Use of agla gene in preparation of l-amino acid

Abstract

Disclosed is a use of α-glucosidase gene and α-glucosidase or a substance for regulating the activity and / or content of α-glucosidase in the preparation of L-amino acids.

Description

Application of aglA gene in the preparation of L-amino acids Technical Field

[0001] The present application relates to the application of the aglA gene in the preparation of L-amino acids in the field of biotechnology. Background Art

[0002] L-amino acids have been used in the animal feed, pharmaceutical, and cosmetic industries, and are primarily produced by fermentation using bacteria such as Corynebacterium or Escherichia. Currently, the production of L-amino acids is primarily limited by low acid production and conversion. Improving substrate utilization efficiency, thereby increasing L-amino acid production during the fermentation process, remains a key issue that needs to be addressed.

[0003] SUMMARY OF THE INVENTION

[0004] The technical problem to be solved by this application is how to increase the yield of L-amino acids. The technical problem to be solved is not limited to the technical subject described above, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0005] To solve the above technical problems, this application provides the following technical solutions:

[0006] This application provides the following applications:

[0007] U1) Use of α-glucosidase in the preparation of L-amino acids;

[0008] U2) Use of the substance that regulates the activity of α-glucosidase in the preparation of L-amino acids;

[0009] U3) Use of the substance for regulating the content of α-glucosidase in the preparation of L-amino acids.

[0010] The above-mentioned α-glucosidase includes A1), A2) or A3):

[0011] A1) a protein having an amino acid sequence containing SEQ ID No. 5;

[0012] A2) a protein having the same function as that obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of SEQ ID No. 5, which is derived from A1) or has 45% or more identity with the protein of A1);

[0013] 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).

[0014] The above-mentioned 45% or more identity includes 47%-65% identity, 65% or more identity, 70% or more identity or 80% or more identity. The 47%-65% identity specifically includes 47% identity, 54% identity, 57% identity or 62% identity.

[0015] In the above applications, the greater than 45% identity can be at least 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In the above applications, the above 47%-65% identity may be 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% identity. In the above applications, the above 65% identity can be at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In the above applications, the 70% or greater identity may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In the above applications, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0016] 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 a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0017] In the present application, the regulation may include upregulating, enhancing or increasing the expression level of the aforementioned protein, and also includes downregulating, inhibiting or reducing the expression level of the aforementioned protein.

[0018] In the present application, the substance includes a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the cell nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated by the gene).

[0019] The L-amino acids in the above applications include polar neutral amino acids, acidic amino acids, basic amino acids and / or non-polar hydrophobic amino acids. The basic amino acids include lysine, arginine and / or histidine. The non-polar hydrophobic amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine and / or proline. The polar neutral amino acids include tryptophan, tyrosine, serine, threonine, cysteine, methionine (methionine), glutamine or asparagine. The acidic amino acids include glutamic acid or aspartic acid.

[0020] In the above application, the protein in A2) includes a protein having an amino acid sequence containing SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0021] The substance in the above application is a biological material related to the above protein, and the biological material includes B1), B2), B3), B4) or B5):

[0022] B1) a nucleic acid molecule encoding the protein; B2) an expression cassette containing the nucleic acid molecule of 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); 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); 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).

[0024] The expression cassette described in the biological material above refers to DNA capable of expressing the proteins described above in host cells. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for expressing any of the aforementioned proteins. The regulatory sequences are capable of directing the coding sequence to express any of the aforementioned 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 must include a promoter and termination signals for transcription and translation. 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 provided with a linker. 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 transcriptional 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 this application. 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 this application. 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 this application. 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 systems 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. In these instances, the protein-encoding nucleic acid sequence should be operably linked to the regulatory sequences.

[0025] The recombinant vector may include a nucleic acid molecule encoding the above-mentioned protein, a promoter, and transcription and translation termination signals. When preparing the recombinant vector, the nucleic acid molecule encoding the above-mentioned protein can be located in the vector so as to be operably linked to an appropriate expression control sequence. The recombinant vector can be any vector (e.g., a plasmid or virus) that is convenient for recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be a linear or closed-loop plasmid. The vector can be an autonomously replicating vector (i.e., a complete structure present outside the chromosome that can be replicated independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may include any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will be integrated into the genome and replicated together with the chromosome into which it is integrated. In addition, a single vector or plasmid can be used, or two or more vectors or plasmids, or transposons, that collectively comprise the entire DNA that will be introduced into the host cell genome can be used. The vector contains one or more selectable markers that are convenient for selecting transformed cells. A selectable marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or prototrophy to an auxotroph. Examples of bacterial selectable markers include the dal genes of Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. Vectors contain elements that enable the vector to be stably integrated into the host cell genome or to ensure autonomous replication of the vector in the cell, independent of the cellular genome. In the case of autonomous replication, the vector may also contain an origin of replication, enabling the vector to replicate autonomously in the target host cell. The origin of replication may contain a mutation that renders it temperature-sensitive in the host cell (see, for example, Ehrlich, 1978, Proceedings of the National Academy of Sciences of the United States of America 75:1433). One or more copies of a nucleic acid molecule encoding any of the above-described proteins may be inserted into the host cell to increase the production of the gene product. The number of copies of the nucleic acid molecule can be increased by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or by inserting an amplifiable selectable marker along with the nucleic acid molecule, and selecting cells containing amplified copies of the selectable marker gene and, thereby, the additional copies of the nucleic acid molecule by culturing the cells in the presence of a suitable selective agent. The procedures used to connect the above-mentioned elements to construct the recombinant expression vectors described herein are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0026] The term "operably linked" is defined herein as a configuration in which a regulatory sequence is appropriately positioned relative to the coding sequence of a DNA sequence such that the regulatory sequence directs the expression of a protein.

[0027] The whole-cell catalyst described in B5) above includes cells, which include microbial cells, plant cells, or animal cells. The animal cells do not include human reproductive cells, fertilized eggs, embryos, or individuals. The animal cells include animal somatic cells or cell lines.

[0028] The nucleic acid molecule described in B1) above includes any one of C1) to C6):

[0029] C1) a cDNA molecule or a DNA molecule whose coding sequence comprises SEQ ID No. 6;

[0030] C2) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 1;

[0031] C3) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 2;

[0032] C4) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 3;

[0033] C5) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 4;

[0034] C6) A cDNA molecule or DNA molecule that hybridizes with the cDNA or DNA molecule encoding the gene defined in C1) or C2) or C3) or C4) or C5) and encodes a protein having the same function.

[0035] The above proteins also fall within the protection scope of this application.

[0036] The above-mentioned biological materials also fall within the protection scope of this application.

[0037] The present application also provides a method for producing L-amino acids, comprising fermenting the aforementioned protein or the aforementioned biological material to prepare L-amino acids.

[0038] In the above method, the L-amino acid comprises L-glutamic acid, L-lysine or L-threonine.

[0039] The use of the above-mentioned protein or the above-mentioned biological material in the preparation of food, feed, medicine, fertilizer or daily chemical products containing L-amino acids also falls within the scope of protection of this application. Beneficial effects

[0040] The present application demonstrates through experiments that, compared to the target microorganism wild-type Corynebacterium glutamicum ATCC13032 and L-lysine-producing strain YP097158, the L-lysine production of recombinant Corynebacterium glutamicum YPL-aglA-1, YPL-aglA-2, YPL-aglA-3, and YPL-aglA-4 overexpressing the aglA-5 gene is significantly increased. Compared to the target microorganism wild-type Escherichia coli W3110 and Escherichia coli CGMCC No. 25404, the L-threonine production of recombinant Corynebacterium glutamicum YPT-aglA-1, YPT-aglA-2, YPT-aglA-3, and YPT-aglA-4 overexpressing the aglA-5 gene is significantly increased. Compared with the target microorganism wild-type Corynebacterium glutamicum ATCC13869 and L-lysine-producing strain CGMCC No.21220, the L-glutamic acid production of recombinant Corynebacterium glutamicum YPG-aglA-1, YPG-aglA-2, YPG-aglA-3 and YPG-aglA-4 overexpressing the aglA-5 gene was significantly improved.

[0041] Preservation Instructions

[0042] Bacterial species: Corynebacterium glutamicum

[0043] Latin name: Corynebacterium glutamicum

[0044] Strain ID: YP097158

[0045] Depository: General Microbiology Center of China Culture Collection Administration

[0046] Abbreviation of depository institution: CGMCC

[0047] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0048] Deposit date: August 16, 2016

[0049] Deposit number: CGMCC No.12856.

[0050] Bacterial species name: Corynebacterium glutamicum

[0051] Latin name: Corynebacterium glutamicum

[0052] Strain ID: YPGLU001

[0053] Depository: General Microbiology Center of China Culture Collection Administration

[0054] Abbreviation of depository institution: CGMCC

[0055] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0056] Date of deposit: November 23, 2020

[0057] Deposit number: CGMCC No.21220. Modes for Carrying Out the Invention

[0058] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0059] 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.

[0060] Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "a", "an" and "an" include plural referents. As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide, which is composed of a heterocyclic base, a sugar and one or more phosphate groups. The naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T) and uracil (U)) are generally derivatives of purines or pyrimidines, but it is understood that natural and non-naturally occurring base analogs are also included. Naturally occurring sugars are pentoses (five-carbon sugars), deoxyribose (which forms DNA) or ribose (which forms RNA), but it is understood that natural and non-naturally occurring sugar analogs are also included. Nucleic acids are generally linked by phosphodiester bonds to form nucleic acids or polynucleotides, but may also be linked by other bonds known in the art, such as phosphorothioate bonds.

[0061] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid (i.e., a polynucleotide sequence) to which it is attached. As used herein, the term "fermentation" broadly refers to the conversion of an organic material into a target substance by a host cell, for example, by growing a culture of recombinant host cells in a medium that includes a carbon source, such that the recombinant host cells convert the carbon source into lysine.

[0062] Wild-type Corynebacterium glutamicum ATCC13032 strain: a product of the American type culture collection (ATCC).

[0063] The Corynebacterium glutamicum strain YP097158 in the following examples is described in the Chinese patent document entitled "A Recombinant Strain for High-yield L-Lysine, Its Construction Method, and Application" with grant number "CN110607313B." The strain number is YP097158 and was deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection on August 16, 2016, under the deposit number CGMCC No. 12856. Hereinafter, it is referred to as Corynebacterium glutamicum CGMCC No. YP097158.

[0064] The Corynebacterium glutamicum CGMCC No. 21220 (Corynebacterium glutamicum) in the following examples is recorded in the Chinese patent document with authorization number "CN112725253B" and titled "A recombinant strain with modified gene BBD29_14900 and its construction method and application". The strain number is YPGLU001 and was deposited in the General Microbiology Center of the China Culture Collection Administration on November 23, 2020, with the deposit number CGMCC No. 21220. hereinafter referred to as Corynebacterium glutamicum CGMCC No. 21220.

[0065] The data in the following examples were processed using GraphPad Prism 8 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.01 indicated a highly significant difference.

[0066] Example 1: Construction of an engineered strain containing an aglA vector

[0067] 1. Construction of aglA gene vector

[0068] In order to study the utilization effect of aglA on isomaltose, the aglA gene sequence in the Bifidobacterium adolescentis genome published by NCBI was used as a template to analyze its protein function and structure. Five aglA genes were designed and synthesized, namely aglA-1 (nucleotide sequence is SEQ ID No.7, encoding the amino acid shown in SEQ ID No.1), aglA-2 (nucleotide sequence is SEQ ID No.8, encoding the amino acid shown in SEQ ID No.2), aglA-3 (nucleotide sequence is SEQ ID No.9, encoding the amino acid shown in SEQ ID No.3), aglA-4 (nucleotide sequence is SEQ ID No.10, encoding the amino acid shown in SEQ ID No.4), and aglA-5 (nucleotide sequence is shown in SEQ ID No.6, encoding the amino acid shown in SEQ ID No.5).

[0069] The five synthesized aglA genes (with H36 promoter during synthesis, SEQ ID No. 50) were ligated with the expression vector pXMJ19 (purchased from Biovector, catalog number .Biovector pXMJ19, containing chloramphenicol resistance) recovered by digestion with Xbal I and BamH I using NEBuilder enzyme (purchased from NEB) at 50°C for 30 min. The ligation products were transformed into DH5α competent cells, spread onto 2-YT agar plates containing chloramphenicol (34 mg / L), and cultured at 37°C for 12 h. Single clones grown in the culture were identified by PCR using primers MJ19-F (5'-GCGGATAACAATTTCACACAG-3', SEQ ID No. 11) / MJ19-R (5'-CTCTCATCCGCCAAAACAG-3', SEQ ID No. 12). The PCR amplification system was 2× Premix rTaq 12.5 μL, 1 μL of each primer (10 pM), and ddH2O to a total volume of 25 μL. PCR amplification program: 94°C pre-denaturation for 15 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 2 min (30 cycles), and 72°C over-extension for 10 min.

[0070] The plasmids of the identified positive strains were extracted and sent for sequencing, and the correctly sequenced plasmids were named pXMJ19-aglA-1 (containing the gene sequence shown in SEQ ID No.7), pXMJ19-aglA-2 (containing the gene sequence shown in SEQ ID No.8), pXMJ19-aglA-3 (containing the gene sequence shown in SEQ ID No.9), pXMJ19-aglA-4 (containing the gene sequence shown in SEQ ID No.10), and pXMJ19-aglA-5 (containing the gene sequence shown in SEQ ID No.6).

[0071] pXMJ19-aglA-1 is a recombinant expression vector obtained by replacing the sequence between the Xbal I and BamH I restriction enzyme recognition sites of pXMJ19 with SEQ ID No. 7, while maintaining the remaining nucleotide sequence. This vector expresses the protein with the amino acid sequence of SEQ ID No. 1. pXMJ19-aglA-2 is a recombinant expression vector obtained by replacing the sequence between the Xbal I and BamH I restriction enzyme recognition sites of pXMJ19 with SEQ ID No. 8, while maintaining the remaining nucleotide sequence. This vector expresses the protein with the amino acid sequence of SEQ ID No. 2. pXMJ19-aglA-3 is a recombinant expression vector obtained by replacing the sequence between the Xbal I and BamH I restriction enzyme recognition sites of pXMJ19 with SEQ ID No. 9, while maintaining the remaining nucleotide sequence. This vector expresses the protein with the amino acid sequence of SEQ ID No. 3. pXMJ19-aglA-4 is a recombinant expression vector obtained by replacing the sequence between the Xbal I and BamH I restriction enzyme recognition sites of pXMJ19 with SEQ ID No. 10 while keeping other nucleotide sequences unchanged. This vector expresses the protein with the amino acid sequence of SEQ ID No. 4.

[0072] pXMJ19-aglA-5 is a recombinant expression vector obtained by replacing the sequence between the Xbal I and BamH I restriction enzyme recognition sites of pXMJ19 with SEQ ID No. 6 while keeping other nucleotide sequences unchanged. This vector expresses the protein with the amino acid sequence of SEQ ID No. 5.

[0073] 2. Construction of ATCC13032 strain containing aglA gene vector

[0074] Culture plates containing chloramphenicol (34 mg / L): the solvent is water, the solutes and their concentrations are sucrose 10 g / L, polypeptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, urea 2 g / L, sodium chloride 2.5 g / L, agar powder 18 g / L, chloramphenicol 34 mg / L, pH 7.0.

[0075] Rich medium: the solvent is water, the solutes and their concentrations are glucose 25 g / L, isomaltose 5 g / L, (NH4)2SO4 2 g / L, H3PO4 0.5 g / L, KCl 0.8 g / L, MgSO4·7H2O 0.8 g / L, FeSO4·7H2O 0.05 g / L, MnSO4·H2O 0.05 g / L, FM902 yeast powder 1.5 g / L, corn steep liquor 5 g / L, molasses 17 g / L, betaine 0.5 g / L, citric acid 2 g / L, VH 20 mg / L, VB1 1.5 mg / L, VB3 1.5 mg / L, VB 12 1.5g / L, sodium hydroxide was used to adjust pH to 7.0.

[0076] In order to identify the utilization effect of the five pXMJ19-aglA vectors constructed in step 1 on isomaltose, the five vectors were transformed into the wild-type Corynebacterium glutamicum ATCC13032 strain by electroporation (for the specific transformation method, see WO2014121669A1), and PCR identification was performed using primers MJ19-F / MJ19-R to obtain positive transformants, namely Corynebacterium glutamicum ATCC13032-pXMJ19-aglA-1, ATCC13032-pXMJ19-aglA-2, ATCC13032-pXMJ19-aglA-3, ATCC13032-pXMJ19-aglA-4 and ATCC13032-pXMJ19-aglA-5.

[0077] The above five positive transformants were cultured on a culture plate containing chloramphenicol (34 mg / L) at 32°C for 12 h, which was considered as one generation. After three consecutive passages, they were inoculated into a 500 mL Erlenmeyer flask containing 30 mL of rich culture medium and fermented in a shake flask at 30°C for 48 h. After the fermentation culture was completed, the concentration of isomaltose was detected by high performance liquid chromatography (HPLC).

[0078] Table 1 Detection of isomaltose content in ATCC13032-pXMJ19-aglA by HPLC

[0079] The results are shown in Table 1. All five Corynebacterium glutamicum ATCC13032-pXMJ19-aglA strains have the ability to decompose isomaltose, among which ATCC13032-pXMJ19-aglA-5 has the best ability to decompose isomaltose, indicating that the aglA-5 gene has the highest activity in degrading isomaltose.

[0080] 3. Construction of ATCC13869 strain containing aglA gene vector

[0081] In order to identify the utilization effect of the five vectors pXMJ19-aglA constructed in step 1 on isomaltose, the five vectors were transformed into the wild-type Corynebacterium glutamicum ATCC13869 strain (CICC, product number 20216) by electroporation, and PCR identification was performed using primers MJ19-F / MJ19-R to obtain positive transformants ATCC13869-pXMJ19-aglA-1, ATCC13869-pXMJ19-aglA-2, ATCC13869-pXMJ19-aglA-3, ATCC13869-pXMJ19-aglA-4 and ATCC13869-pXMJ19-aglA-5. Five positive transformants were inoculated on a culture plate containing chloramphenicol (34 mg / L). After culturing at 32°C for 12 h, a single clone was picked and streaked for culture. After three consecutive passages, the culture was inoculated into a 500 mL Erlenmeyer flask containing 30 mL of rich culture medium. The culture was shaken at 30°C for 48 h. After the fermentation, the concentration of isomaltose was detected by high performance liquid chromatography (HPLC).

[0082] Table 2 Detection of isomaltose content in ATCC13869-pXMJ19-aglA by HPLC

[0083] The results are shown in Table 2. All five Corynebacterium glutamicum ATCC13869-pXMJ19-aglA strains have the ability to decompose isomaltose, among which ATCC13869-pXMJ19-aglA-5 has the best ability to decompose isomaltose, indicating that the aglA-5 gene has the highest activity in degrading isomaltose.

[0084] 4. Construction of W3110 strain containing aglA gene vector

[0085] In order to identify the utilization effect of the five vectors pXMJ19-aglA constructed in step 1 on isomaltose, the five vectors were transformed into the wild-type Escherichia coli W3110 strain (ATCC, catalog number 27325) by electroporation, and PCR identification was performed using primers MJ19-F / MJ19-R to obtain positive transformants W3110-pXMJ19-aglA-1, W3110-pXMJ19-aglA-2, W3110-pXMJ19-aglA-3, W3110-pXMJ19-aglA-4 and W3110-pXMJ19-aglA-5. After five positive transformants were serially passaged three times on 2YT culture plates (chloramphenicol 34 mg / L), they were inoculated into a 500 mL Erlenmeyer flask containing 30 mL of rich culture medium and fermented at 37°C for 24 h. After the fermentation, the concentration of isomaltose was detected by high performance liquid chromatography (HPLC).

[0086] Table 3 HPLC detection of isomaltose content in W3110-pXMJ19-aglA

[0087] As shown in Table 3, all five E. coli W3110-pXMJ19-aglA strains were able to degrade isomaltose, with W3110-pXMJ19-aglA-5 showing the highest isomaltose degradation activity, indicating that the aglA-5 gene has the highest isomaltose degradation activity. The data in Tables 1, 2, and 3 indicate that the aglA-5 gene is capable of degrading isomaltose, with the highest activity, in all C. glutamicum ATCC13032, ATCC13869, and E. coli W3110.

[0088] Example 2: Application of the aglA-5 gene in preparing L-lysine-producing bacteria

[0089] 1. Construction of an engineered strain expressing the aglA-5 gene on a plasmid

[0090] The pXMJ19-aglA-5 plasmid constructed in Example 1 was electroporated into wild-type Corynebacterium glutamicum ATCC13032 and Corynebacterium glutamicum YP097158 (L-lysine producing strain, deposit number: CGMCC No. 12856, deposit date: August 16, 2016, depositor: General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355), cultured on a culture plate containing chloramphenicol (34 mg / L) for 30 h, and single colonies produced by the culture were identified by PCR using primers MJ19-F / MJ19-R (MJ19-F is located at 6570-6590 bp in the pXMJ19 vector, and MJ19-R is located at 69-87 bp in the pXMJ19 vector). PCR amplified a 2014 bp fragment (wherein bases 1-141 are SEQ ID No. 45, bases 142-1962 are the aglA-5 gene with the nucleotide sequence shown in SEQ ID No. 6, and bases 1963-2014 are SEQ ID No.46) was a positive strain.

[0091] The strains obtained using wild-type Corynebacterium glutamicum ATCC13032 and L-lysine-producing strain YP097158 as starting bacteria were named YPL-aglA-1 and YPL-aglA-2, respectively. The recombinant strain YPL-aglA-1 contains a plasmid encoding the aglA gene shown in SEQ ID No. 6, which significantly and stably increases the expression level of the aglA gene in wild-type Corynebacterium glutamicum ATCC13032. The recombinant strain YPL-aglA-2 contains a plasmid encoding the aglA gene shown in SEQ ID No. 6, which significantly and stably increases the expression level of the aglA gene in L-lysine-producing strain YP097158.

[0092] 2. Construction of an engineered strain expressing the aglA-5 gene

[0093] Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, three pairs of primers were designed and synthesized to amplify upstream and downstream homology arm fragments and the coding region and promoter region of the aglA-5 gene. Copies of the aglA-5 gene were inserted into L-lysine-producing bacteria YP097158 and wild-type Corynebacterium glutamicum ATCC13032 by homologous recombination.

[0094] The primers were designed as follows (synthesized by Shanghai Invitrogen):

[0095] P1: 5′-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATGCGTTCTGGACTGAGG-3′ (the underlined nucleotide sequence is the sequence on pK18, SEQ ID No. 13);

[0096] P2:5'-CGTTTAGGGCACCAGATAGAgtgcaccgagaacagatg-3' (SEQ ID No. 14);

[0097] P3:5'-catctgttctcggtgcacTCTATCTGGTGCCCTAAACG-3' (SEQ ID No. 15);

[0098] P4:5'-cttgatttaattgcgccatctgTTACAGCTGCACTGCTGCTGCTC-3' (SEQ ID No. 16);

[0099] P5:5'-GAAGCAGCAGCAGTGCAGCTGTAAcagatggcgcaattaaatcaag-3' (SEQ ID No. 17);

[0100] P6: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCTATGACACCTTCAACGGATC-3' (SEQ ID No. 18).

[0101] In the above primers, the underlined nucleotide sequence is the homologous sequence on the pK18mobsacB plasmid, which is used to integrate the DNA fragment (upstream homologous arm, promoter-aglA-5 gene, downstream homologous arm) into the pK18mobsacB plasmid (Addgene) by homologous recombination. The bases indicated by lowercase letters on P2 and P3 are used to connect the upstream homologous arm and the promoter-aglA-5 gene by homologous recombination. The lowercase letters on P4 and P5 are used to connect the promoter-aglA-5 gene and the downstream homologous arm by homologous recombination.

[0102] Construction method: The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and PCR amplification was performed with primers P1 / P2 and P5 / P6, respectively, to obtain an upstream homology arm fragment of 763 bp (corresponding to positions 1928220 to 1928982 of the genome sequence of Corynebacterium glutamicum ATCC13032 (GenBank: CP025533.1, 27-DEC-2017), i.e., SEQ ID No. 47) and a downstream homology arm fragment of 596 bp (corresponding to positions 1929024 to 1929619 of the genome sequence of Corynebacterium glutamicum ATCC13032 (GenBank: CP025533.1, 27-DEC-2017), i.e., SEQ ID No. 49). Using plasmid pXMJ19-aglA-5 as a template, PCR amplification with primers P3 / P4 yielded a 1901-bp fragment of the aglA-5 gene and its promoter (i.e., promoter-aglA-5 gene). Positions 1-80 correspond to SEQ ID No. 48, and positions 81-1901 correspond to the aglA-5 gene (SEQ ID No. 6). After completion of the PCR reaction, the three amplified fragments were recovered by electrophoresis using a column-based DNA gel recovery kit. The three recovered fragments were ligated with the pK18mobsacB plasmid (Addgene) purified after digestion with Xbal I and BamH I using NEBuilder enzyme (NEB) at 50°C for 30 min. The monoclonal clone grown after transformation of the ligation product was identified by PCR using primers M13F (5′-TGTAAAACGACGGCCAGT-3′, SEQ ID No. 19) / M13R (5′-CAGGAAACAGCTATGACC-3′, SEQ ID No. 20) to obtain a positive integration plasmid (recombinant vector). The resulting recombinant vector is pK18-aglA(L). This positive integration plasmid contains a kanamycin resistance marker, and recombinants with the plasmid integrated into the genome can be obtained by kanamycin screening. The pK18-aglA(L) plasmid is a recombinant expression vector in which the sequence between the Xbal I and BamH I restriction enzyme recognition sites of the pK18mobsacB plasmid is replaced by a DNA fragment, while other sequences remain unchanged. The DNA fragment is formed by homologous recombination of an upstream homology arm (SEQ ID No. 47, 763 bp), the aglA-5 gene (SEQ ID No. 6) and its promoter fragment (SEQ ID No. 48, 80 bp), and a downstream homology arm (partial coding region of the NCgl1742 gene, 596 bp) in a 5'-3' direction.

[0103] PCR amplification system: 5×HiFi with Mg 2+Buffer 10μL, dNTP Mixture (10mM) 1.5μL, primers (10pM) 1.6μL each, KAPA HiFi HotStart (1U / μL) 0.5μL, add ddH2O to a total volume of 50μL.

[0104] PCR amplification program: pre-denaturation at 95°C for 5 min, (denaturation at 98°C for 20 s; annealing at 56°C for 15 s; extension at 72°C for 60 s; 30 cycles), over-extension at 72°C for 5 min.

[0105] The correctly sequenced integration plasmid pK18-aglA (L) was electrotransformed into Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum ATCC13032, respectively, and cultured on a culture plate containing chloramphenicol (34 mg / L) for 30 hours. The single colonies produced by the culture were subjected to PCR identification using P7 / P8 primers. The strains that amplified a fragment of 1488 bp (sequence: SEQ ID No. 50) by PCR were positive, while those that failed to amplify the fragment were the original strains. The positive strains were streaked and cultured on a solid culture plate containing 15% sucrose for 30 hours. The single colonies produced by the culture were further subjected to PCR identification using P9 / P10 primers. The strains that amplified a fragment of 1537 bp (sequence: SEQ ID No. 51) were positive strains in which the aglA-5 gene and its promoter were integrated into the spacer region between the homology arm NCgl1741 and the lower homology arm NCgl1742 on the Corynebacterium glutamicum genome. The strains obtained using wild-type Corynebacterium glutamicum ATCC13032 and lysine-producing strain YP097158 as starting bacteria were named YPL-aglA-3 and YPL-aglA-4, respectively.

[0106] The recombinant bacteria YPL-aglA-3 and YPL-aglA-4 contain the aglA gene shown in SEQ ID No. 6; specifically, the recombinant bacteria YPL-aglA-3 is obtained by replacing the spacer region of the upper homologous arm NCgl1741 and the lower homologous arm NCgl1742 in the genome of wild-type Corynebacterium glutamicum ATCC13032 with the aglA-5 gene and its promoter (SEQ ID No. 48 (80 bp) and SEQ ID No. 6 connected in the 5'-3' direction), that is, the nucleotide sequence from positions 1928983 to 1929023 of the genome sequence of Corynebacterium glutamicum ATCC13032 (GenBank: CP025533.1, 27-DEC-2017) is replaced by the H36 promoter sequence (SEQ ID No. 48, 80 bp) and SEQ ID The recombinant strain YPL-aglA-4 is a recombinant strain obtained by replacing the spacer region of the upper homology arm NCgl1741 and the lower homology arm NCgl1742 in the genome of Corynebacterium glutamicum YP097158 (i.e., the nucleotide sequence 1928983-1929023 corresponding to the genome sequence of Corynebacterium glutamicum ATCC13032 (GenBank: CP025533.1, 27-DEC-2017)) with the aglA-5 gene and its promoter (the sequence is a DNA fragment formed by connecting the H36 promoter sequence (SEQ ID No. 48, 80 bp) and SEQ ID No. 6 in the 5'-3' direction), while keeping the other nucleotides in the genome of Corynebacterium glutamicum YP097158 unchanged.

[0107] PCR identification primers are as follows:

[0108] P7: 5′-TCCAAGGAAGATACACGCC-3′ (corresponding to the outer side of the upper homology arm NCgl1740, located at 1928088-1928106 bp in the genome of Corynebacterium glutamicum ATCC13032, SEQ ID No. 21);

[0109] P8: 5′-GCTACCACCAAAGTAAGAGCC-3′ (corresponding to the interior of the aglA gene, located at 493-513 bp of the coding region, SEQ ID No. 22);

[0110] P9: 5′-TTGTGATGGCGTGAAGTG-3′ (corresponding to the interior of the aglA gene, located at 972-989 bp of the coding region, SEQ ID No. 23);

[0111] P10: 5′-TGGTCGTTGGAATCTTGC-3′ (corresponding to the outer side of the lower homology arm NCgl1742, located at 1929693-1929710 bp in the genome of Corynebacterium glutamicum ATCC13032, SEQ ID No. 24).

[0112] 3. L-lysine fermentation experiment

[0113] YPL-aglA-1 and YPL-aglA-2 constructed in step 1, YPL-aglA-3 and YPL-aglA-4 constructed in step 2, L-lysine-producing strain YP097158, and wild-type Corynebacterium glutamicum ATCC13032 were fermented in a BLBIO-5GC-4-H model fermentor (Shanghai Bailun Biotechnology Co., Ltd.) using the following culture medium and L-lysine fermentation process. After the fermentation, the L-lysine content was detected using an SBA biosensor (purchased from the Institute of Biology, Shandong Academy of Sciences). Each strain was repeated three times, and the results are shown in Table 4. Fermentation medium formula (the rest is water): starch hydrolyzed sugar 30g / L, ammonium sulfate 12g / L, magnesium sulfate 0.87g / L, molasses 20g / L, acidified corn syrup 3mL / L, phosphoric acid 0.4mL / L, potassium chloride 0.53g / L, defoamer (2% flocculant) 4mL / L, ferrous sulfate 120mg / L, manganese sulfate 120mg / L, nicotinamide 42mg / L, calcium pantothenate 6.3mg / L, vitamin B16.3mg / L, copper and zinc salt solution 0.6g / L, biotin 0.88mg / L.

[0114] L-lysine fermentation process: Fermentation conditions: temperature 37°C, time 48h, pH 6.9±0.05, dissolved oxygen DO 10-30%, inoculation size 10% (volume ratio); initial control conditions: temperature 37°C, pH 6.9, tank pressure 0 MPa, air volume 3 L / min, speed 550 rpm; dissolved oxygen control: when dissolved oxygen <30%, increase the speed in sequence from 750 rpm → 800 rpm → 850 rpm → 950 rpm, air volume 4 L / min; tank pressure control: increase the tank pressure by 0.01 MPa after 6h of fermentation; increase the tank pressure by 0.02 MPa → 0.03 MPa → 0.04 MPa → 0.05 MPa after 12h; residual sugar control: 0.1-0.2% before 12h of fermentation; after 12h of fermentation, control the residual sugar to 0.1-0.05% based on DO requirements; ammonia nitrogen control: 0.1-0.15 before 12h of fermentation; after 12h-32h of fermentation 0.15-0.25; 0.1-0.15 after 32 hours of fermentation; fed materials: 25% ammonia water, 70% concentrated sugar, 50% ammonium sulfate, 10% antifoaming agent.

[0115] Table 4 L-lysine fermentation data of aglA engineered strain

[0116] The results are shown in Table 4. Overexpression of the aglA gene in wild-type Corynebacterium glutamicum ATCC13032 and L-lysine-producing strain YP097158 both contributed to the improvement of L-lysine production.

[0117] Example 3: Application of the aglA-5 gene in preparing L-threonine-producing bacteria

[0118] 1. Construction of an engineered strain expressing the aglA-5 gene on a plasmid

[0119] 2YT agar plate formula: yeast 10 g / L, peptone 16 g / L, sodium chloride 5 g / L, agarose 18 g / L, adjust the pH to 7.0 with NaOH, and autoclave at 121°C for 15 min.

[0120] The pXMJ19-aglA-5 plasmid successfully constructed in Example 1 was electroporated into wild-type Escherichia coli W3110 (ATCC, Catalog No. 27325) and Escherichia coli CGMCC No. 25404 (i.e., Escherichia coli, deposit number: CGMCC No. 25404, deposit date: July 25, 2022, deposited by: General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807288), and cultured on 2YT agar plates for 18 h. The single colonies produced by the culture were identified by PCR using primers MJ19-F / MJ19-R, and a 2014 bp fragment (wherein bases 1-141 are SEQ ID No. 45, and bases 142-1962 are nucleotide sequences as shown in SEQ ID Strains expressing the aglA-5 gene shown in SEQ ID No. 6, wherein bases 1963-2014 are SEQ ID No. 46, were positive. Strains obtained using wild-type Escherichia coli W3110 and threonine-producing strain CGMCC25404 as starting strains were named YPT-aglA-1 and YPT-aglA-2, respectively. Recombinant strain YPT-aglA-1 contained a plasmid containing the aglA gene shown in SEQ ID No. 6, and was able to significantly and stably increase the expression level of the aglA gene in wild-type Escherichia coli W3110. Recombinant strain YPT-aglA-2 contained a plasmid containing the aglA gene shown in SEQ ID No. 6, and was able to significantly and stably increase the expression level of the aglA gene in L-threonine-producing strain CGMCC25404.

[0121] 2. Construction of an engineered strain expressing the aglA-5 gene

[0122] Based on the Escherichia coli W3110 genome sequence published by NCBI, the aglA-5 gene was integrated into the yaiT gene coding region of the L-threonine-producing strain CGMCC25404 and wild-type Escherichia coli W3110 using CRISPR / Cas9 gene editing technology to further study the effect of the aglA-5 gene on L-threonine synthesis.

[0123] (1) Construction of sgRNA

[0124] Based on the Escherichia coli W3110 genome sequence published by NCBI (genbank accession number AP009048.1), sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting an appropriate sgRNA target sequence, linearized pGRB cloning vector end sequences were added to the 5' and 3' ends of the target sequence to form a complete sgRNA plasmid through recombination.

[0125] To amplify the sgRNA fragment, no template is required; only PCR annealing is required. The system and procedure are as follows. The PCR reaction system: 10 μL sgRNA-2F, 10 μL sgRNA-2R; PCR procedure: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute. After annealing, the target fragment was recovered using a DNA purification kit, the DNA concentration was determined, and the sample was diluted to 100 ng / μL.

[0126] The pGRB plasmid was extracted and digested with Spe I and dephosphorylated to prevent self-ligation. The digestion system consisted of 5 μL of 10x Buffer, 2.5 μL of Spe I, 3000-5000 ng of pGRB plasmid DNA, and ddH2O supplemented to 50 μL. After digestion at 37°C for 3 hours, the gel was excised and recovered by agarose gel electrophoresis. The dephosphorylation reaction was performed using the following system: 5 μL of 10x Buffer, 1000-2000 ng of pGRB plasmid DNA, 2.5 μL of CIAP, and ddH2O supplemented to 50 μL. After treatment at 37°C for 1 hour, the linearized pGRB plasmid was recovered using a DNA purification kit. The sgRNA and pGRB plasmid were then recombined using the Gibson Assembly Kit (New England Biolabs). The recombination system consisted of 2.5 μL of NEB assembly enzyme, 2 μL of linearized cloning vector, and 0.5 μL of sgRNA. After assembling at 50°C for 30 minutes, the product was transformed into DH5α competent cells, and the plasmid was extracted and sequenced using sequencing primers sgRNA-PF / sgRNA-PR. The correctly sequenced plasmid was named pGRB-sgRNA-1.

[0127] The primers used in this experiment were designed as follows (synthesized by Shanghai Invitrogen). The underlined bases are the homology arm sequences of the pGRB cloning vector, and the bases in lowercase are the sgRNA sequences, targeting positions 389640 to 389659 of the W3110 genomic sequence (GenBank ID AP009048.1):

[0128] sgRNA-2F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTggcaactatgtaaactatagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID No. 25);

[0129] sgRNA-2R:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACctatagtttacatagttgccACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3'' (SEQ ID No. 26);

[0130] sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3' (SEQ ID No. 27);

[0131] sgRNA-PR: 5'-ACTGGCACTGCTGTGCGCCG-3' (SEQ ID No. 28).

[0132] (2) PCR amplification of homologous DNA fragments

[0133] Based on the Escherichia coli W3110 genome sequence published by NCBI, three pairs of primers were designed and synthesized to amplify upstream and downstream homology arm sequences and the coding and promoter regions of the aglA-5 gene. The aglA-5 gene was introduced into the coding regions of the L-threonine-producing strain CGMCC25404 and Escherichia coli W3110yaiT, respectively, using CRISPR / Cas9 gene editing.

[0134] The primers were designed as follows (synthesized by Shanghai Invitrogen):

[0135] P11:5'-AAGAGAATGGAAGAGGCC-3' (SEQ ID No. 29);

[0136] P12:5'-CGTTTAGGGCACCAGATAGAcccaatcaagtgctgtaacg-3' (SEQ ID No. 30);

[0137] P13:5'-cgttacagcacttgattgggTCTATCTGGTGCCCTAAACG-3' (SEQ ID No. 31);

[0138] P14:5'-cggtagtgtaggtttcgttgTTACAGCTGCACTGCTGCTGCTTC-3' (SEQ ID No. 32);

[0139] P15:5'-GAAGCAGCAGCAGTGCAGCTGTAAcaacgaaacctacactaccg-3' (SEQ ID No. 33);

[0140] P16: 5'-CGACCTGTAGTATCCCATTC-3' (SEQ ID No. 34).

[0141] Using W3110 genomic DNA as a template, PCR amplification was performed with primers P11 / P12 and P15 / P16, respectively, and the enzyme used was KAPA HiFi HotStart (KAPA, Catalog No. KR1501), obtaining an upper homology arm fragment of 570 bp (SEQ ID No. 52, i.e., positions 389041 to 389610 of the W3110 genome) and a lower homology arm fragment of 585 bp (SEQ ID No. 53, i.e., positions 390148 to 390733 of the W3110 genome). Using plasmid pXMJ19-aglA-5 as a template, PCR amplification was performed with primers P13 / P14 to obtain a 1901 bp fragment of the aglA-5 gene and its promoter (of which positions 1-80 are the H36 promoter (80 bp), and positions 81-1901 are the aglA-5 gene (SEQ ID No. 6)). After the PCR reaction, the three recovered DNA fragments were recovered by agarose gel electrophoresis using a column-based DNA gel recovery kit. Overlap PCR with primers P11 and P16 yielded the 3056-bp recombinant DNA fragment Up-aglA-Down (the sequence of which is composed of SEQ ID No. 52, SEQ ID No. 48, SEQ ID No. 6 (1821 bp), and SEQ ID No. 53 connected in a 5'-3' direction).

[0142] PCR amplification system: 5×HiFi with Mg 2+ Buffer 10μL, dNTP Mixture (10mM) 1.5μL, primers (10pM) 1.6μL each, KAPA HiFi HotStart (1U / μL) 0.5μL, add ddH2O to a total volume of 50μL.

[0143] PCR amplification program: pre-denaturation at 95°C for 5 min, (denaturation at 98°C for 20 s; annealing at 56°C for 15 s; extension at 72°C for 60 s; 30 cycles), over-extension at 72°C for 5 min.

[0144] (3) Preparation and transformation of competent cells

[0145] 2-YT agar plate containing spectinomycin (100 mg / L): yeast 10 g / L, peptone 16 g / L, sodium chloride 5 g / L, agarose 18 g / L, spectinomycin 100 mg / L, adjust the pH to 7.0 with NaOH, and autoclave at 121°C for 15 min.

[0146] The pREDCas9 plasmid (containing the spectinomycin resistance gene) (Addgene, Catalog No. 71541) was extracted and transformed into L-threonine-producing bacteria CGMCC25404 and Escherichia coli W3110 competent cells, respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies resistant to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF (5'-GCAGTGGCGGTTTTCATG-3', SEQ ID No. 35) / pRedCas9-PR (5'-CCTTGGTGATCTCGCCTTTC-3', SEQ ID No. 36). SEQ ID No. 54 was obtained, which was the CGMCC25404-Cas9 and W3110-Cas9 transformants containing the pREDCas9 plasmid.

[0147] Prepare L-threonine production bacteria CGMCC25404-Cas9 and W3110-Cas9 competent cells. When the bacteria grow to OD 600nm = 0.1, add IPTG to a final concentration of 0.1 mM to induce λ-Red-mediated homologous recombination. 600nm =0.4, the bacteria were collected to prepare competent cells, and the pGRB-sgRNA-1 plasmid and the genomic recombinant fragment Up-aglA-Down were transformed respectively. The cells were spread on 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 hours. After subculturing the single colonies produced by the culture, PCR identification was performed using primers P11 / P16. A fragment containing 3056 bp (whose sequence was connected in the 5'-3' direction by SEQ ID No.52 (570 bp), SEQ ID No.48 (80 bp), SEQ ID No.6 (1821 bp) and SEQ ID No.53 (585 bp)) was amplified by PCR as a positive transformant.

[0148] PCR amplification system: 12.5 μL of 2× Premix r Taq, 1 μL of each primer (10 pM), supplemented with ddH2O for a total volume of 25 μL. PCR amplification program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 3 min (30 cycles), and overextension at 72°C for 10 min.

[0149] Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg / L) and arabinose at a final concentration of 0.2% to eliminate the plasmid pGRB-sgRNA-1. Colonies that grew on spectinomycin (100 mg / L) but not on ampicillin (100 mg / L) were selected, and these colonies were transferred to 2-YT medium and cultured at 42°C to eliminate the pREDCas9 plasmid. Colonies that did not grow on spectinomycin (100 mg / L) but grew on antibiotic-free 2-YT were selected and again identified by PCR using primers P11 / P16. A positive amplification of 3056 bp was identified, and the positive amplification was sent for sequencing. Strains with correct sequencing results were named YPT-aglA-3 (starting with Escherichia coli W3110) and YPT-aglA-4 (starting with L-threonine-producing strain CGMCC25404).

[0150] The recombinant bacteria YPT-aglA-3 and YPT-aglA-4 contain the aglA-5 gene shown in SEQ ID No. 6; specifically, the recombinant bacteria YPT-aglA-3 is a wild-type Escherichia coli W3110 genome yaiT partial coding region (i.e., W3110 genome sequence (genbank number AP009048.1) from 389611 to 390147) replaced with the aglA-5 gene and its promoter (wherein positions 1-80 are H36 promoter (SEQ ID No. 48, 80 bp), positions 81-1901 are aglA-5 gene (SEQ ID No. 6), while maintaining the other nucleotides in its genome unchanged. The recombinant strain YPT-aglA-4 is obtained by replacing the yaiT coding region (corresponding to positions 389611 to 390147 of the W3110 genome sequence (genbank number AP009048.1)) in the genome of the L-threonine-producing strain CGMCC25404 with the aglA-5 gene and its promoter (wherein positions 1-80 are the H36 promoter (SEQ ID No. 48, 80 bp) and positions 81-1901 are the aglA-5 gene (SEQ ID No. 6)), while maintaining the other nucleotides in its genome unchanged. Recombinant strains containing the aglA-5 gene can significantly and stably increase the expression level of the aglA gene.

[0151] 3. L-threonine fermentation experiment

[0152] YPT-aglA-1 and YPT-aglA-2 constructed in step 1, YPT-aglA-3 and YPT-aglA-4 constructed in step 2, wild-type Escherichia coli W3110, and L-threonine-producing bacteria CGMCC25404 were inoculated into a 5L fermenter of the BLBIO-5GC-4-H model (Shanghai Bailun Biotechnology Co., Ltd.) using L-threonine fermentation medium and culture conditions for fermentation experiments, and each strain was repeated three times.

[0153] L-threonine fermentation medium: the solvent is water, the solutes and their concentrations are glucose 13 g / L, (NH4)2SO4 1 g / L, H3PO4 0.5 g / L, KCl 0.8 g / L, MgSO4·7H2O 0.8 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·H2O 0.01 g / L, ZnSO4 0.05 g / L, FM902 yeast powder 1.5 g / L, corn steep liquor 5 g / L, molasses 17 g / L, and the pH is adjusted to 7.0 by adding ammonia.

[0154] L-threonine fermentation culture conditions: Calibration of DO 100%: temperature 37°C, air volume 5 L / min, rotation speed 800 rpm, tank pressure 0 MPa, calibration after 5 minutes; inoculum size 10%; initial conditions: pH 7.0, culture temperature 37°C, tank pressure 0 MPa, air volume 0.5 L / min, rotation speed 400 rpm; full-process control: 1. When dissolved oxygen is <30%, increase the rotation speed in the following order: 500 rpm → 600 rpm → air volume 1 L / min → 700 rpm → 800 rpm; 2. After 8 hours of fermentation, the tank pressure is increased by 0.01Mpa; after 12 hours, the tank pressure is increased by 0.02Mpa→0.03Mpa→0.04Mpa→0.05Mpa; residual sugar control: 0.1-0.5% before 12 hours of fermentation; after 12 hours of fermentation, the residual sugar is controlled at 0.1-0.3% based on DO requirements; fed materials: 25% ammonia water, 55% concentrated sugar, 10% folate; fermentation cycle: about 30 hours, and the control process uses 20-30% dissolved oxygen as the standard for increasing and decreasing air volume.

[0155] Table 5 L-threonine fermentation data of aglA engineered strain

[0156] The results are shown in Table 5. Overexpression of the aglA-5 gene in wild-type Escherichia coli W3110 and L-threonine-producing strain CGMCC25404 both contributed to the improvement of L-threonine production.

[0157] Example 4: Application of the aglA-5 gene in preparing L-glutamic acid-producing bacteria

[0158] 1. Construction of an engineered strain expressing the aglA-5 gene on a plasmid

[0159] The pXMJ19-aglA-5 plasmid successfully constructed in Example 1 was electroporated into wild-type Corynebacterium glutamicum ATCC13869 and Corynebacterium glutamicum CGMCC No.21220 (glutamate-producing bacteria, deposit number: CGMCC No.21220, deposit date: November 23, 2020, deposit unit: General Microbiology Center of China Culture Collection Administration Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807288), and cultured on a culture plate containing chloramphenicol (34 mg / L) for 30 h. The single colonies produced by the culture were identified by PCR using primers MJ19-F / MJ19-R, and a 2014 bp fragment (wherein bases 1-141 are SEQ ID No.45, and bases 142-1962 are nucleotide sequences such as SEQ ID Strains with the aglA-5 gene shown in No. 6, where bases 1963-2014 are SEQ ID No. 46, were positive. Strains obtained using wild-type Corynebacterium glutamicum ATCC13869 and glutamic acid-producing strain CGMCC No. 21220 as starting bacteria were named YPG-aglA-1 and YPG-aglA-2, respectively.

[0160] The recombinant bacterium YPG-aglA-1 contains a plasmid carrying the aglA gene shown in SEQ ID No. 6, and can significantly and stably increase the expression level of the aglA gene in wild-type Corynebacterium glutamicum ATCC13869; the recombinant bacterium YPG-aglA-2 contains a plasmid carrying the aglA gene shown in SEQ ID No. 6, and can significantly and stably increase the expression level of the aglA gene in glutamate-producing bacteria CGMCC No. 21220.

[0161] 2. Construction of an engineered strain expressing the aglA-5 gene

[0162] Based on the genome sequence of Corynebacterium glutamicum ATCC13869 published by NCBI, three pairs of primers were designed and synthesized to amplify upstream and downstream homology arm fragments and the coding region and promoter region of the aglA-5 gene. Copies of the aglA-5 gene were inserted into the glutamate-producing strain CGMCC No. 21220 and the wild-type Corynebacterium glutamicum ATCC13869 by homologous recombination.

[0163] The primers were designed as follows (synthesized by Shanghai Invitrogen):

[0164] P17:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGACCCGCTTGCCATACGAAG-3' (SEQ ID No. 37);

[0165] P18:5'-CGTTTAGGGCACCAGATAGAatctactcatctgaagaatc-3' (SEQ ID No. 38);

[0166] P19:5'-gattcttcagatgagtagatTCTATCTGGTGCCCTAAACG-3' (SEQ ID No. 39);

[0167] P20:5'-caaaccagagtgcccacgaaTTACAGCTGCACTGCTGCTGCTTC-3' (SEQ ID No. 40);

[0168] P21:5'-GAAGCAGCAGCAGTGCAGCTGTAAttcgtgggcactctggtttg-3' (SEQ ID No. 41);

[0169] P22: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCATAAGAAACAACCACTTCC-3' (SEQ ID No. 42).

[0170] In the above primers, the underlined nucleotide sequence is the homologous sequence on pK18, which is used to integrate the DNA fragment (upstream homologous arm, promoter-aglA-5 gene, downstream homologous arm) into the pK18mobsacB plasmid (Addgene) by homologous recombination. The bases indicated by lowercase letters on P18 and P19 are used to connect the upstream homologous arm and the promoter-aglA-5 gene by homologous recombination. The lowercase letters on P20 and P21 are used to connect the promoter-aglA-5 gene and the downstream homologous arm by homologous recombination.

[0171] Construction method: The genome of Corynebacterium glutamicum CGMCC No. 21220 was used as a template, and PCR amplification was performed with primers P17 / P18 and P21 / P22, respectively, to obtain a 750 bp upstream homology arm fragment (corresponding to positions 731131 to 731880 of the genome sequence of Corynebacterium glutamicum ATCC13869 (genbank number CP016335.1), i.e., the BBD29_03485 gene, whose sequence is SEQ ID No. 55) and a 731 bp downstream homology arm fragment (corresponding to positions 732071 to 732801 of the genome sequence of Corynebacterium glutamicum ATCC13869 (genbank number CP016335.1), i.e., the BBD29_03490 gene, whose sequence is SEQ ID No. 56). Using plasmid pXMJ19-aglA-5 as a template and primers P19 / P20, PCR amplification was performed to obtain a 1901 bp fragment of the aglA-5 gene and its promoter (i.e., promoter-aglA-5 gene). Positions 1-80 correspond to the H36 promoter (SEQ ID No. 48, 80 bp), and positions 81-1901 correspond to the aglA-5 gene (SEQ ID No. 6). After the PCR reaction, the three amplified fragments were recovered by electrophoresis using a column-based DNA gel recovery kit. The three recovered fragments were ligated with the pK18mobsacB plasmid (Addgene) purified after digestion with Xbal I and BamH I using NEBuilder enzyme (NEB) at 50°C for 30 min. The monoclonal clone grown after transformation of the ligation product was identified by PCR using primers M13F (5′-TGTAAAACGACGGCCAGT-3′, SEQ ID No. 19) / M13R (5′-CAGGAAACAGCTATGACC-3′, SEQ ID No. 20) to obtain a positive integration plasmid (recombinant vector). The resulting recombinant vector is pK18-aglA(G). This positive integration plasmid contains a kanamycin resistance marker, and recombinants with the plasmid integrated into the genome can be obtained by kanamycin screening. The pK18-aglA(G) plasmid is a recombinant expression vector in which the sequence between the Xbal I and BamH I restriction enzyme recognition sites of the pK18mobsacB plasmid is replaced by a DNA fragment while keeping other sequences unchanged. The DNA fragment is formed by homologous recombination of SEQ ID No. 55 (750 bp), the aglA-5 gene and its promoter fragment (1901 bp), and the base sequence SEQ ID No. 56 (731 bp) in the 5'-3' direction.

[0172] PCR amplification system: 5×HiFi with Mg 2+Buffer 10 μL, dNTP Mix (10 mM) 1.5 μL, primers (10 pM) 1.6 μL each, KAPA HiFi HotStart (1 U / μL) 0.5 μL, and ddH₂O to a total volume of 50 μL. PCR amplification program: pre-denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 98°C for 20 s, annealing at 56°C for 15 s, and extension at 72°C for 60 s, followed by over-extension at 72°C for 5 min.

[0173] The integration plasmid pK18-aglA (G) with correct sequencing was electrotransformed into Corynebacterium glutamicum CGMCC No. 21220 and wild-type Corynebacterium glutamicum ATCC 13869, respectively. The cells were cultured on a culture plate containing chloramphenicol (34 mg / L) for 30 h. Single colonies produced by the culture were identified by PCR using P23 / P8 primers. A positive strain was identified by PCR amplification of a 1423 bp fragment (identification primers P23 and P8 amplified fragment), while a positive strain was identified by PCR amplification of no fragment. The positive strain was streaked on a solid culture plate containing 15% sucrose for 30 h. Single colonies produced by the culture were further identified by PCR using P9 / P24 primers. A positive strain was identified by PCR amplification of a 1640 bp fragment (sequence as SEQ ID No. 58) containing the aglA-5 gene and its promoter integrated into the spacer region of the homology arm BBD29_03485 and the lower homology arm BBD29_03490 on the Corynebacterium glutamicum genome. The strains obtained using wild-type Corynebacterium glutamicum ATCC13869 and glutamate-producing bacteria CGMCC No.21220 as starting bacteria were named YPG-aglA-3 and YPG-aglA-4, respectively.

[0174] The recombinant bacteria YPG-aglA-3 and YPG-aglA-4 contain the aglA gene shown in SEQ ID No. 6; specifically, the recombinant bacteria YPG-aglA-3 is obtained by replacing the spacer region of the upper homologous arm BBD29_03485 and the lower homologous arm BBD29_03490 in the wild-type Corynebacterium glutamicum ATCC13869 genome with the aglA-5 gene and its promoter (wherein positions 1-80 are the H36 promoter (SEQ ID No. 48, 80 bp), and positions 81-1901 are the aglA-5 gene (SEQ ID No. 8)), that is, the nucleotide sequence from positions 731881 to 732070 of the Corynebacterium glutamicum ATCC13869 genome sequence (genbank number CP016335.1) is replaced with the nucleotide sequence of SEQ ID No. 48 and SEQ ID No.6 is a recombinant bacterium obtained by connecting the DNA molecules in the 5'-3' direction while keeping the other nucleotides in the genome of Corynebacterium glutamicum ATCC13869 unchanged. The recombinant bacterium YPG-aglA-4 is obtained by replacing the spacer region between the upper homology arm BBD29_03485 and the lower homology arm BBD29_03490 in the genome of Corynebacterium glutamicum CGMCC No. 21220 (corresponding to positions 731881 to 732070 of the genome sequence of Corynebacterium glutamicum ATCC13869) with the aglA-5 gene and its promoter (wherein positions 1-80 are the H36 promoter (SEQ ID No. 48, 80 bp), and positions 81-1901 are the aglA-5 gene (SEQ ID No. 8)), while keeping the other nucleotides in the genome of Corynebacterium glutamicum CGMCC No. 21220 unchanged. Recombinant bacteria that overexpress the aglA-5 gene in the genome can significantly and stably increase the expression level of the aglA gene.

[0175] PCR identification primers are as follows:

[0176] P23: 5′-GTCCAAGGTGACGGCCGCAC-3′ (corresponding to the outer side of the upper homology arm BBD29_03485, SEQ ID No. 43);

[0177] P8: 5′-GCTACCACCAAAGTAAGAGCC-3′ (corresponding to the interior of the aglA gene, SEQ ID No. 22);

[0178] P9: 5′-TTGTGATGGCGTGAAGTG-3′ (corresponding to the interior of the aglA gene, SEQ ID No. 23);

[0179] P24: 5′-ATATTCGGCCCAGCAGCAGC-3′ (corresponding to the outer side of the lower homology arm BBD29_03490, SEQ ID No. 44).

[0180] 3. L-glutamic acid fermentation experiment

[0181] Fermentation experiments were performed using YPG-aglA-1 and YPG-aglA-2 constructed in step 1, YPG-aglA-3 and YPG-aglA-4 constructed in step 2, wild-type Corynebacterium glutamicum ATCC13869, and glutamate-producing bacteria CGMCC No. 21220 in a BLBIO-5GC-4-H fermentor (purchased from Shanghai Bailun Biotechnology Co., Ltd.) using the following culture medium and the control process shown in Table 6. Each strain was replicated three times. Fermentation medium formula (the rest is water): glucose 5.0g / L, phosphoric acid 0.38g / L, magnesium sulfate 1.85g / L, potassium chloride 1.6g / L, biotin 550μg / L, vitamin B1 300μg / L, ferrous sulfate 10mg / L, manganese sulfate 10g / dl, KH2PO4 2.8g / L, vitamin C 0.75mg / L, vitamin B12 2.5μg / L, p-aminobenzoic acid 0.75mg / L, defoaming agent 0.0015ml / dl, betaine 1.5g / L, cane molasses 7ml / L, corn steep liquor 77ml / L, aspartic acid 1.7g / L, hair powder 2g / L.

[0182] Table 6 Fermentation control process

[0183] Table 7 L-glutamic acid fermentation data of aglA engineered strain

[0184] The results are shown in Table 7. Overexpression of the aglA gene in the wild-type Corynebacterium glutamicum ATCC13869 and the L-glutamic acid producing strain CGMCC No. 21220 contributes to the improvement of L-glutamic acid production.

[0185] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experiments, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In short, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including changes that depart from the disclosed scope in the present application and are made using conventional techniques known in the art.

[0186] Cross-reference to related applications:

[0187] This application claims priority to a Chinese patent application filed with the Patent Office of China on December 8, 2023, with application number 202311679969.1 and invention name "Application of aglA gene in the preparation of L-amino acids". The entire contents of the patent application are hereby incorporated herein by reference. Industrial Applicability

[0188] The present application can significantly increase the yield of L-amino acids.

Claims

1. Application, characterized in that, The application includes any of the following: U1) Application of α-glucosidase in the preparation of L-amino acids; U2) Use of the substance that regulates the activity of α-glucosidase in the preparation of L-amino acids; U3) Use of the substance for regulating the content of α-glucosidase in the preparation of L-amino acids.

2. The use according to claim 1, characterized in that: The regulation includes upregulating, enhancing or increasing the expression level of the α-glucosidase.

3. The use according to claim 2, characterized in that: The α-glucosidase includes any one of the following: A1) a protein whose amino acid sequence is SEQ ID No. 5; A2) a protein having the same function as that obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 5, and derived from A1) or having 45% or more identity with the protein shown 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).

4. The use according to claim 3, characterized in that: The L-amino acids include polar neutral amino acids, acidic amino acids, basic amino acids and / or non-polar hydrophobic amino acids.

5. The use according to claim 4, characterized in that: The basic amino acids include lysine, arginine and / or histidine, or the polar neutral amino acids include tryptophan, tyrosine, serine, threonine, cysteine, methionine, glutamine or asparagine.

6. The use according to claim 4, characterized in that: The acidic amino acid includes glutamic acid or aspartic acid.

7. The use according to claim 3, characterized in that: A2) The protein comprises a protein having an amino acid sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No.

4.

8. The use according to claim 7, characterized in that: The substance is a biological material related to the protein, and the biological material includes any one of the following: B1) a nucleic acid molecule encoding the protein; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); 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); 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).

9. The use according to claim 8, characterized in that: B1) The nucleic acid molecule comprises any one of C1) to C6): C1) the coding sequence comprises SEQ ID No.6; C2) the coding sequence comprises a coding gene whose amino acid sequence is SEQ ID No. 1; C3) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 2; C4) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 3; C5) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 4; C6) A cDNA molecule or a DNA molecule that hybridizes with the cDNA or DNA molecule defined in C1) or C2) or C3) or C4) or C5) and encodes a protein having the same function.

10. A protein, characterized in that The protein includes any of the following: A1) a protein whose amino acid sequence is SEQ ID No. 5; A2) a protein having the same function as that obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 5, and derived from A1) or having 45% or more identity with the protein shown 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).

11. The protein according to claim 10, characterized in that A2) The protein comprises a protein having an amino acid sequence of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No.

4.

12. Biomaterial, characterized in that The biological material is a biological material related to the protein according to claim 10, and the biological material includes any one of the following: B1) a nucleic acid molecule encoding the protein; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); 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); 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).

13. The biomaterial according to claim 12, characterized in that B1) The nucleic acid molecule comprises any of the following: C1) the coding sequence comprises SEQ ID No.6; C2) the coding sequence comprises a coding gene whose amino acid sequence is SEQ ID No. 1; C3) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 3; C4) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 4; C5) the coding sequence comprises a gene encoding an amino acid sequence of SEQ ID No. 5; C6) A cDNA molecule or a DNA molecule that hybridizes with the cDNA or DNA molecule defined in C1) or C2) or C3) or C4) or C5) and encodes a protein having the same function.

14. A method for producing L-amino acids, characterized in that: The method comprises using the biological material according to claim 12 to ferment and prepare L-amino acids.

15. The method according to claim 14, characterized in that The L-amino acid includes L-glutamic acid, L-lysine or L-threonine.

16. A method for producing L-amino acids, characterized in that: The method comprises using the protein of claim 10 to prepare L-amino acids.

17. The method according to claim 16, characterized in that The L-amino acid includes L-glutamic acid, L-lysine or L-threonine.

18. Use of the protein according to claim 10 in the preparation of food, feed, medicine, fertilizer or / and daily chemical products containing L-amino acids.

19. Use of the biomaterial according to claim 12 in the preparation of food, feed, medicine, fertilizer or / and daily chemical products containing L-amino acids.

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