CN113493785A

CN113493785A - High-strength promoter suitable for corynebacterium glutamicum and application

Info

Publication number: CN113493785A
Application number: CN202010263319.9A
Authority: CN
Inventors: 周景文; 陈坚; 李宁; 曾伟主; 堵国成
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2021-10-12
Anticipated expiration: 2040-04-07
Also published as: CN113493785B

Abstract

The invention discloses a high-strength promoter suitable for corynebacterium glutamicum and application thereof, and belongs to the technical field of biological engineering. The invention obtains 18 promoters which can improve the protein expression strength by screening from corynebacterium glutamicum. The promoter obtained by screening can be introduced into microorganisms for producing amino acids, and the promoter can enhance the expression amount of genes in corynebacterium glutamicum, so that the yield of related target products is improved by 2-10 times or even higher, and the promoter has important industrial application value.

Description

High-strength promoter suitable for corynebacterium glutamicum and application

Technical Field

The invention relates to a high-strength promoter suitable for corynebacterium glutamicum and application thereof, belonging to the technical field of biological engineering.

Background

Corynebacterium glutamicum (Corynebacterium glutamicum) is an important industrial model microorganism capable of synthesizing many useful compounds using a crude substrate. However, the wild type Corynebacterium glutamicum is difficult to directly use and must be modified as necessary. Mutation breeding has achieved the fermentative production of a number of compounds in C.glutamicum, but the production and conversion of some compounds is still very limited. For this reason, researchers have turned to applications in metabolic engineering and synthetic biology. Overexpression of the relevant genes by plasmid expression or genomic integration of strong promoters often allows more metabolic flux to the target product. For the polygenic pathway, plasmid expression is likely to cause burden on the bacteria, and ultimately affects the synthesis of the product, and in this case, the use of genome integration expression is highly advantageous.

Whether expressed by plasmid or integrated genome, the copy number of a gene has a great effect on the level of up-regulation of the gene. The number of copies for genomic integration is generally low, and it is difficult to achieve integration of multiple copies, so the use of a strong promoter is a simpler and more efficient means. In previous studies, C.glutamicum already possesses several strong promoters (e.g.P)_sod、P_tuf) And later on is supplemented with P_H36、P₄₅Isopotent promoters, however, these promoters are still not satisfactory. Therefore, screening for higher strength promoters is of great interest to complete the synthetic biology toolkit for C.glutamicum.

Disclosure of Invention

In order to solve the problems, the invention takes a corynebacterium glutamicum genome as a template, designs different primers to amplify DNA fragments, takes fluorescent protein as a reporter gene, constructs recombinant plasmids, transforms a host to obtain a recombinant strain, compares the fluorescence intensity of the recombinant strain, and screens strong promoters.

The first purpose of the invention is to provide a high-strength promoter, the nucleotide sequence of which is shown as SEQ ID NO. 1.

The second purpose of the invention is to provide an expression vector containing a promoter shown in SEQ ID NO. 1.

In one embodiment, the expression vector is obtained by operably linking the promoter represented by SEQ ID NO.1 with a plasmid; the operable linkage means that a nucleotide having promoter activity is functionally linked to a gene sequence to initiate and mediate transcription of a target gene, and the linkage process can be achieved using a gene recombination technique known in the art, and site-specific DNA cleavage and linkage can be performed by using restriction enzymes and ligase known in the art.

In one embodiment, the plasmids include, but are not limited to, pEC-XK99E, pXZ10145, pXMJ19, pJYW-4, pJYW-5.

The third purpose of the invention is to provide a microbial cell containing the high-strength promoter or the expression vector.

In one embodiment, the microbial cell is a recombinant corynebacterium glutamicum.

In one embodiment, the recombinant corynebacterium glutamicum is hosted in corynebacterium glutamicum ATCC13032, and the promoter is introduced into the genome.

In one embodiment, the recombinant corynebacterium glutamicum is hosted in corynebacterium glutamicum ATCC13032, and the expression vector is transformed into corynebacterium glutamicum ATCC13032 cells.

In one embodiment, the recombinant Corynebacterium glutamicum is transformed into Corynebacterium glutamicum ATCC13032 cells by linking the promoter shown in SEQ ID NO.1 and the gene coding for homoserine acetyltransferase shown in SEQ ID NO.20 with the vector pEC-XK 99E.

The fourth purpose of the invention is to provide the application of the high-strength promoter in enhancing gene or protein expression.

In one embodiment, the use is for enhancing the expression of genes in C.glutamicum.

In one embodiment, the gene includes, but is not limited to, a gene involved in the growth or metabolism of a microorganism.

In one embodiment, the gene is the red fluorescent protein gene mCherry.

In one embodiment, the protein is an endogenous protein or an exogenous protein.

In one embodiment, the protein is a homoserine acetyltransferase and the amino acid sequence comprises the amino acid sequence shown in SEQ ID No. 21.

The fifth purpose of the invention is to provide the application of the high-strength promoter in improving the production of O-acetyl-L-homoserine by Corynebacterium glutamicum.

In one embodiment, the application is that the promoter shown in SEQ ID NO.1 and the gene which is shown in SEQ ID NO.20 and codes for homoserine acetyltransferase are connected with a vector pEC-XK99E and transformed into corynebacterium glutamicum capable of producing homoserine to obtain recombinant corynebacterium glutamicum; the recombinant Corynebacterium glutamicum was then cultured at 25-37 ℃ at 150-.

In one embodiment, the application is that the promoter shown in SEQ ID NO.1 and the gene which is shown in SEQ ID NO.20 and codes for homoserine acetyltransferase are connected with a vector pEC-XK99E and transformed into corynebacterium glutamicum capable of producing homoserine to obtain recombinant corynebacterium glutamicum; culturing the recombinant Corynebacterium glutamicum for 2-4 days, transferring the recombinant Corynebacterium glutamicum to a CGXII culture medium containing 40-60g/L glucose and 0.1-1.0g/L yeast extract, culturing for 12-24h, transferring the recombinant Corynebacterium glutamicum to a 48-well or 96-well deep-well plate containing 1-2 mL fermentation medium in an inoculation amount of 1-2%, and culturing for at least 36h under the conditions of 25-37 ℃ and 150-250 rpm.

In one embodiment, the fermentation medium is a medium comprising: 60-120g/L D-glucose, 10-30g/L corn steep liquor, 10-30g/L (NH)₄)₂SO₄，0.5-2g/L KH₂PO₄，0.2-0.6g/L MgSO₄,，0.05-0.15g/L MnSO₄·H₂O，0.005-0.012g/L FeSO₄·7H₂O, 0.5-1.5mg/L vitamin B₁4-8mg/L vitamin B₆2-6mg/L vitamin B₁₂0.02-0.04mg/L biotin.

Has the advantages that: the promoter obtained by screening can be introduced into microorganisms for producing amino acids, and the promoter can enhance the expression quantity of genes in corynebacterium glutamicum so that the yield of related target products is improved by 2-10 times or even higher, and has important industrial application value.

Drawings

FIG. 1: a flow chart for constructing the ptrc-mCherry recombinant plasmid.

FIG. 2: after culturing the recombinant bacterium pEC-XK99E (A) and the recombinant bacterium ptrc-mCherry (B) for 72 hours, observing the results in a plate culture medium, an optical microscope and a fluorescence microscope respectively.

FIG. 3: scheme for constructing pX-mCherry library.

FIG. 4: (A) the growth curve of the recombinant bacterium containing the ptrc-mCherry plasmid constructed in example 2 is shown; (B) the fluorescence intensity/OD of the recombinant bacterium pX-mCherry containing different promoters₆₀₀Values, wherein the numerical value of the horizontal axis represents the promoter number, and the 1 st to 18 th correspond to the promoters of SEQ ID NO.1 to 18, respectively.

Detailed Description

In order to make the above-mentioned objects of the present invention comprehensible, embodiments of the present invention are described in detail below with reference to specific examples.

The culture medium:

LB culture medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride. An LB solid medium was prepared by adding 20g/L agar strips.

LBB medium: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and 18.5g/L of brain-heart leaching solution; adding 20g/L agar strips to obtain LBB solid culture medium.

CGXII medium: urea 5g/L, (NH)₄)₂SO₄ 20g/L，KH₂PO₄ 1g/L，K₂HPO₄ 1g/L，MgSO₄·7H₂O 0.25g/L，MOPS 42g/L，CaCl₂ 0.01g/L，MnSO₄0.01g/L, 0.02mg/L sodium citrate, FeSO₄·7H₂O 0.01g/L，ZnSO₄·7H₂O 0.01g/L，CuSO₄·5H₂O 0.2mg/L，NiCl₂·6H₂O0.02 mg/L, biotin 25 mug/L。

O-acetyl-L-homoserine fermentation medium: 100g/L D-glucose, 20g/L corn steep liquor, 20g/L (NH)₄)₂SO₄，1g/L KH₂PO₄，0.5g/L MgSO₄,，0.01g/L MnSO₄·H₂O，0.01g/L FeSO₄·7H₂O, 1mg/L vitamin B₁6mg/L vitamin B₆4mg/L vitamin B₁₂0.025mg/L Biotin (pH adjusted to 7.0 with Ammonia)

(II) a fluorescence intensity detection method: sampling at regular intervals of 6-12h with the switching starting time of 0h, sucking 200 μ L of bacterial liquid into a 96 shallow pore plate, and detecting the light absorption value (OD) at 600nm₆₀₀) (ii) a The fluorescence values were then detected: excitation wavelength of 587nm, emission wavelength of 610nm, and absorbance (OD)₆₁₀)；OD₆₁₀/OD₆₀₀The value is the fluorescence intensity of the recombinant bacteria.

The partial promoter information in the (third) example is shown in Table 1.

TABLE 1 promoters and corresponding sequences

Example 1 construction of recombinant plasmid ptrc-mCherry

Plasmid pEC-XK99E is used as a template, and a primer P is used_trc-F (CGGCATGCATTTACGTCGACGCGCAACGCAATTAATGTGAGTTAGGC) and P_trc-R (TCGCCCTTGTCTCACTCTAGAGATTACTAGATTCAGGTCTCCTTCTGGATCCCCGGGTACCGAGC) is amplified to obtain P_trcFragment (shown as SEQ ID NO. 19).

Linearizing plasmid pEC-XK99E by using primers XK99E-F (ATGGACGAGCTGTTACAAGTAACTAGCAGGCATGCAAGCTTG) and XK99E-R (GTCGACACGTAAATGCATGCCGC) to obtain XK 99E-XXH; then using XK99E-XXH as a template, and using a primer mCherry-F (ATGTCTAGAGTGAGCAAGGGCGAGGAG) and a primer mCherry-R (TTACTTGTACAGCTCGTCTCTCTCATGCCATGCCG) to amplify the mCherry gene to obtain an mCherry gene fragment; connecting P using Gibson assembly_trcThe recombinant plasmid ptrc-mCherry is obtained by the fragment, XK99E-XXH and mCherry gene fragment.

Example 2 construction of recombinant bacterium ptrc-mCherry for expressing fluorescent protein

And transforming the recombinant plasmid ptrc-mCherry to Corynebacterium glutamicum ATCC13032 by using an electric shock transformation method and other modes to obtain the ptrc-mCherry recombinant strain.

The recombinant strain is streaked on an LBB solid medium plate, is transferred to a CGXII medium containing 40-60g/L glucose after being cultured for 3d, is transferred to a 48-well or 96-well deep-well plate containing 1.5-2mL of the medium by 1-2 percent of inoculum size after being cultured for 18-24h, is cultured under the conditions of 25-37 ℃ and 250rpm for 150-4 hours, and is selected for timing sampling for 6-12 h.

The fluorescence intensity in the culture solution of the cells cultured for 36h was measured, and the result showed that the fluorescence intensity after 36h was 81361.82. Example 3 construction of recombinant pX-mCherry library containing different promoters

The Corynebacterium glutamicum ATCC13032 is streaked and cultured by using LBB solid medium at the temperature of 25-37 ℃, inoculated to LBB liquid medium after 2-4d culture, cultured for 12-48h and collected to extract genome. Using a corynebacterium glutamicum ATCC13032 genome as a template, designing primers and amplifying to obtain different promoter fragments pX (pX1, pX2 and pX3 … … pXn); wherein the nucleotide sequence of part of the promoter is shown in SEQ ID NO. 1-18.

Digesting the recombinant plasmid ptrc-mCherry constructed in the example 1 by using restriction enzymes SalI and XbaI to obtain a linearized fragment p-mCherry-XXH; and connecting pX with p-mCherry-XXH by using a Gibson assembly mode to obtain a recombinant plasmid pX-mCherry. The recombinant plasmid is transformed into Corynebacterium glutamicum ATCC13032 by using an electric shock transformation method and the like, so that a recombinant pX-mCherry library can be obtained.

The strains in the recombinant pX-mCherry library are respectively and independently streaked on an LBB solid medium plate, the strains are transferred to a CGXII medium containing 40g/L glucose and 0.5g/L yeast powder after being cultured for 3 days, the strains are transferred to a 48-hole or 96-hole deep-hole plate containing 2mL CGXII medium supplemented with 40g/L glucose in an inoculation amount of 2 percent after being cultured for 20 hours, and the strains are cultured under the conditions of 30 ℃ and 220rpm and are sampled at regular intervals of 6-12 hours.

The fluorescence intensity in the culture solution of the cells cultured for 36h is detected, and the result shows that the promoter fragment pNCgl1676 shown in SEQ ID NO.1 is constructedThe obtained recombinant strain pNCgl1676-mCherry OD₆₁₀/OD₆₀₀The highest value is 275880.91, which is about 5 times of that of the recombinant bacterium psod-mCherry.

Example 4

A recombinant plasmid pNCgl1676-metX expressing metX gene using promoter pNCgl1676 shown in SEQ ID No.1 was constructed according to the same strategy as in examples 1 to 3, except that mCherry gene was replaced with metX gene encoding homoserine acetyltransferase shown in SEQ ID No.20, and the recombinant plasmid pNCgl1676-metX was transformed into L-homoserine producing Corynebacterium glutamicum to obtain recombinant strain pNCgl 1676-metX.

The recombinant plasmid psod-metX is transformed by the same host cell (the promoter pNCgl1676 is replaced by the SOD promoter (NCgl2826) shown in SEQ ID NO.9, and the mCherry gene is replaced by the metX gene), so that the recombinant bacterium psod-metX is obtained.

The plasmid pEC-XK99E is transformed by the same host cell to obtain the recombinant bacterium pEC-XK 99E.

The contents of recombinant bacteria pNCgl1676-metX and control in O-acetyl-L-homoserine fermentation medium after culturing at 30 ℃ and 220rpm for 48h were determined according to standard amino acid determination method using recombinant bacteria psod-metX and pEC-XK99E as control, and the results showed that: the recombinant strains pNCgl1676-metX, psod-metX and pEC-XK99E have O-acetyl-L-homoserine yields of 1.57g/L, 0.82g/L and 0.13 g/L.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> high-strength promoter suitable for corynebacterium glutamicum and application thereof

<160> 21

<170> PatentIn version 3.3

<210> 1

<211> 295

<212> DNA

<213> Corynebacterium glutamicum

<400> 1

atgcgacagt acttttcatt aagcctaaga aaattccttt aattgacact taattgacca 60

ataagagtcg attagattgc attattaggt aatctagtga tttaatggag aataagagca 120

actggtgaag aaaaggcttg atgaaagaag ttttttatct agctagatgt tcaatcacga 180

gctttaagaa agtatgtcaa taactttgac ataacctaaa cacaataaat tatgtagtat 240

tatgtgacac taagttatta catttattat atgattggtt aggactatgg acatg 295

<210> 2

<211> 222

<212> DNA

<213> Corynebacterium glutamicum

<400> 2

tggatctgta tttgacgtgg ttcgagaccc cgcggtgttg cacaaagtgg aagtcagtgg 60

aggaatcctc gagcctgaat gtgctgcctt gatgaccgaa tttttcgaac ttcacaggta 120

acggatttat atcaatttca gggcgtggcg agcttttagt gattcacgct cctacggtgg 180

gtatcacaaa tacctcaact agaagtagga gatgagcacc ac 222

<210> 3

<211> 192

<212> DNA

<213> Corynebacterium glutamicum

<400> 3

gcttgaaccg gcatgaaaat ctcgtaccgg ttttgggctc acaaggccat ataggaactt 60

tgtaattagt tgcaggttcc aattttgggt caatgtagcg taatattgtt caaggcccat 120

gtgcgggctg tggaggacgt gcattcacgt tctggtcaaa tgaaaaacgg tgaaagggat 180

tgaacgcagc ag 192

<210> 4

<211> 219

<212> DNA

<213> Corynebacterium glutamicum

<400> 4

ctcaacgcga taggtttcaa ccataggcct gacctggctg agatgttttt ggtagaaaaa 60

ccgagtgccc gaattgtttt gtgggtgccc ggttttttct gatttaagca cgtcagaggc 120

gtagaacatt gtctgttcac actctgggtc gcaagattca tcgagaatta atggtagtac 180

ctgtggcttg agggggaatg acgtactagg cttatgggc 219

<210> 5

<211> 255

<212> DNA

<213> Corynebacterium glutamicum

<400> 5

ttatgtgtcg aggtgaatct ccggtgaatt cttatagata acttgttttt gcaggtcagg 60

acggggttaa ggggatgggt gttatctgtc agtatgtgag gagatcaagg tgttgggggt 120

tctagttgct aagatggtga aaacccgtga ggccaaaatc caactgggtg aattacccct 180

gcataaatgc atgagggctt tatacttgtc ttattattaa acttttaggg ttttgatgca 240

ggaaggtgcg agaac 255

<210> 6

<211> 206

<212> DNA

<213> Corynebacterium glutamicum

<400> 6

gcgttttcag atcatgattg attgggcgtg cctgcttttg tgttttttag ggaccccaat 60

gcgcgtgatt caactcatgt ttgatatgtg ctcctaaggt gtgtaaccta tatcgatggt 120

gtgcgtacat cttgagtgac gcaaccattt tgaagtggaa aaacttaagg cctcccgcag 180

gggagtgttc tggaaaagcg gaggat 206

<210> 7

<211> 247

<212> DNA

<213> Corynebacterium glutamicum

<400> 7

cttgattcag ggtagttgac taaagagttg ctcgcgaagt agcacctgtc acttttgtct 60

caaatattaa atcgaatatc aatatatggt ctgtttattg gaacgcgtcc cagtggctga 120

gacgcatccg ctaaagcccc aggaaccctg tgcagaaaga aaacactcct ctggctaggt 180

agacacagtt tataaaggta gagttgagcg ggtaactgtc agcacgtaga tcgaaaggtg 240

cacaaag 247

<210> 8

<211> 187

<212> DNA

<213> Corynebacterium glutamicum

<400> 8

ctacacttct ggagcgttac ggtgcttccg aagacacccc agtggtgtcc ttcaactaag 60

cccgaagttt tttaaccgcc gcattcgatc accaaatgtg gcggttttgc gtcgaaaagc 120

gtgctctttc tacacctctt tgaggttcat tttcgcggtt tcctcacaat cgcctattgt 180

taagtac 187

<210> 9

<211> 192

<212> DNA

<213> Corynebacterium glutamicum

<400> 9

tagctgccaa ttattccggg cttgtgaccc gctacccgat aaataggtcg gctgaaaaat 60

ttcgttgcaa tatcaacaaa aaggcctatc attgggaggt gtcgcaccaa gtacttttgc 120

gaagcgccat ctgacggatt ttcaaaagat gtatatgctc ggtgcggaaa cctacgaaag 180

gattttttac cc 192

<210> 10

<211> 292

<212> DNA

<213> Corynebacterium glutamicum

<400> 10

atagcggaca ttttttgacg cagatcacct tactctgaag gataaggatt cttagtgtcg 60

gtgcactttt actgatgttt cactgtggag gtcaacgact caaagtcgag aattggtggc 120

gcgtgtcact ggaattgacg cgtgaatggg gtggaagtgg acgtcgaaaa gcatttttga 180

gacgtttatg tgagcaatgt cccattttcc ctgctcacct gtatgggcac ccgcggcgga 240

agtggaattg catatggagt tttgatgata tttagcgtaa cttaaaggaa ca 292

<210> 11

<211> 161

<212> DNA

<213> Corynebacterium glutamicum

<400> 11

cccccttttg ggtgtccaga atccaaaatt ccgggcacaa aagtgcaaca atagatgacg 60

tgcgggttga tacagcccaa gcgccgatac atttataatg cgcctagata cgtgcaaccc 120

acgtaaccag gtcagatcaa gtgccccagg aggcccttca g 161

<210> 12

<211> 202

<212> DNA

<213> Corynebacterium glutamicum

<400> 12

acctactcct acgaccccga agtcaggttc cgctccatag ctgcacttgg cacggcatgg 60

aattagtgtc aaaagcctca aaaatactgg tactaaccag ctgtgcggat cgggtatccg 120

cgctacactt agaggtgtta gagatcatga gtttccacga actgtaacgc aggattcacc 180

aatcaatgaa aggtcgaccg ac 202

<210> 13

<211> 213

<212> DNA

<213> Corynebacterium glutamicum

<400> 13

atcaccgtgg aacaaggacg attcggcgca atgatgaagg tcacatcggt taacgaaggc 60

cccttcaccg ttttggtcga gtgctagcca gtcaatccta agagcttgaa acgccccaat 120

gtgggggtgt taagaactcc ataaaagcgc ttgggaactt tttgtggaag cagtccgttg 180

aacctcttga accgcgaatt taggaggcca gtt 213

<210> 14

<211> 300

<212> DNA

<213> Corynebacterium glutamicum

<400> 14

cagtgttacc ccctaagact acccctttcc attgcataca aaggaaatac atatagactt 60

ttgggcatta gattacctcg ataaaagttt agggaatcta aattcattga tcaagacttg 120

ctgtcgccta gctctaattc acttgagccc ggctgctaaa ggtcaagatc attgaatgca 180

ctacttgcta gcagtcatct gaaaaaacga cgttggttcg tagtcgctgg aaatttaata 240

attcctccgt ccccttcaac tagggggtgg aaacccgact atttccgaag gactattctc 300

<210> 15

<211> 251

<212> DNA

<213> Corynebacterium glutamicum

<400> 15

tttttgaatg tgtctgtatg attttgcatc tgctgcgaaa tctttgtttc cccgctaaag 60

ttgaggacag gttgacacgg agttgactcg acgaattatc caatgtgagt aggtttggtg 120

cgtgagttgg aaaaattcgc catactcgcc cttgggttct gtcagctcaa gaattcttga 180

gtgaccgatg ctctgattga cctaactgct tgacacattg catttcctac aatctttaga 240

ggagacacaa c 251

<210> 16

<211> 246

<212> DNA

<213> Corynebacterium glutamicum

<400> 16

cagtctatat cgaaccgatg agagcaatcc ccaagtattt cccacccctg tttttaggta 60

cacctaccgc cgaattttgg acgttaaaca agcctgcacc cccacattta ggagtggatt 120

gtgccttatt tctcacactt tctattaccc acctcactct aggggtggac tccagtgttt 180

cgcgacaaca caatgagtaa gcttgtgaca gccgtattta attctcagta agaaatgagt 240

gatttc 246

<210> 17

<211> 233

<212> DNA

<213> Corynebacterium glutamicum

<400> 17

ggactgttgt gcgggtgtgt aaattaattc cagtcagcgc gaccaacaac gccccaacac 60

ataagagatt atgtggacag tgaggcggat ctaggaaaac aaacgctcga caaaccaaca 120

acacttcatc gaagtcaaca aacaccgatt tggtgaaagt aaacaagatg aagtaacgtt 180

gaacaagctg ccaacaagac accaacacaa acaaatgttg atgctcttat ggt 233

<210> 18

<211> 202

<212> DNA

<213> Corynebacterium glutamicum

<400> 18

gcgtggtgct tctgtgaata gagttgtttg agcgactaga gttaaggcca tgactgtcag 60

aaacggcgca cccaagcggg gaagctcacg atcatcggga aagtcgacgg ggccaaaaac 120

caccgcgacg accaggcgaa gcaaaccaac aaccagcacc tctcggggtg gccggtcaga 180

gactgctgcg tttacaacat cg 202

<210> 19

<211> 350

<212> DNA

<213> Artificial sequence

<400> 19

gcgcaacgca attaatgtga gttagcgcga attgatctgg tttgacagct tatcatcgac 60

tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg 120

caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg 180

ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt 240

aatcatccgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac 300

agaccatgga attcgagctc ggtacccggg gatccagaag gagactagta 350

<210> 20

<211> 1134

<212> DNA

<213> Corynebacterium glutamicum

<400> 20

atgcccaccc tcgcgccttc aggtcaactt gaaatccaag cgatcggtga tgtctccacc 60

gaagccggag caatcattac aaacgctgaa atcgcctatc accgctgggg tgaataccgc 120

gtagataaag aaggacgcag caatgtcgtt ctcatcgaac acgccctcac tggagattcc 180

aacgcagccg attggtgggc tgacttgctc ggtcccggca aagccatcaa cactgatatt 240

tactgcgtga tctgtaccaa cgtcatcggt ggttgcaacg gttccaccgg acctggctcc 300

atgcatccag atggaaattt ctggggtaat cgcttccccg ccacgtccat tcgtgatcag 360

gtaaacgccg aaaaacaatt cctcgacgca ctcggcatca ccacggtcgc cgcagtactt 420

ggtggttcca tgggtggtgc ccgcacccta gagtgggccg caatgtaccc agaaactgtt 480

ggcgcagctg ctgttcttgc agtttctgca cgcgccagcg cctggcaaat cggcattcaa 540

tccgcccaaa ttaaggcgat tgaaaacgac caccactggc acgaaggcaa ctactacgaa 600

tccggctgca acccagccac cggactcggc gccgcccgac gcatcgccca cctcacctac 660

cgtggcgaac tagaaatcga cgaacgcttc ggcaccaaag cccaaaagaa cgaaaaccca 720

ctcggtccct accgcaagcc cgaccagcgc ttcgccgtgg aatcctactt ggactaccaa 780

gcagacaagc tagtacagcg tttcgacgcc ggctcctacg tcttgctcac cgacgccctc 840

aaccgccacg acattggtcg cgaccgcgga ggcctcaaca aggcactcga atccatcaaa 900

gttccagtcc ttgtcgcagg cgtagatacc gatattttgt acccctacca ccagcaagaa 960

cacctctcca gaaacctggg aaatctactg gcaatggcaa aaatcgtatc ccctgtcggc 1020

cacgatgctt tcctcaccga aagccgccaa atggatcgca tcgtgaggaa cttcttcagc 1080

ctcatctccc cagacgaaga caacccttcg acctacatcg agttctacat ctaa 1134

<210> 21

<211> 377

<212> PRT

<213> Corynebacterium glutamicum

<400> 21

Met Pro Thr Leu Ala Pro Ser Gly Gln Leu Glu Ile Gln Ala Ile Gly

1 5 10 15

Asp Val Ser Thr Glu Ala Gly Ala Ile Ile Thr Asn Ala Glu Ile Ala

20 25 30

Tyr His Arg Trp Gly Glu Tyr Arg Val Asp Lys Glu Gly Arg Ser Asn

35 40 45

Val Val Leu Ile Glu His Ala Leu Thr Gly Asp Ser Asn Ala Ala Asp

50 55 60

Trp Trp Ala Asp Leu Leu Gly Pro Gly Lys Ala Ile Asn Thr Asp Ile

65 70 75 80

Tyr Cys Val Ile Cys Thr Asn Val Ile Gly Gly Cys Asn Gly Ser Thr

85 90 95

Gly Pro Gly Ser Met His Pro Asp Gly Asn Phe Trp Gly Asn Arg Phe

100 105 110

Pro Ala Thr Ser Ile Arg Asp Gln Val Asn Ala Glu Lys Gln Phe Leu

115 120 125

Asp Ala Leu Gly Ile Thr Thr Val Ala Ala Val Leu Gly Gly Ser Met

130 135 140

Gly Gly Ala Arg Thr Leu Glu Trp Ala Ala Met Tyr Pro Glu Thr Val

145 150 155 160

Gly Ala Ala Ala Val Leu Ala Val Ser Ala Arg Ala Ser Ala Trp Gln

165 170 175

Ile Gly Ile Gln Ser Ala Gln Ile Lys Ala Ile Glu Asn Asp His His

180 185 190

Trp His Glu Gly Asn Tyr Tyr Glu Ser Gly Cys Asn Pro Ala Thr Gly

195 200 205

Leu Gly Ala Ala Arg Arg Ile Ala His Leu Thr Tyr Arg Gly Glu Leu

210 215 220

Glu Ile Asp Glu Arg Phe Gly Thr Lys Ala Gln Lys Asn Glu Asn Pro

225 230 235 240

Leu Gly Pro Tyr Arg Lys Pro Asp Gln Arg Phe Ala Val Glu Ser Tyr

245 250 255

Leu Asp Tyr Gln Ala Asp Lys Leu Val Gln Arg Phe Asp Ala Gly Ser

260 265 270

Tyr Val Leu Leu Thr Asp Ala Leu Asn Arg His Asp Ile Gly Arg Asp

275 280 285

Arg Gly Gly Leu Asn Lys Ala Leu Glu Ser Ile Lys Val Pro Val Leu

290 295 300

Val Ala Gly Val Asp Thr Asp Ile Leu Tyr Pro Tyr His Gln Gln Glu

305 310 315 320

His Leu Ser Arg Asn Leu Gly Asn Leu Leu Ala Met Ala Lys Ile Val

325 330 335

Ser Pro Val Gly His Asp Ala Phe Leu Thr Glu Ser Arg Gln Met Asp

340 345 350

Arg Ile Val Arg Asn Phe Phe Ser Leu Ile Ser Pro Asp Glu Asp Asn

355 360 365

Pro Ser Thr Tyr Ile Glu Phe Tyr Ile

370 375

Claims

1. A promoter, characterized in that, the nucleotide sequence is shown in any of SEQ ID NO. 1-18.

2. An expression vector comprising the promoter of claim 1.

3. The expression vector according to claim 2, wherein the promoter shown in SEQ ID NO.1 is operably connected to a plasmid to obtain the expression vector; the plasmid includes but is not limited to pEC-XK99E , pXZ10145, pXMJ19, pJYW-4 or pJYW-5.

4. A microbial cell comprising the promoter of claim 1 or the expression vector of any one of claims 2 to 3.

5 . A recombinant Corynebacterium glutamicum, characterized in that the promoter according to claim 1 or the expression vector according to any one of claims 2 to 3 is introduced into the genome of Corynebacterium glutamicum .

6. The recombinant Corynebacterium glutamicum according to claim 5, characterized in that, taking Corynebacterium glutamicum ATCC 13032 as a host cell, containing the promoter shown in SEQ ID NO.1, SEQ ID NO.20 The vector pEC-XK99E for the indicated homoserine acetyltransferase gene.

7. Use of the promoter of claim 1 in enhancing gene or protein expression.

8. The application according to claim 7, wherein the genes include but are not limited to genes involved in the growth or metabolism of microorganisms; the proteins are endogenous proteins or exogenous proteins.

9. The application of the promoter of claim 1 in improving the production of O-acetyl-L-homoserine by Corynebacterium glutamicum.

10. application according to claim 9 is characterized in that, the promoter shown in SEQ ID NO.1, the gene encoding homoserine acetyltransferase shown in SEQ ID NO.20 are connected with carrier pEC-XK99E, Transform into a homoserine-producing Corynebacterium glutamicum to obtain a recombinant Corynebacterium glutamicum; and then cultivate the recombinant Corynebacterium glutamicum at 25-37° C. and 150-250 rpm for at least 36 hours.