Mutant of Pyruvate Carboxylase Gene Promoter and Use Thereof

Abstract

Disclosed are a mutant of a pyruvate carboxylase gene promoter of Corynebacterium glutamicum and applications thereof. The mutant has improved promoter activity compared with a wild-type promoter, and can be used for enhancing expression of a target gene, for example, operably ligating the mutant to a pyruvate carboxylase gene enhances the expression intensity of the pyruvate carboxylase, thereby improving the production efficiency of amino acids of the strain.

Claims

1. A mutant of a pyruvate carboxylase gene promoter in Corynebacterium glutamicum, wherein the mutant is any one selected from the group consisting of the following (i) to (iv): (i) the mutant has one or more mutated nucleotides in a core region corresponding to position 279 to position 317 of a promoter having a nucleotide sequence set forth in SEQ ID NO: 21; (ii) comprising a reverse complementary sequence to the nucleotide sequence set forth in (i); (iii) comprising a reverse complementary sequence to a sequence capable of hybridizing with the nucleotide sequence set forth in (i) or (ii) under high-stringent hybridization conditions or very high-stringent hybridization conditions; and (iv) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in (i) or (ii), wherein the nucleotide sequence of the mutant set forth in any one of (i) to (iv) is not CGATGTTTGATTGGGGGAATCGGGGGTTACGATACTAGG at positions corresponding to position 279 to position 317 of the sequence set forth in SEQ ID NO: 21; and, the mutant set forth in any one of (i) to (iv) has an enhanced promoter activity as compared to a pyruvate carboxylase gene promoter having the sequence set forth in SEQ ID NO: 21.

2. The mutant of the pyruvate carboxylase gene promoter according to claim 1, wherein the nucleotide sequence of the mutant at positions corresponding to position 279 to position 317 of the nucleotide sequence set forth in SEQ ID NO: 21 is as follows: NNNNNNTTGATTNNNNNNNNNNNNNNNTANNATNNNNNN, where N is selected from A, T, C, or G; the mutant has an enhanced promoter activity of 1 to 17 folds or more as compared to the pyruvate carboxylase gene promoter having the sequence set forth in SEQ ID NO: 21.

3. The mutant of the pyruvate carboxylase gene promoter according to claim 1, wherein the nucleotide sequence of the core region of position 279 to position 317 of the mutant is one of the following sequences: TABLE-US-00009 1) CTAATTTTGATTCGTACTGATTTCTGCTACGATGAGTCA; 2) GGATTGTTGATTTGAGCTTGATGAGCGTACAATCAACTT; 3) TTCTCCTTGATTGCGCCTTAACCGTGGTATGATTCGATA; 4) ATTGATTTGATTGGAACCTTACTGTGCTATGATTTGGTA; 5) TCGAGTTTGATTTCACAACGTGTGTGATAGGATATAATA; 6) TTGCGTTTGATTAAAGTATGCAAGGGCTAGTATGGTGAT; 7) ATCATTTTGATTCCGGCGCACATGTGGTAATATGGTATT; 8) TCGCCATTGATTGCCCGCCATCCATGCTATAATCGGAAG; 9) TTCCGCTTGATTGTGGCCATAGTATGATATTATTAATTA; 10) CGGATCTTGATTTTATGATGGGTATTGTATAATCTTGGT; 11) CGGATATTGATTTGGCCGGTGTTGTGGTAGTATCGTGTT; 12) AGGGGTTTGATTGGCCGCTCGGTGTGTTATCATGGAGAG; 13) GAGTTGTTGATTTCGTTGGTGCACGTATACAATGGTTTT; 14) CTTGGCTTGATTTTTGTTTGAGGGTTGTATAATGTTATT; 15) GACTAGTTGATTTCCGCCCTTGGTTGATATTATGCTTGA; 16) ATCCGCTTGATTTAGGCGTACGTTTAATAGTATATTGAA; 17) CGGGGCTTGATTTCCTTGTCGTGGCGTTATTATAATGGA; 18) ATGGAGTTGATTATACGATACTACAGATACTATACTGGT; 19) CCGTAGTTGATTGACTTGGGCAGTATATAGTATAATGAA; or 20) CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG.

4. The mutant of the pyruvate carboxylase gene promoter according to claim 1, wherein the nucleotide sequence of the mutant is set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 20.

5. An expression cassette, comprising the mutant of the pyruvate carboxylase gene promoter according to claim 1.

6. An expression vector, comprising the mutant of the pyruvate carboxylase gene promoter according to claim 1.

7. A recombinant host cell, comprising the mutant of the pyruvate carboxylase gene promoter according to claim 1.

8. The recombinant host cell according to claim 7, the host cell is a genus Enterobacter or a genus Corynebacterium.

9. A method of enhancing a transcriptional level of a gene or preparing a reagent or kit for enhancing a transcriptional level of a gene, which comprises utilizing the mutant of the pyruvate carboxylase gene promoter according to claim 1.

10. A method of producing a target product or preparing a reagent or kit for use in the production of a target product, which comprises utilizing the mutant of the glutamate dehydrogenase gene promoter according to claim 1, wherein the target product is selected from at least one of amino acids and derivatives thereof.

11. A method for enhancing expression of a target gene, wherein the method comprises operably ligating the mutant of the pyruvate carboxylase gene promoter according to claim 1 to a target gene or a target RNA; optionally, the target RNA comprises at least one of tRNA or sRNA, and the target gene comprises at least one of a coding gene of a target product synthesis-associated protein, a coding gene of a gene expression regulatory protein, or a coding gene of a membrane transport-associated protein.

12. A method for preparing a protein, wherein the method comprises a step of expressing the protein using the expression cassette according to claim 5; wherein the protein is a target product synthesis-associated protein, a membrane transport-associated protein, or a gene expression regulatory protein; and a step of isolating or purifying the protein.

13. A method for producing a target product, wherein the method comprises culturing a host cell containing the mutant of the pyruvate carboxylase gene promoter according to claim 1 that is operably ligated to a target product synthesis-associated gene, and collecting the resulting target product; wherein the target product is an amino acid.

14. The method according to claim 13, wherein the target product uses oxaloacetic acid as a precursor.

15. The recombinant host cell according to claim 8, wherein the genus Corynebacterium is selected from the group consisting of Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum B253, Corynebacterium glutamicum ATCC 14067, and derived strains thereof.

16. A method of preparing a protein, or preparing a reagent or kit for use in the preparation of a protein, comprising utilizing the mutant of the pyruvate carboxylase gene promoter according to claim 1; wherein the protein is a target product synthesis-associated protein, a membrane transport-associated protein, or a gene expression regulatory protein.

17. The method according to claim 10, wherein the amino acids and derivatives thereof are one or a combination of two or more selected from: proline, hydroxyproline, lysine, glutamic acid, arginine, ornithine, glutamine, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, methionine, aspartic acid, asparagine, histidine, phenylalanine, tyrosine, tryptophan, 5-aminolevulinic acid, and derivatives of any one of the amino acids described above.

18. The method according to claim 11, wherein the target gene comprises at least one of the following coding genes of enzymes: pyruvate carboxylase pyc gene, glutamate dehydrogenase gdh gene, aspartate kinase lysC gene, threonine operon thrABC gene, aspartate-semialdehyde dehydrogenase asd gene, aspartate-ammonia lyase aspB gene, homoserine dehydrogenase hom gene, homoserine O-acetyltransferase metX gene, dihydrodipicolinate synthase dapA gene, dihydrodipicolinate reductase dapB gene, meso-diaminopimelate dehydrogenase ddh gene, glutamate kinase proB gene, glutamate-5-semialdehyde dehydrogenase proA gene, pyrroline-5-carboxylate dehydrogenase proC gene, proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase putA gene, glutamyl-t-RNA reductase hemA gene, phosphoenolpyruvate carboxylase ppc gene, amino acid transport protein lysE gene, ptsG system-associated coding gene, pyruvate dehydrogenase aceE gene, glyceraldehyde-3-phosphate dehydrogenase gapN gene, and lysine decarboxylase cadA/ldcC gene.

19. The method according to claim 13, wherein the target product synthesis-associated gene is a gene associated with synthesis of the amino acid or a derivative thereof, wherein the amino acid and a derivative thereof are selected from one or a combination of two or more of: proline, hydroxyproline, lysine, glutamic acid, arginine, ornithine, glutamine, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, methionine, aspartic acid, asparagine, histidine, phenylalanine, tyrosine, tryptophan, 5-aminolevulinic acid, and derivatives of any one of the amino acids described above.

20. The method according to claim 14, wherein, the target product using the oxaloacetic acid as a precursor includes one or more of: lysine, threonine, isoleucine, methionine, glutamic acid, proline, hydroxyproline, arginine, glutamine, 5-aminolevulinic acid, pentanediamine, 5-aminovaleric acid, and a derivative of any one of the above.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0089] FIG. 1: Plasmid vector map of pEC-XK99E-Ppyc-rfp plasmid.

[0090] FIG. 2: Fluorescent screening plate of pyc promoter mutants.

DETAILED DESCRIPTION

Definitions of Terms

[0091] When used in combination with the term “including” in the claims and/or specification, the word “a” or “an” may refer to “one”, or refer to “one or more”, “at least one”, and “one or more than one”.

[0092] As used in the claims and specification, the term “including”, “having”, “comprising”, or “containing” is intended to be inclusive or open-ended, and does not exclude additional or unrecited elements or methods and steps.

[0093] Throughout the application document, the term “about” means that a value includes the error or standard deviation caused by the device or method used to measure the value.

[0094] It is applicable to the content disclosed herein that the term “or” is defined only as alternatives and “and/or”, but the term “or” used herein refers to “and/or” unless otherwise expressly stated to be only alternatives or mutual exclusion between alternatives.

[0095] The selected/optional/preferred “numerical range”, when used in the claims or the specification, includes not only the numerical endpoints at both ends of the range, but also all natural numbers covered between the above numerical endpoints with respect to these numerical endpoints.

[0096] As used herein, the term “nucleic acid molecule” or “polynucleotide” refers to a polymer composed of nucleotides. A nucleic acid molecule or polynucleotide may be in the form of an individual fragment, or may be a constituent part of a larger nucleotide sequence structure, which is derived from the nucleotide sequence that has been isolated at least once in number or concentration, and could be recognized, operated, and sequence recovered as well as nucleotide sequence recovered by a standard molecular biological method (e.g., using a cloning vector). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes an RNA sequence (i.e., A, U, G, C), where “U” substitutes for “T”. In other words, “polynucleotide” refers to a nucleotide polymer knocked out from an additional nucleotide (an individual fragment or an entire fragment), or may be a constituent part or component of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0097] As used herein, the terms “target gene” and “gene of interest” can be used interchangeably.

[0098] As used herein, the term “wild-type” refers to an object that can be found in nature. For example, a polypeptide or a polynucleotide sequence present in an organism that can be isolated from one source in nature and has not been intentionally modified by human in the laboratory is naturally occurring. As used herein, “naturally occurring” and “wild-type” are synonymous.

[0099] As used herein, the terms “sequence identity” and “percent identity” refer to the percentage of nucleotides or amino acids that are the same (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides may be determined by aligning the nucleotide sequences of polynucleotides or the amino acid sequences of polypeptides and scoring the number of positions at which nucleotide or amino acid residues are identical in the aligned polynucleotides or polypeptides, and comparing the number of these positions with the number of positions at which nucleotide or amino acid residues are different in the aligned polynucleotides or polypeptides. Polynucleotides may differ at one position by, e.g., containing a different nucleotide (i.e., substitution or mutation) or deleting a nucleotide (i.e., insertion or deletion of a nucleotide in one or two polynucleotides). Polypeptides may differ at one position by, e.g., containing a different amino acid (i.e., substitution or mutation) or deleting an amino acid (i.e., insertion or deletion of an amino acid in one or two polypeptides). The sequence identity may be calculated by dividing the number of positions at which nucleotide or amino acid residues are identical by the total number of nucleotide or amino acid residues in the polynucleotides or polypeptides. For example, the percent identity may be calculated by dividing the number of positions at which nucleotide or amino acid residues are identical by the total number of nucleotide or amino acid residues in the polynucleotides or polypeptides, and multiplying the result by 100.

[0100] In some embodiments, two or more sequences or subsequences, when compared and aligned at maximum correspondence by the sequence alignment algorithm or by the visual inspection measurement, have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% “sequence identity” or “percent identity” of nucleotides. In some embodiments, the sequence is substantially identical over the full length of either or both of the compared biopolymers (e.g., polynucleotides).

[0101] As used herein, the term “complementary” refers to hybridization or base pairing between nucleotides or nucleotides, e.g., between two chains of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleotide to be sequenced or amplified.

[0102] As used herein, the term “high-stringent conditions” means that following the standard DNA blotting procedures, a probe of at least 100 nucleotides in length pre-hybridizes or hybridizes for 12 to 24 hours at 42° C. in 5×SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 μg/ml of cleavaged and denatured salmon sperm DNA, and 50% formamide. Finally, the vector material is washed three times at 65° C. with 2×SSC and 0.2% SDS, each for 15 min.

[0103] As used herein, the term “very high-stringent conditions” means that following the standard DNA blotting procedures, a probe of at least 100 nucleotides in length pre-hybridizes or hybridizes for 12 to 24 hours at 42° C. in 5×SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 μg/ml of cleavaged and denatured salmon sperm DNA, and 50% formamide. Finally, the vector material is washed three times at 70° C. with 2×SSC and 0.2% SDS, each for 15 min.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as typically understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein may be used to implement or test the present disclosure, the methods and materials described herein are preferred.

[0105] The present disclosure will be further illustrated with reference to specific examples. It should be appreciated that these examples are merely intended to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental techniques and experimental methods used in the examples, unless otherwise specified, are all conventional techniques and methods, for example, experimental methods for which no specific conditions are indicated in the following examples are generally performed according to conventional conditions such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by manufacturers. The materials, reagents, etc. used in the examples, unless otherwise specified, are all commercially available.

Example 1. Construction of Plasmid Characterizing the Strength of Pyc Gene Promoter of Corynebacterium glutamicum

[0106] In order to characterize the strength of the pyc gene promoter of Corynebacterium glutamicum, the present disclosure firstly constructed a characterizing vector, and based on the skeleton of the pEC-XK99E plasmid, 60 amino acids at the N-terminus of the pyc gene, a C-peptide, and a red fluorescence protein gene were expressed by the pyc gene promoter. Based on the disclosed genome sequence and pyc gene annotation information of Corynebacterium glutamicum ATCC13032, pyc-FIR primers were designed, and a 180-bp DNA fragment of the pyc gene promoter and the N-terminus was obtained by PCR amplification using the ATCC13032 genome as a template. The pEC-XK99E-rfp.sup.[3] plasmid reported in the literature was used as a template and pEC-F/R as primers to amplify the DNA fragment of the skeleton of the pEC-XK99E plasmid, the C-peptide, and the red fluorescence protein gene. The above two fragments were ligated via Vazyme's One Step Cloning Kit to obtain a pEC-XK99E-Ppyc-rfp characterizing vector. The plasmid vector map was as shown in FIG. 1. The sequences of the primers as used above were listed in Table 1.

TABLE-US-00002 TABLE 1 Primer Nucleotide Sequence SEQ ID NO. pyc-F CCTGATGCGGTATTTTCTCCGAAAACCC SEQ ID NO: 22 AGGATTGCTTTGTG pyc-R GCGGACAGCTTCAGAAGCAAAAG SEQ ID NO: 23 pEC-F TTGCTTCTGAAGCTGTCCGCGGCGGTGG SEQ ID NO: 24 CTCTGGAGGTGGTGGGTCCGGCGGTGGC TCTGCTTCCTCCGAAGACGTTATCAAAG pEC-R GGAGAAAATACCGCATCAGGC SEQ ID NO: 25

Example 2. Screening and Strength Characterization of Pyc Gene Promoter Mutants of Corynebacterium glutamicum

[0107] (1) Construction of Library of Pyc Gene Promoter Mutants of Corynebacterium glutamicum

[0108] The present disclosure performed mutations on the core region of the pyc gene promoter of Corynebacterium glutamicum: “CGATGTTTGATTGGGGGAATCGGGGGTTACGATACTAGG”, where the underlined parts were the main sequences of the −35 region and the −10 region of the promoter. The present disclosure performed mutations on the corresponding positions of the above core region, obtaining “NNNNNNTTGATTNNNNNNNN NNNNTANNATNNNNNN”. The two fragments of the plasmid were amplified using pyc-M1/M2 and pyc-M3/M4 primers respectively, and ligated via Vazyme's One Step Cloning Kit. All of the resulting cloned bacteria were collected and plasmids were extracted to obtain a library of the pyc gene promoter mutants. The above library and the wild-type control pEC-XK99E-Ppyc-rfp obtained in Example 1 were transformed into the Corynebacterium glutamicum ATCC13032 respectively, and coated on a TSB plate. The plate, on which hundreds of clones were grown, was fluorescence photographed with a fluorescence imaging system. Mutants with improved expression intensity were preliminarily screened according to the fluorescence brightness of the clones. The ingredients (g/L) of the medium of the TSB plate were as follows: glucose, 5 g/L; yeast powder, 5 g/L; soy peptone, 9 g/L; urea, 3 g/L; succinic acid, 0.5 g/L; K.sub.2HPO.sub.4.Math.3H.sub.2O, 1 g/L; MgSO.sub.4.Math.7H.sub.2O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; MOPS, g/L; and agar powder, 15 g/L. The present disclosure performed initial screening on more than 10,000 clones, and as shown in FIG. 2, about 30 mutants with enhanced fluorescence intensity were obtained. The sequences of the primers as used above were listed in Table 2.

TABLE-US-00003 TABLE 2 Primer Nucleotide Sequence SEQ ID NO. pyc-M1 CCCGAAAACATTGAGAGGAAAACAAAAA SEQ ID NO: 26 CNNNNNNTTGATTNNNNNNNNNNNNNNN TANNATNNNNNNCGCAGTGACTGCTATC ACCC pyc-M2 AACCTTCCATACGAACTTTGAAACG SEQ ID NO: 27 pyc-M3 CAAAGTTCGTATGGAAGGTTCCG SEQ ID NO: 28 pyc-M4 TTCCTCTCAATGTTTTCGGGC SEQ ID NO: 29
(2) Screening of Library of Pyc Gene Promoter Mutants of Corynebacterium glutamicum

[0109] All mutants with the enhanced fluorescence intensity observed in the plate as described above were cultured in a 96-well plate to characterize the strength of the promoters. The ingredients (g/L) of the TSB liquid medium were as follows: glucose, 5 g/L; yeast powder, 5 g/L; soy peptone, 9 g/L; urea, 3 g/L, succinic acid, 0.5 g/L; K.sub.2HPO.sub.4.Math.3H.sub.2O, 1 g/L; MgSO.sub.4.Math.7H.sub.2O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; and MOPS, 20 g/L. The strains resulting from the plate were inoculated with toothpicks into a 96-well plate containing 200 μl of TSB liquid medium in each well. Three samples were set for each strain. The rotating speed of the plate shaker was 800 rpm. After culturing at 30° C. for 24 h, the fluorescence intensities of the strains were detected, and the strains with improved fluorescence intensity as compared to the wild-type control were sequenced. The results were listed in Table 3. Some of the promoter mutants have the same sequence. Finally, the present disclosure successfully obtained 20 different promoter mutants (the nucleotide sequences of the corresponding mutated promoters were numbered as SEQ ID NOS: 1 to 20) with improved expression intensity as compared to the wild-type promoter, and the range of the increased folds was from 1.8 folds to 16.1 folds, which could provide abundant elements for modifying the expression of genes such as pyc.

TABLE-US-00004 TABLE 3 Folds Fluorescence Increased SEQ ID NO. of Promoter Intensity Than Complete Sequence No. (RFU/OD.sub.600) Wild-Type Core Region Sequence of Promoter of Promoter WT 208 ± 3 CGATGTTTGATTGGGGGAATCGGGGGTTACGATACTAGG SEQ ID NO: 21 P.sub.pyc-1  578 ± 13 1.8 CTAATTTTGATTCGTACTGATTTCTGCTACGATGAGTCA SEQ ID NO: 1 P.sub.pyc-2 822 ± 5 3.0 GGATTGTTGATTTGAGCTTGATGAGCGTACAATCAACTT SEQ ID NO: 2 P.sub.pyc-3 833 ± 7 3.0 TTCTCCTTGATTGCGCCTTAACCGTGGTATGATTCGATA SEQ ID NO: 3 P.sub.pyc-4 1185 ± 34 4.7 ATTGATTTGATTGGAACCTTACTGTGCTATGATTTGGTA SEQ ID NO: 4 P.sub.pyc-5 1203 ± 27 4.8 TCGAGTTTGATTTCACAACGTGTGTGATAGGATATAATA SEQ ID NO: 5 P.sub.pyc-6 1208 ± 28 4.8 TTGCGTTTGATTAAAGTATGCAAGGGCTAGTATGGTGAT SEQ ID NO: 6 P.sub.pyc-7 1495 ± 46 6.2 ATCATTTTGATTCCGGCGCACATGTGGTAATATGGTATT SEQ ID NO: 7 P.sub.pyc-8 1498 ± 23 6.2 TCGCCATTGATTGCCCGCCATCCATGCTATAATCGGAAG SEQ ID NO: 8 P.sub.pyc-9 1544 ± 25 6.4 TTCCGCTTGATTGTGGCCATAGTATGATATTATTAATTA SEQ ID NO: 9 P.sub.pyc-10  1776 ± 127 7.5 CGGATCTTGATTTTATGATGGGTATTGTATAATCTTGGT SEQ ID NO: 10 P.sub.pyc-11 1819 ± 14 7.7 CGGATATTGATTTGGCCGGTGTTGTGGTAGTATCGTGT SEQ ID NO: 11 P.sub.pyc-12 1834 ± 67 7.8 AGGGGTTTGATTGGCCGCTCGGTGTGTTATCATGGAGAG SEQ ID NO: 12 P.sub.pyc-13 2066 ± 15 8.9 GAGTTGTTGATTTCGTTGGTGCACGTATACAATGGTTTT SEQ ID NO: 13 P.sub.pyc-14  2248 ± 142 9.8 CTTGGCTTGATTTTTGTTTGAGGGTTGTATAATGTTATT SEQ ID NO: 14 P.sub.pyc-15 2305 ± 87 10.1 GACTAGTTGATTTCCGCCCTTGGTTGATATTATGCTTGA SEQ ID NO: 15 P.sub.pyc-16  2725 ± 160 12.1 ATCCGCTTGATTTAGGCGTACGTTTAATAGTATATTGAA SEQ ID NO: 16 P.sub.pyc-17  2987 ± 103 13.4 CGGGGCTTGATTTCCTTGTCGTGGCGTTATTATAATGGA SEQ ID NO: 17 P.sub.pyc-18  3093 ± 159 13.9 ATGGAGTTGATTATACGATACTACAGATACTATACTGGT SEQ ID NO: 18 P.sub.pyc-19  3382 ± 211 15.3 CCGTAGTTGATTGACTTGGGCAGTATATAGTATAATGAA SEQ ID NO: 19 P.sub.pyc-20  3560 ± 202 16.1 CGGGCCTTGATTGTAAGATAAGACATTTAGTATAATTAG SEQ ID NO: 20

Example 3. Application of Pyc Gene Promoter Mutants of Corynebacterium glutamicum of Production of Target Products

[0110] (1) Construction of Recombinant Vector of Pyc Gene Promoter Mutant of Corynebacterium glutamicum

[0111] Based on the reported genome sequence of Corynebacterium glutamicum ATCC3032, the upstream and downstream homologous arms of the P.sub.pyc-1, P.sub.pyc-9, P.sub.pyc-16, and P.sub.pyc-20 promoter mutants were subjected to PCR amplification using the ATCC13032 genome as a template and using pyc-UF/pyc-UR1 and pyc-DF1/pyc-DR, pyc-UF/pyc-UR9 and pyc-DF9/pyc-DR, pyc-UF/pyc-UR16 and pyc-DF16/pyc-DR, and pyC-UF/pyc-UR20 and pyc-DF20/pyc-DR as primers, respectively; and at the same time, the pK18mobsacB skeleton was amplified using pK18-1/2 as primers. The above PCR fragments were recovered and then ligated via Vazyme's One Step Cloning Kit, and recombinant vectors pK18-P.sub.pyc-1, pK18-P.sub.pyc-9, pK18-P.sub.pyc-16, and pK18-P.sub.pyc-20 with mutated promoters were obtained, respectively. The sequences of the primers as used above were listed in Table 4.

TABLE-US-00005 TABLE 4 Primer Nucleotide Sequence SEQ ID NO. pyc-UF CAGGAAACAGCTATGACATGGTATCGCC SEQ ID NO: 30 ATGTATCACGCACTC pyc-UR1 TCAGTACGAATCAAAATTAGGTTTTTGT SEQ ID NO: 31 TTTCCTCTCAATGTTTTC pyc-UR9 ATGGCCACAATCAAGCGGAAGTTTTTGT SEQ ID NO: 32 TTTCCTCTCAATGTTTTC pyc-UR16 TACGCCTAAATCAAGCGGATGTTTTTGT SEQ ID NO: 33 TTTCCTCTCAATGTTTTC pyc-UR20 TATCTTACAATCAAGGCCCGGTTTTTGT SEQ ID NO: 34 TTTCCTCTCAATGTTTTC pyc-DF1 CTAATTTTGATTCGTACTGATTTCTGCT SEQ ID NO: 35 ACGATGAGTCAACGCAGTGACTGCTATC ACCC pyc-DF9 TTCCGCTTGATTGTGGCCATAGTATGAT SEQ ID NO: 36 ATTATTAATTAACGCAGTGACTGCTATC ACCC pyc-DF16 ATCCGCTTGATTTAGGCGTACGTTTAAT SEQ ID NO: 37 AGTATATTGAAACGCAGTGACTGCTATC ACCC pyc-DF20 CGGGCCTTGATTGTAAGATAAGACATTT SEQ ID NO: 38 AGTATAATTAGACGCAGTGACTGCTATC ACCC pyc-DR TGTAAAACGACGGCCAGTGCCTAATTTG SEQ ID NO: 39 CGAAGCTCATCAGGTG pK18-1 GCACTGGCCGTCGTTTTAC SEQ ID NO: 40 pK18-2 CATGTCATAGCGTGTTTCCTGTGTG SEQ ID NO: 41

[0112] (2) Construction of Pyc Gene Promoter Mutants of Lysine-Producing Strain of Corynebacterium glutamicum

[0113] The recombinant vectors pK18-P.sub.pyc-1, pK18-P.sub.pyc-9, pK18-P.sub.pyc-16, and pK18-P.sub.pyc-20 as constructed above were transformed into the lysine-producing strain SCgL30 (in which Thr at position 311 of the aspartate kinase of Corynebacterium glutamicum ATCC13032 was mutated as Ile.sup.[4]) of Corynebacterium glutamicum, respectively. The strain was coated on LBHIS solid media containing 5 g/L of glucose and 25 μg/mL of kanamycin and cultured at 30° C. to obtain the first recombinant transformants. The correct first recombinant transformants were inoculated into LB media containing 5 g/L of glucose respectively, and cultured overnight. Thereafter, 1% of the culture was transferred into an LB medium containing 100 g/L of sucrose and cultured overnight at 30° C., and then coated respectively on LB solid media containing 100 g/L of sucrose for screening. Sequencing was performed for confirmation to obtain strains SCgL33, SCgL34, SCgL35, and SCgL36 with the mutated pyc promoters, respectively.

[0114] (3) Evaluation of Lysine Productivity of Pyc Gene Promoter Mutants in Lysine-Producing Strain of Corynebacterium glutamicum

[0115] To test the effect of applying the mutation of the pyc promoter in Corynebacterium glutamicum on production of lysine by strains, fermentation tests were performed in SCgL30, SCgL33, SCgL34, SCgL35, and SCgL36, respectively. The ingredients of the fermentation medium were as follows: glucose, 80 g/L; yeast powder, 1 g/L; soy peptone, 1 g/L; NaCl, 1 g/L; ammonium sulfate, 1 g/L; urea, 8 g/L; K.sub.2HPO.sub.4.Math.3H.sub.2O, 1 g/L; MgSO.sub.4.Math.7H.sub.2O, 0.45 g/L; FeSO4.Math.7H.sub.2O, 0.05 g/L; biotin, 0.4 mg/L; vitamin B1, 0.1 mg/L; MOPS, 40 g/L; and initial pH7.2. The strains were firstly inoculated into TSB liquid media and cultured for 8 h. The cultures were inoculated as seeds into a 24-well plate containing 800 μl of fermentation media in each well with the initial OD.sub.600 controlled at about 0.1, and cultured at 30° C. for 20 h. The rotating speed of the plate shaker was 800 rpm. Three samples were set for each strain. After the fermentation, the lysine yields were measured. The results were listed in Table 5. The lysine yields of the strains after mutation of the pyc promoter were all significantly increased, and the increase was more significant as the promoter strength was enhanced.

TABLE-US-00006 TABLE 5 Strain Lysine Yield (g/L) SCgL30 1.67 ± 0.03 SCgL33 1.77 ± 0.04 SCgL34 2.07 ± 0.06 SCgL35 2.37 ± 0.15 SCgL36 3.27 ± 0.15

Example 4. Application of Pyc Gene Promoter Mutants of Corynebacterium glutamicum for Proline Production

[0116] (1) Application of P.sub.pyc-20 in Construction of Proline-Producing Strains

[0117] In the present disclosure, the G149K mutation was introduced in the Corynebacterium glutamicum ATCC13032 strain, and the codon was mutated from GGT to AAG, thereby obtaining a SLCgP1 strain. The SLCgP1 strain was further applied in the present disclosure to integrate the expression cassette, which was over-expressed using the P.sub.pyc-20 promoter, of glutamate kinase proB.sup.G149K which relieves the feedback inhibition, glutamate-5-semialdehyde dehydrogenase proA along with pyrroline-5-carboxylate dehydrogenase proC into the putA gene, to obtain a proline-producing strain designated as a SLCgP2 strain.

[0118] The SLCgP2 strain was specifically constructed as follows: 1) Firstly, constructing the P.sub.pyc-20 promoter on the pEC-XK99E plasmid to express the expression cassette of proB.sup.G149K, proA, along with proC. The P.sub.pyc-20 promoter fragment was amplified using the genome of the SCgL36 strain as a template and pyc-a/b as primers; the fragments of proB.sup.G149K, proA, and proC were amplified using the genome of the SLCgP1 strain as a template and proB-1/2, proA-1/2, and proC-1/2 as primers, respectively; and at the same time, the pEC-XK99E skeleton was amplified using pEC-1/2 as primers. The above 5 fragments were ligated via Vazyme's One Step Cloning Kit to obtain a pEC-proB.sup.G149KproAproC plasmid. 2) Constructing a recombinant vector comprising the above expression cassette inserted into the putA gene on the chromosome. The upstream and downstream homologous arms inserted into the putA gene were amplified using the genome of the SCgL36 strain as a template and putA-1/2 and putA-3/4 as primers, respectively; the expression cassette fragment was amplified using the pEC-proB.sup.G149KproAproC plasmid as a template and ABC-FIR as primers; and at the same time, the pK18mobsacB skeleton was amplified using pK18-1/2 as primers. The above PCR fragments were ligated via Vazyme's One Step Cloning Kit to obtain a pK18-proB.sup.G149KproAproC recombinant vector. 3) The pK18-proB.sup.G149KproAproC recombinant vector was transformed into the SLCgP1 strain. The strain was coated on an LBHIS solid medium containing 5 g/L of glucose and 25 μg/mL of kanamycin and cultured at 30° C. to obtain the first recombinant transformants. The correct first recombinant transformants were inoculated into LB media containing 5 g/L of glucose respectively, and cultured overnight. Thereafter, 1% of the culture was transferred into an LB medium containing 100 g/L of sucrose and cultured overnight at 30° C., and then coated respectively on an LB solid medium containing 100 g/L of sucrose for screening. Correct mutants were confirmed by PCR with universal and specific primers and sequencing to obtain SLCgP2 strains, respectively. The sequences of the primers as used above were listed in Table 6.

TABLE-US-00007 TABLE 6 Primer Nucleotide Sequence SEQ ID NO. pEC-1 GGAGAAAATACCGCATCAGGC SEQ ID NO: 42 pEC-2 CTGTTTTGGCGGATGAGAGAAG SEQ ID NO: 43 pyc-a CCTGATGCGGTATTTTCTCCGAAAACCCA SEQ ID NO: 44 GGATTGCTTTGTG pyc-b TTGGAGATGCGCTCACGCATTAGAGTAAT SEQ ID NO: 45 TATTCCTTTCAACAAGAG proB-1 ATGCGTGAGCGCATCTCCAAC SEQ ID NO: 46 proB-2 TTACGCGCGGCTGGCGTAGTTG SEQ ID NO: 47 proA-1 ACTACGCCAGCCGCGCGTAAGCCTTTTAT SEQ ID NO: 48 GGTGTGATCCGAC proA-2 TTAAGGCCTAATTTGTCCTGTGCC SEQ ID NO: 49 proC-1 CAGGACAAATTAGGCCTTAATTTGTCGTT SEQ ID NO: 50 TTGGGCCCCC proC-2 TCTCTCATCCGCCAAAACAGCTAGCGCTT SEQ ID NO: 51 TCCGAGTTCTTCAG putA-1 CAGGAAACAGCTATGACATGTTCTAGGGC SEQ ID NO: 52 ATCGACGAACCAG putA-2 GAAATTGTTAAAAGCGCAGCGC SEQ ID NO: 53 ABC-F GCTGCGCTTTTAACAATTTCGAAAACCCA SEQ ID NO: 54 GGATTGCTTTGTG ABC-R TCGAAGCCGCACGTCATCTAG SEQ ID NO: 55 putA-3 TAGATGACGTGCGGCTTCGATCCGTGAACG SEQ ID NO: 56 CCTATCTGTACG putA-4 TGTAAAACGACGGCCAGTGCGATCGATTCC SEQ ID NO: 57 ACGCCCAAAC pK18-1 GCACTGGCCGTCGTTTTAC SEQ ID NO: 58 pK18-2 CATGTCATAGCTGTTTCCTGTGTG SEQ ID NO: 59

[0119] (2) Evaluation of Proline Productivity of Strains Modified by P.sub.pyc-20 Promoter Mutant

[0120] To test the effect of applying the P.sub.pyc-20 promoter mutant in Corynebacterium glutamicum on production of proline by strains, fermentation tests were performed in SLCgP1 and SLCgP2, respectively. The ingredients of the fermentation medium were as follows: glucose, 72 g/L; yeast powder, 1 g/L; soy peptone, 1 g/L; NaCl, 1 g/L; ammonium sulfate, 1 g/L; urea, 10 g/L; K.sub.2HPO.sub.4.Math.3H.sub.2O, 1 g/L; MgSO.sub.4.Math.7H.sub.2O, 0.45 g/L; FeSO.sub.4.Math.7H.sub.2O, 0.05 g/L; biotin, 0.4 mg/L; vitamin B1, 0.1 mg/L; MOPS, 40 g/L; and initial pH7.2. The strains were firstly inoculated into TSB liquid media and cultured for 8 h. The cultures were inoculated as seeds into a 24-well plate containing 800 μl of fermentation media in each well at an inoculation amount of 12 μl, and cultured at 30° C. for 18 h. The rotating speed of the plate shaker was 800 rpm. Three samples were set for each strain. After the fermentation, the proline yields were measured. The results were listed in Table 7. The proline yields of the strains were significantly increased after the P.sub.pyc-20 promoter expressing the expression cassette of proB.sup.G149K, proA along with proC was inserted in the chromosome, and SLCgP2 was increased by 77% as compared to SLCgP1.

TABLE-US-00008 TABLE 7 Strain Proline Yield (g/L) SLCgP1 3.10 ± 0.15 SLCgP2 5.48 ± 0.23

[0121] The above results suggest that the mutants with enhanced pyc gene promoter may be used to express different target genes in Corynebacterium glutamicum, and applied to production of various products. Firstly, the mutants with enhanced pyc gene promoter may be used to enhance the expression of the pyc gene per se in Corynebacterium glutamicum to enhance the activity of Pyc, thereby reinforcing the synthesis from pyruvic acid to oxaloacetic acid, which may be applied to the production of target products dependent upon supply of oxaloacetic acid as the precursor, including biological production of amino acids with oxaloacetic acid as a major metabolic precursor, such as amino acids of the aspartic acid family (lysine, threonine, isoleucine, and methionine), amino acids of the glutamic acid family (glutamic acid, proline, hydroxyproline, arginine, and glutamine), and 5-aminolevulinic acid. The present disclosure has confirmed in the Examples that the mutants with enhanced pyc gene promoter can be used for production of lysine as an amino acid of the aspartic acid family and proline as an amino acid of the glutamic acid family. It has been verified that enhanced expression and activity of Pyc are useful for production of the above products, for example: 1) Peters-Wendisch P G et al. have reported that overexpression of Pyc could improve the yields of glutamic acid, lysine, and threonine.sup.[5]; 2) Pyc is a major enzyme in Corynebacterium glutamicum to catalyze the production of C4 oxaloacetic acid from C3 (PEP or pyruvic acid), and overexpression and enhanced activity of Pyc are important targets for production of amino acids with oxaloacetic acid as a precursor.sup.[6]; 3) the Patent reference.sup.[7] has disclosed that enhanced activities of enzymes (phosphoenolpyruvate carboxylase or pyruvate carboxylase) that facilitate synthesis of oxaloacetic acid can improve the yield of 5-aminolevulinic acid. All pyc gene promoter mutants in the pyruvate carboxylase of Corynebacterium glutamicum in the present disclosure can be used to enhance the expression and activity of Pyc. Therefore, the pyc gene promoter mutants of the present disclosure may also be used for production of 5-aminolevulinic acid. Next, the Examples of the present disclosure also demonstrate that the mutants with enhanced pyc gene promoter may also be used for expression of genes such as proB, proA, and proC, indicating that the pyc gene promoter mutants of the present disclosure has good universality, which can be used for expression of more genes and thus production of more products.

REFERENCES

[0122] [3] Wang, Y C et al. Screening efficient constitutive promoters in Corynebacterium glutamicum based on time-series transcriptome analysis. Chinese Journal of Biotechnology, 2018, 34(11):1760-1771. [0123] [4] CN112877269A [0124] [5] Peters-Wendisch P G, Schiel B, Wendisch V F, et al. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. [J]. J Mol Microbiol Biotechnol, 2001, 3(2):295-300. [0125] [6] Uwe Sauer, Bernhard J. Eikmanns. The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria [M]//FEMS Microbiology Reviews. [0126] [7] CN103981203B