PROTEÍNA, BIOMATERIAL ASSOCIADO À PROTEÍNA, MÉTODO PARA PREPARAR L-LISINA, PRODUTO PARA PREPARAÇÃO DE L-LISINA , APLICAÇÕES DA PROTEÍNA, APLICAÇÃO DO BIOMATERIAL, APLICAÇÃO DO MÉTODO E APLICAÇÃO DO PRODUTO

Abstract

Provided are a TKT gene mutant and an application thereof in the preparation of L-lysine. The TKT gene mutant is a DNA molecule as represented by SEQ ID NO: 3, 5, 7, 9, 11, or 13 in the sequence listing, which encodes a protein as represented by SEQ ID NO: 4, 6, 8, 10, 12, or 14, and a wild type TKT gene is a DNA molecule as represented by SEQ ID NO: 1, which encodes a protein represented by SEQ ID NO: 2. It has been experimentally verified that the TKT gene and the mutant thereof can improve the yield of L-lysine and can be used for the production of L-lysine, and same have good application prospects.

Description

1 / 48 “PROTEIN, PROTEIN-ASSOCIATED BIOMATERIAL, METHOD FOR PREPARING L-LYSINE, PRODUCT FOR PREPARING L-LYSINE, APPLICATIONS OF THE PROTEIN, APPLICATIONS OF THE BIOMATERIAL, APPLICATION OF THE METHOD AND APPLICATION OF THE PRODUCT” Cross-References to Related Requests

[001] This application claims priority over the Chinese patent application (application number: 202310261458.1), filed on March 17, 2023, the full content of which is incorporated herein by reference. Field of Invention

[002] The present invention relates to the field of biotechnology and, more particularly, to a mutant of the TKT gene and an application thereof in the preparation of L-lysine. Background of the Invention

[003] L-lysine has physiological effects such as promoting development, strengthening immunity, and improving the functions of the central nervous system. It is one of the eight essential amino acids that humans and animals cannot synthesize on their own and are necessary for growth. Currently, L-lysine, the second largest variety of amino acids in the world, is mainly produced by fermentation, with Corynebacterium glutamicum being an important lysine-producing strain. L-lysine, responsible for approximately 90% of industrial production, is used as a nutritional enhancer in the animal feed industry, 10% as an umami agent and a sweetener in the food industry, and as an intermediate for drugs in the pharmaceutical industry.

[004] Improvements in L-lysine production by fermentation methods may involve fermentation techniques such as agitation and oxygen supply; or involve the composition of a nutrient medium, such as sugar concentration during fermentation; or involve processing. Petition 870250093954, dated 10 / 14 / 2025, pp. 69 / 122 2 / 48 of the fermentation broth in suitable product forms, for example, by drying and granulating the fermentation broth or ion-exchange chromatography; or involve the intrinsic performance properties of the related microorganisms themselves.

[005] The methods used to improve the performance properties of these microorganisms include mutagenesis, mutant selection, and screening. Strains obtained in this way are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and producers of L-lysine. Brief Description of the Invention

[006] An object of the present invention is to provide a protein that can be used for the production of L-lysine, the protein being called TKT protein, and the TKT protein being A1) or A2) or A3) as follows: A1) a protein comprising (or being) SEQ ID No. 2, or a mutant protein obtained by mutating an alanine residue at position 327 in SEQ ID No. 2 into a threonine residue, a serine residue, a cysteine ​​residue, a proline residue, an asparagine residue, a glutamine residue, a phenylalanine residue, a leucine residue, a valine residue, an isoleucine residue, an aspartic acid residue, a methionine residue, an arginine residue, a glutamic acid residue, a glycine residue, a histidine residue, a lysine residue, a tryptophan residue, or a tyrosine residue; A2) a protein that is obtained by substitution and / or deletion and / or addition of one or more amino acid residues in an amino acid sequence of the protein from A1) except for position 327 and has the same function as A1); and A3) a fusion protein obtained by attaching a tag to an N-terminal and / or a C-terminal of A1) or A2). Petition 870250093954, dated 10 / 14 / 2025, pp. 70 / 122 3 / 48

[007] To facilitate the purification of the A1 protein), the following tag: Poly-Arg, Poly-His, FLAG, Strep-tag II or c-myc can be attached to an amino terminus or a carboxyl terminus of the protein.

[008] Protein A2) above is a protein that has 75% or more identity with an amino acid sequence of protein A1) and has the same function as A1). The 75% or more identity can be 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

[009] The protein above A2) can be synthesized artificially or it can be obtained by synthesizing its coding gene first and then performing biological expression.

[0010] The gene encoding the protein above (A2) can be obtained by deleting codons from one or more amino acid residues in a DNA sequence represented by SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 or SEQ ID No. 1, and / or by nonsense mutations of one or more base pairs, and / or by ligating coding sequences of markers shown in the table above to a 5' end and / or a 3' end of the protein. Wherein, the DNA molecules represented by SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, and SEQ ID No. 1 encode proteins represented by SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, and SEQ ID No. 2, respectively.

[0011] The present invention further provides a biomaterial associated with a TKT protein. The biomaterial is any one of B1) to B4), as follows: B1) a nucleic acid molecule that codes for the TKT protein; B2) an expression cassette containing the nucleic acid molecule from B1); B3) a recombinant vector containing the nucleic acid molecule Petition 870250093954, dated 10 / 14 / 2025, pp. 71 / 122 4 / 48 of B1), or a recombinant vector containing the B2 expression cassette); and B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2, or a recombinant microorganism containing the recombinant vector of B3).

[0012] In the biomaterial above, the nucleic acid molecule of B1) can be any one of b11)-b19) as follows: b11) a DNA molecule as represented by SEQ ID No. 3 in a sequence listing; b12) a DNA molecule as represented by SEQ ID No. 5 in the sequence listing; b13) a DNA molecule as represented by SEQ ID No. 7 in the sequence listing; b14) a DNA molecule as represented by SEQ ID No. 9 in the sequence listing; b15) a DNA molecule as represented by SEQ ID No. 11 in the sequence listing; b16) a DNA molecule as represented by SEQ ID No. 13 in the sequence listing; b17) a DNA molecule as represented by SEQ ID No. 1 in the sequence listing; b18) a DNA molecule that has 75% or more identity with a nucleotide sequence defined by either b11)-b17) and encodes the TKT protein; and b19) a genomic DNA molecule that hybridizes with the nucleotide sequence defined by either b11)-b18) under stringent conditions and encodes the TKT protein.

[0013] The nucleic acid molecule can be DNA, such as cDNA, Petition 870250093954, dated 10 / 14 / 2025, pp. 72 / 122 5 / 48 Genomic DNA or recombinant DNA; and the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0014] A person skilled in the art can easily mutate the nucleotide sequence encoding the TKT protein of the present invention using known methods, such as directed evolution or point mutation. Artificially modified nucleotides that exhibit identity of 75% or more with the nucleotide sequence of the TKT protein of the present invention, provided they are capable of encoding the TKT protein and have a function for it, are all derived from the nucleotide sequences of the present invention and are equivalent to the sequences of the present invention.

[0015] The term “identity,” as used herein, refers to the similarity of a sequence to a natural nucleic acid sequence. “Identity” includes a nucleotide sequence having 75% or more identity, or 85% or more, or 90% or more, or 95% or more with the nucleotide sequence encoding the TKT protein of the present invention. Identity can be assessed by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

[0016] For the biomaterial above, the stringent conditions may be as follows: hybridization at 50 °C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 and 1 mM EDTA and rinsing at 50 °C in 2xSSC and 0.1% SDS; or hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M NaPO4 and 1 mM EDTA and rinsing at 50 °C in 1xSSC and 0.1% SDS; or hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M NaPO4 and 1 mM EDTA and rinsing at 50 °C in 0.5xSSC and 0.1% SDS; or hybridize at 50 °C in a mixed solution of 7% SDS, 0.5 M NaPO4 and 1 mM EDTA, and rinse at 50 °C in 0.1x SSC and 0.1% SDS; or hybridize at 50 °C in a Petition 870250093954, dated 10 / 14 / 2025, pp. 73 / 122 6 / 48 mixed solution of 7% SDS, 0.5 M NaPO4 and 1 mM EDTA, and rinse at 65 °C in 0.1xSSC and 0.1% SDS; or hybridize at 65 °C in a solution of 6xSSC and 0.5% SDS, and then wash a film once with 2xSSC and 0.1% SDS, and 1xSSC and 0.1% SDS, respectively; or hybridize at 68 °C in a solution of 2xSSC and 0.1% SDS, and wash a film twice, 5 minutes each time, and then hybridize at 68 °C in a solution of 0.5xSSC and 0.1% SDS, and wash a film twice, 15 minutes each time; or hybridize at 65 °C in a solution of 0.1xSSPE (or 0.1xSSC) and 0.1% SDS, and wash a film.

[0017] An identity above 75% or more may be an identity of 80%, 85%, 90% or 95% or more.

[0018] In the biomaterial above, the B2 expression cassette (TKT gene expression cassette), which contains the nucleic acid molecule encoding the TKT protein, refers to the DNA capable of expressing the TKT protein in a host cell; and this DNA may include not only a promoter that initiates transcription of a TKT gene, but also a terminator that stops transcription of the TKT gene. In addition, the expression cassette may also include an enhancer sequence.

[0019] In the biomaterial above, the promoter in the B2 expression cassette is a DNA molecule shown by positions 35-437 in SEQ ID No. 15.

[0020] In the biomaterial above, the vector can be a plasmid, a cosmid, a bacteriophage, or a viral vector. The plasmid can specifically be a pXMJ19 or pK18mobsacB plasmid.

[0021] The recombinant B3 vector) can be pXMJ19-TKT, pXMJ19-TKTA327T, pXMJ19-TKTA327S, pXMJ19-TKTA327C, pXMJ19-TKTA327P, pXMJ19-TKTA327N, pXMJ19-TKTA327Qor pK18-TKTA327T; pXMJ19-TKT is a recombinant vector obtained by replacing a DNA fragment between the recognition sequences. Petition 870250093954, dated 10 / 14 / 2025, pp. 74 / 122 7 / 48 Xbal I and BamH I of pXMJ19 by a DNA fragment as represented by SEQ ID No. 15; pXMJ19-TKTA327T differs from pXMJ19-TKT where pXMJ19-TKTA327T is a recombinant vector that is obtained by replacing a gene as represented by SEQ ID No. 1 in pXMJ19-TKT with a gene as represented by SEQ ID No. 3; pXMJ19-TKTA327S differs from pXMJ19-TKT where pXMJ19TKTA327S is a recombinant vector that is obtained by replacing the gene represented by SEQ ID No. 1 in pXMJ19-TKT with a gene represented by SEQ ID No. 5; pXMJ19-TKTA327C differs from pXMJ19-TKT where pXMJ19TKTA327C is a recombinant vector that is obtained by replacing the gene represented by SEQ ID No. 1 in pXMJ19-TKT with a gene represented by SEQ ID No. 7; pXMJ19-TKTA327P differs from pXMJ19-TKT where pXMJ19TKTA327P is a recombinant vector that is obtained by replacing the gene represented by SEQ ID No. 1 in pXMJ19-TKT with a gene represented by SEQ ID No. 9; pXMJ19-TKTA327N differs from pXMJ19-TKT where pXMJ19TKTA327N is a recombinant vector that is obtained by replacing the gene represented by SEQ ID No. 1 in pXMJ19-TKT with a gene represented by SEQ ID No. 11; pXMJ19-TKTA327Q differs from pXMJ19-TKT where pXMJ19TKTA327Q is a recombinant vector obtained by replacing the gene represented by SEQ ID No. 1 in pXMJ19-TKT with a gene represented by SEQ ID No. 13; and pK18-TKTA327T is a recombinant vector obtained by replacing a fragment (small fragment) between the sites of Petition 870250093954, dated 10 / 14 / 2025, pp. 75 / 122 8 / 48 recognition Xbal I and BamH I of a pK18mobsacB vector by a DNA fragment as represented by SEQ ID No. 6 in the sequence listing, while keeping other sequences of the pK18mobsacB vector unchanged.

[0022] In the above biomaterial, the microorganism can be yeast, bacteria, algae or fungi. The bacteria can be Escherichia coli, Corynebacterium glutamicum, Brevibacterium lactofermentum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum, Pantoea, Pantoea ananatis, Bacillus brevis, Lactobacillus brevis or Brevibacterium flavum. The yeast can be Saccharomyces cerevisiae or Pichia pastoris.

[0023] In an example of the present invention, Corynebacterium glutamicum is Corynebacterium glutamicum YP097158 or Corynebacterium glutamicum ATCC13032.

[0024] The recombinant microorganism of B4) is a recombinant microorganism that is obtained by replacing a TKT gene in the microorganism containing the tkt gene, as represented by SEQ ID No. 1, with that represented by SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13, or a recombinant microorganism that is obtained by introducing the nucleic acid molecule of B1) into the microorganism and by expressing the nucleic acid molecule of B1).

[0025] In an example of the present invention, the recombinant microorganism is the recombinant bacteria ATCC13032-pXMJ19-TKTA327T, ATCC13032-pXMJ19-TKTA327S, ATCC13032-pXMJ19-TKTA327C, ATCC13032pXMJ19-TKTA327P, ATCC13032-pXMJ19-TKTA327N, ATCC13032-pXMJ19TKTA327Q, YPL-TKT-1, TKT-1, YPL-TKT-2, YPL-TKT-3, TKT-2, TKT-3, YPL-TKT4, TKT-4, YPL-TKT-5 or TKT-5; ATCC13032-pXMJ19-TKTA327Té is a recombinant bacterium obtained by introducing pXMJ19-TKTA327Tem Corynebacterium glutamicum. Petition 870250093954, dated 10 / 14 / 2025, pp. 76 / 122 9 / 48 ATCC13032; ATCC13032-pXMJ19-TKTA327 is a recombinant bacterium obtained by introducing pXMJ19-TKTA327 into Corynebacterium glutamicum ATCC13032; ATCC13032-pXMJ19-TKTA327C is a recombinant bacterium obtained by introducing pXMJ19-TKTA327C into Corynebacterium glutamicum ATCC13032; ATCC13032-pXMJ19-TKTA327P is a recombinant bacterium obtained by introducing pXMJ19-TKTA327P into Corynebacterium glutamicum ATCC13032; ATCC13032-pXMJ19-TKTA327N is a recombinant bacterium obtained by introducing pXMJ19-TKTA327N into Corynebacterium glutamicum ATCC13032; ATCC13032-pXMJ19-TKTA327Q is a recombinant bacterium obtained by introducing pXMJ19-TKTA327Q into Corynebacterium glutamicum ATCC13032; YPL-TKT-1 differs from Corynebacterium glutamicum YP097158 only in that YPL-TKT-1 is a strain obtained by replacing a gene from Corynebacterium glutamicum YP097158, as represented by SEQ ID No. 1, with a gene as represented by SEQ ID No. 3, while keeping other sequences unchanged; and TKT-1 differs from Corynebacterium glutamicum ATCC13032 only in that TKT-1 is a strain obtained by replacing one gene of Corynebacterium glutamicum ATCC13032, represented by SEQ ID No. 1, with a gene represented by SEQ ID No. 3, while keeping other sequences unchanged; YPL-TKT-2 is a recombinant bacterium obtained by replacing a spacer region between an NCgl1741 upper homologous arm and a Petition 870250093954, dated 10 / 14 / 2025, p. 77 / 122 10 / 48 lower homologous arm NCgl1742 in a Corynebacterium glutamicum YP097158 genome by a DNA fragment as represented by SEQ ID No. 16 in the sequence listing, and retaining other nucleotides unchanged; YPL-TKT-3 is a recombinant bacterium obtained by replacing the spacer region between an upper homologous arm NCgl1741 and a lower homologous arm NCgl1742 in the genome of Corynebacterium glutamicum YP097158 with a DNA fragment as represented by SEQ ID No. 17 in the sequence listing, while keeping other nucleotides unchanged; TKT-2 is a recombinant bacterium obtained by replacing a spacer region between an upper homologous arm NCgl1741 and a lower homologous arm NCgl1742 in a Corynebacterium glutamicum ATCC13032 genome with a DNA fragment as represented by SEQ ID No. 16 in the sequence listing, while leaving other nucleotides unchanged; TKT-3 is a recombinant bacterium obtained by replacing the spacer region between the upper homologous arm NCgl1741 and the lower homologous arm NCgl1742 in the genome of Corynebacterium glutamicum ATCC13032 with a DNA fragment as represented by SEQ ID No. 17 in the sequence listing, while keeping other nucleotides in the genome of Corynebacterium glutamicum ATCC13032 unchanged; YPL-TKT-4 is a recombinant bacterium obtained by introducing pXMJ19-TKT into Corynebacterium glutamicum YP097158; YPL-TKT-5 is a recombinant bacterium obtained by introducing pXMJ19-TKTA327Tem Corynebacterium glutamicum YP097158; TKT-4 is a recombinant bacterium obtained by introducing pXMJ19-TKT into Corynebacterium glutamicum ATCC13032; and TKT-5 is a recombinant bacterium obtained by introducing pXMJ19-TKTA327Tem Corynebacterium glutamicum ATCC13032. Petition 870250093954, dated 10 / 14 / 2025, pp. 78 / 122 11 / 48

[0026] The present invention further provides a method for the preparation of L-lysine. The method includes: expressing a TKT protein in a recipient biological cell, or increasing the content or activity of the TKT protein in the recipient biological cell, or increasing the content or activity of the protein represented by SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 2 in the recipient biological cell to obtain a recombinant biological cell; and culturing the recombinant biological cell to obtain L-lysine.

[0027] In the method above, the biological cell can be yeast, bacteria, algae, fungus, a plant cell, or an animal cell that can synthesize L-lysine.

[0028] A biological cell is any biological cell that can synthesize a target amino acid.

[0029] In the method above, the bacterium is Corynebacterium glutamicum.

[0030] The bacteria of the present invention include, but are not limited to, Corynebacterium glutamicum. L-lysine can be produced from any mutant TKT protein, as represented by SEQ ID No. 2, 4, 6, 8, 10, 12 or 14, and associated biomaterial of the present invention, which contains the TKT gene, as represented by SEQ ID No. 1 in the sequence listing, and is capable of synthesizing L-lysine. The bacteria may be Escherichia coli, Escherichia coli, Corynebacterium glutamicum, Brevibacterium lactofermentum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum, Pantoea, Pantoea ananatis, Bacillus brevis, Lactobacillus brevis, or Brevibacterium flavum. The yeast could be Saccharomyces cerevisiae or Pichia pastoris.

[0031] In an example of the present invention, Corynebacterium glutamicum is Corynebacterium glutamicum YP097158 or Corynebacterium Petition 870250093954, dated 10 / 14 / 2025, pp. 79 / 122 12 / 48 glutamicum ATCC13032.

[0032] The above method can be implemented by introducing a gene encoding the TKT protein into the recipient biological cell and expressing this encoding gene, or by introducing the gene encoding the protein represented by SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 2 into the recipient biological cell and expressing this encoding gene; or the recipient biological cell contains the DNA molecule as represented by SEQ ID No. 1, and the method is implemented by replacing a DNA molecule represented by SEQ ID No. 1 in the recipient biological cell with a DNA molecule represented by SEQ ID No. 3 or SEQ ID No. 5 or SEQ ID No. 7 or SEQ ID No. 9 or SEQ ID No. 11 or SEQ ID No. 13.

[0033] In the above method, the recombinant biological cell can be cultured using a medium that allows the recombinant biological cell to grow; and / or the recombinant biological cell is cultured under conditions that allow the recombinant biological cell to grow.

[0034] The recombinant biological cell may be the recombinant microorganism described above.

[0035] The present invention also provides a product for preparing L-lysine, the product containing (or its active ingredient being) TKT or the biomaterial.

[0036] The application of TKT or the biomaterial in the production of L-lysine, or in the preparation of a product for the production of L-lysine, also falls within the scope of protection of the present invention.

[0037] The application of TKT or of the biomaterial or of the method for preparing L-lysine or of the product for preparing L-lysine in the preparation of foods, feeds or medicines containing L-lysine also falls under Petition 870250093954, dated 10 / 14 / 2025, pp. 80 / 122 13 / 48 scope of protection of the present invention.

[0038] TKT and the biomaterial of the present invention can be used to produce a variety of products, including, but not limited to, lysine, in one example. The resulting products can also be glutamic acid, threonine, tryptophan, arginine, valine, glycine, alanine, leucine, isoleucine, methionine, proline, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, aspartic acid, histidine, shikimic acid, protocatechuic acid, succinic acid, α-ketoglutarate, citric acid, ornithine, citrulline, etc.

[0039] The present invention is described in greater detail below, together with specific embodiments, and the examples provided are for the purpose of clarifying the present invention only, but not of limiting its scope. The examples provided below may be used as a guide for further improvements by a person skilled in the art and do not in any way constitute a limitation of the present invention.

[0040] Instructions for preserving biomaterials: Classification and nomenclature: Corynebacterium glutamicum Strain number: YP097158 Title of the preservation unit: China General Center for Collection of Microbiological Cultures Abbreviation for conservation unit: CGMCC Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101 Preservation date: August 16, 2016 Registration number of the Preservation Center: CGMCC No. 12856. Detailed Description of the Invention

[0041] The experimental methods used in the following examples are conventional methods, unless otherwise specified. The Petition 870250093954, dated 10 / 14 / 2025, pp. 81 / 122 14 / 48 materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, may be commercially available. In the quantitative testing of the following examples, three replicated experiments were performed and the average of the results was calculated. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing was a nucleotide from the 5' end of the corresponding DNA, and the last position was a nucleotide from the 3' end of the corresponding DNA.

[0042] The Corynebacterium glutamicum YP097158 in the following example was preserved at the General Center for Microbiological Culture Collection of China (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology of the Chinese Academy of Sciences) on August 16, 2016, with preservation number CGMCC No. 12856. NINGXIA EPPEN NEW MATERIALS., LTD., depositor of Corynebacterium glutamicum YP097158, authorized HEILONGJIANG EPPEN NEW MATERIALS., LTD. to use this strain. Example 1: Construction of the ATCC13032 strain containing a mutant of the TKT I gene. Construction of the mutant plasmid of the TKT gene.

[0043] First, a wild-type TKT gene (with the sequence represented by SEQ ID No. 1) and its promoter were cloned into a pXMJ19 expression vector. A wild-type TKT promoter and a 2689 bp coding region fragment (with the sequence represented by SEQ ID No. 15) were obtained by using a Corynebacterium glutamicum ATCC13032 genomic sequence published by NCBI as a template and performing PCR amplification using TKT-F / TKT-R primers. The recovered fragment was ligated into a pXMJ19 expression vector (TaKaRa, chloramphenicol-resistant) recovered by enzymatic digestion with Xbal I and BamH I enzymes using a NEBuilder (NEB) enzyme at 50 °C for Petition 870250093954, dated 10 / 14 / 2025, p. 82 / 122 15 / 48 min; the ligation product was transformed into DH5a-competent cells, coated on a 2-YT agar plate containing chloramphenicol (34 mg / L) and cultured at 37 °C for 12 h; and the monoclonal cells that grew in culture were subjected to identification by PCR using TKT-F / TKT-R primers. Those that managed to produce a 2689 bp fragment by PCR amplification were the positive transformant pXMJ19-TKT, which contained a TKT promoter and a coding region sequence.

[0044] To obtain a mutant encoding the TKT gene, a random mutagenesis kit (Agilent Technologies, USA) was used to prepare a plasmid of the mutant TKT gene. Using the constructed pXMJ19TKT plasmid as a template, PCR amplification was performed using TKTF / TKT-R primers to obtain a coding region of the TKT gene and a fragment of the promoter region (with a sequence represented by SEQ ID No. 15, but with random point mutations in the TKT coding region) containing random point mutations.

[0045] The recovered DNA fragment was ligated to a pXMJ19 expression vector (TaKaRa, containing chloramphenicol resistance) recovered by digestion of Xbal I and BamH I enzymes via a NEBuilder (NEB) enzyme at 50 °C for 30 min; and the ligation product was transformed into DH5α, coated onto a 2-YT agar plate containing chloramphenicol (34 mg / L) and cultured at 37 °C for 12 h. Monoclones that grew in culture were subjected to identification by PCR using TKT-F / TKT-R primers. Those that could produce a fragment size of 2689 bp (having a sequence as represented by SEQ ID No. 15, but there were random point mutations in a TKT coding region) were a positive transformant for pXMJ19-TKT-MT containing random mutations of the TKT gene.

[0046] Positions 35-437 in SEQ ID No. 15 were shown Petition 870250093954, dated 10 / 14 / 2025, pp. 83 / 122 16 / 48 as a promoter of the TKT gene.

[0047] The primer design was as follows (synthesized by Shanghai Invitrogen Company): TKT-F: 5'CAGAATAATTAAGCTTGCATGCCTGCAGGTCGACTTCACAGCGGACGATTT C-3' (the underlined nucleotide sequence was a homologous sequence of pXMJ19, SEQ ID No. 18), and TKT-R: 5'CCAAAACAGCCAAGCTGAATTCGAGCTCGGTACCAGGCAAGTAAGGGATG TGC-3' (the underlined nucleotide sequence was a homologous sequence of pXMJ19, SEQ ID No. 19). II. Construction of a strain containing a mutant TKT gene plasmid

[0048] To identify the production performance of the pXMJ19-TKT-MT mutant vector constructed in Step I on L-amino acid, specifically, different random TKT mutant plasmids constructed in Step I were transformed into a wild-type Corynebacterium glutamicum ATCC13032 strain by electric shock (see WO 2014 / 121669 A1 for a specific transformation method) and subjected to identification by PCR using TKT-F / TKT-R primers. Those that managed to produce a fragment size of 2689 bp via PCR amplification were a positive transformant. The positive transformant was consecutively passed three times on a culture plate (see Table 1 for medium components and culture conditions) containing chloramphenicol (34 mg / L), then inoculated into a 500 mL triangular flask filled with 30 mL of rich medium and fermented at 30 °C for 48 h under agitation.The concentration of L-amino acid was detected by high-performance liquid chromatography (HPLC) after fermentation culture. As shown in Table 2, each strain, i.e., a mutant strain ATCC13032-pXMJ19-TKT, with superior L-amino acid production capacity. Petition 870250093954, dated 10 / 14 / 2025, page 84 / 122 17 / 48 amino acids, compared to the wild-type control Corynebacterium glutamicum ATCC13032, was selected.

[0049] Rich medium: water was used as solvent; solute and its concentration were 30 g / L of glucose, 2 g / L of (NH4)2SO4, 0.5 g / L of H3PO4, 0.8 g / L of KCl, 0.8 g / L of MgSO4 . 7H2O, 0.05 g / L of FeSO4 . 7H2O, 0.05 g / L of MnSO4 . H2O, 1.5 g / L FM902 baking powder, 1.5 g / L corn syrup, 17 g / L molasses, 0.5 g / L betaine, 2 g / L citric acid, 20 mg / L VH, 1.5 mg / L VB1, 1.5 mg / L VB3, 1.5 g / L VB12 and sodium hydroxide (adjusted to pH 7.0). Table 1: Compositions and growing conditions of the media Ingredients: Sucrose 10 g / L, Polypeptone 10 g / L, Beef extract 10 g / L, Baking powder 5 g / L, Urea 2 g / L, Sodium chloride 2.5 g / L, Agar powder 18 g / L, pH 7.0, Culture temperature 32 °C, Culture time 12 hours Table 2: Results of high-performance liquid chromatography analysis OF THE MUTANT STRAINS ATCC13032-PXMJ19-TKT L-amino acid names | L-amino acid content (g / 100mL) | ATCG13032 | Mutant strain ATCC13032-TKT 1 | Mutant strain ATGC13032-TKT 2 | Mutant strain ATCC13032-TKT 3 | Mutant strain ATCC13032-TKT 4 | Mutant strain ATCG13032-TKT 5 | Laspartic acid | Not detected | Not detected | Not detected | Not detected | Not detected | L-glutamic acid | 0.005 | 0.004 | 0.007 | 0.003 | 0.003 | 0.001 | L-serine | Not detected | Not detected | Not detected | Not detected | Not detected | Not detected | L-arginine | 0.004 | 0.001 | 0.001 | 0.007 | 0.014 | 0.021 | L-glycine | Not detected | Not detected | Not detected | Not detected | Not detected | Not detected L-threonine 0.008 No 0.001 No 0.001 0.015 Petition 870250093954, dated 10 / 14 / 2025, page 85 / 122 18 / 48 L-amino acid names | L-amino acid content (g / 100mL) | ATCG13032 | Mutant strain ATCC13032-TKT 1 | Mutant strain ATGC13032TKT 2 | Mutant strain ATCC13032-TKT 3 | Mutant strain ATCC13032TKT 4 | Mutant strain ATCG13032-TKT 5 | Detected | Detected | L-lysine | 0.004 | 0.161 | 0.078 | 0.597 | 0.045 | 0.014 | L-proline | Not detected | Not detected | Not detected | Not detected | Not detected | Not detected | L-alanine | Not detected | Not detected | Not detected | Not detected | Not detected | Not detected | L-valine | 0.006 | 0.005 | 0.005 | 0.006 | Not detected | Not detected | L-methionine | 0.001 | Not detected | Not detected | Not detected | Not detected | Not detected | L-cysteine ​​| Not detected | Not detected | Not detected | Detected Not detected Not detected Not detected L-isoleucine Not detected Not detected 0,002 Not detected Not detected Not detected L-leucine Not detected Not detected Not detected Not detected Not detected Not detected L-phenylalanine Not detected Not detected Not detected Not detected Not detected Not detected L-tyrosine Not detected Not detected Not detected Not detected Not detected Not detected,

[0050] As shown in Table 2, the mutant strain Corynebacterium glutamicum ATCC13032-pXMJ19-TKT had the ability to produce some L-lysine, while the mutant strain ATCC13032-pXMJ19-TKT 3 had a superior ability to produce L-lysine, indicating that the mutant strain of the TKT 3 gene had the activity to synthesize L-lysine.

[0051] A plasmid was extracted from the mutant strain ATCC13032pXMJ19-TKT 3, and the TKT gene was sequenced. The results confirmed that guanine (G) at position 979 in a nucleotide sequence of a coding region of the TKT gene mutated to adenine (A) (with a sequence as represented by SEQ ID No. 3, the gene containing this mutation being named gene TKTA327T); alanine (A) at position 327 in a corresponding amino acid sequence mutated to threonine (T) (with a sequence represented by SEQ ID No. 4, the protein containing this mutation being named gene TKTA327T). Petition 870250093954, dated 10 / 14 / 2025, pp. 86 / 122 19 / 48 mutation named protein TKTA327T); and this plasmid was named pXMJ19-TKTA327T, and a mutant strain containing this plasmid was renamed ATCC13032-pXMJ19-TKTA327T.

[0052] pXMJ19-TKTA327T differed from pXMJ19-TKT in the following aspects: pXMJ19-TKTA327Tera was a recombinant vector obtained by replacing a wild-type TKT gene in pXMJ19-TKT with a sequence of the mutant TKTA327T gene, and pXMJ19-TKTA327Tera was capable of expressing a mutant TKTA327T protein. The wild-type TKT gene differed from the mutant TKTA327T gene only in the following aspects: GCT was located at positions 979-981 of the wild-type TKT gene, and ACT was located at positions 979-981 of the mutant TKTA327T gene. The wild-type TKT protein differed from the mutant TKTA327T protein only in: an alanine A residue was located at position 327 in the wild-type TKT protein, and a threonine T residue was located at position 327 in the mutant TKTA327T protein. III. Construction of mutant TKT gene strains and vector RECOMBINANT

[0053] An ATCC13032-pXMJ19-TKTA327T mutant strain was obtained using wild-type Corynebacterium glutamicum ATCC13032 via random mutation. In order to obtain more TKT mutant strains to improve their L-lysine production abilities, a mutant with the same mutation site as the aforementioned TKT, but with a different amino acid, was constructed. Specifically, taking the pXMJ19TKTA327T plasmid sequenced in step II as a template, five mutants were constructed in which the amino acid at position 327 of TKT was replaced with different amino acids. The substituted amino acids were all hydrophilic amino acids. The names of the substituted amino acids in the mutants and the primers used in the respective mutants are shown in Table 3. Petition 870250093954, dated 10 / 14 / 2025, p. 87 / 122 20 / 48 Table 3: Names of the substituted amino acids in the TKT mutants and primers USED ​​IN THEIR RESPECTIVE MUTANTS Genes Substitute Aminoacids Primers Mutant Vector Names pXMJ19-TKT TKT A327S TKT-F / S-PR-1, S-PR-2 / TKT-R pXMJ19-TKTA327S A327C TKT-F / C-PR-1, C-PR-2 / TKT-R pXMJ19-TKTA327C A327P TKT-F / P-PR-1, P-PR-2 / TKT-R pXMJ19-TKTA327P A327N TKT-F / N-PR-1, N-PR-2 / TKT-R pXMJ19-TKTA327N A327Q TKT-F / Q-PR-1, Q-PR-2 / TKT-R pXMJ19-TKTA327Q

[0054] This is the primer you want (sent) Shanghai Invitrogen Company): S-PR-1: 5'-CCATGCAGCCTTCTTCTGTGCAGAGCGCTCTG-3' (SEQ ID No. 20), S-PR-2: 5'-CAGAGCGCTCTGCACAGAAAGAAGGCTGCATGG-3' (SEQ ID No. 21), C-PR-1: 5'-CCATGCAGCCTTCTTCTGTGCACAGCGCTCTG-3' (SEQ ID No. 22), C-PR-2:5'-CAGAGCGCTGTGCACAGAAAGAAGGCTGCATGG3'(SEQ ID No. 23), P-PR-1: 5'-CCATGCAGCCTTCTTCTGTGCAGGGCGCTCTG-3' (SEQ ID No. 24), P-PR-2: 5'-CAGAGCGCCCTGCACAGAAAGAAGGCTGCATGG-3' (SEQ ID No. 25), N-PR-1: 5'-CCATGCAGCCTTCTTCTGTGCATTGCGCTTCTG-3' (SEQ ID No. 26), N-PR-2: 5'-CAGAGCGCAATGCACAGAAGAAGGCTGCATGG-3' (SEQ ID No. 27), Q-PR-1: 5'-CCATGCAGCCTTCTTCTGTGCCTGGCGCTCTG-3' (SEQ ID No. 28), Q-PR-2: 5'-CAGAGCGCCAGGCACAGAAGAAGGCTGCATGG-3' Petition 870250093954, dated 10 / 14 / 2025, pp. 88 / 122 21 / 48 (SEQ ID No. 29).

[0055] Using a wild-type Corynebacterium glutamicum ATCC13032 genome as a template, PCR amplification was performed using the TKT-F / S-PR-1 and KAPA HiFi HotStart primers in Table 2 to obtain an Up DNA fragment (1405 bp) with a TKT mutant base. PCR amplification was performed using the S-PR-2 / TKT-R and KAPA HiFi HotStart primers to obtain a Down DNA fragment (1248 bp) with a TKT mutant base. After the PCR reaction, recovery by agarose gel electrophoresis was performed using a column DNA gel recovery kit, respectively. The recovered DNA fragment was ligated to a pXMJ19 expression vector recovered by enzymatic digestion with Xbal I and BamH I using a NEBuilder (NEB) enzyme at 50 °C for 30 min; and the binding product was transformed into DH5α, coated onto a 2-YT agar plate containing chloramphenicol (34 mg / L) and cultured at 37 °C for 12 h.The monoclones that grew in culture were subjected to identification by PCR using the TKT-F / TKT-R primers, and those that managed to produce a 2689 bp fragment were a positive transformant ATCC13032-pXMJ19-TKTA327S in which the alanine at position 327 of a protein encoding the TKT gene was mutated into tryptophan. The other four lines were constructed in the same way: five pXMJ19TKT mutant vectors and lines with alanine at position 327 replaced by each of the amino acids in Table 3 were named according to the names listed in Table 3. The resulting mutant lines were ATCC13032-pXMJ19TKTA327C, ATCC13032-pXMJ19-TKTA327P, ATCC13032-pXMJ19-TKTA327N and ATCC13032-pXMJ19-TKTA327Q.

[0056] ATCC13032-pXMJ19-TKTA327S contained a recombinant vector pXMJ19-TKTA327S. pXMJ19-TKTA327S differed from pXMJ19-TKT in that: pXMJ19-TKTA327S was a recombinant vector obtained by replacing a wild-type TKT gene in pXMJ19-TKT with a Petition 870250093954, dated 10 / 14 / 2025, pp. 89 / 122 The 22 / 48 sequence of the tktA327S mutant gene (as represented by SEQ ID No. 5), and pXMJ19-TKTA327Sera capable of expressing a mutant TKTA327S protein (as represented by SEQ ID No. 6). The wild-type TKT gene differed from the TKTA327S mutant gene only in the following aspects: the GCT was located at positions 979-981 of the wild-type TKT gene, and the TCT was located at positions 979-981 of the TKTA327S mutant gene. The wild-type TKT protein differed from the mutant tktA327S protein only in the following aspects: an alanine A residue was located at position 327 of the wild-type TKT protein, and a serine S residue was located at position 327 of the mutant TKTA327S protein.

[0057] ATCC13032-pXMJ19-TKTA327C contained a recombinant vector pXMJ19-TKTA327C. pXMJ19-TKTA327C differed from pXMJ19-TKT where: pXMJ19-TKTA327C was a recombinant vector that is obtained by replacing a wild-type TKT gene in pXMJ19-TKT with a mutant TKTA327C gene sequence (as represented by SEQ ID No. 7), and pXMJ19-TKTA327C was capable of expressing a mutant TKTA327C protein (as represented by SEQ ID No. 8). The wild-type TKT gene differed from the mutant tktA327C gene only in the following aspects: the GCT was located at positions 979-981 in the wild-type TKT gene, and the TCT was located at positions 979-981 in the mutant tktA327C gene. The wild-type TKT protein differed from the mutant tktA327C protein only in the following aspects: an alanine A residue was located at position 327 in the wild-type TKT protein, and a cysteine ​​C residue was located at position 327 in the mutant TKTA327C protein.

[0058] ATCC13032-pXMJ19-TKTA327P contained a recombinant vector pXMJ19-TKTA327P. pXMJ19-TKTA327P differed from pXMJ19-TKT where: pXMJ19-TKTA327P was a recombinant vector that was obtained by replacing a wild-type TKT gene in pXMJ19-TKT with a Petition 870250093954, dated 10 / 14 / 2025, pp. 90 / 122 The 23 / 48 sequence of the mutant tktA327P gene (as represented by SEQ ID No. 9), and the pXMJ19-TKTA327P capable of expressing a mutant tktA327P protein (as represented by SEQ ID No. 10). The wild-type TKT gene differed from the mutant tktA327P gene only in the following aspects: the GCT was located at positions 979-981 of the wild-type TKT gene, and the CCT was located at positions 979-981 of the mutant TKTA327P gene. The wild-type TKT protein differed from the mutant tktA327P protein only in the following aspects: an alanine A residue was located at position 327 of the wild-type TKT protein, and a proline P residue was located at position 327 of the mutant TKTA327P protein.

[0059] ATCC13032-pXMJ19-TKTA327N contained a recombinant vector pXMJ19-TKTA327N. pXMJ19-TKTA327N differed from pXMJ19-TKT where: pXMJ19-TKTA327Ne was a recombinant vector that was obtained by replacing a wild-type TKT gene in pXMJ19-TKT with a mutant TKTA327N gene sequence (as represented by SEQ ID No. 11), and pXMJ19-TKTA327Ne was capable of expressing a mutant TKTA327N protein (as represented by SEQ ID No. 12). The wild-type TKT gene differed from the mutant TKTA327N gene only in the following aspects: GCT was located at positions 979-981 of the wild-type TKT gene, and AAT was located at positions 979-981 of the mutant TKTA327N gene. The wild-type TKT protein differed from the mutant TKTA327N protein only in the following aspects: an alanine A residue was located at position 327 of the wild-type TKT protein, and an asparagine N residue was located at position 327 of the mutant TKTA327N protein.

[0060] ATCC13032-pXMJ19-TKTA327Q contained a recombinant vector pXMJ19-TKTA327Q. pXMJ19-TKTA327Q differed from pXMJ19-TKT where: pXMJ19-TKTA327Q was a recombinant vector that was obtained by replacing a wild-type TKT gene in pXMJ19-TKT with a Petition 870250093954, dated 10 / 14 / 2025, pp. 91 / 122 The 24 / 48 sequence of the tktA327Q mutant gene (as represented by SEQ ID No. 13), and pXMJ19-TKTA327Q was capable of expressing a mutant protein TKTA327Q (as represented by SEQ ID No. 14). The wild-type TKT gene differed from the tktA327Q mutant gene only in the following aspects: GCT was located at positions 979-981 of the wild-type TKT gene, and CAG was located at positions 979-981 of the TKTA327Q mutant gene. The wild-type TKT protein differed from the mutant tktA327Q protein only in the following aspects: an alanine A residue was located at position 327 of the wild-type TKT protein, and a glutamine Q residue was located at position 327 of the mutant TKTA327Q protein. IV. Detection of L-lysine production capacity in TKT mutant cell lines.

[0061] To identify the productive performance of the mutant vector constructed in step III against L-lysine, specifically, five recombinant bacteria constructed in step III and ATCC13032-pXMJ19-TKTA327T in step II were consecutively passed three times on a culture plate containing chloramphenicol (34 mg / L), then inoculated into a 500 mL triangular flask with 30 mL of rich medium and fermented at 37 °C for 48 h under agitation. After fermentation, the L-lysine concentration was analyzed by high-performance liquid chromatography (HPLC), and the wild-type Corynebacterium glutamicum ATCC13032 was used as a control.

[0062] The results were shown in Table 4. The L-lysine yield of each mutant strain was significantly higher than that of the wild-type Corynebacterium glutamicum ATCC13032, while the ability of the mutant strain ATCC13032-pXMJ19-TKTA327T to produce L-lysine was superior to that of ATCC13032-pXMJ19-TKTA327S, ATCC13032-pXMJ19-TKTA327C, ATCC13032pXMJ19-TKTA327P, ATCC13032-pXMJ19-TKTA327N and ATCC13032-pXMJ19TKTA327Q, indicating that alanine (A) at position 327 of the TKT protein was mutated. Petition 870250093954, dated 10 / 14 / 2025, pp. 92-122 25 / 48 in threonine (T), which was more conducive to lysine accumulation. Table 4: L-tyrosine detection results for W3110TKT mutant strains by high-performance liquid chromatography. Strains L-lysine (g / 100mL) ATCC13032 0.002 ATCC13032-pXMJ19-TKTA327T 0.585 ATCC13032-pXMJ19-TKTA327S 0.138 ATCC13032-pXMJ19-tktA327C 0.076 ATCC13032-pXMJ19-TKTA327P 0.084 ATCC13032-pXMJ19-TKTA327N 0.113 ATCC13032-pXMJ19-TKTA327Q 0.071 Example 2: Construction of a recombinant vector containing a region TKT GENE CODING MACHINE CONTAINING POINT MUTATIONS

[0063] Based on the genomic sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two primer pairs that amplify a coding region of the TKT gene were designed and synthesized. A point mutation was introduced into the coding region of the TKT gene (SEQ ID No. 1) of Corynebacterium glutamicum YP097158 (preservation number: CGMCC No. 12856, preservation date: August 16, 2016, preservation unit: Institute of Microbiology of the Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing, Tel.: 01064807355) using an allelic substitution method. The point mutation consisted of mutating guanine (G) at position 979 in the nucleotide sequence (SEQ ID No. 1) of the TKT gene to adenine (A) to obtain a DNA molecule represented by SEQ ID No. 3 (a mutated TKT gene, named gene TKTA327T).

[0064] The DNA molecule, as represented by SEQ ID No. 1, encoded a protein with an amino acid sequence as represented by SEQ ID No. 2. The DNA molecule (gene TKTA327T), as represented by SEQ ID No. 3, encoded a mutant protein (the mutant protein being named protein TKTA327T) with a sequence of Petition 870250093954, dated 10 / 14 / 2025, pp. 93 / 122 26 / 48 amino acids as represented by SEQ ID No. 4. The threonine (T) at position 327 in the amino acid sequence (SEQ ID No. 4) of the mutant protein TKTA32?T was mutated from alanine (A).

[0065] A recombinant vector was constructed using NEBuilder recombination technology, and the primers were designed as follows (synthesized by Shanghai Invitrogen Company). The bases in bold represent the mutation sites: P1: 5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGTCATTGCTTTCTGA TGG-3' (the underlined nucleotide sequence was a sequence in pK18, SEQ ID No. 30), P2: 5'-CCATGCAGCCTTCTTCTGTGCAGTGCGCTCTG-3' (SEQ ID No. 31), P3: 5'-CAGAGCGCACTGCACAGAAGAAGGCTGCATGG-3' (SEQ ID No. 32), P4: 5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCGGAGAAGATGAG GAAGGTTC-3' (the underlined nucleotide sequence was a sequence in pK18, SEQ ID No. 33).

[0066] Construction method: using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed using primers P1 and P2, P3 and P4, respectively, to obtain two DNA fragments (TKTUp and TKTDown) from the coding region of the TKT gene, which carried mutant bases and had sizes of 514 bp and 482 bp, respectively.

[0067] The two DNA fragments (TKTUp and TKTDown) were separated and purified by agarose gel electrophoresis and then ligated to a pK18mobsacB plasmid (Addgene) purified by digestion. Petition 870250093954, dated 10 / 14 / 2025, pp. 94 / 122 27 / 48 enzyme (Xbal I / BamH I) via a NEBuilder enzyme (NEB) at 50 °C for 30 min. Monoclones that grew after transformation of the ligation product were subjected to identification by PCR using primers P1 / P4. Those that could produce a 964 bp fragment were a positive recombinant pK18-TKTA327T vector. This recombinant vector contained a kanamycin resistance marker. The correctly enzymatically digested recombinant pK18-TKTA327T vector was delivered to the sequencing company for sequencing and verification. A recombinant pK18-TKTA327T vector containing a correct point mutation (G979A) was preserved for future use.

[0068] This recombinant pK18-TKTA327T vector contained a mutation site (G979A), which will cause guanine (G) at position 979 in a coding region of the TKT gene (SEQ ID No. 1) of the Corynebacterium glutamicum YP097158 strain to be mutated to adenine (A), ultimately resulting in the change of alanine (A) at position 327 of the coding protein to threonine (T).

[0069] The recombinant vector pK18-TKTA327Tera is a recombinant vector that was obtained by replacing a fragment (small fragment) between the Xbal I and BamH I recognition sites of a pK18mobsacB vector with a DNA fragment, as represented by SEQ ID No. 6 in the sequence listing, while keeping the other sequences of the pK18mobsacB vector unchanged. The recombinant vector pK18-TKTA327T contained a mutation site (G979A) of the mutant gene TKTA327T, as represented by SEQ ID No. 3. Example 3: Construction of a modified strain containing the TKTA327T gene

[0070] The allelic substitution plasmid (pK18-TKTA327T) constructed in Example 2 was transformed by electric shock into a Corynebacterium glutamicum YP097158 strain (after sequencing, it was confirmed Petition 870250093954, dated 10 / 14 / 2025, pp. 95 / 122 28 / 48 (a coding region of the wild-type TKT gene was retained in the chromosomes of this strain) and a wild-type strain of Corynebacterium glutamicum ATCC13032, and cultured on a solid culture plate containing kanamycin (50 mg / L) for 40 h. Single colonies produced in culture were identified using primers P1 / P4 in Example 2, respectively. A strain that managed to amplify a 964 bp strip was a positive strain. The positive strain was subjected to strip culture in a medium containing 15% sucrose (this medium was obtained by increasing the sucrose concentration in the medium in Table 1 to 150 g / L). Individual colonies produced in culture were cultured in medium containing kanamycin and in kanamycin-free medium, respectively. Strains that grew in kanamycin-free medium but did not grow in kanamycin-containing medium were subsequently subjected to PCR amplification using primers P1 / P4.The resulting plurality of DNA fragments (964 bp) were sequenced. Through sequence alignment, the strains with the mutation in the base sequence (G979A) were positive strains that had successfully undergone allelic substitution. The positive strains obtained from Corynebacterium glutamicum YP097158 and the wild-type strain Corynebacterium glutamicum ATCC13032 were named YPLTKT-1 and TKT-1, respectively.

[0071] Both recombinant bacteria YPL-TKT-1 and TKT-1 contained a mutated TKTA327T gene, represented by SEQ ID No. 3, and could express the TKTA327T protein, represented by SEQ ID No. 4. The recombinant bacterium YPL-TKT-1 differed from Corynebacterium glutamicum YP097158 only in the following: YPL-TKT-1 was a strain obtained by replacing a TKT gene of Corynebacterium glutamicum YP097158 with a TKTA327T gene, keeping the other sequences unchanged. The recombinant bacterium TKT-1 differed from wild-type Corynebacterium glutamicum ATCC13032 only in this: TKT-1 was a strain that was obtained by replacing a TKT gene of type Petition 870250093954, dated 10 / 14 / 2025, pp. 96 / 122 29 / 48 wild-type Corynebacterium glutamicum ATCC13032 with a TKTA327Te gene retaining other sequences unchanged. Recombinant bacteria containing the mutated TKTA327T gene can significantly and stably increase the amount of TKTA327T gene expression. Example 4: Construction of a modified lineage overexpressing the TKT gene or the TKTA327T gene in the genome.

[0072] According to a genomic sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, three primer pairs were designed and synthesized to amplify upstream and downstream homologous arm fragments, as well as a coding region of the TKT gene or TKTA327T and a promoter region. Copies of the TKT gene or TKTA327T were inserted into wild-type Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum ATCC13032 via homologous recombination.

[0073] The primer design was as follows (synthesized by Shanghai Invitrogen Company): P5: 5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATGCGTTCTGGACT GAGG-3' (the underlined nucleotide sequence was a sequence in pK18, SEQ ID No. 34), P6: 5'-GAAATCGTCCGCTGTGAAGTGCACCGAGAACAGATG-3' (SEQ ID No. 35), P7: 5'-CATCTGTTCTCGGTGCACTTCACAGCGGACGATTTC-3' (SEQ ID No. 36), P8: 5-GATTTAATTGCGCCATCTGAGGCAAGTAAGGGATGTGC3' (SEQ ID No. 37), P9: 5'-GCACATCCCTTACTTGCCTCAGATGGCGCAATTAAATC3' (SEQ ID No. 38), and Petition 870250093954, dated 10 / 14 / 2025, pp. 97 / 122 30 / 48 P10: 5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCTATGACACCTTC AACGG ATC-3' (the underlined nucleotide sequence was a sequence in pK18, SEQ ID No. 39).

[0074] Construction method: taking a genome from Using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed using primers P5 / P6, P7 / P8, and P9 / P10, respectively, to obtain a 763 bp upstream homologous arm fragment (corresponding to a partial coding region of NCgl1740 and an NCgl1741 gene and its promoter region from Corynebacterium glutamicum ATCC13032), a 2621 bp TKT gene and its promoter fragment (having a sequence as represented by SEQ ID No. 16), and a 596 bp downstream homologous arm fragment (corresponding to a partial coding region of the NCgl1742 gene from Corynebacterium glutamicum ATCC13032). After the PCR reaction, three amplified fragments from each template were recovered by electrophoresis using a column-based DNA gel recovery kit. Three recovered fragments were ligated to a pK18mobsacB plasmid (Addgene), which was purified after digestion with Xbal I and BamH I enzymes at 50 °C for 30 minutes using a NEBuilder (NEB) enzyme.Monoclones grown after transformation of the ligation product were subjected to identification by PCR using M13F / M13R primers to obtain a positive integrated plasmid (recombinant vector). The resulting recombinant vector was pK18-TKTOE. This positive integrated plasmid contained a kanamycin resistance marker, and a recombinant in which a plasmid was integrated into a genome was obtained by kanamycin screening. In SEQ ID No. 16, positions 1-403 were promoters of the TKT gene, and positions 404-2506 were the TKT gene.

[0075] M13F: 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID No. 40), Petition 870250093954, dated 10 / 14 / 2025, pp. 98 / 122 31 / 48

[0076] M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID No. 41).

[0077] Taking a Corynebacterium glutamicum YPLTKT-1 genome as a template, PCR amplification was performed using primers P5 / P6, P7 / P8 and P9 / P10, respectively, to obtain a 763 bp upstream homologous arm fragment (corresponding to a partial coding region of NCgl1740 and an NCgl1741 gene and its promoter region of Corynebacterium glutamicum ATCC13032), a 2621 bp TKTA327T gene and its promoter fragment (having a sequence as represented by SEQ ID No. 17), and a 596 bp downstream homologous arm fragment (corresponding to a partial coding region of the NCgl1742 gene of Corynebacterium glutamicum ATCC13032). Following the PCR reaction, three amplified fragments from each template were recovered by electrophoresis using a column-based DNA gel recovery kit.Three recovered fragments were ligated to a pK18mobsacB plasmid (Addgene), which was purified after digestion with Xbal I and BamH I enzymes at 50 °C for 30 minutes using a NEBuilder (NEB) enzyme. Monoclones grown after transformation of the ligation product were subjected to identification by PCR using M13F / M13R primers to obtain a positive integrated plasmid (recombinant vector). The resulting recombinant vector was pK18-TKTA327TOE. This positive integrated plasmid contained a kanamycin resistance marker, and a recombinant in which a plasmid was integrated into a genome was obtained by kanamycin screening. In SEQ ID No. 17, positions 1403 were promoters of the TKTA327T gene, and positions 404-2506 were the TKTA327T gene.

[0078] The correctly sequenced integrated plasmids (pK18-TKTOE, pK18-TKTA327TOE) were electrotransformed into wild-type Corynebacterium glutamicum YP097158 and Corynebacterium glutamicum ATCC13032, respectively, and cultured in medium for 30 h. Individual colonies Petition 870250093954, dated 10 / 14 / 2025, pp. 99 / 122 32 / 48 cultured strains were subjected to PCR identification using primers P11 / P12, and those that produced a 1559 bp fragment by PCR amplification were considered positive strains, while those that did not produce any fragment were considered probiotic. Positive strains were cultured on a solid streak plate containing 15% sucrose for 30 h. Individual colonies produced in culture were subsequently subjected to PCR identification using primers P13 / P14. The bacterium that showed a size of 1559 bp by amplification was a positive strain in which a TKT gene or TKTA327Te and its promoter were integrated into a spacer region between an upper homologous arm NCgl1741 and a lower homologous arm NCgl1742 in a Corynebacterium glutamicum genome.The strains obtained from Corynebacterium glutamicum YP097158 as starter bacteria were named YPL-TKT-2 (without mutation points) and YPL-TKT-3 (with mutation points), respectively. The strains obtained from Corynebacterium glutamicum ATCC13032 as starter bacteria were named TKT-2 (without mutation points) and TKT-3 (with mutation points), respectively.

[0079] The recombinant bacterium YPL-TKT-2 contained double copies of the TKT gene, as represented by SEQ ID No. 1. Specifically, the recombinant bacterium YPL-TKT-2 was a recombinant bacterium obtained by replacing a spacer region between an upper homologous arm NCgl1741 and a lower homologous arm NCgl1742 in a Corynebacterium glutamicum YP097158 genome with a DNA fragment, as represented by SEQ ID No. 16 (positions 1-403 of SEQ ID No. 16 are shown as promoters and positions 404-2506 are shown as the TKT gene), while leaving other nucleotides in the Corynebacterium glutamicum YP097158 genome unchanged. Recombinant bacteria containing double copies of the TKT gene can significantly and stably increase the Petition 870250093954, dated 10 / 14 / 2025, pp. 100 / 122 33 / 48 quantity of TKT gene expression.

[0080] The recombinant bacterium TKT-2 contained double copies of the TKT gene, as represented by SEQ ID No. 1. Specifically, the recombinant bacterium TKT-2 was obtained by replacing a spacer region between an upper homologous arm NCgl1741 and a lower homologous arm NCgl1742 in a Corynebacterium glutamicum ATCC13032 genome with a DNA fragment, as represented by SEQ ID No. 16, while keeping the remaining nucleotides of the Corynebacterium glutamicum ATCC13032 genome unchanged. Recombinant bacteria containing double copies of the TKT gene could significantly and stably increase the amount of TKT gene expression.

[0081] The recombinant bacterium YPL-TKT-3 contained the TKTA327T gene, as represented by SEQ ID No. 3. Specifically, the recombinant bacterium YPL-TKT-3 was a recombinant bacterium obtained by replacing the spacer region between the upper homologous arm NCgl1741 and the lower homologous arm NCgl1742 in the genome of Corynebacterium glutamicum YP097158 with a DNA fragment, as represented by SEQ ID No. 17 (positions 1-403 of SEQ ID No. 17 were shown as promoters and positions 404-2506 were shown as the TKTA327T gene) and leaving other nucleotides in the genome of Corynebacterium glutamicum YP097158 unchanged.

[0082] The recombinant bacterium TKT-3 contained the mutated TKTA327T gene, as represented by SEQ ID No. 3. Specifically, the recombinant bacterium TKT-3 was a recombinant bacterium that was obtained by replacing a spacer region between the upper homologous arm NCgl1741 and the lower homologous arm NCgl1742 in the genome of Corynebacterium glutamicum ATCC13032 with a DNA fragment, as represented by SEQ ID No. 17, while retaining other nucleotides. Petition 870250093954, dated 10 / 14 / 2025, pp. 101 / 122 34 / 48 in the genome of unchanged Corynebacterium glutamicum ATCC13032.

[0083] The primers for identification by PCR were as follows: P11: 5'-TCCAAGGAAGATACACGCC-3' (corresponding to the outer part of the upper homologous arm NCgl1740, SEQ ID No. 42), P12: 5'-GCCACAAGAAAGAAGAGAAG-3' (corresponding to the interior of the TKT gene, SEQ ID No. 43), P13: 5'-CATCCTCAACGGCATTTC-3' (corresponding to the interior of the TKT gene, SEQ ID No. 44), and P14: 5'-TGGTCGTTGGAATCTTGC-3' (corresponding to the outer part of the lower homologous arm NCgl1742, SEQ ID No. 45). Example 5: Construction of a modified lineage overexpressing the TKT gene or the TKTA327T gene in plasmids.

[0084] The pXMJ19-TKT and pXMJ19-TKTA327T plasmids, successfully constructed in Example 1, were electrotransformed into Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum ATCC13032, respectively, and cultured in medium (see Table 1 for medium components) for 30 h. Individual colonies produced in culture were subjected to identification by PCR using TKT-F / TKT-R primers, and those that produced a 2689 bp fragment (with the sequence represented by SEQ ID No. 15) by PCR amplification were considered positive strains. The strains obtained from Corynebacterium glutamicum YP097158 as starter bacteria were named YPL-TKT-4 (containing the pXMJ19-TKT plasmid) and YPL-TKT-5 (containing the pXMJ19-TKTA327T plasmid), respectively. The strains obtained from Corynebacterium glutamicum ATCC13032 as starter bacteria were named TKT-4 (containing the pXMJ19-TKT plasmid) and TKT-5 (containing the pXMJ19-TKTA327T plasmid), respectively.

[0085] Recombinant bacteria YPL-TKT-4 and TKT-4 containing Petition 870250093954, dated 10 / 14 / 2025, pages 102 / 122 35 / 48 the TKT gene plasmid, as represented by SEQ ID No. 1, can significantly and stably increase the amount of TKT gene expression.

[0086] Recombinant bacteria YPL-TKT-5 and TKT-5 containing the TKTA327T gene plasmid, as represented by SEQ ID No. 3, can significantly and stably increase the amount of TKT gene expression. Example 6: Construction of a modified strain with a deficient TKT gene in GENOME

[0087] According to a genomic sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two primer pairs that amplified fragments at both ends of a coding region of the TKT gene were synthesized as upstream and downstream homologous arm fragments. The primer design was as follows (synthesized by Shanghai Invitrogen Company): P15:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGG GAAATAGATG GGT GTAGACG-3' (the underlined nucleotide sequence was a sequence in pK18, SEQ ID No. 46), P16: P16:5'GCAAGGAACGGAAACAACGCCTTCAGGTCATCCATCTC-3' (SEQ ID No. 47), P17: 5'-GAGATGGATGACCTGAAGGCGTTGTTTCCGTTCCTTGC-3' (SEQ ID No. 48), and P18:5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCATCAACCTTTGCC CACAG-3' (the underlined nucleotide sequence was a sequence in pK18, SEQ ID No. 49).

[0088] Construction method: taking the genome of Petition 870250093954, dated 10 / 14 / 2025, pp. 103 / 122 36 / 48 Using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed using primers P15 / P16 and P17 / P18, respectively, to obtain an upstream homologous arm fragment (772 bp) and a downstream homologous arm fragment (824 bp) from which TKT was eliminated. The amplified product was subjected to electrophoresis and purified using a column DNA gel recovery kit. The recovered DNA fragments were ligated to a pK18mobsacB plasmid (Addgene) purified by Xbal I / BamH I enzymatic digestion using NEBuilder (NEB) enzyme at 50 °C for 30 min. A monoclone grown after transformation of the ligation product was subjected to identification by PCR using primers M13F / M13R to obtain a positive elimination vector ρK18-ΔTKT. This plasmid had resistance to kanamycin as a screening marker. This plasmid was submitted for sequencing.

[0089] The correctly sequenced knockout plasmid ρK18-ΔTKT was electrotransformed into Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum ATCC13032, and cultured in culture medium for 30 h. Individual colonies produced in culture were subjected to identification by PCR using P15 / P18 primers. Strains that amplified both the 1596 bp and 3106 bp bands simultaneously were both positive, and strains that amplified only one 3106 bp band were probiotic. Positive strains were screened on solid medium with 15% sucrose and then cultured in medium containing kanamycin and in medium without kanamycin for 30 h, respectively. Strains that grew in medium without kanamycin but did not grow in medium containing kanamycin were selected and subsequently subjected to identification by PCR using P15 / P18 primers.The strains that amplified a 1596 bp band were positive strains whose partial coding region of the TKT gene was inactivated. TKT fragments from the positive strains were submitted. Petition 870250093954, dated 10 / 14 / 2025, pp. 104 / 122 37 / 48 again to PCR amplification using P15 / P18 primers for sequencing. The correctly sequenced strains were named YPL-tk-6 (the TKT gene in the genome of Corynebacterium glutamicum YP097158 was inactivated) and TKT-6 (the TKT gene in the genome of wild-type Corynebacterium glutamicum ATCC13032 was inactivated). Example 7: L-lysine fermentation experiment

[0090] The strains constructed in Examples 2 to 6, the original strain YP097158 of Corynebacterium glutamicum and the wild strain Corynebacterium glutamicum ATCC13032, were fermented in a BLBIO-5GC-4-H model fermentation tank (Shanghai Bailun Biotechnology Co., Ltd.) using the media shown in Table 5 and the control process shown in Table 6. After fermentation, the L-lysine yield was detected by ninhydrin colorimetry. This process was repeated three times for each strain. The results are presented in Table 7. Table 5: Fermentation media formulas Ingredients Formula Hydrolyzed starch sugar 30 g / L Ammonium sulfate 12 g / L Magnesium sulfate 0.87 g / L Molasses 20 g / L Acidified corn syrup 3 mL / L Phosphoric acid 0.4 mL / L Potassium chloride 0.53 g / L Antifoaming agent (2% Paodi) 4 mL / L Ferrous sulfate 120 mg / L Manganese sulfate 120 mg / L Nicotinamide 42 mg / L Calcium pantothenate 6.3 mg / L Petition 870250093954, dated 10 / 14 / 2025, pp. 105 / 122 38 / 48 Vitamin B1 6.3 mg / L Copper and zinc salt solution 0.6 g / L Biotin 0.88 mg / L Table 6: Fermentation control process Corrected to D0100% Temperature of 37 °C, air volume of 4 L / min, speed of 1000 rpm, tank pressure of 0 Mpa and calibrated after 5 min Inoculation quantity 10% Culture temperature °C 37 °C pH 6.9±0.05 Dissolved oxygen DO 10-30% Initial conditions Temperature of 37 °C, pH of 6.9, tank pressure of 0 Mpa, air volume of 3 L / min and speed of 550 rpm Control of the whole process control of the whole process 1, dissolved oxygen < 30% and the speed was increased sequentially to 750 rpm ^ 800 rpm ^ air volume of 4 L / min ^ 850 rpm ^ 950 rpm; 2. The tank pressure was increased to 0.01 MPa after 6 h of fermentation and the tank pressure was increased to 0.02 MPa ^ 0.03 MPa ^ 0.04 MPa ^ 0.05 MPa after 12 h of fermentation. Residual sugar control: 0.1-0.2% before F12h; residual sugar was controlled to 0.1-0.05% after F12h in combination with D0 requirements. Ammonia-nitrogen control: 0.1-0.15% before F12h; 0.15-0.25% for F12-F32h;0.1-0.15 after F32h Batch feed material: 25% ammonia water, 70% concentrated sugar, 50% ammonium sulfate and 10% Paodi Fermentation cycle Approximately 48 h; Table 7: Yield analysis and significance of L-lysine in strains MODIFIED TICKETS Strains L-lysine concentration (g / 100mL) Average (g / L) Significance analysis Result of the first fermentation Result of the second fermentation Result of the third fermentation ATCC13032 0.006 0.003 0.007 0.0053±0.0017 TKT-1 0.422 0.392 0.513 0.442±0.051 P<0.01 TKT-2 0.322 0.348 0.313 0.327±0.015 P<0.01 TKT-3 0.518 0.472 0.556 0.515±0.034 P<0.01 TKT-4 0.442 0.472 0.523 0.479±0.033 P<0.01 TKT-5 0.502 0.592 0.573 0.556±0.039 P<0.01 Petition 870250093954, dated 10 / 14 / 2025, pp. 106 / 122 39 / 48 Strains L-lysine concentration (g / 100mL) Average (g / L) Significance analysis Result of the first fermentation Result of the second fermentation Result of the third fermentation TKT-6 0.004 0.003 0.006 0.0043±0.0012 Not significant YP097158 18.62 18.46 17.74 18.27±0.38 YPL-TKT-1 19.64 19.08 19.84 19.52±0.32 P<0.01 YPL-TKT-2 19.32 18.88 19.84 19.35±0.39 P<0.05 YPL-TKT-3 19.64 20.26 20.06 19.99±0.26 P<0.01 YPL-TKT-4 19.42 18.85 19.35 19.21±0.25 P<0.05 YPL-TKT-5 20.34 19.61 20.67 20.21±0.44 P<0.01 YPL-TKT-6 17.64 16.97 17.33 17.31±0.27 P<0.01

[0091] The results were shown in Table 7. The point mutation (G979A) in the coding region of the tkt gene and overexpression of the TKT gene or TKTA327Tem Corynebacterium glutamicum contributed to an increase in L-lysine yield, while weakening or deletion of the TKT gene were not conducive to L-lysine accumulation.

[0092] The present invention is detailed above. To a person skilled in the art, the present invention can be implemented over a wide range, under the same parameters, concentrations and conditions, without departing from the purpose and scope of the present invention and without unnecessary experimentation. Although special examples are provided in the present invention, it should be understood that further improvements can be made to the present invention. In summary, according to the principles of the present invention, the present application is intended to include any alteration, use or improvement of the present invention, including alterations that deviate from the scope disclosed in the present application and are made by conventional technologies known in the art. According to Petition 870250093954, dated 10 / 14 / 2025, pp. 107 / 122 40 / 48 within the scope of the claims attached below, some basic features can be applied. Industrial Applicability

[0093] The protein and related biomaterial of the present invention can increase the yield of L-lysine and can be used for the production of L-lysine, and have good application prospects.

[0094] The following sequences are involved in the present invention:

[0095] SEQ ID No. 1: Wild-type ORF(CDS) sequence (2103 bp nucleotide sequence) of the TKT gene TTGACCACCTTGACGCTGTCACCTGAACTTCAGGCGCTCACTGTACGCAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTA GACACTGTTCGTGTCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTCCGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATAC ACCTTGTACCAGCGGGTTATGAACGTAGATCCACAGGACACCACAACTGGGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCT TTGACCCAGTACATCCAGCTTTACTTGGGTGGATTCGGCCTTGAGATGGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCA GGACACCCTGAGTACCGCCACACCAAGGGCGTTGAGATCACCACTGGCCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATG GCTGCTCGTCGTGAGCGTGGCCTATTCGACCCAACCGCTGCTGAGGGCGAATCCCCATTCGACCACCACATCTACGTCATTGCTTCTGAT GGTGACCTGCAGGAAGGTGTCACCTCTGAGGCATCCTCCATCGCTGGCACCCAGCAGCTGGGCAACCTCATCGTGTTCTGGGATGACAAC CGCATCTCCATCGAAGACAACACTGAGATCGCTTTCAACGAGGACGTTGTTGCTCGTTACAAGGCTTACGGCTGGCAGACCATTGAGGTT GAGGCTGGCGAGGACGTTGCAGCAATCGAAGCTGCAGTGGCTGAGGCTAAGAAGGACACCAAGCGACCTACCTTCATCCGCGTTCGCACC ATCATCGGCTTCCCAGCTCCAACTATGATGAACACCGGTGCTGTGCACGGTGCTGCTCTTGGCGCAGCTGAGGTTGCAGCAACCAAGACT GAGCTTGGATTCGATCCTGAGGCTCACTTCGCGATCGACGATGAGGTTATCGCTCACACCCGCTCCCTCGCAGAGCGCGCTGCACAGAAG AAGGCTGCATGGCAGGTCAAGTTCGATGAGTGGGCAGCTGCCAACCCTGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTT CCAGCGGGCTACGCTGACGAGCTCCCAACATGGGATGCAGATGAGAAGGGCGTCGCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCA CTGGGCAAGACCCTTCCTGAGCTGTGGGGCGGTTCCGCTGACCTCGCAGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTTCGGCCCTGAGTCCATCTCCACCGAGACCTGGTCTGCTGAGCCTTACGGCCGTAACCTGCACTTCGGTATCCGTGAGCACGCTATGGGATCCATC CTCAACGGCATTTCCCTCCACGGTGGCACCCGCCCATACGGCGGAACCTTCCTCATCTTCTCCGACTACATGCGTCCTGCAGTTCGTCTT GCAGCTCTCATGGAGACCGACGCTTACTACGTCTGGACCCACGACTCCATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAA ACCTTGGCTGCACTGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCCTGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTT GAGTACAAGGAAGGCCCTAAGGGTCTTGCACTGACCCGCCAGAACGTTCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTT CGCCGCGGTGGCTACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGATGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTT AACGCTGCGAAGGCTCTGGAAGCTGAGGGCGTTGCAGCTCGCGTTGTTTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAG TACATCGAGTCCGTTCTGCCTGCAGCTGTGACCGCTCGTGTGTCTGTTGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACC CAGGGCCGTGCTGTCTCCCTTGAGCACTTCGGTGCTTCTGCGGATTACCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTC GTGGCAGCGGCCAAGGACTCCATTAACGGTTAA

[0096] SEQ ID No. 2: sequência de proteína TKT (ou seja, sequência de aminoácidos 700aa codificada pela SEQ ID No. 1) Petition 870250093954, dated 10 / 14 / 2025, pp. 108 / 122 41 / 48 MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSS LTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIYVIASD GDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVAEAKKDTKRPTFIRVRT IIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERAAQKKAAWQVKFDEWAAANPENKALFDRLNSREL PAGYADELPTWDADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPES1STETWSAEPYGRNLHFIREHAMGS1 LNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLRPADANETAQAWAAAL EYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVVSVPCMDWFQEQDAE YIESVLPAAVTARVSVEAG1AMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDS1NG

[0097] SEQ ID No. 3: TKTA327T genetic mutation sequence ORF(CDS) (2103 bp nucleotide sequence) TTGACCACCTTGACGCTGTCACCTGAACTTCAGGCGCTCACTGTACGCAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTA GACACTGTTCGTGTCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTCCGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATAC ACCTTGTACCAGCGGGTTATGAACGTAGATCCACAGGACACCAACTGGGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCT TTGACCCAGTACATCCAGCTTTACTTGGGTGGATTCGGCCTTGAGATGGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCA GGACACCCTGAGTACCGCCACACCAAGGGCGTTGAGATCACCACTGGCCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATG GCTGCTCGTCGTGAGCGTGGCCTATTCGACCCAACCGCTGCTGAGGGCGAATCCCCATTCGACCACCACATCTACGTCATTGCTTCTGAT GGTGACCTGCAGGAAGGTGTCACCTCTGAGGCATCCTCCATCGCTGGCACCCAGCAGCTGGGCAACCTCATCGTGTTCTGGGATGACAAC CGCATCTCCATCGAAGACAACACTGAGATCGCTTTCAACGAGGACGTTGTTGCTCGTTACAAGGCTTACGGCTGGCAGACCATTGAGGTT GAGGCTGGCGAGGACGTTGCAGCAATCGAAGCTGCAGTGGCTGAGGCTAAGAAGGACACCAAGCGACCTACCTTCATCCGCGTTCGCACC ATCATCGGCTTCCCAGCTCCAACTATGATGAACACCGGTGCTGTGCACGGTGCTGCTCTTGGCGCAGCTGAGGTTGCAGCAACCAAGACT GAGCTTGGATTCGATCCTGAGGCTCACTTCGCGATCGACGATGAGGTTATCGCTCACACCCGCTCCCTCGCAGAGCGCACTGCACAGAAGAAGGCTGCATGGCAGGTCAAGTTCGATGAGTGGGCAGCTGCCAACCCTGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTT CCAGCGGGCTACGCTGACGAGCTCCCAACATGGGATGCAGATGAGAAGGGCGTCGCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCA CTGGGCAAGACCCTTCCTGAGCTGTGGGGCGGTTCCGCTGACCTCGCAGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTTCGGC CCTGAGTCCATCTCCACCGAGACCTGGTCTGCTGAGCCTTACGGCCGTAACCTGCACTTCGGTATCCGTGAGCACGCTATGGGATCCATC CTCAACGGCATTTCCCTCCACGGTGGCACCCGCCCATACGGCGGAACCTTCCTCATCTTCTCCGACTACATGCGTCCTGCAGTTCGTCTT GCAGCTCTCATGGAGACCGACGCTTACTACGTCTGGACCCACGACTCCATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAA ACCTTGGCTGCACTGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCCTGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTT GAGTACAAGGAAGGCCCTAAGGGTCTTGCACTGACCCGCCAGAACGTTCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTT CGCCGCGGTGGCTACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGATGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTT AACGCTGCGAAGGCTCTGGAAGCTGAGGGCGTTGCAGCTCGCGTTGTTTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAG TACATCGAGTCCGTTCTGCCTGCAGCTGTGACCGCTCGTGTGTCTGKGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACCCAGGGCCGTGCTGTCTCCCTTGAGCACTTCGGTGCTTCTGCGGATTACCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTC GTGGCAGCGGCCAAGGACTCCATTAACGGTTAA

[0098] SEQ ID No. 4: mutant gene protein sequence TKTA327T (i.e., amino acid sequence 700aa encoded by SEQ ID No. 3) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSS LTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIYVIASD GDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVAEAKKDTKRPTFIRVRT IIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERTAQKKAAWQVKFDEWAAANPENKALFDRLNSREL PAGYADELPTTOADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFIREHAMGSI LNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLRPADANETAQAWAAAL EYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVVSVPCMDWFeEQDAE YIESVLPAAVTARVSVEAGIAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDSING Petition 870250093954, dated 10 / 14 / 2025, pp. 109 / 122 42 / 48

[0099] SEQ ID No. 5: TKTA327S genetic mutation sequence ORF(CDS) (2103 bp nucleotide sequence) TTGACCACCTTGACGCTGTCACCTGAACTTCAGGCGCTCACTGTACGCAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTA GACACTGTTCGTGTCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTCCGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATAC ACCTTGTACCAGCGGGTTATGAACGTAGATCCACAGGACACCAACTGGGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCT TTGACCCAGTACATCCAGCTTTACTTGGGTGGATTCGGCCTTGAGATGGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCA GGACACCCTGAGTACCGCCACACCAAGGGCGTTGAGATCACCACTGGCCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATG GCTGCTCGTCGTGAGCGTGGCCTATTCGACCCAACCGCTGCTGAGGGCGAATCCCCATTCGACCACCACATCTACGTCATTGCTTCTGAT GGTGACCTGCAGGAAGGTGTCACCTCTGAGGCATCCTCCATCGCTGGCACCCAGCAGCTGGGCAACCTCATCGTGTTCTGGGATGACAAC CGCATCTCCATCGAAGACAACACTGAGATCGCTTTCAACGAGGACGTTGTTGCTCGTTACAAGGCTTACGGCTGGCAGACCATTGAGGTT GAGGCTGGCGAGGACGTTGCAGCAATCGAAGCTGCAGTGGCTGAGGCTAAGAAGGACACCAAGCGACCTACCTTCATCCGCGTTCGCACC ATCATCGGCTTCCCAGCTCCAACTATGATGAACACCGGTGCTGTGCACGGTGCTGCTCTTGGCGCAGCTGAGGTTGCAGCAACCAAGACT GAGCTTGGATTCGATCCTGAGGCTCACTTCGCGATCGACGATGAGGTTATCGCTCACACCCGCTCCCTCGCAGAGCGCTCTGCACAGAAGAAGGCTGCATGGCAGGTCAAGTTCGATGAGTGGGCAGCTGCCAACCCTGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTT CCAGCGGGCTACGCTGACGAGCTCCCAACATGGGATGCAGATGAGAAGGGCGTCGCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCA CTGGGCAAGACCCTTCCTGAGCTGTGGGGCGGTTCCGCTGACCTCGCAGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTTCGGC CCTGAGTCCATCTCCACCGAGACCTGGTCTGCTGAGCCTTACGGCCGTAACCTGCACTTCGGTATCCGTGAGCACGCTATGGGATCCATC CTCAACGGCATTTCCCTCCACGGTGGCACCCGCCCATACGGCGGAACCTTCCTCATCTTCTCCGACTACATGCGTCCTGCAGTTCGTCTT GCAGCTCTCATGGAGACCGACGCTTACTACGTCTGGACCCACGACTCCATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAA ACCTTGGCTGCACTGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCCTGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTT GAGTACAAGGAAGGCCCTAAGGGTCTTGCACTGACCCGCCAGAACGTTCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTT CGCCGCGGTGGCTACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGATGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTT AACGCTGCGAAGGCTCTGGAAGCTGAGGGCGTTGCAGCTCGCGTTGTTTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAG TACATCGAGTCCGTTCTGCCTGCAGCTGTGACCGCTCGTGTGTCTGTTGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACCCAGGGCCGTGCTGTCTCCTTGAGCACTTCGGTGCTTCTGCGGATTACCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTC GTGGCAGCGGCCAAGGACTCCATTAACGGTTAA

[00100] SEQ ID no. 6: protein sequence of the TKTA327S mutant gene (ie, 700aa amino acid sequence encoded by SEQ ID No. 5) LTQYIQLYLGGFGLEMDDLKALRTTOSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIYVIASD GDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVAEAKKDTKRPTFTRVRT I lGFPAP1WTGAVEGAAl.GAAEVAATKTEl.GFI)PEAHFarDI)IWIAHTLAERSAQKTEl^O^K PAGYADEI.PTWDADEKGVATRKASEAAl.Qal.GKTI.Pel.WGGSARI.AGSNNTVIKGSPSFGPESISTETWSAEPYGRNl.HFGI REHAMGSI LNGISLUGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTIIDSIGLGEDGPTIIQPVETLAALRAIPGLSVLRPADANETAQAWAAAL EYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVMRVVSVPCMDWFQDAE YIESVLPAAVTARVVEAGAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTVAAKDSING

[00101] SEQ ID No. 7: TKTA327C mutant gene sequence ORF(CDS) (2103 bp nucleotide sequence) Petition 870250093954, dated 10 / 14 / 2025, pp. 110 / 122 43 / 48 TTGACCACCTTGACGCTGTCACCTGAACTTCAGGCGCTCACTGTACGCAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTA GACACTGTTCGTGTCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTCCGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATAC ACCTTGTACCAGCGGGTTATGAACGTAGATCCACAGGACACCAACTGGGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCT TTGACCCAGTACATCCAGCTTTACTTGGGTGGATTCGGCCTTGAGATGGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCA GGACACCCTGAGTACCGCCACACCAAGGGCGTTGAGATCACCACTGGCCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATG GCTGCTCGTCGTGAGCGTGGCCTATTCGACCCAACCGCTGCTGAGGGCGAATCCCCATTCGACCACCACATCTACGTCATTGCTTCTGAT GGTGACCTGCAGGAAGGTGTCACCTCTGAGGCATCCTCCATCGCTGGCACCCAGCAGCTGGGCAACCTCATCGTGTTCTGGGATGACAAC CGCATCTCCATCGAAGACAACACTGAGATCGCTTTCAACGAGGACGTTGTTGCTCGTTACAAGGCTTACGGCTGGCAGACCATTGAGGTT GAGGCTGGCGAGGACGTTGCAGCAATCGAAGCTGCAGTGGCTGAGGCTAAGAAGGACACCAAGCGACCTACCTTCATCCGCGTTCGCACC ATCATCGGCTTCCCAGCTCCAACTATGATGAACACCGGTGCTGTGCACGGTGCTGCTCTTGGCGCAGCTGAGGTTGCAGCAACCAAGACT GAGCTTGGATTCGATCCTGAGGCTCACTTCGCGATCGACGATGAGGTTATCGCTCACACCCGCTCCCTCGCAGAGCGCTGTGCACAGAAG AAGGCTGCATGGCAGGTCAAGTTCGATGAGTGGGCAGCTGCCAACCCTGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTT CCAGCGGGCTACGCTGACGAGCTCCCAACATGGGATGCAGATGAGAAGGGCGTCGCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCA CTGGGCAAGACCCTTCCTGAGCTGTGGGGCGGTTCCGCTGACCTCGCAGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTTCGGC CCTGAGTCCATCTCCACCGAGACCTGGTCTGCTGAGCCTTACGGCCGTAACCTGCACTTCGGTATCCGTGAGCACGCTATGGGATCCATC CTCAACGGCATTTCCCTCCACGGTGGCACCCGCCCATACGGCGGAACCTTCCTCATCTTCTCCGACTACATGCGTCCTGCAGTTCGTCTT GCAGCTCTCATGGAGACCGACGCTTACTACGTCTGGACCCACGACTCCATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAA ACCTTGGCTGCACTGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCCTGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTT GAGTACAAGGAAGGCCCTAAGGGTCTTGCACTGACCCGCCAGAACGTTCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTT CGCCGCGGTGGCTACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGATGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTTAACGCTGCGAAGGCTCTGGAAGCTGAGGCGTTGCAGCTCGCGTTGTTTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAG TACATCGAGTCCGTTCTGCCTGCAGCTGTGACCGTCCGTGTGTCTGTTGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACC CAGGGCCGTGCTGTCTCCCTTGAGCACTTCGGTGCTTCTGCGGATTACCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTC GTGGCAGCGGCCAAGGACTCCATTAACGGTTAA

[00102] SEQ ID No. 8: protein sequence of the TKTA327C mutant gene (i.e., 700aa amino acid sequence encoded by SEQ ID No. 7) MTTLTLSPELQALTVRNYPSDWSDVDTKÀVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSS LTQYIQLYLGGFGLERODLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIYVIASD GDLQEGVTSEASSIAGTQQLGNLIVTWDDNRISIEDNTEIAFNEDmRYKAYGWQnEVEAGEDVAAIEAAVAEAKKDTKRPTFIRVRT IIGFPAPTWNTGAVHG.ULGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERCAQKKAAWQVKFDEWAAANPENKALFDRLNSREL PAGYADELPTWDADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFIREHAMGSI LNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLRPADANETAQAWAAAL EYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARYVSVPCMDWFQEQDAE YIESVLPAAVTARVSVEAGIAMPWWLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDSING

[00103] SEQ ID No. 9: Genetic mutation sequence TKTA327PORF(CDS) (nucleotide sequence of 2103 bp) Petition 870250093954, dated 10 / 14 / 2025, pages 111 / 122 44 / 48 TTGACCACCTTGACGCTGTCACCTGAACTTCAGGCGCTCACTGTACGCAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTA GACACTGTTCGTGTCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTCCGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATAC ACCTTGTACCAGCGGGTTATGAACGTAGATCCACAGGACACCAACTGGGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCT TTGACCCAGTACATCCAGCTTTACTTGGGTGGATTCGGCCTTGAGATGGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCA GGACACCCTGAGTACCGCCACACCAAGGGCGTTGAGATCACCACTGGCCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATG GCTGCTCGTCGTGAGCGTGGCCTATTCGACCCAACCGCTGCTGAGGGCGAATCCCCATTCGACCACCACATCTACGTCATTGCTTCTGAT GGTGACCTGCAGGAAGGTGTCACCTCTGAGGCATCCTCCATCGCTGÍXACCCAGCAGCTGGGCAACCTCATCGTGTTCTGGGATGACAAC CGCATCTCCATCGAAGACAACACTGAGATCGCTTTCAACGAGGACGnGTTGCTCGTTACAAGGCTTACGGCTGGCAGACCATTGAGGTT GAGGCTGGCGAGGACGTTGCAGCAATCGAAGCTGCAGTGGCTGAGGCTAAGAAGGACACCAAGCGACCTACCTTCATCCGCGTTCGCACC ATCATCGGCnCCCAGCTCCAACTATGATGAACACCGGTGCTGTGCACGGTGCTGCTCTTGGCGCAGCTGAGGTTGCAGCAACCAAGACT GAGCTTGGATTCGATCCTGAGGCTCACTTCGCGATCGACGATGAGGTTATCGCTCACACCCGCTCCCTCGCAGAGCGCCCTGCACAGAAGAAGGCTGCATGGCAGGTCAAGTTCGATGAGTGGGCAGCTGCCAACCCTGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTT CCAGCGGGCTACGCTGACGAGCTCCCAACATGGGATGCAGATGAGAAGGGCGTCGCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCA CTGGGCAAGACCCTTCCTGAGCTGTGGGGCGGTTCCGCTGACCTCGCAGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTTCGGC CCTGAGTCCATCTCCACCGAGACCTGGTCTGCTGAGCCTTACGGCCGTAACCTGCACTTCGGTATCCGTGAGCACGCTATGGGATCCATC CTCAACGGCATTTCCCTCCACGGTGGCACCCGCCCATACGGCGGAACCTTCCTCATCTTCTCCGACTACATGCGTCCTGCAGTTCGTCTT GCAGCTCTCATGGAGACCGACGCTTACTACGTCTGGACCCACGACTCCATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAA ACCTTGGCTGCACTGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCCTGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTT GAGTACAAGGAAGGCCCTAAGGGTCTTGCACTGACCCGCCAGAACGTTCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTT CGCCGCGGTGGCTACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGATGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTT AACGCTGCGAAGGCTCTGGAAGCTGAGGGCGTTGCAGCTCGCGTTGTTTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAG TACATCGAGTCCGTTCTGCCTGCAGCTGTGACCGCTCGTGTGTCTGTTGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACC CAGGGCCGTGCTGTCTCCCTTGAGCACTTCGGTGCTTCTGCGGATTACCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTC GTGGCAGCGGCCAAGGACTCCATTAACGGTTAA

[00104] SEQ ID No. 10: sequência de proteína do gene mutante TKTA327P(ou seja, sequência de aminoácidos 700aa codificada pela SEQ ID No. 9) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSS I.TQYIQLYI.GGFGl.EMDDi.KALRTffDSETPGHPEYRHTKGVETTTGPI.GQGI.ASAVCTMAMARRERGI.FDPTAAEGESPFDHHTYVIASP GDLQEGVTSEASS IAGTQQLGNLIVFWDDNRISIEDNTE1AFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVAEAKKDTKRPTFIRVRT IIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERPAQKKAAWQVKFDEWANKPEHFAIDREFARVRT PAGYADEI.PTWDADEKfAATRKASEAAEQAI.Gm.Pel.WGGSAIWTSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREHAMGSl LNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVALPAVALMETDAY EYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVSVPCMDWFQEQDAE ΥΙΕ3νΕΡΑΑνΤΑΕν3νΕΑ0ΙΑΜΡΪΥΕΡΕ0Τς6ΕΑν5 1.ΕΗΡΕ.Α5ΑϋΥΰΤΕΡΕΚΡ01ΤΤ0ΑννΑΑΑΚ03ΙΝ0

[00105] SEQ ID no. 11: TKTA327N mutant gene sequence ORF(CDS) (nucleotide sequence of 2103 bp) Petition 870250093954, of 14 / 10 / 2025, p. 112 / 122 45 / 48 TTGACCACCTTGACGCTGTCACCTGAACTTCAGGCGCTCACTGTACGCAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTA GACACTGTTCGTGTCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTCCGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATAC ACCTTGTACCAGCGGGTTATGAACGTAGATCCACAGGACACCAACTGGGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCT TTGACCCAGTACATCCAGCTTTACTTGGGTGGATTCGGCCTTGAGATGGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCA GGACACCCTGAGTACCGCCACACCAAGGGCGTTGAGATCACCACTGGCCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATG GCTGCTCGTCGTGAGCGTGGCCTATTCGACCCAACCGCTGCTGAGGGCGAATCCCCATTCGACCACCACATCTACGTCATTGCTTCTGAT GGTGACCTGCAGGAAGGTGTCACCTCTGAGGCATCCTCCATCGCTGGCACCCAGCAGCTGGGCAACCTCATCGTGTTCTGGGATGACAAC CGCATCTCCATCGAAGACAACACTGAGATCGCTTTCAACGAGGACGTTGTTGCTCGTTACAAGGCTTACGGCTGGCAGACCATTGAGGTT GAGGCTGGCGAGGACGTTGCAGCAATCGAAGCTGCAGTGGCTGAGGCTAAGAAGGACACCAAGCGACCTACCTTCATCCGCGTTCGCACC ATCATCGGCTTCCCAGCTCCAACTATGATGAACACCGGTGCTGTGCACGGTGCTGCTCTTGGCGCAGCTGAGGTTGCAGCAACCAAGACT GAGCTTGGATTCGATCCTGAGGCTCACTTCGCGATCGACGATGAGGTTATCGCTCACACCCGCTCCCTCGCAGAGCGCAATGCACAGAAGAAGGCTGCATGGCAGGTCAAGTTCGATGAGTGGGCAGCTGCCAACCCTGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTT CCAGCGGGCTACGCTGACGAGCTCCCAACATGGGATGCAGATGAGAAGGGCGTCGCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCA CTGGGCAAGACCCTTCCTGAGCTGTGGGGCGGTTCCGCTGACCTCGCAGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTTCGGC CCTGAGTCCATCTCCACCGAGACCTGGTCTGCTGAGCCTTACGGCCGTAACCTGCACTTCGGTATCCGTGAGCACGCTATGGGATCCATC CTCAACGGCATTTCCCTCCACGGTGGCACCCGCCCATACGGCGGAACCTTCCTCATCTTCTCCGACTACATGCGTCCTGCAGTTCGTCTT GCAGCTCTCATGGAGACCGACGCTTACTACGTCTGGACCCACGACTCCATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAA ACCTTGGCTGCACTGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCCTGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTT GAGTACAAGGAAGGCCCTAAGGGTCTTGCACTGACCCGCCAGAACGTTCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTT CGCCGCGGTGGCTACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGATGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTT AACGCTGCGAAGGCTCTGGAAGCTGAGGGCGTTGCAGCTCGCGTTGTTTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAG TACATCGAGTCCGTTCTGCCTGCAGCTGTGACCGCTCGTGTGTCTGTTGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACCCAGGGCCGTGCTGTCTCCTTGAGCACTTCGGTGCTTCTGCGGATTACCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTC GTGGCAGCGGCCAAGGACTCCATTAACGGTTAA

[00106] SEQ ID no. 12: protein sequence of TKTA327Ndo mutant gene (i.e., 700aa amino acid sequence encoded by SEQ ID No. 11) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAMAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSS LTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIYVIASD GDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVAEAKKDTKRPTFIRVRT IIGFPÀPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERNAQREKVREKWANKREQFALQFALGANFARFARV PAGYADELPTWDADEKGVATRCASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREHAMGSI LNGISLHGGTRPYGGTFLIFSDYMRPAVRLAAUIETDAYYVWTHDSIGLGEDGPTHQPVETLAAUIETDAYYVWTHDSIGLGEDGPTHQPVETLAAUIETDAYYVWTHDSIGLGEDGPTHQPVETLAAUIETDAYVWTHDSIGLGEDGPTHQPVETLAAUIETDAYYVWTHDSIGLGEDGPTHQPVETLAAUIETDAYV EYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVSVPCMDWFQEQDAE YIESVTPAAVTARVWEGIAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVAKDSING

[00107] SEQ ID No. 13: mutant gene sequence TKTA327QORF(CDS) (nucleotide sequence of 2103 bp) Petition 870250093954, dated 10 / 14 / 2025, pp. 113 / 122 46 / 48 TTGACCACCTTGACGCTGTCACCTGAACTTCAGGCGCTCACTGTACGCAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTA GACACTGTTCGTGTCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTCCGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATAC ACCTTGTACCAGCGGGTTATGAACGTAGATCCACAGGACACCAACTGGGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCT TTGACCCAGTACATCCAGCTTTACTTGGGTGGATTCGGCCTTGAGATGGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCA GGACACCCTGAGTACCGCCACACCAAGGGCGTTGAGATCACCACTGGCCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATG GCTGCTCGTCGTGAGCGTGGCCTATTCGACCCAACCGCTGCTGAGGGCGAATCCCCATTCGACCACCACATCTACGTCATTGCTTCTGAT GGTGACCTGCAGGAAGGTGTCACCTCTGAGGCATCCTCCATCGCTGGCACCCAGCAGCTGGGCAACCTCATCGTGTTCTGGGATGACAAC CGCATCTCCATCGAAGACAACACTGAGATCGCTTTCAACGAGGACGTTGTTGCTCGTTACAAGGCTTACGGCTGGCAGACCATTGAGGTT GAGGCTGGCGAGGACGTTGCAGCAATCGAAGCTGCAGTGGCTGAGGCTAAGAAGGACACCAAGCGACCTACCTTCATCCGCGTTCGCACC ATCATCGGCTTCCCAGCTCCAACTATGATGAACACCGGTGCTGTGCACGGTGCTGCTCTTGGCGCAGCTGAGGTTGCAGCAACCAAGACT GAGCTTGGATTCGATCCTGAGGCTCACTTCGCGATCGACGATGAGGTTATCGCTCACACCCGCTCCCTCGCAGAGCGCCAGGCACAGAAGAAGGCTGCATGGCAGGTCAAGTTCGATGAGTGGGCAGCTGCCAACCCTGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTT CCAGCGGGCTACGCTGACGAGCTCCCAACATGGGATGCAGATGAGAAGGGCGTCGCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCA CTGGGCAAGACCCTTCCTGAGCTGTGGGGCGGTTCCGCTGACCTCGCAGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTTCGGC CCTGAGTCCATCTCCACCGAGACCTGGTCTGCTGAGCCTTACGGCCGTAACCTGCACTTCGGTATCCGTGAGCACGCTATGGGATCCATC CTCAACGGCATTTCCCTCCACGGTGGCACCCGCCCATACGGCGGAACCTTCCTCATCTTCTCCGACTACATGCGTCCTGCAGTTCGTCTT GCAGCTCTCATGGAGACCGACGCTTACTACGTCTGGACCCACGACTCCATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAA ACCTTGGCTGCACTGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCCTGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTT GAGTACAAGGAAGGCCCTAAGGGTCTTGCACTGACCCGCCAGAACGTTCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTT CGCCGCGGTGGCTACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGATGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTT AACGCTGCGAAGGCTCTGGAAGCTGAGGGCGTTGCAGCTCGCGTTGTTTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAG TACATCGAGTCCGTTCTGCCTGCAGCTGTGACCGCTCGTGTGTCTGTTGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACCCAGGGCCGTGCTGTCTCCTTGAGCACTTCGGTGCTTCTGCGGATTACCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTC GTGGCAGCGGCCAAGGACTCCATTAACGGTTAA

[00108] SEQ ID no. 14: protein sequence of TKTA327Qdo mutant gene (i.e., 700aa amino acid sequence encoded by SEQ ID No. 13) MTTLSLSPELQALTVRNYPSDWSDVDTKAVDTVRVIAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSS LTQYIQLYLGGFGLEMDDLKALRTTOSLTPGHPEYRHTKGVEITTGPLGQGLAERGLAGLAGLAGFLAGFADPHYFADPH GDLQEGVTSESIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAIEAAVAEAanDTKRPTFIRVRT IIGFPAPTMMNTGAVHGAALGAAEVAATKTELGDPPEAHFADDEVIAHTRSLAERQAQKKAWKDEFKVALKVALQLPEAHFIRVRT PAGYADELPTWDADEKGVATRCASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREEAMGSI LNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVTLAALMETDAYYVWTHDSIGLGEDGPTHQPVTLRAIPLAAPAGALQAVAL EYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSEVQLAVNAAKALEAEGVAARWSVPMCMDWFQEQDAE YIESVLPAAVTARVVEAGAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAWAAKDSING

[00109] SEQ ID No. 15: sequência de amplificação do primer TKT-F / TKT-R (tamanho de 2689 pb) CAGAATAATTAAGCTTGCATGCCTGCAGGTCGACTTCACAGCGGACGATTTCAGGCCCTCGTAGCTCGAGAGTTTGAAGGGGTCCGATTC GTTCCGTTCGTGACGCTTTGTGAGGTTTTTTGACGTTGCACCGTATTGCTTGCCGAACATTTTTCTTTTCCTTTCGGTTTTTCGAGAATT TTCACCTACAAAAGCCCACGTCACAGCTCCCAGACTTAAGATTGATCACACCTTTGACACATTTGAACCACAGTTGGTTATAAAATGGGT TCAACATCACTATGGTTAGAGGTGTTGACGGGTCAGATTAAGCAAAGACTACTTTCGGGGTAGATCACCTTTGCCAAATTTGAACCAATT AACCTAAGTCGTAGATCTGATCATCGGATCTAACGAAAACGAACCAAAACTTTGGTCCCGGTTTAACCCAGGAAGGATTGACCACCTTGA Petição 870250093954, de 14 / 10 / 2025, pág. 114 / 122 47 / 48 CGCTGTCACCTGAACTTCAGGCGCTCACTGTACGCAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTAGACACTGTTCGTG TCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTCCGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATACACCTTGTACGAGC GGGTTATGAACGTAGATCCACAGGACACCAACTGGGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCTTTGACCCAGTACA TCCAGCTTTACTTGGGTGGATTCGGCCTTGAGATGGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCAGGACACCCTGAGT ACCGCCACACCAAGGGCGTTGAGATCACCACTGGCCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATGGCTGCTCGTCGTG AGCGTGGCCTATTCGACCCAACCGCTGCTGAGGGCGAATCCCCATTCGACCACCACATCTACGTCATTGCTTCTGATGGTGACCTGCAGG AAGGTGTCACCTCTGAGGCATCCTCCATCGCTGGCACCCAGCAGCTGGGCAACCTCATCGTGTTCTGGGATGACAACCGCATCTCCATCG AAGACAACACTGAGATCGCTTTCAACGAGGACGTTGTTGCTCGTTACAAGGCTTACGGCTGGCAGACCATTGAGGTTGAGGCTGGCGAGG ACGTTGCAGCAATCGAAGCTGCAGTGGCTGAGGCTAAGAAGGACACCAAGCGACCTACCTTCATCCGCGTTCGCACCATCATCGGCTTCC CAGCTCCAACTATGATGAACACCGGTGCTGTGCACGGTGCTGCTCTTGGCGCAGCTGAGGTTGCAGCAACCAAGACTGAGCTTGGATTCG ATCCTGAGGCTCACTTCGCGATCGACGATGAGGTTATCGCTCACACCCGCTCCCTCGCAGAGCGCGCTGCACAGAAGAAGGCTGCATGGCAGGTCAAGTTCGATGAGTGGGCAGCTGCCAACCCTGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTTCCAGCGGGCTACG CTGACGAGCTCCCAACATGGGATGCAGATGAGAAGGGCGTCGCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCACTGGGCAAGACCC TTCCTGAGCTGTGGGGCGGTTCCGCTGACCTCGCAGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTTCGGCCCTGAGTCCATCT CCACCGAGACCTGGTCTGCTGAGCCTTACGGCCGTAACCTGCACTTCGGTATCCGTGAGCACGCTATGGGATCCATCCTCAACGGCATTT CCCTCCACGGTGGCACCCGCCCATACGGCGGAACCTTCCTCATCTTCTCCGACTACATGCGTCCTGCAGTTCGTCTTGCAGCTCTCATGG AGACCGACGCTTACTACGTCTGGACCCACGACTCCATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAAACCTTGGCTGCAC TGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCCTGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTTGAGTACAAGGAAG GCCCTAAGGGTCTTGCACTGACCCGCCAGAACGTTCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTTCGCCGCGGTGGCT ACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGATGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTTAACGCTGCGAAGG CTCTGGAAGCTGAGGGCGTTGCAGCTGGCGTTGTTTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAGTACATCGAGTCCG TTCTGCCTGCAGCTGTGACCGCTCGTGTGTCTGTTGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACCCAGGGCCGTGCTGTCTCCCTTGAGCACTTCGGTGCTTCTGCGGATTACCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTCGTGGCAGCGGCCA AGGACTCCATTAACGGTTAATTGCCCTGCTGTTTTAGCTTCAACCCGGGGCAATATCTCCGGAATTTTATTGCCCGGGTTGTTG TTCTTAATCGGTACAAAGGGTCTTAAGCACATCCCTTACTTGCCTGGTACCGAGCTCGAATTCAGCTTGGCTGTTTTGG

[00110] SEQ ID no. 16: P7 / P8TKT integrated into the genome and its promoter sequence (2621 bp) TTCACAGCGGACGATTTCAGGCCCTCGTAGCTCGAGAGTTTGAAGGGGTCCGATTCGTTCCGTTCGTGACGCTTTGTGAGGTTTTTTGAC GTTGCACCGTATTGCTTGCCGAACATTTTTCTTTTCCTTTCGGTTTTTCGAGAATTTTCACCTACAAAAGCCCACGTCACAGCTCCCAGA CTTAAGATTGATCACACCTTTGACACATTTGAACCACAGTTGGTTATAAAATGGGTTCAACATCACTATGGTTAGAGGTGTTGACGGGTC AGATTAAGCAAAGACTACTTTCGGGGTAGATCACCTTTGCCAAATTTGAACCAATTAACCTAAGTCGTAGATCTGATCATCGGATCTAAC GAAAACGAACCAAAACTTTGGTCCCGGTTTAACCCAGGAAGGATTGACCACCTTGACGCTGTCACCTGAACTTCAGGCGCTCACTGTACG CAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTAGACACTGTTCGTGTCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTC CGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATACACCTTGTACCAGCGGGTTATGAACGTAGATCCACAGGACACCAACTG GGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCTTTGACCCAGTACATCCAGCTTTACTTGGGTGGATTCGGCCTTGAGAT GGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCAGGACACCCTGAGTACCGCCACACCAAGGGCGTTGAGATCACCACTGG CCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATGGCTGCTCGTCGTGAGCGTGGCCTATTCGACCCAACCGCTGCTGAGGG CGAATCCCCATTCGACCACCACATCTACGTCATTGCTTCTGATGGTGACCTGCAGGAAGGTGTCACCTCTGAGGCATCCTCCATCGCTGGCACCCAGCAGCTGGGGCAACCTCATCGTGTTCTGGATGACAACCGCATCTCCATCGAAGACAACACTGAGATCGCCTTTCAACGAGGACGGT TGTTGCTCGnACAAGGCTTACGGCTGGCAGACCATGAGGTTGAGGCTGGCGAGGACGTTGCAGCAATCGAAGCTGCAGTGGC TAAGAAGGACACCAAGCCACCTACCTCTCTCGCAGCTCCCAACCATCGGCCTTCCCAGCTCCCAACCATGGGATGCGAGTGTGGA TATCGCTCACACCCGCTCCCCCGCAGAGCGCGCTGCACAGAAGAGCTGCATGGCAGGTCGAGTCAAGTTCGATGGGCAGCTGCCAACC TGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTTCCACGGGCTACGAGCTCCCAACATGGGATGCAGATGGAGAA GGGCGTCCGCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCACTGGGCAAGACCCTTCCTGAGCTGTGGGGCGGTTCCGCTGACCTCGC AGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTCGGCCTGAGTCCATCTCCACCGAGACCTGGTCTGCTGAGCCTTACGGCCG TAACCTGCACTTCGGTATCCTGAGCACGCTATGGATCCATCCTCACGGCATTTCCCTCCACGGTGCACCGCCCATACGGCGGAAC CTTCCTCATCTTCTCGACTACATGCGTCCTGCAGTTCGTCTTGCAGCTCTCATGGAGACCGACGCTTACTACGTCTGGACCCACGACTC CATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAAACCTTGGCTGCACTGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCC Petição 870250093954, de 14 / 10 / 2025, pág. 115 / 122 48 / 48 TGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTTGAGTACAAGGAAGGCCCTAAGGGTCTTGCACTGACCCGCCAGAACGT TCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTTCGCCGCGGTGGCTACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGA TGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTTAACGCTGCGAAGGCTCTGGAAGCTGAGGGCGTTGCAGCTCGCGTTGT TTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAGTACATCGAGTCCGTTCTGCCTGCAGCTGTGACCGCTCGTGTGTCTGT TGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACCCAGGGCCGTGCTGTCTCCCTTGAGCACTTCGGTGCTTCTGCGGATTA CCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTCGTGGCAGCGGCCAAGGACTCCATTAACGGTTAATTGCCCTGCTGTTT TTAGCTTCAACCCGGGGCAATATGATTCTCCGGAATTTTATTGCCCCGGGTTGTTGTTGTTAATCGGTACAAAGGGTCTTAAGCACATCC CTTACTTGCCT

[00111] SEQ ID No. 17: P7 / P8 TKTA327Tintegrado ao genoma e sua sequência promotora (2621 pb) TTCACAGCGGACGATTTCAGGCCCTCGTAGCTCGAGAGTTTGAAGGGGTCCGATTCGTTCCGTTCGTGACGCTTTGTGAGGTTTTTTGAC GTTGCACCGTATTGCTTGCCGAACATTTTTCTTTTCCTTTCGGTTTTTCGAGAATTTTCACCTACAAAAGCCCACGTCACAGCTCCCAGA CTTAAGATTGATCACACCTTTGACACATTTGAACCACAGTTGGTTATAAAATGGGTTCAACATCACTATGGTTAGAGGTGTTGACGGGTC AGATTAAGCAAAGACTACTTTCGGGGTAGATCACCTTTGCCAAATTTGAACCAATTAACCTAAGTCGTAGATCTGATCATCGGATCTAAC GAAAACGAACCAAAACTTTGGTCCCGGTTTAACCCAGGAAGGATTGACCACCTTGACGCTGTCACCTGAACTTCAGGCGCTCACTGTACG CAATTACCCCTCTGATTGGTCCGATGTGGACACCAAGGCTGTAGACACTGTTCGTGTCCTCGCTGCAGACGCTGTAGAAAACTGTGGCTC CGGCCACCCAGGCACCGCAATGAGCCTGGCTCCCCTTGCATACACCTTGTACCAGCGGGTTATGAACGTAGATCCACAGGACACCAACTG GGCAGGCCGTGACCGCTTCGTTCTTTCTTGTGGCCACTCCTCTTTGACCCAGTACATCCAGCTTTACTTGGGTGGATTCGGCCTTGAGAT GGATGACCTGAAGGCTCTGCGCACCTGGGATTCCTTGACCCCAGGACACCCTGAGTACCGCCACACCAAGGGCGTTGAGATCACCACTGGCCCTCTTGGCCAGGGTCTTGCATCTGCAGTTGGTATGGCCATGGCTGCTCGTCGTGAGCGTGGCCTATTCGACCCAACCGCTGCTGAGGGG CGAATCCCCATTCGCCACCACATCTACGTCATTGCTGTGATGGTGACCTGCAGGAGGTGTTCACCTCTGAGGCATCCTCCATCGCTGG CACCCAGCGAGCTGGGCAACCTCATCGTGTTCTGGATGACAACCGCATCTCCATCGAAGACAACACTGAGATGGCCTTTCAACGGAGGAGGTTGCTGAGGC TAAGAAGGACACCAGCGACCTACCTTCATCCGCGTTCGCACCATCATCGGCTTCCCAGCTCCAACTATGATACACACCGGTGCTTGGA CGGTGCTCTTGGGCGCAGCTGAGGGTTGCAGCAACCAAGACTGAGCTTGGATTCGATCCTGAGGCTCACTTCGCGATCGACGATGAGCGTGACGATGAGGGT TATCGCTCACACCCGCTCCCTCGCAGAGCGCACTGCACAGAAGAAGGCTGCATGGCAGGTCAAGTTCGATGAGTGGGCAGCTGCCAACCC TGAGAACAAGGCTCTGTTCGATCGCCTGAACTCCCGTGAGCTTCCAGCGGGCTACGCTGACGAGCTCCCAACATGGGATGCAGATGAGAA GGGCGTCCAACTCGTAAGGCTTCCGAGGCTGCACTTCAGGCACTGGGCAAGACCCTTCCTGAGCTGGGGCGGTTCCGCTGACCTGC. AGGTTCCAACAACACCGTGATCAAGGGCTCCCCTTCCTTCGGCCCTGAGTCCATCTCCACCGAGACCTGGTCTGCTGAGCCTTACGGCCG TAACCTGCACTTCGGTATCCGTGAGCACGCTATGGGATCCATCTCAACGGCATTTCCCTCCACGGTGGCACCCGCCCATACGCGGAACCTTCCTCATCTTCTCCGACTACATGCGTCCTGCAGTTCGTCTTGCAGCTCTCATGGAGACCGACGCTTACTACGTCTGGACCCACGACTC CATCGGTCTGGGCGAAGATGGCCCAACCCACCAGCCTGTTGAAACCTTGGCTGCACTGCGCGCCATCCCAGGTCTGTCCGTCCTGCGTCC TGCAGATGCGAACGAGACCGCCCAGGCTTGGGCTGCAGCACTTGAGTACAAGGAAGGCCCTAAGGGTCTTGCACTGACCCGCCAGAACGT TCCTGTTCTGGAAGGCACCAAGGAGAAGGCTGCTGAAGGCGTTCGCCGCGGTGGCTACGTCCTGGTTGAGGGTTCCAAGGAAACCCCAGA TGTGATCCTCATGGGCTCCGGCTCCGAGGTTCAGCTTGCAGTTAACGCTGCGAAGGCTCTGGAAGCTGAGGGCGTTGCAGCTCGCGTTGT TTCCGTTCCTTGCATGGATTGGTTCCAGGAGCAGGACGCAGAGTACATCGAGTCCGTTCTGCCTGCAGCTGTGACCGCTCGTGTGTCTGT TGAAGCTGGCATCGCAATGCCTTGGTACCGCTTCTTGGGCACCCAGGGCCGTGCTGTCTCCCTTGAGCACTTCGGTGCTTCTGCGGATTA CCAGACCCTGTTTGAGAAGTTCGGCATCACCACCGATGCAGTCGTGGCAGCGGCCAAGGACTCCATTAACGGTTAATTGCCCTGCTGTTT TTAGCTTCAACCCGGGGCAATATGATTCTCCGGAATTTTATTGCCCCGGGTTGTTGTTGTTAATCGGTACAAAGGGTCTTAAGCACATCC CTTACTTGCCT Petição 870250093954, de 14 / 10 / 2025, pág. 116 / 122

Claims

1 / 4 Claims 1. PROTEIN, characterized by being (A1) or (A2) or (A3) as follows: (A1) a protein comprising SEQ ID No. 2, or a mutant protein that is obtained by mutation of an alanine residue at position 327 in SEQ ID No.2 in a threonine residue, a serine residue, a cysteine ​​residue, a proline residue, an asparagine residue, a glutamine residue, a phenylalanine residue, a leucine residue, a valine residue, an isoleucine residue, an aspartic acid residue, a methionine residue, an arginine residue, a glutamic acid residue, a glycine residue, a histidine residue, a lysine residue, a tryptophan residue, or a tyrosine residue; (A2) a protein obtained by substitution and / or deletion and / or addition of one or more amino acid residues in an amino acid sequence of the protein of (A1), except for position 327, and having the same function as (A1); and (A3) a fusion protein obtained by attaching a tag to an N-terminus and / or a C-terminus of (A1) or (A2).

2. PROTEIN-ASSOCIATED BIOMATERIAL, as defined in claim 1, characterized by being any one of (B1) to (B4) as follows: (B1) a nucleic acid molecule encoding the protein, as defined in claim 1; (B2) an expression cassette containing the nucleic acid molecule of (B1); (B3) a recombinant vector containing the nucleic acid molecule of (B1), or a recombinant vector containing the expression cassette of (B2); and (B4) a recombinant microorganism containing the nucleic acid molecule of (B1), or a recombinant microorganism containing the expression cassette of (B2), or a recombinant microorganism containing the recombinant vector of (B3).

3. BIOMATERIAL, according to claim 2, characterized in that the nucleic acid molecule of (B1) is any one of (b11)-(b19) as follows: (b11) a DNA molecule as represented by SEQ ID No. 3 in the sequence listing; (b12) a DNA molecule as represented by SEQ ID No. 5 in the sequence listing; (b13) a DNA molecule as represented by SEQ ID No. 7 in the sequence listing; (b14) a DNA molecule as represented by SEQ ID No. 9 in the sequence listing; (b15) a DNA molecule as represented by SEQ ID No. 11 in the sequence listing; (b16) a DNA molecule as represented by SEQ ID No. 13 in the sequence listing; (b17) a DNA molecule as represented by SEQ ID No.1 in the sequence listing; (b18) a DNA molecule that has 75% or more identity with a nucleotide sequence defined by any one of (b11) (b17) and that encodes the protein as defined in claim 1; and (b19) a genomic DNA molecule that hybridizes with the nucleotide sequence defined by any one of (b11) - (b18) under stringent conditions and that encodes the protein as defined in claim 1.

4. BIOMATERIAL, according to any of the Petition 870250093954, dated 10 / 14 / 2025, pp. 118 / 122 3 / 4 claims 2 to 3, characterized by a promoter in the (B2) expression cassette being a DNA molecule shown by positions 35-437 in SEQ ID No. 15; and the recombinant microorganism of (B4) is a recombinant microorganism that is obtained by replacing a TKT gene in the microorganism containing the tkt gene, as represented by SEQ ID No. 1, with that as represented by SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13, or a recombinant microorganism that is obtained by introducing the nucleic acid molecule of (B1) into the microorganism and by expressing the nucleic acid molecule of (B1).

5. METHOD FOR PREPARING L-LYSINE, characterized by comprising: expressing the protein, as defined in claim 1, in a recipient biological cell, or increasing the content or activity of the protein, as defined in claim 1, in the recipient biological cell, or increasing the content or activity of the protein as represented by SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 2 in the recipient biological cell to obtain a recombinant biological cell; and culturing the recombinant biological cell to obtain L-lysine.

6. METHOD, according to claim 5, characterized in that the biological cell is a yeast, bacterium, alga, fungus, plant cell or animal cell that is capable of synthesizing L-lysine.

7. METHOD, according to claim 6, characterized in that the bacteria are Corynebacterium glutamicum.

8. PRODUCT FOR PREPARING L-LYSINE, characterized by containing the protein, as defined in claim 1, or the biomaterial, as defined in any one of claims 2 to 4.

9. APPLICATION OF THE PROTEIN, as defined in Petition 870250093954, dated 10 / 14 / 2025, page 119 / 122 4 / 4 claim 1, characterized by being in the production of L-lysine, or in the preparation of a product for the production of L-lysine.

10. APPLICATION OF THE BIOMATERIAL, as defined in any one of claims 2 to 4, characterized by being in the production of L-lysine, or in the preparation of a product for the production of L-lysine.

11. APPLICATION OF THE PROTEIN, as defined in claim 1, characterized by being in the preparation of foods, feeds or medicines containing L-lysine.

12. APPLICATION OF THE BIOMATERIAL, as defined in any one of claims 2 to 4, characterized by being in the preparation of foods, feeds or medicines containing L-lysine.

13. APPLICATION OF THE METHOD, as defined in any one of claims 5 to 7, characterized by being in the preparation of foods, feeds or medicines containing L-lysine.

14. APPLICATION OF THE PRODUCT, as defined in claim 8, characterized by being in the preparation of food, animal feed or medicines containing L-lysine. Petition 870250093954, dated 10 / 14 / 2025, pp. 120 / 122