is integrated into the yncI site of the Escherichia coli pseudogene. The nucleotide sequence encoding the CyoB protein mutant is shown in SEQ ID NO. 3; the original nucleotide sequence encoding the CyoB protein is shown in SEQ ID NO. 4.

[0012] In one embodiment, the cytochrome bd oxidase cydAB gene cluster is derived from Escherichia coli, and the nucleotide sequence encoding the cydAB gene cluster is shown in SEQ ID NO. 5. The cytochrome bd oxidase cydAB gene cluster is integrated into the Escherichia coli pseudogene mbhA site.

[0013] In one embodiment, the gene encoding the polyphosphate kinase (PPK) mutant is derived from Deinococcus radiodurans; the amino acid at position 131 of the polyphosphate kinase (PPK) mutant is mutated from threonine to alanine; and the polyphosphate kinase (PPK) mutant is integrated into the yjiP pseudogene site of Escherichia coli. The nucleotide sequence encoding the PPK protein mutant is shown in SEQ ID NO. 6, and the original nucleotide sequence encoding the PPK protein is shown in SEQ ID NO. 7.

[0014] Preferably, the Escherichia coli described in the present invention is the L-threonine producing strain LMT4 (patent number: CN115011620 B2023-11-07).

[0015] The present invention also provides a cytochrome bo oxidase subunit CyoA mutant, which is obtained by mutating the amino acid at position 161 of the cytochrome bo oxidase subunit CyoA shown in SEQ ID NO.9 from valine to leucine.

[0016] The present invention also provides a cytochrome bo oxidase subunit CyoB mutant, which is obtained by mutating the amino acid at position 126 of the cytochrome bo oxidase subunit CyoB shown in SEQ ID NO.10 from alanine to aspartic acid.

[0017] The present invention also provides a polyphosphate kinase PPK mutant, which is obtained by mutating the amino acid at position 131 of the carbonic anhydrase with an amino acid sequence as shown in SEQ ID NO. 11 from threonine to alanine.

[0018] The present invention also provides a gene encoding the mutant, a recombinant vector carrying the gene, or a recombinant strain expressing the mutant.

[0019] The present invention also provides a recombinant Escherichia coli that overexpresses the aforementioned cytochrome bo oxidase subunit CyoA mutant, the aforementioned cytochrome bo oxidase subunit CyoB mutant, and the aforementioned polyphosphate kinase PPK mutant. The recombinant Escherichia coli is based on the L-threonine-producing strain LMT4, described in Chinese invention patent publication number CN 115011620 B.

[0020] In one embodiment, the recombinant E. coli also overexpresses the cytochrome bd oxidase cydAB gene cluster from E. coli, and simultaneously knocks out the amn gene on the genome;

[0021] In one embodiment, the cytochrome bo oxidase subunit CyoA mutant, the cytochrome bo oxidase subunit CyoB mutant, the polyphosphate kinase PPK mutant, and the cytochrome bd oxidase cydAB gene cluster are expressed by a Trc promoter;

[0022] In one embodiment, the nucleotide sequence encoding the Trc promoter is shown in SEQ ID NO.8;

[0023] In one embodiment, yeep on the E. coli genome is knocked out and the cytochrome bo oxidase subunit CyoA mutant is integrated at the yeep site; the pseudogene yncI on the E. coli genome is knocked out and the cytochrome bo oxidase subunit CyoB mutant is integrated at the yncI site; the pseudogene mbhA on the E. coli genome is knocked out and the cytochrome bd oxidase cydAB gene cluster is integrated at the mbhA site; the pseudogene yjiP on the E. coli genome is knocked out and the polyphosphate kinase PPK mutant is integrated at the yjiP site;

[0024] In one embodiment, the yeep gene ID on NCBI is 946524; the yncI gene ID on NCBI is 945121; the mbhA gene ID on NCBI is 944921; the yjiP gene ID on NCBI is 38094982; the amn gene gene ID on NCBI is 946508;

[0025] In one embodiment, the nucleotide sequence encoding the cydAB gene cluster is shown as SEQ ID NO.5.

[0026] The present invention also provides a method for enhancing the efficiency of electron respiration transfer and energy transfer in Escherichia coli. The method comprises overexpressing the above-mentioned cytochrome b oxidase subunit CyoA mutant, the above-mentioned cytochrome b oxidase subunit CyoB mutant and the above-mentioned polyphosphate kinase PPK mutant in the recombinant Escherichia coli, and also overexpressing the cytochrome bd oxidase cydAB gene cluster derived from Escherichia coli, and simultaneously knocking out the amn gene on the genome; the recombinant Escherichia coli is based on the L-threonine production strain LMT4 as a chassis cell.

[0027] In one embodiment, the cytochrome bo oxidase subunit CyoA mutant, the cytochrome bo oxidase subunit CyoB mutant, the polyphosphate kinase PPK mutant, and the cytochrome bd oxidase cydAB gene cluster are expressed by a Trc promoter;

[0028] In one embodiment, the nucleotide sequence encoding the Trc promoter is shown in SEQ ID NO.8;

[0029] In one embodiment, yeep on the E. coli genome is knocked out and the cytochrome bo oxidase subunit CyoA mutant is integrated at the yeep site; the pseudogene yncI on the E. coli genome is knocked out and the cytochrome bo oxidase subunit CyoB mutant is integrated at the yncI site; the pseudogene mbhA on the E. coli genome is knocked out and the cytochrome bd oxidase cydAB gene cluster is integrated at the mbhA site; the pseudogene yjiP on the E. coli genome is knocked out and the polyphosphate kinase PPK mutant is integrated at the yjiP site;

[0030] In one embodiment, preferably, the yeep gene ID on NCBI is 946524; the yncI gene ID on NCBI is 945121; the mbhA gene ID on NCBI is 944921; the yjiP gene ID on NCBI is 38094982; the amn gene gene ID on NCBI is 946508;

[0031] In one embodiment, the nucleotide sequence encoding the cydAB gene cluster is shown as SEQ ID NO.5.

[0032] The present invention also provides a method for preparing threonine, which comprises fermenting the threonine using the above-mentioned recombinant Escherichia coli;

[0033] In one embodiment, the recombinant Escherichia coli seed liquid is inoculated into a fermentation medium for fermentation; the fermentation medium is 20 g / L glucose, 2 g / L potassium dihydrogen phosphate, 3 g / L yeast powder, 1 g / L betaine, 1 g / L magnesium sulfate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 8 g / L corn steep liquor powder, and 10 mg / L vitamin B1.

[0034] In one embodiment, the inoculation amount of the seed solution is: 20%;

[0035] In one embodiment, the fermentation conditions are: 37° C., 30% dissolved oxygen in the fermentation liquid, and a fermentation period of at least 48 hours.

[0036] The present invention also provides a recombinant nucleic acid of Escherichia coli, which includes a gene encoding the above-mentioned cytochrome bo oxidase subunit CyoA mutant, a gene encoding the above-mentioned cytochrome bo oxidase subunit CyoB mutant, and a gene encoding the above-mentioned polyphosphate kinase PPK mutant; the recombinant Escherichia coli is based on the L-threonine production strain LMT4 as a chassis cell.

[0037] In one embodiment, the nucleotide sequence encoding the cytochrome bo oxidase subunit CyoA mutant is shown as SEQ ID NO.1, the nucleotide sequence encoding the cytochrome bo oxidase subunit CyoB mutant is shown as SEQ ID NO.3, and the nucleotide sequence encoding the polyphosphate kinase PPK mutant is shown as SEQ ID NO.6.

[0038] The present invention also provides the use of the recombinant Escherichia coli in preparing L-threonine or in improving the L-threonine yield and / or sugar-acid conversion rate.

[0039] Beneficial effects

[0040] (1) Overexpressing a cytochrome b oxidase subunit CyoA mutant from Escherichia coli, a cytochrome b oxidase subunit CyoB mutant from Pseudomonas aeruginosa, a cytochrome bd oxidase cydAB gene cluster from Escherichia coli, a polyphosphate kinase PPK mutant from Radiodurans deinococcus, and knocking out the amn gene. The genome of Escherichia coli is modified using the recombinant nucleic acid to obtain a recombinant Escherichia coli strain ECTHRS-5 that uses glucose as a substrate.

[0041] (2) The recombinant Escherichia coli ECTHRS-5 was used for fermentation production, and the L-threonine yield and conversion rate were significantly improved, laying the foundation for the industrial production of L-threonine.

[0042] In the embodiment of the present invention, the recombinant Escherichia coli ECTHRS-5 was used, glucose was used as a substrate, and fermentation was carried out in a 5L fermentor for 48 hours to produce 160.8g / L L-threonine, with a sugar-acid conversion rate of 0.62g / g, indicating that the method of the present invention for enhancing the electron respiration transfer and energy transfer efficiency of Escherichia coli to increase L-threonine production has broad industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : Reaction scheme of the present invention.

[0044] Figure 2 : Schematic diagram of the effect of recombinant Escherichia coli ECTHRS-5. DETAILED DESCRIPTION

[0045] The fermentation medium involved in the following examples is 20 g / L glucose, 2 g / L potassium dihydrogen phosphate, 3 g / L yeast powder, 1 g / L betaine, 1 g / L magnesium sulfate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 8 g / L corn steep liquor powder, and 10 mg / L vitamin B1.

[0046] The LMT4 strain involved in the following examples is described in the Chinese invention patent publication number CN 115011620 B. In the present invention, the primers shown in Table 1 were used to construct the recombinant E. coli.

[0047] Table 1: Primer information

[0048]

[0049] The primers for the mutants involved in the following examples are shown in Table 2:

[0050] Table 2: Mutant primers

[0051]

[0052]

[0053] The present invention provides an Escherichia coli recombinant nucleic acid, which comprises an overexpressed Escherichia coli-derived cytochrome bo oxidase subunit CyoA mutant, a Pseudomonas aeruginosa-derived cytochrome bo oxidase subunit CyoB mutant, an Escherichia coli-derived cytochrome bd oxidase cydAB gene cluster, a Radiodurans deinococcus radiodurans-derived polyphosphate kinase PPK mutant, and a knocked-out amn gene.

[0054] The present invention uses the L-threonine producing strain LMT4 of Escherichia coli as the starting strain, and knocks out the pseudogene yeep of the starting strain by gene editing and integrates the cytochrome bo oxidase subunit CyoA mutant (CyoA) derived from Escherichia coli at the yeep site. V161L ) encoding gene; knocking out the pseudogene yncI of the starting strain and integrating the cytochrome bo oxidase subunit CyoB mutant (CyoB) from Pseudomonas aeruginosa at the yncI site A126D ) encoding gene; and knocking out the pseudogene mbhA of the starting strain and integrating the cytochrome bd oxidase cydAB gene cluster from Escherichia coli at the mbhA site; knocking out the pseudogene yjiP of the starting strain and integrating the polyphosphate kinase PPK mutant (PPK) from Radiodurans deinococcus at the yjiP site. T131A ) encoding gene; knock out the ackA gene.

[0055] In the present invention, the overexpression of CyoA V161L 、CyoB A126D , cydAB gene cluster, PPK T131A The coding genes are preferably expressed by the Trc promoter. The present invention does not specifically limit the gene editing method, but preferably includes CRISPR-Cas9.

[0056] The present invention also provides a method for constructing the recombinant Escherichia coli, preferably using the CRISPR-Cas9 method, more preferably comprising: (1) PCR amplifying the upstream homology arm and downstream homology arm of the pseudogene yeep from the genome of Escherichia coli K-12MG1655; (2) amplifying the upstream homology arm and downstream homology arm of the pseudogene yeep from the genome of Escherichia coli containing the cytochrome bo oxidase subunit CyoA mutant (CyoA V161L ) plasmid PCR amplified the trc promoter driven cytochrome bo oxidase subunit CyoA mutant (CyoA V161L ) encoding gene; (3) the upstream and downstream homology arms of yeep and the CyoA driven by Trc promoter V161L Fragment fusion to obtain U-CyoA V161L -D fragment; (4) the obtained fusion fragment U-CyoA V161L -D and a vector containing yeep-sgRNA were transformed into the L-threonine production strain LMT4 containing a Cas9 plasmid, resulting in the knockout of the pseudogene yeep and the integration of CyoA driven by the Trc promoter at the yeep site. V161L recombinant strains;

[0057] According to the above steps, CyoB can be integrated into the pseudogene yncI site. A126D ; cydAB gene cluster integrated at mbhA site; PPK integrated at yjiP site T131A ; and construction of recombinant strains with amn gene knockout.

[0058] The detection methods involved in the following embodiments are as follows:

[0059] Determination method of L-threonine:

[0060] 1) Sample Preparation: Take 1 mL of fermentation broth after 48 hours of fermentation and centrifuge at 12,000 rpm for 10 minutes to remove the bacterial cells and collect the supernatant. Dilute the supernatant appropriately with deionized water and filter through a 0.22 μm pore size filter membrane.

[0061] 2) Analytical method: OPA pre-column derivatization

[0062] 3) Chromatographic Conditions: Column: C18 (250×4.6) mm; Column Temperature: 40°C; Mobile Phase A: Weigh 3.01 g of anhydrous sodium acetate into a beaker, dissolve in ultrapure water, and dilute to 1 L. Then, add 200 μL of triethylamine and adjust the pH to 7.20±0.05 with 5% acetic acid. After filtration, add 5 mL of tetrahydrofuran, mix, and filter using a 0.22 μm inorganic filter membrane. Place in an ultrasonic cleaning pot and degas for 20 minutes before use. Mobile Phase B: Weigh 3.01 g of anhydrous sodium acetate into a beaker, dissolve in ultrapure water, and dilute to 200 mL. Adjust the pH to 7.20±0.05 with 5% acetic acid. Then, add 400 mL of acetonitrile and 400 mL of methanol to this solution, mix, filter, and degas for 20 minutes in an ultrasonic cleaning pot before use. Flow rate: 1.0 ml / min; UV detector: 338 nm; column temperature: 40°C.

[0063] The method for enhancing the electron respiration transfer and energy transfer efficiency of Escherichia coli and improving the L-threonine production provided by the present invention is described in detail below with reference to the examples.

[0064] Example 1: Screening of mutant enzymes

[0065] 1. Screening of cytochrome bo oxidase subunit CyoA mutants

[0066] Site 161 of CyoA was selected for site-directed mutagenesis, and the primers involved are shown in Table 2.

[0067] The amino acid sequence of the wild type of the cytochrome bo oxidase subunit CyoA is as follows:

[0068] MRLRKYNKSLGWLSLFAGTVLLSGCNSALLDPKGQIGLEQRSLILTAFGLMLIVVIPAILMAVGFAWKYRASNKDAKYSPNWSHSNKVEAVVWTVPILIIIFLAVLTWKTTHALEPSKPLAHDEKPITIEVVSMDWKWFFIYPEQGIATVNEIAFPA NTPVYFKVTSNSVMNSFFIPRLGSQIYAMAGMQTRLHLIANEPGTYDGISASYSGPGFSGMKFKAIATPDRAAFDQWVAKAKQSPNTMSDMAAFEKLAAPSEYNQVEYFSNVKPDLFADVINKFMAHGKSMDMTQPEGEHSAHEGMEGMDMSHAESAH

[0069] (1) Using the pTrc99A-CyoA plasmid as a template, the cytochrome bo oxidase subunit CyoA on the plasmid was site-directed mutagenesis using different primer sequences, and then introduced into Escherichia coli JM109 to obtain cells containing the pTrc99A-CyoAmut plasmid containing the cytochrome bo oxidase subunit CyoA mutant. The pTrc99A-CyoAmut plasmid was obtained using a plasmid extraction kit;

[0070] Recombinant vectors were prepared: pTrc99A-CyoA V161L 、pTrc99A-CyoA V161A 、pTrc99A-CyoA V161R .

[0071] (2) The prepared recombinant vectors were introduced into Escherichia coli LMT4 to obtain the recombinant strain: LMT4 / pTrc99A-CyoA V161L 、LMT4 / pTrc99A-CyoA V161A 、LMT4 / pTrc99A-CyoA V161R A single clone of the prepared recombinant E. coli was inoculated into 10 mL of LB liquid medium and cultured at 37°C and 220 rpm for 10 hours to prepare a seed solution. The seed solution was inoculated into a fermentation medium at a 10% (v / v) inoculum and cultured at 37°C and 220 rpm for 36 hours. The L-threonine production of each strain was then measured.

[0072] The results showed that CyoA was present in shake flask fermentation. V161L 、CyoA V161A 、CyoA V161R The recombinant strains of the mutants produced 20.3 g / L, 17.6 g / L, and 18.7 g / L of L-threonine, respectively, while the LMT4 / pTrc99A-CyoA strain containing the wild-type cytochrome bo oxidase subunit CyoA produced 17.5 g / L of L-threonine according to the above method.

[0073] Therefore, choose CyoA V161L Conduct follow-up research.

[0074] 2. Screening of cytochrome bo oxidase subunit CyoB mutants

[0075] Site 126 of CyoB was selected for site-directed mutagenesis, and the primers involved are shown in Table 2.

[0076] The amino acid sequence of the wild type of the cytochrome bo oxidase subunit CyoB is as follows:

[0077] MFGKLSLEAIPYHEPIVMVTLAMIALGGIAVVGLITYFRKWTYLWSEWLTTVDHKKIGVMYIIVAMVMLLRGFADAIMMRTQLAAATGGSEGYLPPEHYDQIFTAHGVIMIIFMAMPFFTGLMNLAVPLQIGARDVAFPFLNSLSFYLLLAGVLLVNISLGVGEFAKT GWVAYPPLAGIQYSPGVGVDYYIWALQLSGLGTTLTGVNFLVTVMKMRAPGMKLMDMPIFTWTCTWANVLIVASFPILTAALALLTVDRYLDFHIFTNELGGNPMMYVNLFWAWGHPEVYILILPAFGVFSEVTSTFSGKRLFGHHSMIYASGAIAILGFAVWLHHFF TMGAGASVNTFFGLATMLISIPTGVKLFNWLFTMYQGRVRFTAPMLWTLGFMVTFSIGGMTGVLLAVPGADFVLHNSLFVIAHFHNVIIGGAVFGYIAGFAFWFPKAFGFTLNEKWGKAAFWFWLSGFYVAFMPLYALGFMGMTRRLNHSDNPLWEPYLYVAVVGAVL ILFGIACQLIQIVVSVRDRNQNLDVTGDPWGGRTLEWSTSSPPPFYNFAHMPEKVGLDCWHEAKEAGVAYKAPAKYEAIHMPSNTATGLFMGLFLTVFGFAFIWHIWWLVGASLVATIAVFVRHAARDDQGYMVPAEEVARIEGERMKALAKAGALPAGARVESFERV

[0078] (1) Using the pTrc99A-CyoB plasmid as a template, the cytochrome bo oxidase subunit CyoB on the plasmid was site-directed mutagenesis using different primer sequences, and then introduced into Escherichia coli JM109 to obtain cells containing the pTrc99A-CyoBmut plasmid containing the cytochrome bo oxidase subunit CyoB mutant. The pTrc99A-CyoBmut plasmid was obtained using a plasmid extraction kit;

[0079] Recombinant vectors were prepared separately: pTrc99A-CyoB A126D 、pTrc99A-CyoB A126V 、pTrc99A-CyoB A126T .

[0080] (2) The prepared recombinant vectors were introduced into Escherichia coli LMT4 to obtain the recombinant strain: LMT4 / pTrc99A-CyoB A126D 、LMT4 / pTrc99A-CyoB A126V 、LMT4 / pTrc99A-CyoB A126T ; Pick a single clone of the prepared recombinant Escherichia coli and inoculate it into 10 mL of LB liquid culture medium, and culture it at 37°C and 220 rpm for 10 h; prepare a seed solution.

[0081] The seed liquid was inoculated into the fermentation medium at an inoculum rate of 10% (v / v). After culturing at 37° C. and 220 rpm for 36 h, the L-threonine production of each strain was detected.

[0082] The results showed that overexpression of CyoB in shake flask fermentation A126D 、CyoB A126V 、CyoB A126T The recombinant strains of the mutants produced 22.8 g / L, 21.6 g / L, and 20.2 g / L of L-threonine, respectively, while the LMT4 / pTrc99A-CyoB strain containing the wild-type cytochrome bo oxidase subunit CyoB produced 19.7 g / L of L-threonine according to the above method.

[0083] Therefore, choose CyoB A126D Conduct follow-up research.

[0084] 3. Screening of polyphosphate kinase (PPK) mutants

[0085] The 131 site of PPK was selected for site-directed mutagenesis, and the primers involved are shown in Table 2.

[0086] The amino acid sequence of the wild type polyphosphate kinase PPK is as follows:

[0087] MDIDNYRVKPGKRVKLSDWATNDDAGLSKEEGQAQTAKLAGELAEWQERLYAEGKQSLLLILQARDAAGKDGAVKKVIGAFNPAGVQITSFKQPSAEELSHDFLWRIHQKAPAKGYVGVFNRSQYEDVLVTRV YDMIDDKTAKRRLEHIRHFEELLTDNATRIVKVYLHISPEEQKERLQARLDNPGKHWKFNPGDLKDRSNWDKFNDVYEDALTTSTDDAPWYVVPADRKWYRDLVLSHILLGALKDMNPQFPAIDYDPSKVVIH

[0088] (1) Using the pTrc99A-PPK plasmid as a template, the polyphosphate kinase PPK on the plasmid was site-directed mutagenesis using different primer sequences, and then introduced into Escherichia coli JM109 to obtain cells containing the pTrc99A-PPKmut plasmid containing the polyphosphate kinase PPK mutant. The pTrc99A-PPKmut plasmid was obtained using a plasmid extraction kit;

[0089] Recombinant vectors were prepared: pTrc99A-PPK T131A 、pTrc99A-PPK T131D 、pTrc99A-PPK T131G .

[0090] (2) The prepared recombinant vectors were introduced into Escherichia coli LMT4 to obtain the recombinant strain: LMT4 / pTrc99A-PPK T131A 、LMT4 / pTrc99A-PPK T131D 、LMT4 / pTrc99A-PPK T131G ; Pick a single clone of the prepared recombinant Escherichia coli and inoculate it into 10 mL of LB liquid culture medium, and culture it at 37°C and 220 rpm for 10 h; prepare a seed solution.

[0091] The seed liquid was inoculated into the fermentation medium at an inoculum rate of 10% (v / v). After culturing at 37° C. and 220 rpm for 36 h, the L-threonine production of each strain was detected.

[0092] The results showed that PPK was overexpressed in shake flask fermentation. T131A 、PPK T131D 、PPK T131G The recombinant strains of the mutants produced 24.6 g / L, 22.7 g / L, and 21.3 g / L of L-threonine, respectively, while the LMT4 / pTrc99A-PPK strain containing the wild-type polyphosphate kinase PPK produced 22.4 g / L of L-threonine according to the above method.

[0093] Therefore, choose PPK T131A Conduct follow-up research.

[0094] Example 2: Construction of genetically engineered bacteria

[0095] 1. Construction of ECTHRS-1

[0096] Integrate CyoA at the yeep locus of LMT4 strain (NCBI number: Gene ID: 946524) V161L

[0097] (1) Fusion fragment U-CyoA V161L -D build

[0098] Using the primers yeep-UF, yeep-UR, yeep-DF, and yeep-DR in Table 1, the upstream and downstream homology arms of the yeep gene were amplified from the genome of Escherichia coli K-12MG1655, respectively, to obtain fragments yeep1 and yeep2;

[0099] Using the primers CyoA-F and CyoA-R in Table 1, the mutant containing the cytochrome bo oxidase subunit CyoA prepared in Example 1 (CyoA V161L PCR amplification of CyoA driven by trc promoter on the plasmid V161L The coding gene (SEQ ID NO.1) was obtained by fragment CyoA V161L ;

[0100] Fragments yeep1, CyoA V161L , yeep2 for fusion PCR to obtain the fusion fragment U-CyoA V161L -D.

[0101] (2) Construction of yeep-sgRNA recombinant plasmid

[0102] Using primers PGRB-F and PGRB-R, linearized vector L-PGRB was obtained by PCR from vector PGRB. The designed sgRNA was ligated with the linearized vector L-PGRB to construct the recombinant plasmid yeep-sgRNA.

[0103] (3) Overexpression of CyoA V161L Construction of recombinant Escherichia coli

[0104] The recombinant plasmid yeep-sgRNA and fusion fragment U-CyoA V161L -D was transformed into the L-threonine production strain LMT4 containing the Cas9 plasmid, and the transformants were screened by colony PCR using primers yeep-UF and yeep-DR to confirm the fusion fragment U-CyoA V161L -D was successfully integrated into the yeep site, and 2 mM arabinose was added to culture at 30 °C for 12 h. The recombinant plasmid yeep-sgRNA was removed to obtain the recombinant strain LMT4Δyeep::P trc -CyoA V161L , named ECTHRS-1.

[0105] 2. Construction of ECTHRS-2

[0106] CyoB was integrated into the yncI site of ECTHRS-1 strain (NCBI number: Gene ID: 945121) A126D .

[0107] (1) Fusion fragment U-CyoB A126D -D build

[0108] Using the primers yncI-UF, yncI-UR, yncI-DF, and yncI-DR in Table 1, the upstream and downstream homology arms on both sides of the yncI gene were amplified from the genome of Escherichia coli K-12MG1655, respectively, to obtain fragments yncI1 and yncI2;

[0109] Using the primers CyoB-F and CyoB-R in Table 1, the mutant containing the cytochrome bo oxidase subunit CyoB prepared in Example 1 (CyoB A126D ) plasmid PCR amplified the trc promoter driven cytochrome bo oxidase subunit CyoB mutant (CyoB A126D ) encoding gene (SEQ ID NO.3), and obtained fragment CyoB A126D ;

[0110] The fragment yncI 1, CyoB A126D , yncI 2 for fusion PCR to obtain the fusion fragment U-CyoB A126D -D.

[0111] (2) Construction of yncI-sgRNA recombinant plasmid

[0112] Using primers PGRB-F and PGRB-R, linearized vector L-PGRB was obtained by PCR from vector PGRB. The designed sgRNA was ligated with the linearized vector L-PGRB to construct the recombinant plasmid yncI-sgRNA.

[0113] (3) Overexpression of CyoB A126D Construction of recombinant Escherichia coli

[0114] The recombinant plasmid yncI-sgRNA and fusion fragment U-CyoB A126D -D was transformed into ECTHRS-1 obtained in step 1, and the transformants were screened by colony PCR using primers yncI-UF and yncI-DR to confirm the fusion fragment U-CyoB A126D -D was successfully integrated into the yncI site, 2 mM arabinose was added and cultured at 30°C for 12 h, and the recombinant plasmid yncI-sgRNA was removed to obtain the recombinant strain LMT4Δyeep::P trc -CyoA V161L ΔyncI::Ptrc -CyoB A126D , named ECTHRS-2.

[0115] 3. Construction of ECTHRS-3

[0116] The cytochrome bd oxidase cydAB gene cluster was integrated into the mbhA site of the ECTHRS-2 strain (NCBI number: Gene ID: 944921).

[0117] (1) Construction of the fusion fragment U-cydAB-D

[0118] Using primers mbhA-UF, mbhA-UR, mbhA-DF, and mbhA-DR in Table 1, the upstream and downstream homology arms of the mbhA gene were amplified from the genome of Escherichia coli K-12MG1655, respectively, to obtain fragments mbhA1 and mbhA2;

[0119] Using primers cydAB-F and cydAB-R in Table 1, PCR amplified the gene encoding the cytochrome bo oxidase subunit cydAB driven by the trc promoter (SEQ ID NO. 5) from a plasmid containing the cytochrome bo oxidase subunit cydAB (constructed in the same manner as in Example 1, plasmid: pTrc99A-cydAB) to obtain the fragment cydAB;

[0120] The fragments mbhA1, cydAB, and mbhA2 were subjected to fusion PCR to obtain the fusion fragment U-cydAB-D.

[0121] (2) Construction of mbhA-sgRNA recombinant plasmid

[0122] Using primers PGRB-F and PGRB-R, linearized vector L-PGRB was obtained by PCR from vector PGRB. The designed sgRNA was ligated with the linearized vector L-PGRB to construct the recombinant plasmid mbhA-sgRNA.

[0123] (3) Construction of recombinant Escherichia coli overexpressing the cydAB gene

[0124] The recombinant plasmid mbhA-sgRNA and fusion fragment U-cydAB-D were transformed into ECTHRS-2 obtained in step 2. The transformants were screened by colony PCR using primers mbhA-UF and mbhA-DR to confirm that the fusion fragment U-cydAB-D was successfully integrated into the mbhA site. 2 mM arabinose was added and cultured at 30°C for 12 h. The recombinant plasmid mbhA-sgRNA was removed and the recombinant strain LMT4Δyeep::P was obtained. trc -CyoA V161L ΔyncI::Ptrc -CyoB A126D ΔmbhA::P trc -cydAB, named ECTHRS-3.

[0125] 4. Construction of ECTHRS-4

[0126] The polyphosphate kinase PPK mutant (PPK) was integrated into the yjiP site of ECTHRS-3 strain (NCBI Gene ID: 38094982). T131A ).

[0127] (1) Construction of the fusion fragment U-PPK-D

[0128] Using primers yjiP-UF, yjiP-UR, yjiP-DF, and yjiP-DR in Table 1, the upstream and downstream homology arms of the yjiP gene were amplified from the genome of Escherichia coli K-12MG1655, respectively, to obtain fragments yjiP1 and yjiP2;

[0129] Using the primers PPK-F and PPK-R in Table 1, the polyphosphate kinase PPK mutant (PPK T131A ) plasmid PCR amplified the polyphosphate kinase PPK mutant driven by the trc promoter (PPK T131A ) encoding gene (SEQ ID NO.6), and obtained the fragment PPK T131A ;

[0130] The fragment yjiP 1, PPK T131A , yjiP 2 for fusion PCR to obtain the fusion fragment U-PPK T131A -D.

[0131] (2) Construction of yjiP-sgRNA recombinant plasmid

[0132] Using primers PGRB-F and PGRB-R, the linearized vector L-PGRB was obtained by PCR from the vector PGRB. The designed sgRNA was ligated with the linearized vector L-PGRB to construct the recombinant plasmid yjiP-sgRNA.

[0133] (3) Overexpression of PPK T131A Construction of recombinant Escherichia coli

[0134] The recombinant plasmid yjiP-sgRNA and fusion fragment U-PPK T131A -D was transformed into ECTHRS-3 obtained in step 3, and the transformants were screened by colony PCR using primers yjiP-UF and yjiP-DR to confirm the fusion fragment U-PPKT131A -D was successfully integrated into the yjiP site, and 2 mM arabinose was added to culture at 30°C for 12 h. The recombinant plasmid yjiP-sgRNA was removed and the recombinant strain LMT4Δyeep::P was obtained. trc -CyoA V161L ΔyncI::P trc -CyoB A126D ΔmbhA::P trc -cydABΔyjiP::P trc -PPK T131A , named ECTHRS-4.

[0135] 5. Construction of ECTHRS-5 strain

[0136] Knockout of the amn gene in the ECTHRS-4 strain genome (Gene ID: 946508)

[0137] (1) Using primers amn-UF, amn-UR, amn-DF, and amn-DR in Table 1, the upstream and downstream homology arms of the amn gene were amplified from the genome of Escherichia coli K-12MG1655, respectively, to obtain fragments amn1 and amn2; fragments amn1 and amn2 were fused by PCR to obtain the fusion fragment U-amn-D.

[0138] (2) Construction of amn-sgRNA recombinant plasmid

[0139] Using primers PGRB-F and PGRB-R, linearized vector L-PGRB was obtained by PCR from vector PGRB. The designed sgRNA was ligated with the linearized vector L-PGRB to construct the recombinant plasmid amn-sgRNA.

[0140] (3) The recombinant plasmid amn-sgRNA and the fusion fragment U-amn-D were transformed into the THRHS-4 strain, and the primers amn-UF and amn-DR were used to perform colony PCR to screen the transformants to confirm the knockout of the amn gene. 2 mM arabinose was added and cultured at 30 °C for 12 h. The recombinant plasmid amn-sgRNA was removed, and the recombinant strain LMT4Δyeep::P was obtained. trc -CyoA V161L ΔyncI::P trc -CyoB A126D ΔmbhA::P trc -cydABΔyjiP::P trc -PPK T131A Δamn, named ECTHRS-5.

[0141] Example 3: Preparation of L-threonine under shake flask conditions

[0142] The specific steps are as follows:

[0143] The genetically engineered bacteria ECTHRS-1, ECTHRS-2, ECTHRS-3, ECTHRS-4, and ECTHRS-5 prepared in Example 2 were added to LB liquid culture medium, and cultured at 37° C. and 220 rpm for 10 h to prepare seed solution.

[0144] The seed liquid was inoculated into the fermentation medium at an inoculum rate of 10% (v / v). After culturing at 37° C. and 220 rpm for 36 h, the L-threonine production of each strain was detected.

[0145] The fermentation medium is glucose 20g / L, potassium dihydrogen phosphate 2g / L, yeast powder 3g / L, betaine 1g / L, magnesium sulfate 1g / L, FeSO4·7H2O 10mg / L, MnSO4·H2O 10mg / L, corn steep liquor powder 8g / L, vitamin B1 10mg / L

[0146] The results showed that ECTHRS-1 was 20.1g / L, ECTHRS-2 was 22.4g / L, ECTHRS-3 was 23.0g / L, ECTHRS-4 was 24.7g / L, and ECTHRS-5 was 25.8g / L.

[0147] Example 4: Preparation of L-threonine under 5 L fermentation tank conditions

[0148] The recombinant strain ECTHRS-5 constructed in Example 2 was inoculated into a seed culture medium and cultured under the following conditions. The seed culture was then transferred into a fermentation medium at a 20% inoculum volume and cultured under the following conditions.

[0149] 1. 5L seed tank process control

[0150] a. Adjust the temperature to 37°C, pH to 7.0, speed to 500 rpm, and air volume to 0.3 m 3 / h, the whole process temperature is controlled at 37℃, the tank pressure is 0.05~0.08MPa, and the culture period is 10h;

[0151] b. Transplantation standard: OD600: 12-15.

[0152] c. The seed culture medium is 5 g / L corn steep liquor powder, 20 g / L glucose, 5 g / L yeast powder, 2 g / L KH2PO4, 1 g / L magnesium sulfate, 20 mg / L FeSO4·7H2O, and 20 mg / L MnSO4·H2O.

[0153] 2. 5L fermentation tank fermentation process control

[0154] a. Adjust the temperature to 37°C, pH to 7.0, initial speed to 300 rpm, and air volume to 0.3 m 3 / h, the whole process temperature is controlled at 37℃, and the tank pressure is 0.05~0.08MPa;

[0155] c.DO control: at 0h, air volume 0.3m3 / h, 300rpm, tank pressure 0.05MPa;

[0156] d. When DO drops below 30%, control the dissolved oxygen level at 30% by adjusting the ventilation volume and stirring speed until the fermentation ends;

[0157] e. The fermentation medium is 20 g / L glucose, 2 g / L potassium dihydrogen phosphate, 3 g / L yeast extract, 1 g / L betaine, 1 g / L magnesium sulfate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 8 g / L corn steep liquor powder, and 10 mg / L vitamin B1.

[0158] The results showed that the engineered strain ECTHRS-5 accumulated 160.8 g / L L-threonine in a 5 L fermenter with a fermentation time of 48 h through fed-batch fermentation, and the sugar-acid conversion rate was 0.62 g / g (62%).

[0159] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant nucleic acid of Escherichia coli, comprising a gene encoding a cytochrome bo oxidase subunit CyoA mutant, a gene encoding a cytochrome bo oxidase subunit CyoB mutant, and a gene encoding a polyphosphate kinase PPK mutant; the gene sequence encoding the cytochrome bo oxidase subunit CyoA mutant is shown in SEQ ID NO.1, the gene sequence encoding the cytochrome bo oxidase subunit CyoB mutant is shown in SEQ ID NO.3, and the gene sequence encoding the polyphosphate kinase PPK mutant is shown in SEQ ID NO.

6.

2. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli overexpresses a cytochrome bo oxidase subunit CyoA mutant, a cytochrome bo oxidase subunit CyoB mutant, and a polyphosphate kinase PPK mutant; The cytochrome bo oxidase subunit CyoA mutant is obtained by mutating the amino acid at position 161 of the cytochrome bo oxidase subunit CyoA shown in SEQ ID NO.9 from valine to leucine; The cytochrome bo oxidase subunit CyoB mutant is obtained by mutating the amino acid at position 126 of the cytochrome bo oxidase subunit CyoB shown in SEQ ID NO.10 from alanine to aspartic acid; The polyphosphate kinase PPK mutant is obtained by mutating the amino acid at position 131 of the carbonic anhydrase with the amino acid sequence shown in SEQ ID NO. 11 from threonine to alanine.

3. The recombinant Escherichia coli according to claim 2, characterized in that In the recombinant E. coli, the cytochrome bd oxidase cydAB gene cluster from E. coli is also overexpressed, and the amn gene on the genome is knocked out at the same time; Preferably, the cytochrome bo oxidase subunit CyoA mutant, the cytochrome bo oxidase subunit CyoB mutant, the polyphosphate kinase PPK mutant, and the cytochrome bd oxidase cydAB gene cluster are all expressed by the Trc promoter; Preferably, the nucleotide sequence encoding the Trc promoter is shown in SEQ ID NO.8; Preferably, the overexpression is: knocking out yeep on the E. coli genome and integrating the cytochrome bo oxidase subunit CyoA mutant at the yeep site; knocking out the pseudogene yncI on the E. coli genome and integrating the cytochrome bo oxidase subunit CyoB mutant at the yncI site; knocking out the pseudogene mbhA on the E. coli genome and integrating the cytochrome bd oxidase cydAB gene cluster at the mbhA site; knocking out the pseudogene yjiP on the E. coli genome and integrating the polyphosphate kinase PPK mutant at the yjiP site; Preferably, the yeep gene has a Gene ID of 946524 on NCBI; the yncI gene has a Gene ID of 945121 on NCBI; the mbhA gene has a Gene ID of 944921 on NCBI; the yjiP gene has a Gene ID of 38094982 on NCBI; the amn gene has a Gene ID of 946508 on NCBI; Preferably, the nucleotide sequence encoding the cydAB gene cluster is shown as SEQ ID NO.

5.

4. A cytochrome bo oxidase subunit CyoA mutant, characterized in that: The mutant is obtained by mutating the amino acid at position 161 of the cytochrome bo oxidase subunit CyoA, whose amino acid sequence is shown in SEQ ID NO.9, from valine to leucine.

5. A cytochrome bo oxidase subunit CyoB mutant, characterized in that: The mutant is obtained by mutating the amino acid at position 126 of the cytochrome bo oxidase subunit CyoB, whose amino acid sequence is shown in SEQ ID NO. 10, from alanine to aspartic acid.

6. A polyphosphate kinase (PPK) mutant, characterized in that: The mutant is obtained by mutating the amino acid at position 131 of the carbonic anhydrase with the amino acid sequence shown in SEQ ID NO. 11 from threonine to alanine.

7. A gene encoding the mutant according to any one of claims 4 to 6, a recombinant vector carrying the gene, or a recombinant strain expressing the mutant according to any one of claims 4 to 6.

8. A method for enhancing the efficiency of electron respiration and energy transfer in Escherichia coli, characterized in that: The method comprises overexpressing the cytochrome bo oxidase subunit CyoA mutant according to claim 4, the cytochrome bo oxidase subunit CyoB mutant according to claim 5, and the polyphosphate kinase PPK mutant according to claim 6 in the recombinant Escherichia coli, further overexpressing the cytochrome bd oxidase cydAB gene cluster derived from Escherichia coli, and simultaneously knocking out the amn gene on the genome; Preferably, the cytochrome bo oxidase subunit CyoA mutant of claim 4, the cytochrome bo oxidase subunit CyoB mutant of claim 5, the polyphosphate kinase PPK mutant of claim 6, and the cytochrome bd oxidase cydAB gene cluster are all expressed by the Trc promoter; Preferably, the nucleotide sequence encoding the Trc promoter is shown in SEQ ID NO.8; Preferably, the overexpression is: knocking out yeep on the E. coli genome and integrating the cytochrome bo oxidase subunit CyoA mutant at the yeep site; knocking out the pseudogene yncI on the E. coli genome and integrating the cytochrome bo oxidase subunit CyoB mutant at the yncI site; knocking out the pseudogene mbhA on the E. coli genome and integrating the cytochrome bd oxidase cydAB gene cluster at the mbhA site; knocking out the pseudogene yjiP on the E. coli genome and integrating the polyphosphate kinase PPK mutant at the yjiP site; Preferably, the yeep gene has a Gene ID of 946524 on NCBI; the yncI gene has a Gene ID of 945121 on NCBI; the mbhA gene has a Gene ID of 944921 on NCBI; the yjiP gene has a Gene ID of 38094982 on NCBI; the amn gene has a Gene ID of 946508 on NCBI; Preferably, the nucleotide sequence encoding the cydAB gene cluster is shown as SEQ ID NO.

5.

9. A method for preparing threonine, characterized in that: The method is to prepare the product by fermentation using the recombinant Escherichia coli according to claim 2 or 3; Preferably, the recombinant Escherichia coli seed solution is inoculated into a fermentation medium for fermentation; Preferably, the inoculation amount of the seed solution is: 20%; Preferably, the fermentation conditions are: 37° C., 30% dissolved oxygen in the fermentation liquid, and a fermentation period of at least 48 hours.

10. Use of the recombinant Escherichia coli according to claim 2 or 3 in preparing L-threonine or in improving L-threonine yield and / or sugar-acid conversion rate.

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