CN111041029B - Strong promoter and application thereof in production of vitamin B12Application of strain - Google Patents
Strong promoter and application thereof in production of vitamin B12Application of strain Download PDFInfo
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- CN111041029B CN111041029B CN201911335790.8A CN201911335790A CN111041029B CN 111041029 B CN111041029 B CN 111041029B CN 201911335790 A CN201911335790 A CN 201911335790A CN 111041029 B CN111041029 B CN 111041029B
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/42—Cobalamins, i.e. vitamin B12, LLD factor
Abstract
The invention carries out bioinformatics analysis and functional verification by amplifying a strong promoter in the sword grass adhesion bacteria to obtain the vitamin B which can be widely used for producing the sinorhizobium meliloti, the pseudomonas denitrificans, the sword grass adhesion bacteria and the like12The nucleotide sequence of the strong promoter for gene expression, genetic gene operation and strain improvement in the strain is SEQ ID NO. 1. The invention also relates to a plasmid vector containing the strong promoter, a method for constructing a genetic engineering strain by using the promoter, a corresponding strain and application of a host cell in starting expression of a target gene.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a strong promoter, in particular to a strong promoter obtained by separating and cloning from xietnamese adhesion bacteria, a plasmid containing the strong promoter, a transformant containing a plasmid vector, and a method for producing vitamin B by using the strong promoter in the aspects of heterologous or homologous protein expression and vitamin B production12Application in bacterial strains.
Background
The promoter is a component of a gene and can be recognized by RNA polymerase, and transcription of the gene behind the promoter is initiated, and the strength of the promoter has direct influence on the expression efficiency of an exogenous gene in gene operation. Metabolic engineering often requires the expression of foreign genes or the regulation of endogenous gene expression, and promoter selection is critical to gene expression regulation. The promoter affects the transcription level of genes, influences the coordination among the genes in an artificial synthesis way or an origin way, and then influences the metabolic function of the strain.
Proteobacteria, the largest of bacteria, can be classified into five classes according to 16sRNA, including α -Proteobacteria, β -Proteobacteria, γ -Proteobacteria, δ -Proteobacteria, and ε -Proteobacteria. Among these, α -proteobacteria include many animal and plant pathogenic bacteria, such as: brucella abortus, Agrobacterium tumefaciens, etc., and many strains capable of producing secondary metabolites with certain functions, such as Zymomonas mobilis capable of producing bioethanol, and vitamin B can be produced12Pseudomonas denitrificans, Sinorhizobium meliloti, Ensifer sticktight, etc. To further study the production of vitamin B12Physiological characteristics of strains, and expansion of corresponding strains to produce vitamin B12The capability of (2) is usually required to over-express one or more genes in the thalli, and a high-efficiency promoter original is required as a basis. However, vitamin B is produced12The number of efficient promoters available in a strain is very limited.
The inventor discovers that the vitamin B can be applied to various vitamin B production through long-term research and exploration12A strong promoter in a strain.
Disclosure of Invention
The invention aims to: provides a strong promoter, a plasmid vector containing the strong promoter and vitamin B production thereof12Application in bacterial strains. The invention takes Ensifer adhaerens Casida A (Ensifer armillaria viscosa) as a template, obtains a strong promoter sequence by amplification, and evaluates the promoter by taking green fluorescent protein as a reporter gene. The strong promoter can also be applied to other vitamin B production outside the sword grass stickum12In the strain. The invention expands the production of vitamin B12Promoter elements of bacterial species for vitamin B production12The gene expression, genetic gene manipulation and strain improvement in the strain are of great significance.
In a first aspect, the present invention provides a strong promoter, the nucleotide sequence of which is selected from the following sequences (a), (b) or (c):
(a) the strong promoter is a nucleotide sequence shown in a sequence table SEQ ID NO. 1;
(b) a nucleotide sequence which has more than 75 percent of consistency with SEQ ID NO.1 of a sequence table and has the function of a promoter;
(c) a nucleotide sequence which can be hybridized with the nucleotide sequence of (a) or (b) under high-stringency conditions and has the function of a promoter.
Preferably, the (b) is a nucleotide sequence which has homology of more than 95% with the sequence table SEQ ID NO.1 and has the function of a promoter.
In a second aspect, the present invention provides a plasmid vector containing a strong promoter, which is an episomal or integrative vector. Preferably, the episomal vector is a broad-host shuttle plasmid vector containing replicon and mob genes recognizable by gram-negative bacteria, and further preferably pBBR1MCS 2; the integration vector is a homologous recombination vector carrying a homologous recombination arm with 500-4000 bp flanking target genes, and a pUC series plasmid is further preferred.
The plasmid vector also includes a nucleic acid sequence encoding a polypeptide of interest. The nucleic acid sequence encoding the polypeptide of interest includes a fluorescent protein; preferably, the fluorescent protein is green fluorescent protein.
The promoter nucleotide sequence of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the promoter isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as the promoter activity for expressing the target gene is maintained. The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence that is 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identical to the promoter nucleotide sequence 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 assess the identity between related sequences.
In a third aspect, the present invention provides a host cell comprising the plasmid vector of the second aspect.
Preferably, the host cell is α -proteobacteria; further preferably, it is one of Sinorhizobium meliloti (Sinorhizobium meliloti), Pseudomonas denitrificans (Pseudomonas denitificas) or Ensifer adherens (Sinorhizobium adherens); more preferably Sinorhizobium meliloti (Sinorhizobium meliloti).
In a fourth aspect, the present invention provides the use of a strong promoter according to the first aspect or a plasmid vector according to the second aspect or a host cell according to the third aspect for promoting expression of a gene of interest.
Drawings
FIG. 1: map of plasmid vector pBBR-P29-gfp.
FIG. 2: expression effect of the promoter in different strains.
Detailed Description
The following examples and figures of the present invention are merely illustrative of specific embodiments for carrying out the invention and these should not be construed as limiting the invention and any changes which may be made without departing from the principles and spirit of the invention are within the scope of the invention.
The experimental techniques and experimental methods used in this example are conventional techniques unless otherwise specified. The materials, reagents and the like used in the present examples are all available from normal commercial sources unless otherwise specified.
Example 1: construction of promoter-containing plasmid vector
1. Preparation of reporter Gene-containing vector
Using the primers gfp-EcoRI-F, gfp-KpnI-R of Table 1, the ECE164 plasmid (Yang Song)
Jonas M.Nikoloff
Gang Fu,Jingqi Chen,Qinggang Li,Nengzhong Xie,Ping Zheng,Jibin Sun,Dawei Zhang*.Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology and industrial applications. PLoS One2016Jul 5; 11(7) e0158447.doi: 10.1371/journal.pane.0158447.) as a template, and EcoRI and KpnI enzyme cutting sites are introduced by PCR amplification to obtain a gfp fragment of the green fluorescent protein gene. And (3) carrying out electrophoresis verification, carrying out enzyme treatment on the DpnI, and recovering electrophoresis gel to obtain a purified gfp fragment. The purified gfp fragment and the plasmid pBBR1MCS2 were double digested with EcoRI and KpnI, respectively, and the two double digested products were ligated by T4 ligase overnight at 4 ℃. The ligation product was transformed into E.coli DH5 alpha, spread on LB solid plate containing 50mg/L kanamycin, cultured for 16h and then subjected to colony PCR detection,and (4) carrying out Jinzhi sequencing, and after the sequencing is correct, naming the obtained positive bacterium as E. The plasmid pBBR-gfp was extracted with a plasmid kit for further use.
2. Preparation of plasmid containing strong promoter
The primer pairs P29-XbaI-F and P29-EcoRI-R in the table 1 are respectively utilized, the genome of Ensifer adhaerens Casida A (Ensifer encephalum) is taken as a template, EcoRI and XbaI enzyme cutting sites are introduced through PCR amplification to obtain a promoter P29 fragment, and the purified P29 fragment is obtained after electrophoresis verification, DpnI enzyme method treatment and electrophoretic gel recovery.
The purified P29 fragment and the pBBR-gfp plasmid are subjected to double enzyme digestion by EcoRI and XbaI respectively, and the double enzyme digestion product of the P29 fragment and the double enzyme digestion product of the PBBR-gfp plasmid are connected by T4 ligase at 4 ℃ overnight. The ligation products were transformed into E.coli DH5 α, spread on LB solid plates containing 50mg/L kanamycin, cultured for 16h, and then colony PCR was performed, and after sequencing was performed by Kimura sequencing, the resulting positive bacteria were named E.coli/pBBR-P29-gfp. The plasmid pBBR-P29-gfp was extracted with the plasmid kit and the plasmid map is shown in FIG. 1. The nucleotide sequence of the promoter P29 is shown in SEQ ID NO. 1.
Example 2: promoter P29 for producing vitamin B12Activity determination in bacterial species
1. Transformation-triparental transformation method
Taking Sinorhizobium meliloti as an example, the plasmid pBBR-P29-gfp in example 1 is transferred into Sinorhizobium meliloti according to a three-parent method to obtain the Sinorhizobium meliloti, namely SM/pBBR-P29-gfp. The method comprises the following specific steps:
(1) inoculating newly activated Sinorhizobium meliloti CGMCC NO.9638, Escherichia coli (containing corresponding plasmids) and auxiliary vector MT616, and performing shake culture in culture boxes at 30 deg.C and 37 deg.C respectively until OD value is about 1.0;
(2) separately transferring 500. mu.L of the bacterial liquid of Sinorhizobium meliloti CGMCC NO.9638, MT616 and the bacterial liquid of Escherichia coli to a 1.5mL sterile EP tube under aseptic condition, and centrifuging at 4 ℃ and 12,000rpm for 1 min.
(3) The supernatant was discarded under sterile conditions, and the pellet was suspended with 1mL of 0.85% sterile physiological saline.
(4) Centrifugation was again carried out at 12,000rpm for 1min at 4 ℃ and the supernatant was removed under aseptic conditions.
(5) The recipient cells, E.coli and MT616 pellet were suspended with 500. mu.L of fresh LB liquid medium, respectively.
(6) Three kinds of the bacterial solutions, each 2. mu.L, were dropped on the same position of LB solid medium to which no resistance was added, and carefully mixed. The bacterial liquids of single components and the bacterial liquids mixed between every two components are respectively sampled and used as test control groups.
(7) After the bacterial liquid is naturally air-dried, the bacterial liquid is inversely cultured in an incubator at 37 ℃ for about 1 day until a single bacterial colony grows out.
(8) Different single colonies were picked and streaked onto plates containing the corresponding antibiotics, and the plates were inverted and incubated in an incubator at 30 ℃ until colonies grew out. Meanwhile, different single colonies in the control group are selected and streaked on the plate containing the corresponding antibiotics.
(9) Colonies were picked from the resistant plates and verified by colony PCR. The obtained positive bacterium is named as SM/pBBR-P29-gfp.
2. Preparation of different strains
The plasmid pBBR-P29-gfp of example 1 was transferred into Pseudomonas denitrificans (Pseudomonas denitificas, abbreviated as PD) and Ensifer Adherens (EA), also called Sinorhizobium adherens (Sinorhizobium adherens), by the triparental transformation method to obtain plasmid-containing Pseudomonas denitrificans: PD/pBBR-P29-gfp; carrying out sword fungus adhesion: EA/pBBR-P29-gfp.
3. Activity assay of promoter P29 in Sinorhizobium meliloti
LB/MC medium: LB Medium supplemented with MgCl2(2.5mM)、CaCl2(2.5mM)。
The strain SM/pBBR-P29-gfp in section 1 of example 2 was streaked on LB/MC solid plate containing 100mg/L kanamycin to activate, and a single colony was picked and inoculated into 5mL of LB/MC liquid medium containing 100mg/L kanamycin. After culturing at 28 ℃ at 200r/min for 16 hours, the above-mentioned bacterial suspension was transferred to 1.8mL of a 24-well plate containing 100mg/L of LB/MC liquid medium containing kanamycin in a volume of 10%, and the plate was placed in a plate shaker at 28 ℃ and 700rpm with shaking culture at 80% humidity. Measurement of OD by timed sampling600And fluorescence (cells were centrifuged at 4000 rpm for 10 minutes, supernatant was discarded, and cells were resuspended in an equal amount of double distilled water), and the fluorescence expression intensity of the strain at an absorption wavelength of 488nm and an emission wavelength of 523nm was measured. The starting strain without plasmid is the control. Each experiment was done in 3 replicates. The results are shown in FIG. 2.
4. Activity measurement of promoter P29 in Pseudomonas denitrificans
The strain PD/pBBR-P29-gfp in section 2 of example 2 was streaked on an LB/MC solid plate containing 100mg/L kanamycin and activated, followed by the same procedure as in section 3 of example 2. The starting strain without plasmid is the control. Each experiment was done in 3 replicates. The results are shown in FIG. 2.
5. Activity assay of promoter P29 in Ensifer adhesins
The strain EA/pBBR-P29-gfp of example 2 part 2 was streaked on LB/MC solid plate containing 100mg/L kanamycin and activated, followed by the same procedure as example 2 part 3. The starting strain without plasmid is the control. Each experiment was done in 3 replicates. The results are shown in FIG. 2.
6. Promoter P29 produces vitamin B in 3 different kinds12Analysis of expression intensity in Strain
As shown in FIG. 2, the promoter P29 produces vitamin B in 3 kinds of vitamins12The bacterial strain has stronger starting effect on green fluorescent protein expression genes.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> a strong promoter and application thereof in producing vitamin B12 bacterial strain
<130> 2019.11.10
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1000
<212> DNA
<213> Ensifer adhaerens
<400> 1
cgcctcgcca cccagctctt cgtaaagctt ggcaatcgca ccggcgcagg ggatcagccg 60
gtgaccacgc tcgacgacga ggtccgggtt ggcgcagatg aacgggatct tgcgcttggc 120
aaggcccgtc agggtcgcgc ggtaatgctc gggcgtttcg gtctcgtcgt catagaagcc 180
ggcgcagacg accgtctcgg catcttccga ggaaacgatc tgcgtcccca gcccctcgag 240
caggggcagg tcgcgctcgg cgccgatgaa gaagatcttc ttgtcggcgg cagcgatcag 300
cgcccgggtc acgtcgcccg aagtgacgat ccgatcatag gcttcgtcgg gcacgccgag 360
actgcggatc tgcaccttca ccatcgggct cgggcgcggt gaattggtga tgagcacgac 420
ggtcagtccg gcggcgcgcg cctcggccaa tgcttcacag gcggacgcga aagcctgcac 480
gccgttgtga agcacgcccc agacgtcaca taaaacgaca tcgtagcgct tggcgatttc 540
gcgaaaactg ttgatccttg cggccatcct ggcttcctgc aaacacggtt tccggcttgc 600
atggcaagga agacaggaga ttacaagtct cgtctcccga aaaaggcgca ccctcccaag 660
gattccggcg gtgtcgagaa ccaggaaaaa tgcactgcag atgcggcccg ccgcgtagaa 720
gcacattgtc gtggttatca accgtcaaaa tcccgcccat gccactcatc gacgcgatcg 780
cccacatttc gccaagaatg gggctaggct gcctgtgccc gcgcctgctg cgcccgtcca 840
gggcaactgc cgtcagcaaa aaatctaggc gccgatcctt gactcacccc atacccaagc 900
ttatctcggc atcgctagca ctcgataaat gagagtgcta acacccatcc atcgggtcga 960
tcagcgatcc gtaatgtcat ttgatcgagg gattagacta 1000
Claims (15)
1. The strong promoter is characterized in that the nucleotide sequence is shown as a sequence table SEQ ID NO. 1.
2. A plasmid vector comprising the strong promoter of claim 1.
3. The plasmid vector according to claim 2, wherein the plasmid vector is an episomal vector or an integrative vector.
4. The plasmid vector according to claim 3, wherein the episomal vector comprises a replicon and a replicon recognized by gram-negative bacteriamobBroad host shuttle plasmid vectors for genes.
5. The plasmid vector according to claim 3 or 4, wherein the episomal vector is pBBR1MCS 2.
6. The plasmid vector as claimed in claim 3, wherein the integrative vector is a homologous recombination vector carrying a homologous recombination arm flanking the target gene with 500-4000 bases.
7. A plasmid vector according to claim 3 or 6, characterized in that the integrative vector is a pUC series plasmid.
8. The plasmid vector of claim 2, further comprising a nucleic acid sequence encoding a polypeptide of interest.
9. The plasmid vector of claim 8 wherein the nucleic acid sequence encoding the polypeptide of interest comprises a fluorescent protein.
10. The plasmid vector of claim 9 wherein the fluorescent protein is green fluorescent protein.
11. A host cell comprising the plasmid vector of any one of claims 2 to 10.
12. The host cell of claim 11, wherein the host cell is a vitamin B-producing host cell12And (3) strain.
13. The host cell of claim 12, wherein the host cell is Sinorhizobium melilotiSinorhizobium melilotiPseudomonas denitrificansPseudomonas denitrificansOr Zygomyces sticklandiiSinorhizobium adhaerens or Ensifer adhaerensOne kind of (1).
14. The host cell of claim 12 or 13, wherein the host cell is Sinorhizobium melilotiSinorhizobium meliloti。
15. Use of a strong promoter according to claim 1 or a plasmid vector according to any one of claims 2 to 10 or a host cell according to any one of claims 11 to 14 for promoting expression of a gene of interest.
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