Method for producing L-threonine by using escherichia coli overexpressing aspartate dehydrogenase

Abstract

The invention discloses a method for producing L-threonine by using escherichia coli overexpressing aspartate dehydrogenase, and belongs to the technical field of genetic engineering technology. According to the invention, aspartate dehydrogenase is inserted into the genome of recipient bacteria to enhance the ability of escherichia coli to synthesize threonine precursor aspartic acid, thereby enhancing the L-threonine production capacity of modified bacteria. According to the application disclosed by the invention, the expression intensity of the aspDH5 gene sourced from Piga ntiphaga sp.H8 is enhanced, so that the accumulation concentration of the L-threonine in the CGMCC25404 strain is promoted to be increased from 30.55 g / L to 32.78 g / L, the yield of the L-threonine is increased by 7.3%, and the difference is extremely significant.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a method for producing L-threonine by using Escherichia coli overexpressing aspartate dehydrogenase. BACKGROUND

[0002] L-threonine belongs to a restricted amino acid, has the effects of promoting the development of mammalian mammary cells, improving the immune state of animals, and accelerating the repair of muscle tissue, and is widely used in the industries of medicine, food, and feed. At present, L-threonine is mainly produced by microbial fermentation in industry. With the increasing market demand for L-threonine, researchers are increasingly concerned about continuously optimizing the fermentation performance of the production strain and developing more economical and efficient fermentation processes to improve the production efficiency of threonine.

[0003] The application of Escherichia coli in threonine production can be traced back to the end of the 20th century and the beginning of the 21st century. With the progress of biotechnology, scientists have found that by modifying Escherichia coli through metabolic engineering, L-threonine can be efficiently produced. The application of metabolic engineering technology makes Escherichia coli become the main strain for industrial production of L-threonine, and the main reasons are that Escherichia coli has the characteristics of short growth cycle, high cell strength, and low requirement for equipment.

[0004] In Escherichia coli, the synthesis of L-threonine starts from aspartate, which is gradually converted into L-threonine through a series of enzyme-catalyzed reactions. These reactions are controlled by multiple enzymes, and the change of each step of the metabolic pathway will affect the system parameters and metabolic flux.

[0005] Aspartate dehydrogenase (aspDH) catalyzes the reversible reaction of oxaloacetate with ammonium and NAD(P)H to generate aspartate and water, and NAD(P)H + The aspDH gene can be introduced into the host cell through an expression vector that can independently replicate in the host cell, or can be integrated into the recipient chromosome through methods such as homologous recombination to construct a threonine production strain.

[0006] However, the effect of enhancing the expression of the above-mentioned genes on L-threonine production has not been studied, and in particular, there is no report on the production of L-threonine by Enterobacteriaceae bacteria overexpressing the above-mentioned genes. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a method for producing L-threonine. The technical problem to be solved is not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0008] To solve the above technical problems, the present application provides the following technical solutions:

[0009] The present invention provides an application, which includes any of the following:

[0010] U1) Use of aspartate dehydrogenase in the preparation of L-threonine;

[0011] U2) Use of a substance for regulating the activity and / or content of aspartate dehydrogenase in the preparation of L-threonine.

[0012] The aspartate dehydrogenase is derived from at least the genus Pusillimonas and / or Pseudomonas ae ruginosa and / or the genus Pigmentiphaga.

[0013] In the above application, the aspartate dehydrogenase comprises at least any one of the following:

[0014] A1) a protein comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5 or SEQ ID NO: 8;

[0015] A2) a protein having aspartate dehydrogenase function obtained by substitution and / or deletion and / or addition of amino acid residues in any one of the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 8, or more thereof, and derived from A1) or having 98% or greater identity with the protein set forth in A1);

[0016] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0017] The tag protein includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0018] As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search to calculate the identity of the amino acid sequence, the value (%) of identity can then be obtained.

[0019] Herein, the greater than 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0020] In the above application, the substance is a biomaterial, and the biomaterial is any of the following:

[0021] B1) a nucleic acid molecule encoding the protein;

[0022] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0023] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0024] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0025] B5) A whole-cell catalyst containing the nucleic acid molecule described in B1), or a whole-cell catalyst containing the expression cassette described in B2), or a whole-cell catalyst containing the recombinant vector described in B3), or a whole-cell catalyst containing the recombinant microorganism described in B4).

[0026] In the above-mentioned biological material, the nucleic acid molecule in B1) comprises any one of the following:

[0027] C1) a cDNA molecule or a DNA molecule whose coding sequence comprises SEQ ID NO: 2;

[0028] C2) a cDNA molecule or a DNA molecule whose coding sequence comprises SEQ ID NO: 6;

[0029] C3) a cDNA molecule or a DNA molecule whose coding sequence comprises SEQ ID NO: 9;

[0030] C4) A cDNA molecule or DNA molecule that hybridizes with the cDNA or DNA molecule defined in C1) or C2) or C3) and encodes a protein having the same function.

[0031] The present invention also provides an application of a biomaterial related to the aforementioned aspartate dehydrogenase, wherein the application is the application of the biomaterial in any one of the following B1) to B3).

[0032] B1) Application of the biomaterial in constructing an engineered bacterium producing L-threonine;

[0033] B2) Application of the biomaterial in the preparation of L-threonine;

[0034] B3) Application of the biomaterial in regulating the production of L-threonine in microorganisms;

[0035] The biological material is at least one of the following C1) to C3):

[0036] C1), a nucleic acid molecule encoding the aforementioned glutamate dehydrogenase,

[0037] C2), an expression cassette containing the nucleic acid molecule described in C1),

[0038] C3), a recombinant vector containing the nucleic acid molecule described in C1) and / or a recombinant vector containing the expression cassette described in C2).

[0039] In the above application, the nucleic acid molecules described in C1) include nucleic acid molecules with nucleotide sequences of SEQ ID NO.2, SEQ ID NO.6, and SEQ ID NO.9.

[0040] The aforementioned biological materials also fall within the protection scope of the present invention.

[0041] The present invention also provides a recombinant microorganism, which is any one of the following:

[0042] D1), a recombinant microorganism containing the aforementioned nucleic acid molecule;

[0043] D2), a recombinant microorganism containing the aforementioned expression cassette;

[0044] D3), ​​a recombinant microorganism containing the aforementioned recombinant vector;

[0045] D4) A recombinant microorganism containing the aforementioned aspartate dehydrogenase.

[0046] The present invention also provides a whole-cell catalyst, including a whole-cell catalyst containing the aforementioned biomaterial, or a whole-cell catalyst containing the aforementioned recombinant microorganism.

[0047] The present invention also provides a method for producing L-threonine, comprising fermenting the protein or the biological material to prepare L-threonine.

[0048] In the above method, the fermentation preparation of L-threonine using the aforementioned protein or the aforementioned biological material includes the following steps: upregulating, enhancing or increasing the content and / or activity of the aforementioned protein or the expression of the encoding gene of the protein in the recipient bacteria to obtain an engineered bacterium, and fermenting the engineered bacterium to obtain a fermentation broth, wherein the fermentation broth contains the L-threonine.

[0049] In the above method, the upregulation, enhancement or increase of the content and / or activity of the aforementioned protein or the expression of the gene encoding the protein in the recipient bacteria includes introducing a substance that upregulates, enhances or increases the expression level of the gene encoding the protein into the recipient bacteria.

[0050] The present invention also provides a method for constructing an engineered bacterium for producing L-threonine, comprising the steps of upregulating, enhancing or increasing the content and / or activity of the aforementioned protein or the expression of the gene encoding the protein in the recipient bacterium to obtain the engineered bacterium.

[0051] In the above method, the upregulation, enhancement or increase of the content and / or activity of the protein or the expression of the gene encoding the protein in the recipient bacteria includes introducing a substance that upregulates, enhances or increases the expression level of the gene encoding the protein into the recipient bacteria.

[0052] In the above-mentioned method for producing L-threonine or the method for constructing an engineered bacterium for producing L-threonine, the substance is a biomaterial, and the biomaterial is any one of the following:

[0053] E1) a nucleic acid molecule encoding the protein;

[0054] E2) an expression cassette containing the nucleic acid molecule described in E1);

[0055] E3) a recombinant vector containing the nucleic acid molecule described in E1), or a recombinant vector containing the expression cassette described in E2);

[0056] E4) a recombinant microorganism containing the nucleic acid molecule described in E1), or a recombinant microorganism containing the expression cassette described in E2), or a recombinant microorganism containing the recombinant vector described in E3);

[0057] E5) A whole-cell catalyst containing the nucleic acid molecule of E1), or a whole-cell catalyst containing the expression cassette of E2), or a whole-cell catalyst containing the recombinant vector of E3), or a whole-cell catalyst containing the recombinant microorganism of E4).

[0058] The expression cassette containing the nucleic acid molecule described in B2) or E2) refers to a DNA capable of expressing the above-mentioned proteins in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary to express the nucleic acid molecule for any of the above-mentioned proteins. The regulatory sequences are capable of directing the coding sequence to express any of the above-mentioned proteins in a suitable host cell under compatible conditions. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequence includes a promoter and termination signals for transcription and translation. The regulatory sequence may be provided with a linker to introduce specific restriction enzyme sites into the vector for ligating the regulatory sequence to the coding region of the protein-encoding nucleic acid sequence. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence recognized by the host cell in which the nucleic acid sequence is to be expressed. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. A regulatory sequence may also be a suitable transcription terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that is functional in the selected host cell may be used in the present invention. A regulatory sequence may also be a suitable leader sequence, i.e., an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence may be operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the selected host cell may be used in the present invention. A regulatory sequence may also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of the protein that directs the encoded protein into the cell's secretory pathway. Any signal peptide coding region that directs the expressed protein into the secretory pathway of the selected host cell may be used in the present invention. It may also be desirable to add regulatory sequences that can regulate protein expression based on the growth conditions of the host cells. Examples of regulatory sequences are those that can turn gene expression on or off in response to chemical or physical stimuli (including in the presence of regulatory compounds). Other examples of regulatory sequences are those that enable gene amplification.

[0059] In the above biological materials, the nucleic acid molecules described in B1) include nucleic acid molecules with nucleotide sequences of SEQ ID NO.2, SEQ ID NO.6, and SEQ ID NO.9.

[0060] The recombinant bacteria prepared by the aforementioned method also fall within the scope of protection of the present invention.

[0061] The content and / or activity of the aforementioned protein in the engineered bacteria or recombinant bacteria or the expression of the coding gene of the protein is higher than that in the recipient bacteria.

[0062] The above-mentioned recipient bacteria include but are not limited to Escherichia coli and CGMCC25404.

[0063] The present invention also provides the use of the aforementioned protein or the aforementioned biological material in the preparation of food, feed, medicine, fertilizer and / or daily chemical products containing L-threonine.

[0064] The present invention provides an improved method for producing L-threonine by fermentation using modified Escherichia coli. Specifically, the ability of Escherichia coli to fully synthesize the threonine precursor aspartate can be enhanced by inserting aspartate dehydrogenase, thereby enhancing the L-threonine production capacity of the modified bacteria.

[0065] The present invention also provides the use of the aforementioned recombinant bacteria or the aforementioned whole-cell catalyst in the preparation of L-amino acids.

[0066] The present invention also provides a method for increasing the L-threonine production of a recipient microorganism, which comprises introducing the aforementioned biological material into the recipient microorganism.

[0067] The recipient microorganism includes Escherichia coli.

[0068] In some specific embodiments of the present invention, the recipient microorganism includes Escherichia coli CGMCC No. 25404 or Escherichia coli MG1655.

[0069] The present invention enhances the expression intensity of the aspDH5 gene derived from Pigmentiphaga sp. H8, thereby increasing the L-threonine accumulation concentration in the CGMCC25404 strain from 30.55 g / L to 32.78 g / L, and increasing the L-threonine yield by 7.3%, with extremely significant differences; enhances the expression intensity of the aspDH17 gene derived from Pusillimonas sp. T2, thereby increasing the L-threonine accumulation concentration in the CGMCC25404 strain from 30.55 g / L to 31.72 g / L, and increasing the L-threonine yield by 3.83%, with significant differences; enhances the expression intensity of the aspDH19 gene derived from Pseudomonas aeruginosa PAO1, thereby increasing the L-threonine accumulation concentration in the CGMCC25404 strain from 30.55 g / L to 31.7 g / L, and increasing the L-threonine yield by 3.76%, with significant differences; enhances the expression intensity of the Delftia sp. The expression intensity of the aspDH20 gene from sp.Cs1-4 increased the L-threonine accumulation concentration in the CGMCC25404 strain from 30.55 to 30.66 g / L, and the L-threonine yield increased by 0.35%, but there was no significant difference.

[0070] Preservation Instructions

[0071] Chinese name of the strain: Escherichia coli

[0072] Latin name: Escherichia coil

[0073] Classification name: Escherichia coli

[0074] Item No.: YP0518

[0075] Depository: General Microbiology Center of China Culture Collection Administration

[0076] Abbreviation of depository unit: CGMCC

[0077] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0078] Date of deposit: July 25, 2022

[0079] The registration number of the CGMCC Collection Center is: CGMCC No.25404. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is the pREDCas9 plasmid map.

[0081] Figure 2 This is the pGRB plasmid map. DETAILED DESCRIPTION

[0082] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0083] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0084] The Escherichia coli MG1655 in the following examples was derived from Beijing Beina Chuanglian Biotechnology Research Institute (BNCC), and the strain number is BNCC363342.

[0085] The following examples were processed using one-way ANOVA to process the data. The experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated a very significant difference.

[0086] Example 1. Construction of genetically engineered bacteria YPThr-Pig-aspDH5 and MG1655-Pig-aspDH5

[0087] 1. Preparation of DNA fragments for homologous recombination

[0088] The recombinant fragment used to overexpress the aspDH5 gene from Pigmentiphaga sp. H8 (its coding region is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is SEQ ID NO: 1) consists of the Pig-aspDH5 gene and its upstream and downstream homology arms (upstream homology arm-Pig-aspDH5-downstream homology arm). Primers were designed using the primer design software Primer5, using the Pig-aspDH5 gene and its upstream and downstream homology arm sequences as templates. The specific primers are as follows:

[0089] aspDH-up-F: 5'-TTTTGCGGGAGACTAAATCACTGG-3';

[0090] aspDH-up-R: 5'-TTGGTCTTACCAATTTgctagcattatacctaggactgagctagctgtcaaAAGAAAACTCCTTTCGAATC-3';

[0091] Pig-aspDH5-F: 5'-aatgctagcAAATTGGTAAGACCAATTAAGCTTAAAGAGGAGAAAgAATTCATGCTAAGGATAACAATGAT-3';

[0092] Pig-aspDH5-R: 5'-caaaggcgcttactttcgccagTTAGATCGCAACTGCGTGC-3';

[0093] Pig-aspDH5-down-F: 5'-GCACGCAGTTGCGATCTAActggcgaaagtaagcgcctttg-3';

[0094] aspDH-down-R: 5'-TTGTGCTTTCGATGCCAGCC-3'.

[0095] The upstream homologous arm of Pig-aspDH5 gene was amplified from the genomic DNA of the engineering strain CGMCC 25404 using aspDH-up-F and aspDH-up-R, and the amplification system is shown in Table 1, and a 500 bp upstream homologous arm (i.e. the 1st to 500th of SEQ ID NO: 3) was obtained. The downstream homologous arm of Pig-aspDH5 gene was amplified from the genomic DNA of the engineering strain CGMCC 25404 using Pig-aspDH5-down-F and aspDH-down-R, and the amplification system is shown in Table 1, and a 500 bp downstream homologous arm (i.e. the 1385th to 1884th of SEQ ID NO: 3) was obtained. In order to amplify the Pig-aspDH5 gene, the Pig-aspDH5 gene was synthesized by whole gene, and the codon optimization was carried out according to the host codon preference, and the part or all of the codons rare to the host in the coding aspDH5 gene were replaced by the host preferred codons. Then the synthesized Pig-aspDH5 gene was used as a template to amplify the promoter PJ23119 and the Pig-aspDH5 gene (i.e. the 501st to 1384th nucleotides of SEQ ID NO: 3) using primers Pig-aspDH5-F and Pig-aspDH5-R, and named as PJ23119-Pig-aspDH5. The amplification system is shown in Table 1.

[0096] Table 1, PCR reaction system

[0097] Components Volume (50 μL) template 2μL Upstream primer (10 μmol L) 1 μL Downstream primer (10 μmol L) 1 μL dNTP mixture (10 mmol / L) 4 μL 5xBuffer 10 μL HS enzyme (5U / μL) 0.5μL <![CDATA[ddH2O]]> 31.5μL

[0098] Overlap PCR was performed using the upstream primer aspDH-up-F for the upstream homology arm and the downstream primer aspDH-down-R for the downstream homology arm as amplification primers, and the upstream and downstream homology arms, along with PJ23119-Pig-aspDH5, as templates to generate recombinant fragments. Overlap PCR reaction conditions (Takara Biotech PrimeSTAR HS enzyme) included initial denaturation (95°C) for 5 minutes, followed by 30 cycles of denaturation (98°C) for 10 seconds, annealing ((Tm-3 / 5)°C) for 15 seconds, and extension at 72°C (this enzyme activity extends approximately 1 kb per minute), followed by a further extension at 72°C for 10 minutes, and a hold (4°C). The overlapping PCR reaction system is shown in Table 2, and a DNA fragment for homologous recombination was obtained. The nucleotide sequence of the DNA fragment PJ23119-Pig-aspDH5 for homologous recombination is SEQ ID NO: 3 (i.e., the donor DNA fragment), wherein nucleotides 1 to 500 are the upstream homology arm of the Pig-aspDH5 gene (500 bp), nucleotides 501 to 577 are the PJ23119 promoter (77 bp), nucleotides 578 to 1384 are the Pig-aspDH5 gene (807 bp), and nucleotides 1385 to 1884 are the downstream homology arm of the Pig-aspDH5 gene (500 bp).

[0099] Table 2. Overlapping PCR reaction system

[0100] Components Volume (50 μL) template 2μL Upstream primer of upstream homology arm (10 μmol L) 1 μL Downstream homology arm upstream primer (10 μmol L) 1 μL dNTP mixture (10 mmol / L) 4 μL 5xBuffer 10 μL HS enzyme (5U / μL) 0.5μL <![CDATA[ddH2O]]> 31.5μL

[0101] Note: The template consists of equimolar amounts of the amplified fragments of the upstream and downstream homology arms and the target gene (PJ23119-Pig-aspDHS), and the total amount does not exceed 10 ng.

[0102] 2. Construction of pGRB-yaeQ plasmid

[0103] pGRB was purchased from Addgene, catalog number #71539, and the map is Figure 2 , with pUC18 as the backbone, including promoter J23119, g RNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100 mg / L), cultured at 37°C.

[0104] The pGRB plasmid was constructed by recombining a DNA fragment containing the target sequence with a linearized vector fragment.

[0105] (1) Target sequence and primer design

[0106] Use CRISPR RGEN Tools to design the target sequence (PAM: 5'-NGG-3'), and design primers for amplifying the sgRNA fragment based on the target sequence (forward primer F structure: 5'-linearized vector end sequence (34 bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector end sequence (34 bp)-3', reverse primer R structure: a primer that is reverse complementary to the forward primer R), as follows:

[0107] gRNA-F: 5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTgaacgtctgcaatttacccgGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3';

[0108] gRNA-R: 5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACcgggtaaattgcagacgttcACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3';

[0109] In the above primers, lowercase letters represent the yaeQ target sequence.

[0110] (2) Preparation of DNA fragments containing target sequences

[0111] Prepare DNA fragments containing the target sequence by annealing single-stranded DNA. Reaction conditions: Pre-denaturation at 95°C for 5 minutes; annealing at 30-50°C for 1 minute. Annealing reaction system: 10 μL gRNA-F (10 μmol / L) and 10 μL gRNA-R (10 μmol / L) to obtain DNA fragments containing the target sequence.

[0112] (3) Preparation of linear vectors

[0113] The vector was linearized using inverse PCR. The primers used were as follows: pGRB-F: 5'-ACTAGTATTATACCTAGGACTGAGC-3'; pGRB-R: 5'-GTTTTAGAGCTAGAAATAGCAAGTT-3'. The PCR reaction system is shown in Table 1. The PCR protocol (Takara Biotech PrimeSTAR HS enzyme) was as follows: initial denaturation (95°C) for 5 minutes, followed by 30 cycles of denaturation (98°C) for 10 seconds, annealing (Tm-3 / 5)°C for 15 seconds, and extension at 72°C (this enzyme activity extends approximately 1 kb per minute); further extension at 72°C for 10 minutes, followed by a hold at 4°C to obtain the linearized cloning vector.

[0114] (4) Recombination reaction

[0115] The recombinant system is shown in the following table. The recombinant enzymes used are II One Step Cloning Kit series enzymes, reconstitution conditions: 37 ° C, 30 min, to obtain a reaction solution.

[0116] Table 3. Recombination system

[0117] Reaction system volume 20 μL 5×CE II Buffer 4 μL Linearized cloning vector 1 μL DNA fragment containing the target sequence 1 μL ExnaseR I II 2μL <![CDATA[ddH2O]]> 12 μL

[0118] (5) Plasmid transformation

[0119] Take 10 μL of the reaction solution from "(4) Recombination Reaction" and add it to 100 mL of DH5α competent cells. Mix gently and place on ice for 20 min. Heat shock at 42°C for 45-90 s. Immediately place on ice for 2-3 min. Add 900 μL of SOC and resuspend at 37°C for 1 h. Centrifuge at 8000 rpm for 2 min. Discard part of the supernatant and resuspend the cells in about 200 μL. Spread the suspension onto a plate containing 100 mg / L ampicillin. Invert the plate and incubate at 37°C overnight.

[0120] (6) Clone identification

[0121] The ampicillin-resistant colony was inoculated into LB medium containing 100 mg / L ampicillin and cultured overnight. The plasmid was extracted and identified by enzyme digestion.

[0122] The plasmid identified as correctly inserted was named pGRB-yaeQ. This pGRB-yaeQ can transcribe the gRNA with the nucleotide sequence of SEQ ID NO: 4 (5'-GAACGUCUGCAAUUUACCCG-3', with T replacing U in the sequence listing), thereby forming a complex with the Cas9 protein and recognizing the target gene site through base pairing and PAM, thereby achieving the target DNA double-strand break.

[0123] Plasmid pGRB-yaeQ is a recombinant vector obtained by inserting the DNA fragment represented by nucleotides 35 to 54 of gRNA-F into the HS enzyme recognition site of pGRB plasmid while keeping other nucleotide sequences unchanged.

[0124] 3. Transformation of plasmids and recombinant DNA fragments

[0125] pREDCas9 was purchased from Addgene, catalog number #71541, and the map is Figure 1 , elimination system carrying gRNA expression plasmid pGRB, Red recombination system of λ phage and Cas9 protein expression system, spectinomycin resistance (working concentration: 100 mg / L), culture at 32°C.

[0126] (1) Transformation with pREDCas9

[0127] The pREDCas9 plasmid was electroporated into the electrocompetent cell of the starting strain. After resuscitation, the cells were plated on LB plates containing spectinomycin and incubated overnight at 32°C. Single colonies growing on the resistant plates were subjected to colony PCR using identification primers to screen for positive recombinants.

[0128] (2) Preparation of electroporation competent cells of target strain containing pREDCas9

[0129] The positive recombinants in "(1) pREDCas9 transformation" were cultured at 32°C until OD 600nm =0.1-0.2, add 0.1M IPTG (to make the final concentration 0.1mM) and continue to culture until OD 600nm Competent cells were prepared when the pH was between 0.6 and 0.7. The purpose of adding IPTG was to induce expression of the recombinase on the pREDCas9 plasmid. The culture medium and preparation process required for competent cell preparation followed standard procedures to obtain electroporated competent cells containing pREDCas9.

[0130] (3) Transformation of pGRB-yaeQ recombinant DNA fragment

[0131] Plasmid pGRB-yaeQ and the donor DNA fragment (SEQ ID NO: 3) were simultaneously electroporated into the pREDCas9-containing electroporation competent cells obtained in "(2) Preparation of electroporation competent cells of target strain containing pREDCas9". The revived cells after electroporation were plated on LB plates containing ampicillin and spectinomycin and cultured overnight at 32°C. Colony PCR verification was performed using the identification primers aspDH5-up-F / Pig-aspDH5-R1 and Pig-aspDH5-F1 / aspDH-down-R, and positive recombinants were screened and maintained.

[0132] aspDH-up-F: 5'-TTTTGCGGGAGACTAAATCACTGG-3';

[0133] Pig-aspDH5-R1: 5'-CCGGGGTATCTTTCCAAGCC-3';

[0134] Pig-aspDH5-F1: 5'-CGCTGTCGGATGTTGAACTG-3';

[0135] aspDH5-down-R: 5'-TTGTGCTTTCGATGCCAGCC-3'.

[0136] (4) Elimination of plasmids

[0137] ① Elimination of pGRB-yaeQ

[0138] The positive recombinants obtained in "(3) Transformation of pGRB-yaeQ and recombinant DNA fragments" were cultured overnight in LB medium containing 0.2% arabinose. After appropriate dilution, they were spread on LB plates containing spectinomycin resistance and cultured at 32°C overnight. Single colonies were picked and streaked onto LB plates containing ampicillin and spectinomycin resistance, respectively. Single colonies that did not grow on the ampicillin plate but grew on the spectinomycin resistance plate were selected and preserved to obtain positive recombinants.

[0139] ② Elimination of pREDCas9 plasmid

[0140] The positive recombinants obtained from "① Elimination of pGRB-yaeQ" were transferred to LB liquid medium without resistance and cultured overnight at 42°C. After appropriate dilution, they were spread onto LB plates without resistance and cultured overnight at 37°C. Single colonies were picked and streaked onto LB plates containing spectinomycin resistance and non-resistance, respectively. Single colonies that did not grow on the spectinomycin resistance plate but grew on the non-resistance plate were selected to obtain positive single colonies.

[0141] The positive colonies obtained above were sent for sequencing, and the strain with the correct sequencing result was named recombinant bacteria YPThr-Pig-aspDH5. The recombinant bacteria YPThr-Pig-aspDH5 is a recombinant Escherichia coli obtained by replacing the 1-414bp coding sequence of the yaeQ gene (genbak number D49445.1) of the genome sequence of strain CGMCC25404 with the DNA fragment sequence shown in nucleotides 501 to 1384 in SEQ ID NO: 3, and keeping the other nucleotide sequences of the CGMCC25404 genome sequence unchanged. It should be understood by those skilled in the art that in Examples 1-4 of the present invention, the yaeQ gene is only inserted into one gene site of each aspartate dehydrogenase gene, and the remaining sites suitable for inserting the aspartate dehydrogenase gene are suitable for use and are not further limited here.

[0142] Using the wild-type strain MG1655 as the starting strain, the recombinant strain MG1655-Pig-aspDH5 was obtained according to the aforementioned method. The recombinant strain MG1655-Pig-aspDH5 was obtained by replacing the coding sequence of the yaeQ gene (genbak number D49445.1) from the MG1655 genome with the DNA fragment represented by nucleotides 501 to 1384 of SEQ ID NO:3, while retaining the remaining nucleotide sequence of the MG1655 genome unchanged.

[0143] Example 2: Construction of genetically engineered bacteria YPThr-Pus-aspDH17 and MG1655-Pus-aspDH17

[0144] 1. Preparation of DNA fragments for homologous recombination

[0145] The recombinant fragment used to overexpress the aspDH17 gene from Pusillimonas sp. T2 (its coding region is SEQ ID NO: 6, and the amino acid sequence of the encoded protein is SEQ ID NO: 5) consists of the Pus-aspDH17 gene and its upstream and downstream homology arms (upstream homology arm-Pus-aspDH17-downstream homology arm). Primers were designed using the primer design software Primer5, using the Pus-aspDH17 gene and its upstream and downstream homology arm sequences as templates, as follows:

[0146] aspDH-up-F: 5'-TTTTGCGGGAGACTAAATCACTGG-3';

[0147] aspDH-up-R: 5'-TTGGTCTTACCAATTTgctagcattatacctaggactgagctagctgtcaaAAGAAAACTC CTTTCGAATC-3';

[0148] Pus-aspDH17-F: 5'-aatgctagcAAATTGGTAAGACCAATTAAGCTTAAAGAGGAGAAAgAATTCATGTCAG TTCAAAGGATAGC-3';

[0149] Pus-aspDH17-R: 5'-caaaggcgcttactttcgccagTTATACTGCCACAGCCGCG-3';

[0150] Pus-aspDH17-down-F: 5'-CGCGGCTGTGGCAGTATAActggcgaaagtaagcgcctttg-3';

[0151] aspDH-down-R: 5'-TTGTGCTTTCGATGCCAGCC-3'.

[0152] Using genomic DNA from the engineered strain CGMCC25404 as a template, the upstream homology arm of the Pus-aspDH17 gene was amplified using aspDH-up-F and aspDH-up-R. The amplification system is shown in Table 1, resulting in a 500 bp upstream homology arm (i.e., positions 1 to 500 of SEQ ID NO: 7). Using genomic DNA from the engineered strain CGMCC25404 as a template, the downstream homology arm of the Pus-aspDH17 gene was amplified using aspDH17-down-F and aspDH-down-R. The amplification system is shown in Table 1, resulting in a 500 bp downstream homology arm (i.e., positions 1397 to 1896 of SEQ ID NO: 7). To amplify the Pus-aspDH17 gene, the Pus-aspDH17 gene was synthesized using the full gene and codon-optimized based on host codon preference. Some or all codons encoding the Pus-aspDH17 gene that are rare to the host were replaced with codons preferred by the host. The synthetic Pus-aspDH17 gene was then used as a template to amplify the promoter PJ23119 and the Pus-aspDH17 gene (i.e., nucleotides 501 to 1396 of SEQ ID NO: 7) with primers Pus-aspDH17-F and Pus-aspDH17-R. The amplification system is shown in Table 1 and is named PJ23119-Pus-aspDH17.

[0153] Overlap PCR was performed using the upstream primer aspDH-up-F for the upstream homology arm and the downstream primer aspDH-down-R for the downstream homology arm as amplification primers, and the upstream and downstream homology arms, along with PJ23119-Pus-aspDH17, as templates to generate recombinant fragments. Overlap PCR reaction conditions (Takara Biotech PrimeSTAR HS enzyme) included initial denaturation (95°C) for 5 minutes, followed by 30 cycles of denaturation (98°C) for 10 seconds, annealing (Tm-3 / 5)°C for 15 seconds, and extension at 72°C (this enzyme activity extends approximately 1 kb per minute), followed by a further extension at 72°C for 10 minutes, and a hold (4°C). The overlapping PCR reaction system is shown in Table 2, and a DNA fragment for homologous recombination was obtained. The nucleotide sequence of the DNA fragment PJ23119-Pus-aspDH17 for homologous recombination is SE Q ID NO: 7 (i.e., the donor DNA fragment), wherein nucleotides 1 to 500 are the upstream homology arm of the Pus-aspDH17 gene (500 bp), nucleotides 501 to 577 are the PJ23119 promoter (77 bp), nucleotides 578 to 1396 are the Pus-aspDH17 gene (819 bp), and nucleotides 1397 to 1896 are the downstream homology arm of the Pus-aspDH17 gene (500 bp).

[0154] 2. Transformation of pGRB-yaeQ plasmid and recombinant DNA fragments

[0155] (1) Transformation of pGRB-yaeQ and recombinant DNA fragments

[0156] Plasmid pGRB-yaeQ and the donor DNA fragment were simultaneously electroporated into the pREDCas9-containing electrocompetent cells obtained in Example 1. Resuscitated cells were plated onto LB plates containing ampicillin and spectinomycin and incubated overnight at 32°C. Colony PCR was performed using the primers aspDH5-up-F / Pus-aspDH17-R1 and Pus-aspDH17-F1 / aspDH-down-R to confirm the recombinants. Positive recombinants were screened and maintained.

[0157] aspDH-up-F: 5'-TTTTGCGGGAGACTAAATCACTGG-3';

[0158] Pus-aspDH17-R1: 5'-CTGCCCTCGAAGAACAATGC-3';

[0159] Pus-aspDH17-F1: 5'-TGCGATTGGCGGTATCGATG-3';

[0160] aspDH5-down-R: 5'-TTGTGCTTTCGATGCCAGCC-3'.

[0161] (2) Elimination of plasmids

[0162] ① Elimination of pGRB-yaeQ

[0163] The positive recombinants obtained in "(3) Transformation of pGRB-yaeQ and recombinant DNA fragments" were cultured overnight in LB medium containing 0.2% arabinose. After appropriate dilution, they were spread on LB plates containing spectinomycin resistance and cultured at 32°C overnight. Single colonies were picked and streaked onto LB plates containing ampicillin and spectinomycin resistance, respectively. Single colonies that did not grow on the ampicillin plate but grew on the spectinomycin resistance plate were selected and preserved to obtain positive recombinants.

[0164] ② Elimination of pREDCas9 plasmid

[0165] The positive recombinants obtained from "① Elimination of pGRB-yaeQ" were transferred to LB liquid medium without resistance and cultured overnight at 42°C. After appropriate dilution, they were spread onto LB plates without resistance and cultured overnight at 37°C. Single colonies were picked and streaked onto LB plates containing spectinomycin resistance and non-resistance, respectively. Single colonies that did not grow on the spectinomycin resistance plate but grew on the non-resistance plate were selected to obtain positive single colonies.

[0166] The positive colonies obtained above were sent for sequencing, and the strain with the correct sequencing results was designated recombinant strain YPThr-Pus-aspDH17. Recombinant strain YPThr-Pus-aspDH17 was obtained by replacing the coding sequence of the 1st to 414th base pairs of the yaeQ gene (genbak number D49445.1) of the genomic sequence of strain CGMCC25404 with the DNA fragment sequence represented by nucleotides 501 to 1396 of SEQ ID NO:7, while maintaining the remaining nucleotide sequences of the CGMCC25404 genomic sequence unchanged.

[0167] Using the wild-type strain MG1655 as the starting strain, recombinant E. coli MG1655-Pus-aspDH17 was obtained using the aforementioned method. Recombinant E. coli MG1655-Pus-aspDH17 is obtained by replacing the coding sequence of the yaeQ gene (genbak number D49445.1) from the MG1655 genome with the DNA fragment represented by nucleotides 501 to 1396 of SEQ ID NO:7, while retaining the remaining nucleotide sequence of the MG1655 genome unchanged.

[0168] Example 3. Construction of genetically engineered bacteria YPThr-Pae-aspDH19 and MG1655-Pae-aspDH19

[0169] 1. Preparation of DNA fragments for homologous recombination

[0170] The recombinant fragment used to overexpress the aspDH19 gene from Pseudomonas aeruginosa PAO1 (coding region: SEQ ID NO: 9, amino acid sequence of the encoded protein: SEQ ID NO: 8) consists of the Pae-aspDH19 gene and its upstream and downstream homology arms (upstream homology arm - Pae-aspDH19 - downstream homology arm). Primers were designed using the primer design software Primer5, using the Pae-aspDH19 gene and its upstream and downstream homology arm sequences as templates. The specific primers are as follows:

[0171] aspDH-up-F: 5'-TTTTGCGGGAGACTAAATCACTGG-3';

[0172] aspDH-up-R: 5'-TTGGTCTTACCAATTTgctagcattatacctaggactgagctagctgtcaaAAGAAAACTC CTTTCGAATC-3';

[0173] Pae-aspDH19-F: 5'-aatgctagcAAATTGGTAAGACCAATTAAGCTTAAAGAGGAGAAAgAATTCATGCTAA ATATTGTAATGAT-3';

[0174] Pae-aspDH19-R: 5'-caaaggcgcttactttcgccagTTAAATGCTGATCGCGTGTG-3';

[0175] Pae-aspDH19-down-F: 5'-CACACGCGATCAGCATTTAActggcgaaagtaagcgcctttg-3';

[0176] aspDH-down-R: 5'-TTGTGCTTTCGATGCCAGCC-3'.

[0177] Using genomic DNA from the engineered strain CGMCC25404 as a template, the upstream homology arm of the Pae-aspDH19 gene was amplified using aspDH-up-F and aspDH-up-R. The amplification system is shown in Table 1, resulting in a 500-bp upstream homology arm (i.e., positions 1 to 500 of SEQ ID NO: 10). Using genomic DNA from the engineered strain CGMCC25404 as a template, the downstream homology arm of the Pae-aspDH19 gene was amplified using aspDH19-down-F and aspDH-down-R. The amplification system is shown in Table 1, resulting in a 500-bp downstream homology arm (i.e., positions 1382 to 1881 of SEQ ID NO: 10). To amplify the Pae-aspDH19 gene, the Pae-aspDH19 gene was synthesized using the full gene and codon-optimized based on host codon preference. Some or all codons encoding the Pae-aspDH19 gene that are rare to the host were replaced with codons preferred by the host. The synthesized Pae-aspDH19 gene was then used as a template to amplify the promoter PJ23119 and the Pae-aspDH19 gene (i.e., nucleotides 501 to 1381 of SEQ ID NO: 10) with primers Pae-aspDH19-F and Pae-aspDH19-R. The amplification system is shown in Table 1 and is named PJ23119-Pae-aspDH19.

[0178] Overlap PCR was performed using the upstream primer aspDH-up-F for the upstream homology arm and the downstream primer aspDH-down-R for the downstream homology arm as amplification primers, and the upstream and downstream homology arms, along with PJ23119-Pae-aspDH19, as templates to generate recombinant fragments. Overlap PCR reaction conditions (Takara Biotech PrimeSTAR HS enzyme) included initial denaturation (95°C) for 5 minutes, followed by 30 cycles of denaturation (98°C) for 10 seconds, annealing ((Tm-3 / 5)°C) for 15 seconds, and extension at 72°C (this enzyme activity extends approximately 1 kb per minute), followed by a further extension at 72°C for 10 minutes, and a hold (4°C). The overlapping PCR reaction system is shown in Table 2, and a DNA fragment for homologous recombination was obtained. The nucleotide sequence of the DNA fragment PJ23119-Pae-aspDH19 for homologous recombination is SE Q ID NO: 10 (i.e., the donor DNA fragment), wherein nucleotides 1 to 500 are the upstream homologous arm of the Pae-aspDH19 gene (500 bp), nucleotides 501 to 577 are the PJ23119 promoter (77 bp), nucleotides 578 to 1381 are the Pae-aspDH19 gene (804 bp), and nucleotides 1382 to 1881 are the downstream homologous arm of the Pae-aspDH19 gene (500 bp).

[0179] 2. Transformation of pGRB-yaeQ plasmid and recombinant DNA fragments

[0180] (1) Transformation of pGRB-yaeQ and recombinant DNA fragments

[0181] Plasmid pGRB-yaeQ and the donor DNA fragment were simultaneously electroporated into the pREDCas9-containing electrocompetent cells obtained in Example 1. Resuscitated cells were plated onto LB plates containing ampicillin and spectinomycin and incubated overnight at 32°C. Colony PCR was performed using the primers aspDH5-up-F / Pae-aspDH19-R1 and Pae-aspDH19-F1 / aspDH-down-R to confirm the recombinants. Positive recombinants were screened and maintained.

[0182] aspDH-up-F: 5'-TTTTGCGGGAGACTAAATCACTGG-3';

[0183] Pae-aspDH19-R1: 5'-TTCGGGTATAAAACGCGCTGC-3';

[0184] Pae-aspDH19-F1: 5'-CGGCTCTCGTATTGAGCTCC-3';

[0185] aspDH5-down-R: 5'-TTGTGCTTTCGATGCCAGCC-3'.

[0186] (2) Elimination of plasmids

[0187] ① Elimination of pGRB-yaeQ

[0188] The positive recombinants obtained in "(3) Transformation of pGRB-yaeQ and recombinant DNA fragments" were cultured overnight in LB medium containing 0.2% arabinose. After appropriate dilution, they were spread on LB plates containing spectinomycin resistance and cultured at 32°C overnight. Single colonies were picked and streaked onto LB plates containing ampicillin and spectinomycin resistance, respectively. Single colonies that did not grow on the ampicillin plate but grew on the spectinomycin resistance plate were selected and preserved to obtain positive recombinants.

[0189] ② Elimination of pREDCas9 plasmid

[0190] The positive recombinants obtained from "① Elimination of pGRB-yaeQ" were transferred to LB liquid medium without resistance and cultured overnight at 42°C. After appropriate dilution, they were spread onto LB plates without resistance and cultured overnight at 37°C. Single colonies were picked and streaked onto LB plates containing spectinomycin resistance and non-resistance, respectively. Single colonies that did not grow on the spectinomycin resistance plate but grew on the non-resistance plate were selected to obtain positive single colonies.

[0191] The positive colonies obtained above were sent for sequencing, and the strain with the correct sequencing results was designated recombinant strain YPThr-Pae-aspDH19. Recombinant strain YPThr-Pae-aspDH19 was obtained by replacing the coding sequence of the 1st to 414th base pairs of the yaeQ gene (genbak number D49445.1) of the genomic sequence of strain CGMCC25404 with the DNA fragment sequence represented by nucleotides 501 to 1381 of SEQ ID NO: 10, while maintaining the remaining nucleotide sequences of the CGMCC25404 genomic sequence unchanged.

[0192] Using the wild-type strain MG1655 as the starting strain, recombinant E. coli MG1655-Pae-aspDH19 was obtained using the aforementioned method. Recombinant E. coli MG1655-Pae-aspDH19 is obtained by replacing the coding sequence of the yaeQ gene (genbak number D49445.1) from the MG1655 genome with the DNA fragment represented by nucleotides 501 to 1381 of SEQ ID NO: 10, while retaining the remaining nucleotide sequence of the MG1655 genome unchanged.

[0193] Example 4. Construction of genetically engineered bacteria YPThr-Del-aspDH20 and MG1655-Del-aspDH20

[0194] 1. Preparation of DNA fragments for homologous recombination

[0195] The recombinant fragment used to overexpress the aspDH20 gene (coding region of SEQ ID NO: 12, amino acid sequence of the encoded protein of SEQ ID NO: 11) from Delftia sp. Cs1-4 consists of the Del-aspDH17 gene and its upstream and downstream homology arms (upstream homology arm - Del-aspDH20 - downstream homology arm). Primers were designed using the primer design software Primer5, using the Del-aspDH20 gene and its upstream and downstream homology arm sequences as templates. The specific primers are as follows:

[0196] aspDH-up-F: 5'-TTTTGCGGGAGACTAAATCACTGG-3';

[0197] aspDH-up-R: 5'-TTGGTCTTACCAATTTgctagcattatacctaggactgagctagctgtcaaAAGAAAACTC CTTTCGAATC-3';

[0198] Del-aspDH20-F: 5'-aatgctagcAAATTGGTAAGACCAATTAAGCTTAAAGAGGAGAAAgAATTCATGAATA TTGCAGTAATAGG-3';

[0199] Del-aspDH20-R:5'-caaaggcgcttactttcgccagTTAGATGGCGATTGCGGT-3';

[0200] Del-aspDH20-down-F:5'-ACCGCAATCGCCATCTAActggcgaaagtaagcgcctttg-3';

[0201] aspDH-down-R: 5'-TTGTGCTTTCGATGCCAGCC-3'.

[0202] Using genomic DNA from the engineered strain CGMCC25404 as a template, the upstream homology arm of the Del-aspDH20 gene was amplified using aspDH-up-F and aspDH-up-R. The amplification system is shown in Table 1, resulting in a 500 bp upstream homology arm (i.e., positions 1 to 500 of SEQ ID NO: 13). Using genomic DNA from the engineered strain CGMCC25404 as a template, the downstream homology arm of the Del-aspDH20 gene was amplified using Del-aspDH20-down-F and aspDH-down-R. The amplification system is shown in Table 1, resulting in a 500 bp downstream homology arm (i.e., positions 1376 to 1875 of SEQ ID NO: 13). To amplify the Del-aspDH20 gene, the Del-aspDH20 gene was synthesized using the full gene and codon-optimized based on the host codon preference. Some or all codons encoding the Del-aspDH20 gene that are rare to the host were replaced with codons preferred by the host. The synthesized Del-aspDH20 gene was then used as a template to amplify the promoter PJ23119 and the Del-aspDH20 gene (i.e., positions 501 to 1375 of SEQ ID NO: 13) using primers Del-aspDH20-F and Del-aspDH20-R, and the resulting fragment was named PJ23119-Del-aspDH20. The amplification system is shown in Table 1.

[0203] Overlap PCR was performed using the upstream primer aspDH-up-F for the upstream homology arm and the downstream primer aspDH-down-R for the downstream homology arm as amplification primers, and the upstream and downstream homology arms, along with PJ23119-Del-aspDH20, as templates to generate recombinant fragments. Overlap PCR reaction conditions (Takara Biotech PrimeSTAR HS enzyme) included initial denaturation (95°C) for 5 minutes, followed by 30 cycles of denaturation (98°C) for 10 seconds, annealing ((Tm-3 / 5)°C) for 15 seconds, and extension at 72°C (this enzyme activity extends approximately 1 kb per minute), followed by a further extension at 72°C for 10 minutes, and a hold (4°C). The overlapping PCR reaction system is shown in Table 2, and a DNA fragment for homologous recombination was obtained. The nucleotide sequence of the DNA fragment PJ23119-Del-aspDH20 for homologous recombination is SE Q ID NO: 13 (i.e., the donor DNA fragment), wherein positions 1 to 500 are the upstream homology arm of the Del-aspDH20 gene (500 bp), nucleotides 501 to 577 are the PJ23119 promoter (77 bp), nucleotides 578 to 1375 are the Del-as pDH20 gene (798 bp), and nucleotides 1376 to 1875 are the downstream homology arm of the Del-aspDH20 gene (500 bp).

[0204] 2. Transformation of pGRB-yaeQ plasmid and recombinant DNA fragments

[0205] (1) Transformation of pGRB-yaeQ and recombinant DNA fragments

[0206] Simultaneously electrotransform pGRB-yaeQ and the donor DNA fragment into the pREDCas9-containing electrocompetent cells obtained in Example 1. Resuscitated cells were plated onto LB plates containing ampicillin and spectinomycin and incubated overnight at 32°C. Colony PCR was performed using the primers aspDH5-up-F / Del-aspDH20-R1 and Del-aspDH20-F1 / aspDH-down-R to confirm the recombinants. Positive recombinants were screened and maintained.

[0207] aspDH-up-F: 5'-TTTTGCGGGAGACTAAATCACTGG-3';

[0208] Del-aspDH20-R1: 5'-TGCATAAAGACGCACGGTGG-3';

[0209] Del-aspDH20-F1:5'-TTCCGGAAGTTACCGATGCC-3';

[0210] aspDH5-down-R: 5'-TTGTGCTTTCGATGCCAGCC-3'.

[0211] (2) Elimination of plasmids

[0212] ① Elimination of pGRB-yaeQ

[0213] The positive recombinants obtained in "(3) Transformation of pGRB-yaeQ and recombinant DNA fragments" were cultured overnight in LB medium containing 0.2% arabinose. After appropriate dilution, they were spread on LB plates containing spectinomycin resistance and cultured at 32°C overnight. Single colonies were picked and streaked onto LB plates containing ampicillin and spectinomycin resistance, respectively. Single colonies that did not grow on the ampicillin plate but grew on the spectinomycin resistance plate were selected and preserved to obtain positive recombinants.

[0214] ② Elimination of pREDCas9 plasmid

[0215] The positive recombinants obtained from "① Elimination of pGRB-yaeQ" were transferred to LB liquid medium without resistance and cultured overnight at 42°C. After appropriate dilution, they were spread onto LB plates without resistance and cultured overnight at 37°C. Single colonies were picked and streaked onto LB plates containing spectinomycin resistance and non-resistance, respectively. Single colonies that did not grow on the spectinomycin resistance plate but grew on the non-resistance plate were selected to obtain positive single colonies.

[0216] The positive colonies obtained above were sent for sequencing, and the strain with the correct sequencing results was designated recombinant strain YPThr-Del-aspDH20. Recombinant strain YPThr-Del-aspDH20 was obtained by replacing the coding sequence of the 1st to 414th base pairs of the yaeQ gene (genbak number D49445.1) of the genomic sequence of strain CGMCC25404 with the DNA fragment sequence represented by nucleotides 501 to 1375 of SEQ ID NO: 13, while maintaining the remaining nucleotide sequences of the CGMCC25404 genomic sequence unchanged.

[0217] Using the wild-type strain MG1655 as the starting strain, recombinant E. coli MG1655-Del-aspDH20 was obtained according to the aforementioned method. Recombinant E. coli MG1655-Del-aspDH20 is obtained by replacing the coding sequence of the yaeQ gene (genbak number D49445.1) from the MG1655 genome with the DNA fragment represented by nucleotides 501 to 1375 of SEQ ID NO: 13, while retaining the remaining nucleotide sequence of the MG1655 genome unchanged.

[0218] Example 5: Verification of L-threonine production performance in strains modified to overexpress aspartate dehydrogenase genes

[0219] The experiment was repeated 3 times, and each repetition was as follows:

[0220] The MG1655, MG1655-Pig-aspDH5, MG1655-Pus-aspDH17, MG1655-Pae-aspDH19, MG1655-Del-aspDH20 strains and the engineered strains of CGMCC25404, YPThr-Pig-aspDH5, YPThr-Pus-aspDH17, YPThr-Pae-aspDH19, and YPThr-Del-aspDH20 were streaked onto slant cultures and cultured at 37°C for 12 h; a ring of slant seeds was scraped with an inoculating loop and inoculated into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium, sealed with nine layers of gauze, and cultured at 37°C and 200 rpm for 7-10 h; then, 1 mL of the culture of each strain was inoculated into 50 mL of shake flask fermentation medium and fermented at 37°C and 200 rpm for 36 h. The L-threonine content in the fermentation broth was determined by HPLC. Three replicates were performed for each strain, and the average value was calculated. The HPLC assay parameters were as follows: The dilution factor was determined based on the sample content. 1.5 mL of the sample dilution was centrifuged at 10,000 rpm for 5 minutes. 10 μL of the supernatant was transferred to a 2.0 mL centrifuge tube. 200 μL of NaHCO₃ solution and 100 μL of 1% DNFB solution were added, sequentially. Mix thoroughly, and then derivatize in a 60°C water bath in the dark for 60 minutes. Cool to room temperature in the dark, add 800 μL of KH₂PO₄ buffer solution (Phase D), mix thoroughly, and filter through a 0.22 μm syringe filter. 15 μL of the sample was sampled for chromatographic analysis.

[0221] Chromatographic column: Agilent ZORBAX Eclipse AAA Analytice 4.6×150mm 5μm (Cat. No.: 993400-902).

[0222] Guard column: Agilent ZORBAX Eclipse AAA Analytice Guard Column 4.6×12.5 mm 5 μm (Cat. No. 820950-931).

[0223] Mobile phase B: 55% acetonitrile-water solution.

[0224] Mobile phase D: 5.44 g / L KH2PO4 solution (pH = 7.2), gradient elution.

[0225] Column temperature: 40℃.

[0226] Flow rate: V = 1.0 mL / min.

[0227] The running time is 30 minutes.

[0228] Injection volume: 15 μL.

[0229] Detection wavelength: 360nm.

[0230] The slant culture medium consists of: peptone 16 g / L, yeast powder 10 g / L, NaCl 5 g / L, agar 15-20 g / L, and the rest is water, pH 7.0-7.2;

[0231] The seed culture medium consists of: glucose 1-5 g / L, peptone 5-10 g / L, beef extract 5-10 g / L, yeast powder 1-5 g / L, NaCl 1-2.5 g / L, and the rest is water, pH 7.0-7.2;

[0232] The shake flask fermentation medium composition was as follows: glucose 40 g / L, (NH4)2SO4 12 g / L, KH2PO4 0.8 g / L, MgSO4·7H2O 0.8 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·H2O 0.01 g / L, FM902 yeast powder 1.5 g / L, calcium carbonate 0.5 g / L, and the rest was water, with sodium hydroxide used to adjust the pH to 7.0.

[0233] Table 4. L-threonine acid production of engineered strains

[0234]

[0235] The results are shown in Table 4. For both the high-L-threonine-producing strains CGMCC25404 and MG1655, enhancing the expression intensity of the aspDH5 gene from Pigmentiphaga sp. H8 increased the L-threonine accumulation concentration in the CGMCC25404 strain from 30.55 g / L to 32.78 g / L, and the L-threonine yield increased by 7.3%, which was a very significant difference. Enhancing the expression intensity of the aspDH17 gene from Pusillimonas sp. T2 increased the L-threonine accumulation concentration in the CGMCC25404 strain from 30.55 g / L to 31.72 g / L, and the L-threonine yield increased by 3.83%, which was a significant difference. Enhancing the expression intensity of the Pseudomonas aeruginosa The expression intensity of the aspDH19 gene derived from PAO1 increased the L-threonine accumulation concentration in the CGMCC25404 strain from 30.55 g / L to 31.7 g / L, and the L-threonine yield increased by 3.76%, which was a significant difference; enhancing the expression intensity of the aspDH20 gene derived from Delftia sp. Cs1-4 increased the L-threonine accumulation concentration in the CGMCC25404 strain from 30.55 to 30.66 g / L, which was not a significant difference and could not improve the L-threonine yield.

[0236] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Application, characterized by: The application includes any of the following: U1) Use of aspartate dehydrogenase in the preparation of L-threonine; U2) Use of a substance for regulating the activity and / or content of aspartate dehydrogenase in the preparation of L-threonine. The aspartate dehydrogenase is derived from at least the genus Pusillimona and / or Pseudomonas aeruginosa and / or the genus Pigmentiphaga.

2. The use according to claim 1, wherein the aspartate dehydrogenase comprises at least any one of the following: A1) a protein comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5 or SEQ ID NO: 8; A2) a protein having aspartate dehydrogenase function obtained by substitution and / or deletion and / or addition of amino acid residues in any one of the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 8, or more thereof, and derived from A1) or having 98% or greater identity with the protein set forth in A1); A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

3. Use of the biomaterial related to aspartate dehydrogenase according to claim 1, characterized in that: The application is the application of the biomaterial in any one of the following B1) to B3), B1) Application of the biomaterial in constructing an engineered bacterium producing L-threonine; B2) Application of the biomaterial in the preparation of L-threonine; B3) Application of the biomaterial in regulating the production of L-threonine in microorganisms; The biological material is at least one of the following C1) to C3): C1), a nucleic acid molecule encoding the glutamate dehydrogenase according to claim 1, C2), an expression cassette containing the nucleic acid molecule described in C1), C3), a recombinant vector containing the nucleic acid molecule described in C1) and / or a recombinant vector containing the expression cassette described in C2).

4. The use according to claim 3, characterized in that: The nucleic acid molecule described in C1) comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO.2, SEQ ID NO.6 or SEQ ID NO.

9.

5. Biomaterial, characterized in that: The biomaterial is the biomaterial according to claim 3 or 4.

6. A recombinant microorganism, characterized in that: The recombinant microorganism is any one of the following: D1), a recombinant microorganism containing the nucleic acid molecule according to D1) of claim 3 or 4; D2), a recombinant microorganism containing the expression cassette according to D2) of claim 3 or 4; D3), ​​a recombinant microorganism containing the recombinant vector described in D3) of claim 3 or 4; D4) A recombinant microorganism containing the aspartate dehydrogenase according to claim 1.

7. A whole-cell catalyst, comprising a whole-cell catalyst comprising the biomaterial according to claim 5, or a whole-cell catalyst comprising the recombinant microorganism according to claim 6.

8. A method for producing L-threonine, characterized in that: The method comprises utilizing the protein described in claim 1 or the biological material described in claim 5 to prepare L-threonine.

9. Use of the recombinant bacterium according to claim 6 or the whole-cell catalyst according to claim 7 in the preparation of L-amino acids.

10. A method for increasing the L-threonine production of a recipient microorganism, characterized in that: The method comprises introducing the biological material of claim 5 into the recipient microorganism.

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