CN117535293A

CN117535293A - BBD29_03870 gene promoter mutant and application thereof

Info

Publication number: CN117535293A
Application number: CN202311462181.5A
Authority: CN
Inventors: 孟刚; 魏爱英; 李峰; 赵春光; 苏厚波; 马文有; 王攀; 张坤; 王吉玮
Original assignee: Ningxia Eppen Biotech Co ltd
Current assignee: Ningxia Eppen Biotech Co ltd
Priority date: 2023-11-06
Filing date: 2023-11-06
Publication date: 2024-02-09

Abstract

The invention discloses a BBD29_03870 gene promoter mutant and application thereof. The invention provides the use of a DNA molecule as a promoter; the DNA molecule comprises a DNA molecule with a nucleotide sequence shown as SEQ ID No.1, or a DNA molecule with a promoter function obtained by performing conservative substitution on a plurality of nucleotides except 290 th site of the SEQ ID No. 1. The BBD29_03870 gene/BBD29_ 09880 gene promoter is replaced by the mutant promoter shown in SEQ ID No.1 in corynebacterium glutamicum, so that the L-glutamic acid yield can be obviously improved. The invention has important significance for improving the yield of L-glutamic acid produced by fermentation of coryneform bacteria.

Description

BBD29_03870 gene promoter mutant and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a BBD29_03870 gene promoter mutant and application thereof.

Background

L-glutamic acid is produced mainly by corynebacterium glutamicum and is widely used in the fields of food, medicine, animal feed and the like. L-glutamic acid is biosynthesized from alpha-ketoglutarate, an intermediate product of citric acid circulation in microbial cells.

The low yield is one of defects limiting the industrial process of L-glutamic acid production. In order to enhance the L-glutamic acid production ability of coryneform bacteria, many studies have been reported to enhance the high yield of L-glutamic acid by using recombinant DNA techniques, by enhancing the anabolic flow of L-glutamic acid, by enhancing the studies of the deficiency pathway to provide sufficient oxaloacetic acid and acetyl CoA, by studying the mechanism of excretion of L-glutamic acid, by studying the mechanism of carbon source substitution, and so on.

The tricarboxylic acid cycle is one of the most important metabolic pathways in cells, and pyruvate carboxylase is a key enzyme in the tricarboxylic acid cycle, capable of producing ATP and other important metabolites. In the tricarboxylic acid cycle, pyruvate carboxylase catalyzes the reaction of pyruvate with CO2 to acetyl CoA. Acetyl-coa is one of the most important metabolites in cells, one of the precursors of tricarboxylic acid cycle.

However, more than 40% of the 3000 genes in the Corynebacterium glutamicum genome still have unknown or lack experimental evidence of gene function. However, it has not been reported that the G mutation at position 107 in the promoter region of pyruvate carboxylase encoded by BBD29_03870 gene is A to increase L-glutamic acid yield during fermentation of glutamic acid.

Disclosure of Invention

In order to better mine the metabolic potential of corynebacterium glutamicum and further improve the efficiency of biological production, the invention has the beneficial effect of mutating G at the 107 th position of the promoter region of the pyruvate carboxylase encoded by BBD29_03870 gene of the strain producing L-glutamic acid into A which is used as corynebacterium type bacteria producing L-glutamic acid.

In a first aspect, the invention claims the use of a DNA molecule, i.e. bbd29_03870 gene promoter mutant, as a promoter to initiate expression of a gene of interest.

Further, the DNA molecule comprises (or is) any one of the following:

(a1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 1;

(a2) And (3) performing conservative substitution on a plurality of nucleotides except the 290 th site (corresponding to the 107 th site of the BBD29_03870 gene promoter region) of SEQ ID No.1 to obtain the DNA molecule with the promoter function.

Wherein, the target gene can be a visual marker gene (such as GUS gene) or a functional gene to be expressed.

In the present invention, the target gene is specifically a BBD29_03870 gene and/or a BBD29_09880 gene, which are functional genes for producing L-amino acids (e.g., L-glutamic acid) in the genome of a microorganism. The microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria can be Corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium lactofermentum, brevibacterium flavum (brevibacterium flavum), brevibacterium beijing (Corynebacterium pekinense), brevibacterium ammoniaphaga, corynebacterium crenatum or Pantoea (Pantoea). Further, the bacterium may be Corynebacterium glutamicum. Still further, the corynebacterium glutamicum is corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 or a wild type corynebacterium glutamicum strain ATCC13869.

In a second aspect, the invention claims a DNA molecule as described in the first aspect above.

In a third aspect, the invention claims an expression cassette, recombinant vector or recombinant bacterium comprising a DNA molecule as described in the second aspect above.

Wherein the vector may be a plasmid, cosmid, phage or viral vector. Further, the recombinant vector may be a recombinant expression vector (plasmid) or a recombinant cloning vector (plasmid).

Wherein the expression cassette may consist of the DNA molecule having a promoter function, a target gene whose expression (transcription) is initiated by the DNA molecule, and a transcription termination sequence for terminating transcription; the DNA molecule is functionally linked to the gene of interest, and the gene of interest is linked to the transcription termination sequence. Further, the expression cassette may further include enhancers and the like.

Wherein the recombinant bacterium may be a recombinant bacterium containing the expression cassette or the recombinant vector.

In a specific embodiment of the invention, the recombinant bacterium is recombinant bacterium a or recombinant bacterium B.

The recombinant bacteria A is specifically obtained by replacing a promoter of BBD29_03870 gene in a bacterial genome with the DNA molecule in the second aspect;

the promoter of the bbd29_03870 gene can comprise (or be) any of the following:

(b1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 2;

(b2) A DNA molecule derived from a bacterium and having 95% or more identity with (b 1) and having a promoter function.

In particular, the replacement of the bbd29_03870 gene promoter in the bacterial genome with the DNA molecule described in the second aspect above may be achieved in particular by i.e. mutating position 290 of SEQ ID No.2 in the bacterial genome from G to a (other nucleotide sequence unchanged).

The recombinant bacterium B is specifically obtained by replacing a promoter of BBD29_09880 gene in a bacterial genome with the DNA molecule in the second aspect;

the promoter of the bbd29_09880 gene can comprise (or be) any of the following:

(c1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 6;

(c2) A DNA molecule derived from a bacterium and having 95% or more identity with (c 1) and having a promoter function.

In the DNA molecules described in the above (b 2) and (c 2), the term "identity" used herein refers to sequence similarity to a natural nucleotide sequence. "identity" includes nucleotide sequences having 95% or 96% or 97% or 98% or 99% or more identity to the nucleotide sequence shown in SEQ ID No.2 or SEQ ID No.6 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences. The following is the same.

Wherein the DNA molecule shown in SEQ ID No.2 is the promoter sequence of BBD29_03870 gene of wild type Corynebacterium glutamicum strain ATCC13869. In the invention, the promoter mutant shown in SEQ ID No.1 is obtained by introducing point mutation (specifically, G at 290 th position of SEQ ID No.2 is mutated into A, and other nucleotide sequences are unchanged).

In a fourth aspect, the invention claims a method for increasing the production of bacterial L-amino acids.

The method for improving the yield of bacterial L-amino acid as claimed in the present invention may comprise the following steps (A) or (B):

(A) Replacing the promoter of the bbd29_03870 gene in the bacterial genome with a DNA molecule as described in the second aspect above, thereby achieving an increase in the yield of said L-amino acids in the bacterium;

the promoter of the bbd29_03870 gene can comprise (or be) any of the following:

(b1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 2;

Further, the replacement of the bbd29_03870 gene promoter in the bacterial genome with the DNA molecule described in the second aspect above may be achieved in particular by i.e. mutating position 290 of SEQ ID No.2 in the bacterial genome from G to a (other nucleotide sequence unchanged).

(B) Replacing the promoter of the bbd29_09880 gene in the bacterial genome with a DNA molecule as described in the second aspect above, thereby achieving an increase in the yield of said L-amino acids in the bacterium;

the promoter of the bbd29_09880 gene can comprise (or be) any of the following:

(c1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 6;

Further, in the method, the step of fermenting and culturing the obtained recombinant bacterium may be further included after replacing the bbd29_03870 gene/bbd29_ 09880 gene promoter in the genome of the bacterium with the DNA molecule described in the second aspect, and the L-amino acid (e.g., L-glutamic acid) may be obtained from the fermentation and culture product.

In a specific embodiment of the present invention, the L-amino acid is L-glutamic acid.

In a fifth aspect, the invention claims a method of producing an L-amino acid.

The method for producing L-amino acid claimed in the present invention may comprise the steps of: introducing the DNA molecule of the second aspect into a biological cell capable of synthesizing the L-amino acid, such that the DNA molecule drives expression of a gene in the L-amino acid synthesis pathway in the biological cell, resulting in a recombinant biological cell; culturing the recombinant biological cell, thereby obtaining the L-amino acid.

Further, in the method, culturing the recombinant biological cell may specifically be: fermenting and culturing the recombinant biological cells.

Wherein the biological cell may be a bacterium, yeast, algae, fungus, plant cell or animal cell capable of synthesizing the L-amino acid, or the like.

In the fourth and fifth aspects of the foregoing, the fermentation culture may be carried out by a person skilled in the art using methods known in the art, optimization and modification of the fermentation process may be carried out by routine experimentation, and the fermentation culture may be carried out in a suitable medium under fermentation conditions known in the art. Wherein the medium may comprise: carbon source, nitrogen source, trace elements and combinations thereof. During the fermentation culture, the pH of the culture may be adjusted (e.g., controlled to a pH of 6.8-7.0). During the fermentation culture, the temperature of the culture may be controlled to 30 to 40 ℃. In the process of carrying out the fermentation culture, the concentration of fed-batch sugar (for example, 50-55% is controlled, namely, the mass of sugar contained in 100mL of liquid is 50-55 g), the residual sugar of a fermentation system (for example, 0.5-1.0% is controlled, namely, the mass of sugar contained in 100mL of liquid is 0.5-1.0 g) and the like can be controlled.

In a specific embodiment of the present invention, the formulation of the medium used in carrying out the fermentation culture and the fermentation control process are described in detail in the examples.

In a sixth aspect, the invention claims any of the following applications:

use of P1, a DNA molecule as described in the second aspect hereinbefore or an expression cassette or recombinant vector or recombinant bacterium as described in the third aspect hereinbefore for the production of L-amino acids;

use of P2, a DNA molecule as described in the second aspect hereinbefore or an expression cassette or recombinant vector as described in the third aspect hereinbefore for the construction of an engineered strain producing L-amino acids;

use of P3, a DNA molecule as described in the second aspect hereinbefore or an expression cassette or recombinant vector as described in the third aspect hereinbefore for the preparation of a food, pharmaceutical, feed or cosmetic product comprising an L-amino acid.

In the above aspects, the bacterium is a bacterium having an ability to produce an L-amino acid (e.g., L-glutamic acid).

Wherein, the "bacterium having an ability to produce an L-amino acid (e.g., L-glutamic acid)" means that the bacterium has the following ability: the ability to produce and accumulate L-amino acids (e.g., L-glutamic acid) in bacteria using foreign substances (e.g., culture medium) may further include the ability to secrete L-amino acids (e.g., L-glutamic acid) into the culture system. Thus, L-amino acids (e.g., L-glutamic acid) can be collected when the bacteria are cultured in a medium.

The bacteria may be naturally collected wild-type bacteria or may be modified bacteria. The term "modified bacteria" refers to modified bacteria obtained by artificial mutation and/or mutagenesis of naturally collected wild-type bacteria.

In particular, the bacteria may be coryneform bacteria, such as, in particular, corynebacterium glutamicum.

In a specific embodiment of the present invention, the corynebacterium glutamicum is Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 or wild type Corynebacterium glutamicum strain ATCC13869.

Correspondingly, the recombinant bacterium claimed in the third aspect is recombinant bacterium YPG-03870, recombinant bacterium G03870 or recombinant bacterium YPG-03870 ^G-107A Or recombinant bacterium G03870 ^G-107A 。

The recombinant strain YPG-03870 is a strain obtained by replacing a BBD29_03870 gene promoter (shown as SEQ ID No. 2) in a corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 genome with a mutated promoter (shown as SEQ ID No. 1) and keeping other sequences unchanged. Wherein, the substitution of the BBD29_03870 gene promoter (shown as SEQ ID No. 2) in the genome of corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 to the mutated promoter (shown as SEQ ID No. 1) can be realized by mutating the 290 th position of SEQ ID No.2 from G to A.

The recombinant strain G03870 is obtained by replacing a BBD29_03870 gene promoter (shown as SEQ ID No. 2) in the genome of the wild Corynebacterium glutamicum strain ATCC13869 with a mutated promoter (shown as SEQ ID No. 1) and keeping other sequences unchanged. Wherein, the "the BBD29_03870 gene promoter (shown as SEQ ID No. 2) in the genome of the wild Corynebacterium glutamicum strain ATCC13869 is replaced by a mutated promoter (shown as SEQ ID No. 1)" can be concretely realized by mutating the 290 th position of the SEQ ID No.2 from G to A.

The recombinant bacterium YPG-03870 ^G-107A In order to replace the self promoter of BBD29_09880 gene on the genome of Corynebacterium glutamicum CGMCC No.21220 (shown as SEQ ID No. 6) with BBD29-03870 ^G-107A MutationThe type promoter (shown as SEQ ID No. 1) is a recombinant bacterium obtained by keeping other nucleotides in the genome of corynebacterium glutamicum CGMCC No.21220 unchanged.

The recombinant bacterium G03870 ^G-107A To replace the self-promoter of the BBD29_09880 gene on the genome of Corynebacterium glutamicum ATCC13869 (as shown in SEQ ID No. 6) with BBD29-03870 ^G-107A A mutant promoter (shown as SEQ ID No. 1) and a recombinant bacterium obtained by keeping other nucleotides in the genome of Corynebacterium glutamicum ATCC13869 unchanged.

In the above aspects, the L-amino acid is L-glutamic acid.

The DNA molecules of the present invention may be used to produce a variety of products including, but not limited to, glutamic acid in the examples, which may also be lysine, valine, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, alpha ketoglutaric acid, citric acid, ornithine, citrulline, and the like. When various products are produced, the DNA molecule of the invention is placed at the upstream of the gene in the synthesis path of the target product, and the DNA molecule drives the synthesis of the gene in the synthesis path of the target product, thus realizing the production of the target product.

The research result of the invention shows that: in corynebacterium glutamicum, the wild type promoter of BBD29_03870 gene shown in SEQ ID No.2 is replaced by the mutant promoter shown in SEQ ID No.1, so that the yield of L-glutamic acid can be remarkably improved (p<0.01). In Corynebacterium glutamicum, the BBD29_09880 gene promoter shown in SEQ ID No.6 was replaced with mutant BBD29_03870 ^G-107A The promoter (SEQ ID No. 1) can also significantly improve the L-glutamic acid yield (p)<0.01). The invention has important significance for improving the yield of L-glutamic acid produced by fermentation of coryneform bacteria.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The corynebacterium glutamicum (Corynebacterium glutamicum) in the following examples is CGMCC No.21220, the strain number is YPHLU 001, and the strain is preserved in China general microbiological culture Collection center (CGMCC No. 21220) at 11/23/2020. Hereinafter abbreviated as Corynebacterium glutamicum CGMCC No.21220.

Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13869 in the examples described below was Corynebacterium glutamicum (Corynebacterium glutamicum) numbered 13869 in ATCC. Hereinafter abbreviated as Corynebacterium glutamicum ATCC13869.

EXAMPLE 1 construction of recombinant vector comprising BBD29_03870 Gene and its mutant promoter

Two pairs of primers for amplifying the BBD29_03870 gene promoter region were designed and synthesized based on the genomic sequence of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13869, and then point mutations were introduced into the BBD29_03870 gene promoter regions of Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 and wild-type Corynebacterium glutamicum strain ATCC13869 by means of allele substitution. The wild BBD29_03870 gene and the promoter thereof are reserved on the chromosome of the corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 through sequencing.

The point mutation is to mutate the 290 th guanine G of the wild type promoter nucleotide sequence (SEQ ID No. 2) of the BBD29_03870 gene into adenine A, and other sequences are unchanged, so as to obtain a mutant promoter nucleotide sequence shown in SEQ ID No. 1. The mutation site corresponds to: the first position of the start codon GTG of the BBD29_03870 gene is marked as +1, and the mutation site is at position-107.

The following primers (the base in bold font is the mutation position) were designed, and then vector construction was performed using NEBuilder recombination technology:

P1：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGCACTCGGTGAAGGCGTG-3'，

P2：

P3：

P4：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCCGCTTCAGCTTCACGAG-3'。

note that: the P1 and P4 underlined portions are the sequences carried on the pK18mobsacB vector.

The specific construction method is as follows: the Corynebacterium glutamicum ATCC13869 is used as a template, and the primer pair P1/P2 and the primer pair P3/P4 are respectively used for PCR amplification, wherein the amplification system and the reaction program are as follows:

the PCR amplification system is as follows: 10 xEx Taq Buffer 5. Mu.L, dNTP mix (2.5 mM each) 4. Mu.L, mg ²⁺ (25 mM) 4. Mu.L, 2. Mu.L each of primer (10 pM), 0.25. Mu.L of Ex Taq (5U/. Mu.L), and a total volume of 50. Mu.L;

the PCR amplification reaction procedure was: pre-denaturation at 94℃for 5min, (denaturation at 94℃for 30s; annealing at 52℃for 30s; extension at 72℃for 40s;30 cycles), over-extension at 72℃for 10min.

As a result of PCR amplification, two DNA fragments (both fragments having mutant bases) of the coding region and the promoter region of BBD29_03870 gene of 745bp and 768bp, respectively, were obtained. The amplified product of the primer pair P1/P2 was designated BBD29_03870Up. The amplified product of the primer pair P3/P4 was designated BBD29_03870Down.

Then, the two DNA fragments (BBD29_ 03870Up and BBD29_03870 Down) were subjected to agarose gel electrophoresis, and after separation and purification, they were ligated with the pK18mobsacB plasmid (Biovector NTCC collection, which contains a kanamycin resistance marker on the plasmid) purified by double digestion with Xba I/BamHI, using NEBuilder enzyme (ligation at 50℃for 30 min). Then connect theThe product is transformed into DH5a, and the grown monoclonal is identified by PCR of M13 primer (M13F: 5'-TGTAAAACGACGGCC AGT-3'; M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain a positive recombinant vector, which is named pK18-BBD29_03870 ^G-107A 。

Recombinant vector pK18-BBD29_03870 ^G-107A Is described in the structure: the small fragment between the Xba I/BamHI cleavage sites of the pK18mobsacB plasmid was replaced with the DNA fragment shown at positions 37-1437 of SEQ ID No.3, and the other sequences of the pK18mobsacB vector were kept unchanged, thus obtaining a recombinant vector.

Recombinant vector pK18-BBD29_03870 ^G-107A Contains the complete SEQ ID No.3. Positions 1-36 and 1438-1475 of SEQ ID No.3 are the self sequence on the pK18mobsacB plasmid; positions 37-439 are the bbd29_03870 promoter upstream sequence; positions 440-835 are the complete promoter sequence after mutation; the 836-1437 position is a partial sequence of the ORF of the BBD29_03870 gene. Wherein, 729 of SEQ ID No.3 is a mutation site which causes guanine (G) at position-107 (the first position of the initiation codon GTG of BBD29-03870 gene is +1) of the promoter region of BBD29-03870 gene in Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 and wild type Corynebacterium glutamicum strain ATCC13869 to be mutated into adenine (A) (corresponding to position 290 of SEQ ID No.1 and SEQ ID No. 2).

Example 2 construction of a cell containing BBD29_03870 ^G-107A Is an engineered strain of (2)

The allelic substitution plasmid pK18-BBD29_03870 obtained in example 1 ^G-107A The strain is transformed into Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 and wild Corynebacterium glutamicum strain ATCC13869 by electric shock. Then culturing in a culture medium, wherein the components of the culture medium and the culture conditions are as follows:

composition of the medium: sucrose 10g/L; 10g/L polypeptone; 10g/L of beef extract; 5g/L yeast powder; urea 2g/L; sodium chloride 2.5g/L; 20g/L of agar powder; the balance being water; pH 7.0.

Culture conditions: 32 ℃.

The single colonies generated by the culture were identified by the primer P1 and the universal primer M13R (5'-CAG GAAACAGCTATGACC-3') in example 1, respectively, and the strain amplified into 1482bp size band (SEQ ID No. 4) was a positive strain. Ext> theext> positiveext> strainsext> areext> cultivatedext> onext> aext> cultureext> mediumext> containingext> 15ext>%ext> ofext> sucroseext>,ext> singleext> coloniesext> generatedext> byext> cultivationext> areext> respectivelyext> cultivatedext> onext> aext> cultureext> mediumext> containingext> kanamycinext> andext> aext> cultureext> mediumext> notext> containingext> kanamycinext>,ext> strainsext> whichext> doext> notext> growext> onext> theext> cultureext> mediumext> containingext> kanamycinext>,ext> namelyext> strainsext> whichext> areext> positiveext> forext> homologousext> recombinationext> withext> genomeext>,ext> theext> obtainedext> eightext> positiveext> strainsext> areext> furtherext> amplifiedext> byext> usingext> Pext> 1ext> andext> Pext> 4ext> primersext> (ext> theext> sequencesext> areext> shownext> inext> exampleext> 1ext>)ext>,ext> thenext> PCRext> productsext> areext> sequencedext>,ext> andext> theext> strainsext> withext> theext> nucleotideext> sequenceext> ofext> theext> positionext> -ext> 107ext> (ext> theext> firstext> positionext> ofext> aext> BBDext> 29_ext> 03870ext> geneext> initiationext> codonext> GTGext> isext> markedext> asext> +1ext>)ext> ofext> aext> BBDext> 29_ext> 03870ext> geneext> promoterext> regionext> areext> obtainedext> throughext> sequenceext> comparisonext>,ext> namelyext> theext> strainsext> withext> successfulext> allelicext> replacementext> (ext> Gext> -ext> Aext>)ext> (ext> theext> positionext> 290ext> ofext> SEQext> IDext> No.ext> 1ext> andext> SEQext> IDext> No.ext> 2ext>)ext> areext> obtainedext>.ext> The resulting positive strains were designated YPG-03870 and G03870, respectively, depending on the original strain.

Recombinant bacteria YPG-03870 and G03870 each contain a fragment shown in SEQ ID No.3 at 37-1437 comprising the mutant promoter of the BBD29_03870 gene.

Recombinant strain YPG-03870 differs from Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 only in that: YPG-03870 is a strain obtained by replacing the wild type promoter of BBD29_03870 gene shown in SEQ ID No.2 in Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220 genome with a mutant promoter shown in SEQ ID No.1 and keeping other sequences unchanged.

Recombinant G03870 differs from wild Corynebacterium glutamicum ATCC13869 in that: g03870 is a strain obtained by replacing the wild-type promoter of BBD29_03870 gene shown in SEQ ID No.2 in ATCC13869 genome with the mutant promoter shown in SEQ ID No.1 and keeping other sequences unchanged.

EXAMPLE 3 construction of PK18-PBBD 29-03870 ^G-107A -bbd29_09880 recombinant vector

To further demonstrate the BBD29_03870 shown in SEQ ID No.1 ^G-107A Efficient promoter action in the present example in Corynebacterium glutamicum ATCC13869 and Corynebacterium glutamicum (Corynebacterium glutamicum) CGMCC No.21220, the promoters were replaced with the promoters themselves of the genes BBD29_09880 encoding glutamate dehydrogenase, respectively, and a high-yield glutamate strain was constructed, which was promoted with the promoters. Wherein SEQ ID No.5 and SEQ ID No.6 are the ORF (CDS) of BBD29_09880 and the promoter sequence thereof, respectively.

The following primers were designed and then the NEBuilder recombination technique was used to construct PBBD29_03870 ^G-107A -bbd29_09880 vector.

P5：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGATGATCTTCTGGCCGCTG-3'，

P6：5'-AAAGGAATAATTACTCTAATGACAGTTGATGAGCAG-3'，

P7：5'-CTGCTCATCAACTGTCATTAGAGTAATTATTCCTTT-3'，

P8：5'-GGTGGGGGAGTTGGTCATTTTTTCTGAGTCTCAGAT-3'，

P9：5'-ATCTGAGACTCAGAAAAAATGACCAACTCCCCCACC-3'，

P10：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGGCTTTGTGCCATCAACTT-3'。

Note that: the P5 and P10 underlined portions are the sequences carried on the pK18mobsacB vector.

The specific construction method is as follows: the Corynebacterium glutamicum ATCC13869 is used as a template, and the primer pair P5/P6 and the primer pair P9/P10 are respectively used for PCR amplification, and the specific amplification system and the reaction program are as follows:

Two PBBD29_03870 with the sizes of 764bp and 797bp are obtained through PCR amplification ^G-107A -DNA fragment of bbd29_ 09880. The amplified product of the primer pair P5/P6 was designated as PBBD29_03870 ^G-107A Up, the amplified product of the primer pair P9/P10 was designated as PBBD 29-03870 ^G-107A Down. With Corynebacterium glutamicum YPG-03870 ^G-107A As template, with primer pair P7-P8 is amplified by PCR to obtain PBBD29_03870 with the size of 432bp ^G-107A Is designated as PBBD29_03870 ^G-107A 。

The three DNA fragments PBBD 29-03870 ^G-107A Up、PBBD29_03870 ^G-107A And PBBD29_03870 ^G ^-107A Down was subjected to agarose gel electrophoresis, separated and purified, and then ligated with pK18mobsacB plasmid (Biovector NTCC collection, containing kanamycin resistance marker) purified by Xba I/BamH I double enzyme digestion, which was subjected to plasmid ligation at 50℃for 30min with NEBuilder enzyme, and the ligation product was transformed into DH5a, and the resulting monoclonal was subjected to PCR identification by M13 primer (M13F: 5'-TGTAAAACGACGGCC AGT-3'; M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain a positive recombinant vector designated pK18-PBBD 29-03870 ^G-107A -BBD29_09880。

Recombinant vector pK18-PBBD29_03870 ^G-107A -structural description of bbd29_ 09880: the small fragment between the Xba I/BamH I cleavage sites of the pK18mobsacB plasmid was replaced with the DNA fragment shown in positions 1-1847 of SEQ ID No.7, and the other sequences of the pK18mobsacB vector were kept unchanged, to obtain a recombinant vector.

Recombinant vector pK18-PBBD29_03870 ^G-107A The BBD29_09880 contains the complete SEQ ID No.7 (BBD29_ 03870 promoter is genomic forward, i.e. BBD29_03870 gene is genomic forward, and BBD29_09880 gene is genomic reverse, so BBD29_03870 is used ^G-107A The mutant promoter should start the BBD29_09880 gene, BBD29_03870 ^G-107A The mutant promoter was integrated into the genome in reverse complement to initiate expression of the bbd29_09880 gene). The 5' -end part sequence of BBD29_09880 at positions 1-710 and BBD29_03870 at positions 711-1106 of SEQ ID No.7 ^G-107A A mutant promoter sequence (wherein position 817 is a mutation site); the 1107-1847 position is the 5' end part sequence of the ORF of BBD29_09885 gene.

Example 4 construction of the inclusion Gene PBBD 29-03870 ^G-107A Engineering strain of BBD29_09880

Recombinant plasmid pK18-PBBD29_03870 obtained in example 3 ^G-107A Bbd29_09880 transformed into glutamate rods by electric shock, respectivelyBacillus (Corynebacterium glutamicum) CGMCC No.21220 and wild type corynebacterium glutamicum strain ATCC13869, and then cultured in a medium having the following composition and culture conditions:

Culture conditions: 32 ℃.

The single colony generated by culture is identified by the primers P11 and P12 respectively, and the strain which can amplify the 1038bp size band (SEQ ID No. 8) is a positive strain. Culturing positive strain on 15% sucrose-containing medium, culturing single colony produced by culture on kanamycin-containing medium and kanamycin-free medium, respectively, growing on kanamycin-free medium, and amplifying strain which does not grow on kanamycin-containing medium by further using primers P13 and P14, wherein strain capable of amplifying 1161bp band (SEQ ID No. 9) is Corynebacterium glutamicum CGMCC No.21220 and BBD29_03870 on ATCC13869 genome ^G-107A Positive strains with promoters replacing the BBD29_09880 original promoters are respectively named YPG-03870 according to the differences of the original strains ^G-107A And G03870 ^G-107A 。

Among them, primers P11 and P12 and primers P13 and P14 are as follows:

p11:5'-TAACGGTTGCGCCGAGTT-3' (corresponding to the upper homology arm BBD29_ 09880),

p12:5'-GGTGGAAGCGTTTAAATGCG-3' (corresponding to BBD 29-03870) ^G-107A A promoter region),

p13:5'-GGGTGATAGCAGTCACTTCG-3' (corresponding to BBD 29-03870) ^G-107A A promoter region),

p14:5'-TGCATGAGGGTGGACAGC-3' (corresponding to the lower homology arm BBD29_ 09885).

Recombinant bacterium YPG-03870 ^G-107A Comprises BBD29-03870 shown in SEQ ID No.1 ^G-107A A promoter; specifically, recombinant bacterium YPG-03870 ^G-107A In order to replace the promoter of BBD29_09880 shown in SEQ ID No.6 on the genome of Corynebacterium glutamicum CGMCC No.21220 with that shown in SEQ ID No.1BBD29-03870 shown ^G-107A A mutant promoter for keeping other nucleotides in the genome of the corynebacterium glutamicum CGMCC No.21220 unchanged.

Recombinant bacterium G03870 ^G-107A Comprises BBD29-03870 shown in SEQ ID No.1 ^G-107A A promoter; specifically, recombinant bacterium G03870 ^G-107A The promoter of BBD 29-09880 shown in SEQ ID No.6 on the genome of Corynebacterium glutamicum ATCC13869 is replaced with BBD29-03870 shown in SEQ ID No.1 ^G-107A A mutant promoter, a recombinant bacterium which retains other nucleotides in the genome of Corynebacterium glutamicum ATCC13869.

Example 5 fermentation production of L-glutamic acid

Recombinant strains YPG-03870 and G03870 constructed in example 2 and recombinant strain YPG-03870 constructed in example 4 ^G-107A And G03870 ^G-107A And the corresponding original strains Corynebacterium glutamicum CGMCC No.21220 and ATCC13869 are test strains.

Each test strain was subjected to fermentation experiments in a BLBIO-5GC-4-H model fermenter (Shanghai Bai Biotechnology Co., ltd.) with the medium shown in Table 1 and the control process shown in Table 2. After the fermentation, the yield and OD (562 nm) of L-glutamic acid were measured by using an SBA biosensing instrument (SBA-40E).

TABLE 1 fermentation Medium formulation (balance water)

TABLE 2 fermentation control Process

Each strain was replicated three times. The L-glutamate production data used single factor analysis of variance, with experimental results P <0.05 (x) indicating significant differences and P <0.01 (x) indicating very significant differences.

The results are shown in tables 3 and 4.

The results show that: in corynebacterium glutamicum, the wild-type promoter of BBD29_03870 gene shown in SEQ ID No.2 in genome is replaced by the mutant promoter shown in SEQ ID No.1, which can remarkably improve the L-glutamic acid yield (p<0.01). In Corynebacterium glutamicum, the BBD29_09880 gene promoter shown in SEQ ID No.6 in the genome was replaced with the mutant BBD29_03870 shown in SEQ ID No.1 ^G-107A The promoter can also significantly improve the L-glutamic acid yield (p<0.01)。

TABLE 3 results of fermentation experiments of Corynebacterium glutamicum CGMCC No.21220 and its mutants

Note that: p in Table<0.01 YPG-04605 and YPG-03870 ^G-107A The L-glutamic acid yield of (C) is very significantly different from that of CGMCC No.21220.

Table 4 results of L-glutamic acid fermentation experiments with wild-type strain ATCC13869 and mutant thereof

Note that: p in Table<0.01 represents G04605 and G03870 ^G-107A The L-glutamic acid production of (C) was very significantly different from that of ATCC13869.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims

Use of a dna molecule as a promoter;

the DNA molecule comprises any one of the following:

(a1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 1;

(a2) And (3) performing conservative substitution on a plurality of nucleotides except 290 th site of SEQ ID No.1 to obtain the DNA molecule with the function of a promoter.
A dna molecule comprising any one of:

(a1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 1;

(a2) And (3) performing conservative substitution on a plurality of nucleotides except 290 th site of SEQ ID No.1 to obtain the DNA molecule with the function of a promoter.
3. An expression cassette, recombinant vector or recombinant bacterium comprising the DNA molecule of claim 2.
4. A recombinant bacterium according to claim 3, wherein: the recombinant bacteria are recombinant bacteria A or recombinant bacteria B;

the recombinant bacterium A is obtained by replacing a promoter of BBD29_03870 gene in a bacterial genome with the DNA molecule of claim 2;

the promoter of the BBD29_03870 gene comprises any one of the following:

(b1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 2;

(b2) A DNA molecule derived from a bacterium and having 95% or more identity to (b 1) and a promoter function;

the recombinant bacterium B is obtained by replacing a promoter of BBD29_09880 gene in a bacterial genome with the DNA molecule of claim 2;

the promoter of the BBD29_09880 gene comprises any one of the following:

(c1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 6;

(c2) A DNA molecule derived from a bacterium and having 95% or more identity with (c 1) and having a promoter function.
5. A method for increasing the production of bacterial L-amino acids comprising the steps of (a) or (B):

(A) Replacing the promoter of the bbd29_03870 gene in the bacterial genome with the DNA molecule of claim 2 to achieve an increase in the L-amino acid yield of the bacterium;

the promoter of the BBD29_03870 gene comprises any one of the following:

(b1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 2;

(b2) A DNA molecule derived from a bacterium and having 95% or more identity to (b 1) and a promoter function;

(B) Replacing the promoter of the bbd29_09880 gene in the bacterial genome with the DNA molecule of claim 2 to achieve an increase in the L-amino acid yield of the bacterium;

the promoter of the BBD29_09880 gene comprises any one of the following:

(c1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 6;

(c2) A DNA molecule derived from a bacterium and having 95% or more identity with (c 1) and having a promoter function.
6. A method for producing an L-amino acid comprising the steps of: introducing the DNA molecule of claim 2 into a biological cell capable of synthesizing said L-amino acid, such that the DNA molecule of claim 1 drives expression of a gene in said L-amino acid synthesis pathway in said biological cell, resulting in a recombinant biological cell; culturing the recombinant biological cell to obtain the L-amino acid.
7. The method according to claim 6, wherein: the biological cell is a bacterium, yeast, algae, fungus, plant cell or animal cell capable of synthesizing the L-amino acid.
8. Any of the following applications:

use of P1, the DNA molecule of claim 2 or the expression cassette or recombinant vector or recombinant bacterium of claim 3 or 4 for the production of L-amino acids;

use of P2, a DNA molecule according to claim 2 or an expression cassette or recombinant vector according to claim 3 or 4 in the construction of an engineering strain producing L-amino acids;

use of P3, a DNA molecule according to claim 2 or an expression cassette or recombinant vector according to claim 3 or 4 for the preparation of a food, pharmaceutical, feed or cosmetic product comprising an L-amino acid.
9. The recombinant bacterium of claim 4 or the method of claim 5 or 7, wherein: the bacteria are coryneform bacteria;

further, the bacterium is Corynebacterium glutamicum.
10. The method or use according to any one of claims 5-9, characterized in that: the L-amino acid is L-glutamic acid.