CN110592081B - Xylose-induced promoter, plasmid vector and application thereof - Google Patents

Abstract

The invention obtains the xylose-induced promoter which can be widely used for gene expression, genetic gene operation and strain improvement in alpha-proteobacteria such as sinorhizobium meliloti, zymomonas mobilis, corynebacterium cresum, pseudomonas denitrificans, agrobacterium tumefaciens, brucella abortus, pseudomonas fluorescens, leguminous rhizobium and sinorhizobium adherens by amplifying the xylose-induced promoter in the sinorhizobium meliloti and carrying out bioinformatics analysis and function verification, and the nucleotide sequence of the xylose-induced promoter is SEQ ID NO.1 or SEQ ID NO. 2. The invention also relates to a plasmid vector containing the xylose-inducible promoter, a method for constructing a genetic engineering strain by using the promoter, a corresponding strain and application of host cells in inducing and starting target gene expression under the xylose-inducible condition.

Description

Xylose-induced promoter, plasmid vector and application thereof

The technical field is as follows: the invention belongs to the technical field of biology, and relates to a xylose-inducible promoter, a plasmid vector and application thereof

Background art:

promoters are classified into Constitutive promoters (Constitutive promoters) and Specific promoters (Specific promoters). Constitutive promoters are capable of transcription in all cells at all times; specific promoters can be further divided into tissue-specific promoters and inducible promoters. The inducible promoter can specifically induce the expression of a target gene and generate a large amount of specific products in an organism, so that a regulation response is made, and the inducible promoter is suitable for the external environment or improves the yield of metabolites.

Sinorhizobium meliloti is a non-model strain. Currently, the R.meliloti inducible promoter is less studied, and P has been studied by Mostafavi et al_araA、P_tauA、P_rhaRAnd P _melA4 promoters and comparison of their function, P_araAPromoter region positionUpstream of the araABCDEF operon, it was shown to be essential for arabinose catabolism and that arabinose was inducible more than 100-fold in a specific medium, however, its background expression was relatively high and not suitable for stringent regulation. P_tauAAnd P_melAHave more strict regulation, but their expression intensity is lower, and are not suitable for over-expressing target genes. P_rhaRThe expression of (1) was the least effective (Mostafavi, Lewis et al 2014). Weakly inducible promoters have certain limitations in their application to increase metabolite production, while strongly inducible promoters with tight regulation have advantages in this regard.

The induced promoter regulates the start and the stop of the transcription of downstream genes under the stimulation of specific physical or chemical signals, and is particularly important for the genetic manipulation of some cytotoxic genes. Inducible promoters are essential for genetic manipulation systems. Metabolic engineering also requires the selection of more inducible promoter elements to achieve precise regulation of the expression levels of genes in a certain metabolic pathway. Strictly regulated promoters express cloned genes, allowing for convenient observation of the desired phenotype, while leaky promoters may affect data analysis. In view of the above, we need to search for highly efficient, tightly regulated inducible promoters in Sinorhizobium meliloti.

Therefore, through long-term research, the inventor finds a strong induction promoter which can be applied to strict regulation in Sinorhizobium meliloti and other various alpha-proteobacteria strains.

Mostafavi,M.,J.C.Lewis,T.Saini,J.A.Bustamante,I.T.Gao,T.T.Tran,S.N.King,Z.Huang and J.C.Chen(2014)."Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression."BMC Microbiol 14:295.

The invention content is as follows:

the invention aims to: provides two strict and high-strength expressed xylose-inducible promoters, a plasmid vector containing the xylose-inducible promoters and application thereof. The invention takes Sinorhizobium meliloti CGMCC NO.9638 as a template, obtains two xylose-induced promoter sequences by amplification, and takes green fluorescent protein as a reportGenes, xylose inducible promoters were evaluated. The invention is compared with arabinose-induced promoters reported in the literature. Subsequently, the hemA gene is respectively overexpressed at the plasmid level and the genome level by a xylose-induced promoter with higher expression intensity, and vitamin B₁₂The yield is greatly improved. Meanwhile, the two xylose-induced promoters can also be applied to other various alpha-proteobacteria strains besides Sinorhizobium meliloti. The invention expands the promoter element of alpha-proteobacteria and has important significance for gene expression, genetic gene operation and strain improvement in alpha-proteobacteria.

In a first aspect, the present invention provides a xylose-inducible promoter, the nucleotide sequence of which is optionally selected from the sequences described in (a), (b), (c), (d), (e) or (f) below:

(a) the xylose-induced 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 is hybridized with the nucleotide sequence of (a) or (b) under high-stringency conditions and has the function of a promoter;

(d) the xylose-induced promoter is a nucleotide sequence shown in a sequence table SEQ ID NO. 2;

(e) a nucleotide sequence which has more than 75 percent of consistency with the sequence table SEQ ID NO.2 and has the function of a promoter;

(f) a nucleotide sequence which can be hybridized with the nucleotide sequence in (d) or (e) under high-stringency conditions and has the function of a promoter.

Preferably, (b) is a nucleotide sequence having a homology of 95% or more with SEQ ID NO.1 of the sequence Listing and having a promoter function, and (e) is a nucleotide sequence having a homology of 95% or more with SEQ ID NO.2 of the sequence Listing and having a promoter function.

In a second aspect, the present invention provides a plasmid vector containing a xylose-inducible promoter, wherein the plasmid vector is an episomal or an 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 xylose is induced, and the concentration of the xylose is 0.1-4%; preferably, the concentration of xylose is 0.5-2%.

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), Zymomonas mobilis (Zymomonas mobilis), Bacillus crescentus (Caulobacter crescentus), Pseudomonas denitrificans (Pseudomonas denitis), Agrobacterium tumefaciens (Agrobacterium tumefaciens), Brucella abortus (Brucella abortus), Pseudomonas fluorescens (Pseudomonas fluorescens), Rhizobium leguminosarum (Rhizobium leguminium) or Sinorhizobium adherens (Sinorhizobium meliloti); more preferably Sinorhizobium meliloti (Sinorhizobium meliloti).

In a fourth aspect, the present invention provides the use of a xylose inducible promoter according to the first aspect, or a plasmid vector according to the second aspect, or a host cell according to the third aspect, to induce and initiate expression of a gene of interest under xylose-inducing conditions.

Drawings

FIG. 1: map of plasmid vector pBBR-Promoter-gfp.

FIG. 2: the construction scheme of plasmid pBBR-Promoter-gfp.

FIG. 3: schematic truncation of the xylose promoter.

FIG. 4: containing promoter P_AAnd hemA Gene pBBR-P_AhemA plasmid map.

FIG. 5: containing promoter P_AAnd pUC18-P of hemA Gene_AhemA plasmid map.

FIG. 6: xylose inducible promoter P_AAnd P_BGraph of induction effect in E.coli.

FIG. 7: the induction effect of different inducible promoters in Sinorhizobium meliloti under different concentrations of an inducer is shown. P_araA(FIG. A) the inducer is arabinose and P_A(FIG. B) and P_B(FIG. C) the inducer is xylose.

FIG. 8: xylose inducible promoter P_AAnd P_BGraph of induction effect in different strains.

FIG. 9: the expression intensity and induction effect of the truncated xylose-induced promoter are shown.

FIG. 10: vitamin B₁₂Standard graph of (2).

FIG. 11: VB of different sinorhizobium meliloti strains fermented for 144h₁₂Yield and biomass results.

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

The primer gfp-EcoRI-F, gfp-XhoI-R in Table 1 was used, ECE164 plasmid was used as a template, and EcoRI and XhoI cleavage sites were 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 plasmid pBBR1MCS2 were double digested with EcoRI and XhoI, respectively, and the two double digested products were ligated by T4 ligase overnight at 4 ℃. The ligation product was transformed into E.coli DH5 α, spread on LB solid plate containing 50mg/L kanamycin, cultured for 16h, and colony PCR was performed, subjected to Kingou sequencing, and after the sequencing was correct, the obtained positive strain was named E.coli/pBBR-gfp. And extracting the plasmid pBBR-gfp by using a plasmid kit for later use.

2. Preparation of plasmid containing arabinose and xylose inducible promoter

Primer pairs P of Table 1 were used_araAXbaI-F and P_araA-EcoRI-R，P_AXbaI-F with P, respectively_AEcoRI-R and P_BEcoRI-R, using genome of Sinorhizobium meliloti CGMCC NO.9638 strain as template, PCR amplifying, introducing EcoRI and XbaI enzyme cutting site to obtain arabinose inducible promoter P_araAFragments and xylose inducible promoter P_A、P_BThe fragment is subjected to electrophoretic verification, DpnI enzymatic treatment and electrophoretic gel recovery to obtain purified P_araA、P_AAnd P_BAnd (3) fragment.

Subjecting the purified P to_araA、P_A、P_BThe promoter fragment and the pBBR-gfp plasmid were digested with EcoRI and XbaI, respectively, and P was digested with EcoRI and XbaI_araA、P_A、P_BDouble cleavage product fraction of promoter fragmentThe double-enzyme-digested products with the PBBR-gfp plasmid are respectively connected with T4 ligase overnight at 4 ℃. The ligation products are transformed into Escherichia coli DH5 alpha, spread on LB solid plate containing 50mg/L kanamycin, cultured for 16h, colony PCR detection is carried out, Jinzhi sequencing is carried out, and after the sequencing is correct, the obtained positive bacteria are named as E_araA-gfp、E.coli/pBBR-P_A-gfp、E.coli/pBBR-P_B-gfp. Extraction of plasmid pBBR-P with plasmid kit_araA-gfp、pBBR-P_A-gfp、pBBR-P_BGfp for standby. The plasmid map is shown in FIG. 1, and the plasmid construction process is shown in FIG. 2. Xylose inducible promoter P_AThe nucleotide sequence of (A) is shown as SEQ ID NO.1, and the xylose-induced promoter P_BThe nucleotide sequence of (A) is shown in SEQ ID NO.2, and the arabinose-induced promoter P_araAThe nucleotide sequence of (A) is shown in SEQ ID NO. 3.

3. Preparation of plasmid containing truncated xylose-inducible promoter

Using the primer P shown in Table 1_B-XbaI-F2、P_B-XbaI-F3、P_B-XbaI-F4、P_B-XbaI-F5、P_BXbaI-F6 and P_BEcoRI-R, as recombinant plasmid pBBR-P_BIntroducing XbaI and EcoRI restriction sites by PCR with gfp as a template to obtain promoter fragments F2, F3, F4, F5 and F6 with different lengths, carrying out electrophoretic verification, carrying out enzymatic treatment on DpnI, and recycling electrophoretic gel to obtain purified F2, F3, F4, F5 and F6 fragments.

Using the primer P shown in Table 1_A-XbaI-F7、P_AXbaI-F8 and P_AEcoRI-R, as recombinant plasmid pBBR-P_AIntroducing XbaI and EcoRI restriction sites by PCR with gfp as a template to obtain promoter F7 and F8 fragments with different lengths, carrying out electrophoretic verification, carrying out enzymatic treatment by DpnI, and recycling electrophoretic gel to obtain purified F7 and F8 fragments.

Using the primer P shown in Table 1_AXbaI-F and P_BEcoRI-R2, as recombinant plasmid pBBR-P_BThe promoter fragment R2 was obtained by introducing XbaI and EcoRI cleavage sites by PCR using gfp as a template. And (3) carrying out electrophoretic verification, carrying out enzymatic treatment on the DpnI, and recovering electrophoretic gel to obtain a purified R2 fragment.

The purified F2, F3, F4, F5, F6, F7, F8 and R2 fragments and the pBBR-gfp plasmid with the fluorescent marker gene obtained in example 1 were digested with XbaI and EcoRI, respectively, and the digested products of the promoter fragment and the pBBR-gfp fragment with the fluorescent marker gene were ligated with T4 ligase overnight at 4 ℃. The ligation products were transformed into E.coli DH5 α, spread on LB solid plate containing 50mg/L kanamycin, cultured for 16h, and subjected to colony PCR detection, subjected to Kinsero sequencing, and after the sequencing was correct, the obtained positive bacteria were named E.coli/pBBR-F2-gfp, E.coli/pBBR-F3-gfp, E.coli/pBBR-F4-gfp, E.coli/pBBR-F5-gfp, E.coli/pBBR-F6-gfp, E.coli/pBBR-F7-gfp, E.coli/pBBR-F8-gfp, and E.coli/pBBR-R2-gfp. Plasmids pBBR-F2-gfp, pBBR-F3-gfp, pBBR-F4-gfp, pBBR-F5-gfp, pBBR-F6-gfp, pBBR-F7-gfp, pBBR-F8-gfp and pBBR-R2-gfp were extracted with a plasmid kit for future use (the schematic diagram of the truncation thereof is shown in FIG. 3). The nucleotide sequences of F2, F3, F4, F5, F6, F7, F8 and R2 are shown as SEQ ID NO.4 to SEQ ID NO.11, respectively.

Example 2: activity assay of xylose-inducible promoter in E.coli

LB medium components: 1% tryptone, 0.5% yeast extract, 1% sodium chloride.

coli/pBBR-P of example 1_A-gfp、E.coli/pBBR-P_BGfp and E.coli/pBBR-gfp (control), after streaking activation on LB solid plate containing 50mg/L kanamycin, single colonies were picked up and inoculated into 5mL LB liquid medium containing 50mg/L kanamycin, and cultured at 37 ℃ for 16 hours at 200 r/min. Transferring the bacterial liquid to a 24-well plate containing 1.8mL LB liquid medium containing 50mg/L kanamycin at an inoculation amount of 1%, placing the plate in a plate shaker at 37 ℃, 700rpm and shaking with 80% humidityCulturing for OD₆₀₀When the concentration reaches 0.4-0.6%, adding a certain amount of xylose inducer to make the final concentration of the inducer be 0% and 1%, respectively, and sampling at regular time to determine OD₆₀₀And 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. Results are shown in FIG. 6, E.coli/pBBR-P after xylose addition_AGfp and E.coli/pBBR-P_BThe fluorescence expression intensity trend of gfp is the same, the fluorescence intensity is obviously improved, and E_AGfp ratio E_BHigh fluorescent expression intensity of-gfp, indicating promoter P_AAnd P_BIt is active in the large intestine.

Example 3: activity determination of arabinose and xylose inducible promoter in Sinorhizobium meliloti CGMCC NO.9638

1. Transformation-triparental transformation method

3 plasmids pBBR-P from example 1_araA-gfp、pBBR-P_A-gfp、pBBR-P_BThe gfp gene was transferred into Sinorhizobium meliloti CGMCC NO.9638 as follows. Obtaining the Rhizobium meliloti SM/pBBR-P_araA-gfp、SM/pBBR-P_A-gfp、SM/pBBR-P_B-gfp。

(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. Obtaining positive bacteria named as SM/pBBR-P_araA-gfp、SM/pBBR-P_A-gfp、SM/pBBR-P_B-gfp。

2. Activity determination of arabinose and xylose inducible promoter in Sinorhizobium meliloti CGMCC NO.9638

LB/MC medium: LB Medium supplemented with MgCl₂(2.5mM)、CaCl₂(2.5mM)。

Three strains SM/pBBR-P from the first part of example 3_araA-gfp、SM/pBBR-P_AGfp and SM/pBBR-P_BThe gfp was streaked on LB/MC solid plate containing 100mg/L kanamycin and a single colony was picked and inoculated into 5ml LB/MC liquid medium containing 100mg/L kanamycin. After culturing for 36h at 30 ℃ and 200r/min, transferring the bacterial liquid to 1.8mL of 24-well plate containing LB/MC liquid culture medium containing 100mg/L kanamycin in an inoculation amount of 10%, placing the plate in a plate shaker for shake culture at 30 ℃, 700rpm and 80% humidity, and taking the initial strain of sinorhizobium meliloti CGMCC NO.9638 as a negative control. To be OD₆₀₀When the concentration reaches 0.4-0.6, a certain amount of arabinose or xylose inducer is correspondingly added for induction, the concentration of the inducer is respectively 0%, 0.5%, 1%, 2% and 4%, and the OD is measured by sampling at regular time₆₀₀And fluorescence (cells were centrifuged at 4000 rpm for 10 min, supernatant discarded, cells resuspended in equal amounts of double distilled water), and assayedThe fluorescence expression intensity of the strain is determined under the absorption wavelength of 488nm and the emission wavelength of 523 nm. The results are shown in FIG. 7, where SM/pBBR-P increased with xylose concentration from 0 to 1%_AGfp and SM/pBBR-P_BFluorescence of-gfp increased. The fluorescence reached a maximum when the final concentration of xylose was 1%. As the concentration of xylose increases, the fluorescence intensity of the fluorescent protein decreases. When the concentration of arabinose is 0.5%, SM/pBBR-P_araAThe fluorescence intensity of gfp is maximal. SM/pBBR-P_araAThe fluorescence intensity of-gfp is significantly lower than that of SM/pBBR-P_AGfp and SM/pBBR-P_BFluorescence intensity at gfp.

Example 4: activity assay of xylose-inducible promoter in other bacteria

1. Preparation of eight strains

The 2 plasmids pBBR-P from example 1 were transformed by the triparental transformation method of the first part of example 3_AGfp and pBBR-P_BGfp is transferred into Zymomonas mobilis (Zymomonas mobilis), corynebacterium crescentis (Caulobacter creescens), Pseudomonas denitrificans (Pseudomonas denitrificans), Agrobacterium tumefaciens (Agrobacterium tumefaciens), Brucella abortus (Brucella abortus), Pseudomonas fluorescens (Pseudomonas claudicans), Rhizobium leguminosum (Rhizobium leguminatum) and fusarium adherens (escherichia adherens), which are called Sinorhizobium adherens (Sinorhizobium mobilis), respectively, to yield plasmid-containing Zymomonas mobilis: ZM/pBBR-P_AGfp and ZM/pBBR-P_B-gfp; bacillus crescentus: CC/pBBR-P_AGfp and CC/pBBR-P_B-gfp; and (3) pseudomonas denitrificans: PS/pBBR-P_AGfp and PS/pBBR-P_B-gfp; agrobacterium tumefaciens: AT/pBBR-P_AGfp and AT/pBBR-P_B-gfp; brucella abortus: BA/pBBR-P_AGfp and BA/pBBR-P_B-gfp; pseudomonas fluorescens: PF/pBBR-P_AGfp and PF/pBBR-P_B-gfp; leguminous rhizobia: RL/pBBR-P_AGfp and RL/pBBR-P_B-gfp; and (3) root adhesion of sinorhizobium: SA/pBBR-P_AGfp and SA/pBBR-P_B-gfp。

2. Activity measurement of xylose-induced promoter in Zymomonas mobilis

The strain ZM/pBBR-P of the first part of example 4_AGfp and ZM/pBBR-P_BStreaking and activating gfp on LB/MC solid plate containing 100mg/L kanamycin, picking single colony and inoculating to 5mL 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. To be OD₆₀₀When the concentration reaches 0.4-0.6%, adding a certain amount of xylose for induction to make the final concentration of the inducer respectively be 0% and 1%, and sampling at regular time to determine OD₆₀₀And 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 results are shown in FIG. 8.

3. Activity assay of xylose-inducible promoter in Bacillus crescentus

PYE medium: peptone 20g/L, yeast extract powder 10g/L, beef extract 2g/L (pH adjusted to 7.5).

The strain CC/pBBR-P in the first part of example 4 was used_AGfp and CC/pBBR-P_BGfp was streaked on a PYE solid plate containing 100mg/L kanamycin to activate, followed by the same operation as the second part of example 4.

4. Activity measurement of xylose-inducible promoter in Pseudomonas denitrificans

Culture medium of Pseudomonas denitrificans: KH (Perkin Elmer)₂PO₄ 2.5g/L、NaCl 2.5g/L、NH₄Cl 0.5g/L、MgSO₄0.13g/L, glucose 1g/L, CaCl₂5g/L (pH adjusted to 7.0-7.2).

The strain PS/pBBR-P from the first part of example 4_AGfp and PS/pBBR-P_BGfp was streaked on a solid culture plate of Pseudomonas denitrificans containing 100mg/L kanamycin, followed by the same operation as in the second part of example 4.

5. Activity assay of xylose-inducible promoter in Agrobacterium tumefaciens

YEB Medium: beef extract 5g/L, yeast extract 1g/L, egg5g/L of white peptone and 5g/L, MgSO of sucrose₄·H₂O0.5 g/L (pH adjusted to 7.0).

The strain AT/pBBR-P in the first part of example 4 was used_AGfp and AT/pBBR-P_BGfp was streaked on YEB solid plate containing 100mg/L kanamycin and activated, followed by the same operation as the second part of example 4.

6. Activity assay of xylose-inducible promoter in Brucella abortus

Brucella abortus culture medium: 10g/L of peptone, 5g/L of beef extract powder, 10g/L of glucose and 5g/L of sodium chloride (pH is adjusted to 7.5).

The strain BA/pBBR-P in the first part of example 4 was used_AGfp and BA/pBBR-P_BGfp was streaked on solid plate containing 100mg/L kanamycin in Brucella abortus medium and the subsequent operation was the same as in the second part of example 4.

7. Activity assay of xylose-inducible promoter in Pseudomonas fluorescens

The strain PF/pBBR-P in the first part of example 4 was used_AGfp and PF/pBBR-P_BGfp was streaked on YEB solid plate containing 100mg/L kanamycin and activated, followed by the same operation as the second part of example 4.

8. Activity assay of xylose-inducible promoter in leguminous Rhizobium

YEM medium: mannitol 5g/L, Yeast extract 0.5g/L, MgSO₄·7H₂O 0.2g/L、NaCl 0.1g/L、K₂HPO₄0.5g/L and 5g/L of sodium gluconate.

The strain RL/pBBR-P from the first part of example 4_AGfp and RL/pBBR-P_BGfp was streaked on YEM solid plate containing 100mg/L kanamycin and activated, followed by the same operation as the second part of example 4.

9. Activity assay of xylose-inducible promoter in Rhizobium adherens

The strain SA/pBBR-P in the first part of example 4 was used_AGfp and SA/pBBR-P_BThe gfp was streaked on an LB/MC solid plate containing 100mg/L kanamycin and activated, followed by the same operation as the second part of example 4.

10. Analysis of expression intensities of xylose-inducible promoter in 8 different strains

As can be seen from FIG. 8, promoter P_AAnd P_BHas stronger starting effect on green fluorescent protein expression genes in eight strains, has the same trend as the green fluorescent protein expression genes in sinorhizobium meliloti CGMCC NO.9638, and is a promoter P_AHas an effect stronger than that of P_BThe effect of (1). The strong and weak sequence of the starting effect of the two promoters in eight strains is to adhere to sinorhizobium japonicum, zymomonas mobilis, pseudomonas denitrificans, pseudomonas fluorescens, pseudomonas crescent moon, pseudomonas fluorescens, agrobacterium tumefaciens, brucella abortus and leguminous rhizobium.

Example 5: exploration of the core region of xylose-inducible promoters

The 9 plasmids pBBR-F2-gfp, pBBR-F3-gfp, pBBR-F4-gfp, pBBR-F5-gfp, pBBR-F6-gfp, pBBR-F7-gfp, pBBR-F8-gfp and pBBR-R2-gfp of example 1 were transformed into Sinorhizobium meliloti CGMCC NO.9638, respectively, according to the triparental transformation method of the first part of example 3. Respectively obtaining the Sinorhizobium meliloti engineering bacteria of SM/pBBR-F2-gfp, SM/pBBR-F3-gfp, SM/pBBR-F4-gfp, SM/pBBR-F5-gfp, SM/pBBR-F6-gfp, SM/pBBR-F7-gfp, SM/pBBR-F8-gfp and SM/pBBR-R2-gfp. These strains were streaked on LB/MC plates containing 100mg/L of solid kanamycin, and the resulting strains were picked upA single colony was inoculated into 5mL of LB/MC liquid medium containing 100mg/L kanamycin. After culturing at 30 ℃ and 200r/min for 36 hours, the above-mentioned bacterial suspension was transferred to a 24-well plate containing 1.8mL of LB/MC liquid medium containing 100mg/L kanamycin in an amount of 10%, and shake-cultured in a plate shaker at 30 ℃ and 700rpm with 80% humidity, to thereby obtain SM/pBBR-P in example 3_AGfp and SM/pBBR-P_BGfp as positive control and the initial strain sinorhizobium meliloti cgmccno.9638 as negative control. To be OD₆₀₀When the concentration reaches 0.4-0.6%, adding a certain amount of xylose for induction to make the final concentration of the inducer respectively be 0% and 1%, and sampling at regular time to determine OD₆₀₀And 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 results are shown in FIG. 9, the eight strains of bacteria containing xylose promoter truncation sequence plasmids have no obvious change under the conditions of adding xylose and not adding xylose, and have the same trend with negative control sinorhizobium meliloti CGMCCNO.9638. Indicating that the core region of the promoter has been disrupted.

Example 6: application of xylose-induced promoter

1. Construction of xylose-inducible promoter overexpression hemA gene strain

Delta-aminolevulinate (ALA) is a synthetic vitamin B₁₂And a common precursor of tetrapyrrole compounds such as heme (heme) and siroheme (siroheme). ALA is synthesized by the C4 or C5 pathway. In the C4 pathway, ALA is produced by the polymerization of glycine and succinyl-CoA under the catalysis of ALA synthetase (HemA). The C5 pathway uses glutamate as a substrate, and glutamate binds to tRNA molecules under the catalysis of glutaminyl-tRNA synthetase (GltX), so that glutaminyl-tRNA is produced. glutamyl-tRNA is reduced into glutaminate 1-semialdehyde under the action of glutamyl-tRNA reductase (HemA), and then ALA is generated under the catalysis of glutaminate aminotransferase (HemL). The C4 pathway exists in Sinorhizobium meliloti, ALA synthetase (HemA) is a key enzyme for ALA synthesis, so that the promoter P is induced by xylose_AThe hemA gene is over-expressed so as to increase the synthesis of ALA and further improve the vitamin B₁₂Of (2)Amount of the compound (A).

(1)SM/pBBR-P_AConstruction of the hemA Strain

Using the primer set pBBR-F, P shown in Table 1_A-R and primer pair hemA-F, hemA-R, respectively, as recombinant plasmid pBBR-P_ATaking gfp and Sinorhizobium meliloti CGMCC NO.9638 genomes as templates, and obtaining linearized pBBR-P by PCR_AAnd hemA fragments. After electrophoretic verification, DpnI enzyme method treatment and electrophoretic gel recovery, the purified and linearized pBBR-P is obtained_AAnd hemA fragments. The two segments were assembled together by Gibson. The ligation products are transformed into Escherichia coli DH5 alpha, coated on a kanamycin 50mg/L plate, cultured for 16h, colony PCR detection is carried out, Jinzhi sequencing is carried out, and after the sequencing is correct, the obtained positive bacteria are named as E_A-hemA. Extraction of plasmid pBBR-P with plasmid kit_AhemA ready for use (plasmid map shown in FIG. 4). Then, the strain is transformed into the Sinorhizobium meliloti CGMCC NO.9638 according to the three-parent transformation method in the example 3. Colonies were picked from the resistant plates and verified by colony PCR. Obtaining positive sinorhizobium meliloti named as SM/pBBR-P_AhemA (SM 1). The nucleotide sequence of the hemA gene is shown in SEQ ID NO. 12.

(2)SM/phbC::P_AConstruction of the hemA Strain

The pUC18 fragment sequence was obtained by PCR using pUC18 plasmid as a template with the primers pUC18-F/R in Table 1. After electrophoresis verification, DpnI enzyme treatment and electrophoretic gel recovery, a purified and linearized pUC18 fragment was obtained. The primers phbC-F1/R1 and P in the table 1 are utilized_A-F1/R1 and hemA-F1/R1, genome of Sinorhizobium meliloti CGMCC NO.9638 strain is used as a template, and phbC and P are obtained by PCR amplification_AAnd hemA structural gene sequences. Obtaining purified phbC and P by glue recovery_AAnd hemA sequences. Connecting the four fragments by a Gibson kit, transforming the connected product into escherichia coli DH5 alpha, coating the escherichia coli DH5 alpha on a 100mg/L ampicillin plate, culturing for 16h, then carrying out colony PCR detection, carrying out Jinwei intelligent sequencing, and obtaining a positive bacterium named as E_A-hemA. Plasmid pUC18-P was extracted with a plasmid kit_A-hemA Standby(the plasmid map is shown in FIG. 5). Then, the strain is transformed into the Sinorhizobium meliloti CGMCC NO.9638 according to the three-parent transformation method in the example 3. Plasmid pUC18-P_AhemA as the source of donor DNA fragment in homologous recombination, phbC gene sequence as the homology arm in homologous recombination, xylose-inducible promoter P by single crossover homologous recombination_AAnd structural gene hemA is integrated into the chromosome genome of the Rhizobium meliloti, and grown clones are picked from a resistant plate and subjected to colony PCR verification. The obtained strain which has undergone single-exchange homologous recombination is preserved and named as SM/phbC:: P_A-hemA(SM2)。

(3 construction of a Positive control Strain

The plasmid pBBR1MCS2 of example 1 was transformed into S.meliloti CGMCC NO.9638 according to the three-parent transformation method of example 3 to obtain the positive control strain S.meliloti SM/pBBR.

2. Culture condition of sinorhizobium meliloti

The strains SM1 and SM2 were streaked on LB/MC solid media containing 100mg/L kanamycin and 200mg/L ampicillin respectively with an inoculating needle under aseptic conditions, and were allowed to stand at a constant temperature of 30 ℃ for 48 hours for culture to obtain single colonies. Single colonies were picked up with an inoculating needle in a test tube containing 5mL of LB/MC liquid medium containing 100mg/L kanamycin and 200mg/L ampicillin, respectively, and cultured at 30 ℃ and 200rpm for 36 hours. The seed medium was inoculated at a ratio of 10% to 30mL of a fermentation medium containing 100mg/L kanamycin and 200mg/L ampicillin, respectively. After shaking (220r/min) culture at 30 ℃ for 144h, the thalli are collected and the yield is detected. SM/pBBR is used as positive control, corresponding culture conditions are the same as SM1, Sinorhizobium meliloti CGMCC NO.9638 is used as negative control, no antibiotic is added during culture, and other conditions are the same as other strains.

3. Detection method

(1) Sample pretreatment

Taking 10mL of fermentation liquor, adding 8% sodium nitrite solution and glacial acetic acid, shaking up by 2.5mL each, and placing in a water bath at 95-100 ℃ for 30-40 min; cooling to room temperature, centrifuging at 10000 rpm for 1min, and passing the supernatant through a 0.22 μm membrane

The filter was filtered into the sample vial and 20. mu.l of 2% NaCN (w/v) was added to 1ml of the supernatant.

(2) Preparation of standards

Configuring gradient vitamin B₁₂Standard substance (20mg/L, 50mg/L, 100mg/L, 150 mg/L).

(3) HPLC detection conditions

C18-250A column (Agilent, 4.6mmid 9X 250mm, 5 μm). The mobile phase comprises 70% organic phase (acetonitrile) and 30% inorganic phase (sodium acetate aqueous solution), the absorption wavelength is 361nm, the column temperature is 35 deg.C, the flow rate is 0.8mL/min, and the sample injection amount is 20 μ L.

(4) Vitamin B₁₂Drawing of standard curve

Performing HPLC detection on the standard substances with different concentrations according to the above conditions, and drawing peak area A-VB₁₂Concentration standard curve. Using the measured peak area A as the ordinate, vitamin B₁₂The mass concentration C (mg/L) is recorded as the abscissa and vitamin B is plotted₁₂A standard curve. See fig. 10, resulting in the regression equation y 19.846x-80.857, R²The absorbance is well linear with mass concentration at 0.999. After the liquid phase is finished, according to vitamin B₁₂The standard curve calculates the sample yield.

4. VB production by different Sinorhizobium meliloti strains₁₂Comparison of

As can be seen in FIG. 11, vitamin B in the negative control₁₂The yield of (a) was 110mg/L, vitamin B was positive control to SM/pBBR₁₂The yield of (5) was 91 mg/L. Laboratory Strain SM1, vitamin B₁₂The yield of the strain is the highest and is 122mg/L, which is 10.9 percent higher than that of a negative control strain and 34.07 percent higher than that of a positive control strain. Experimental Strain SM2 Strain, vitamin B₁₂The yield of the strain is 115mg/L, which is improved by 4.5 percent compared with a negative control strain, is improved by 26.37 percent compared with a positive control strain, and is reduced by 5.74 percent compared with an SM1 strain. OD of negative control₆₀₀OD of 60.81, positive control₆₀₀OD of SM1 at 52.34₆₀₀At an OD of 57.9, SM2₆₀₀Is 64.53. Indicating that the plasmid has a certain metabolic burden on the strain. However, the overexpression of hemA gene on plasmid can obviously improve the production of vitamin by the strainElement B₁₂The ability of the cell to perform. After integration of the hemA gene into the genome, the yield was slightly lower than SM1, but significantly higher than the negative control, due to the higher copy number when no plasmid was expressed. The availability of the xylose inducible promoter of the present invention was further demonstrated.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> xylose-inducible promoter, plasmid vector and application thereof

<130> 2019

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2048

<212> DNA

<213> Sinorhizobium meliloti

<400> 1

cctttcctag gtcgaaaaat ctatctgaat caaatattta aggtgtcgaa tcccctgtct 60

ccggacagcc gtgcggcgct ccgttttcca cgtctcgcgg atttttgatt tacgtacatc 120

agaaattcct ctaaaggcag aacccggagg aaaacatgcg cccgacagtt cacgatatcg 180

ctgctagagc cggggtcagc ctcgcgaccg tcgaccgggt tctgaacaac cggccgggcg 240

tgcgtggtgt cacccgcgac aaggtcgagc gcgccatcga agcgctcggc tatgtgcgcg 300

acgtcgccgc cgccaacctc gccaagagcc ggatctaccc gctggtcttc atcctgccgg 360

agggcgacaa caccttcatg cgcggtcttg cggccgaggt ggaggcggcg aaggcgcgct 420

cggcagtcga gcgcacggcg atcacggtct tgtccgtccg ggccttcgac ccggaagcgt 480

tagctgcagc gcttcataaa gcgcgggcgc ttgacccggc cggcgtcgcg gtcgttgcga 540

tcgatgcgcc cgaggtcgtc agtgcggtag atgcgctgcg tgaagacggc gtcgccgtcg 600

tgacgttggt ttccgacctg ccggggtcgt cgcgcgatca tttcgccggt gtcgacaacg 660

ttgccgccgg ccggaccgcc ggcaccctga tggggcgttt cctcggtggc cgaaacggcc 720

cggtggcgat cgtagccggc tcgatgctgg tgcgcgacca tcgcgaccgt ctcgaaggct 780

ttggcgcggc catggcagaa agtgcgcctg gccgccggct gctgcccgtc gtcgaaggcc 840

atgacgatcc ggctgaggtc gatcgcctcg tctccaacct tctcgccgat caaccggagc 900

ttgccggcat ctacagtctc ggcgccggca atcgcggcct gatcgcggca ctggaaaagg 960

caggtcgcga aaaggctgtc tgcgccatcg ctcatgaact gacgccgcac agccgcgcgg 1020

cacttctgtc cggcacgctc gacgcactcc tcaatcagga cggcggccat gaggtgcgca 1080

gcgccatccg ggtgctgaag gcccgcgccg acggcctttc ggtgatcggg gcgcaggagc 1140

gcatccgcat cgaaattttc ctgaaggaca acctgccggt cgatccggcg tagcgctttg 1200

aatttacgca gttctttcga agaattgctc aatggaggca agcatgtatc tcggactgga 1260

tctcggcacc tcgggcgtca aggcaatgtt gatcgatggc ggccagcgca tcgtcggttc 1320

ggcctccggc gcgctcgacg tctcgcggcc gcatcccggc tggtcggaac aggacccggc 1380

tgattgggtg cgcgccaccg aagaggcaat tgccgggctg aaggcggcac acccggcgga 1440

gctttcggcg gtgcgcggta tcggcctttc cggccagatg cacggtgcga cgctgcttga 1500

tgccgacgac cgggtgcttc ggccctgcat cctctggaac gacacccgca gcttccgaga 1560

ggcggcagcg ctcgatgcgg acccgcagtt ccgcgccatc accggcaaca tcgtcttccc 1620

cggcttcacc gcaccgaagc ttgcctgggt gcgggccaac gagcccgaca tcttcgccaa 1680

ggtgcgctgg gtgctgctgc cgaaggatta tctgcgcctg tggctgaccg gcgagcacat 1740

gtcggaaatg tccgacagcg ccggcactgc ctggctcgat accggcaagc ggcagtggtc 1800

ggaaagcctg cttgccgcca ccgacatgga cgaaaagcag atgccgagcc tggtcgaggg 1860

aaccgatgcg gccgggaagt tgcgttcgga actggccgcg cgctggggca tttccggtga 1920

cgtcgtggtt gccggcggtg caggcgacaa tgcggcttct gcgtgtggca tgggcacggt 1980

cggcgagggc aatgccttcg tctcgctcgg tacctcgggt gttctcttcg cggcgaatgc 2040

aagctact 2048

<210> 2

<211> 1243

<212> DNA

<213> Sinorhizobium meliloti

<400> 2

cctttcctag gtcgaaaaat ctatctgaat caaatattta aggtgtcgaa tcccctgtct 60

ccggacagcc gtgcggcgct ccgttttcca cgtctcgcgg atttttgatt tacgtacatc 120

agaaattcct ctaaaggcag aacccggagg aaaacatgcg cccgacagtt cacgatatcg 180

ctgctagagc cggggtcagc ctcgcgaccg tcgaccgggt tctgaacaac cggccgggcg 240

tgcgtggtgt cacccgcgac aaggtcgagc gcgccatcga agcgctcggc tatgtgcgcg 300

acgtcgccgc cgccaacctc gccaagagcc ggatctaccc gctggtcttc atcctgccgg 360

agggcgacaa caccttcatg cgcggtcttg cggccgaggt ggaggcggcg aaggcgcgct 420

cggcagtcga gcgcacggcg atcacggtct tgtccgtccg ggccttcgac ccggaagcgt 480

tagctgcagc gcttcataaa gcgcgggcgc ttgacccggc cggcgtcgcg gtcgttgcga 540

tcgatgcgcc cgaggtcgtc agtgcggtag atgcgctgcg tgaagacggc gtcgccgtcg 600

tgacgttggt ttccgacctg ccggggtcgt cgcgcgatca tttcgccggt gtcgacaacg 660

ttgccgccgg ccggaccgcc ggcaccctga tggggcgttt cctcggtggc cgaaacggcc 720

cggtggcgat cgtagccggc tcgatgctgg tgcgcgacca tcgcgaccgt ctcgaaggct 780

ttggcgcggc catggcagaa agtgcgcctg gccgccggct gctgcccgtc gtcgaaggcc 840

atgacgatcc ggctgaggtc gatcgcctcg tctccaacct tctcgccgat caaccggagc 900

ttgccggcat ctacagtctc ggcgccggca atcgcggcct gatcgcggca ctggaaaagg 960

caggtcgcga aaaggctgtc tgcgccatcg ctcatgaact gacgccgcac agccgcgcgg 1020

cacttctgtc cggcacgctc gacgcactcc tcaatcagga cggcggccat gaggtgcgca 1080

gcgccatccg ggtgctgaag gcccgcgccg acggcctttc ggtgatcggg gcgcaggagc 1140

gcatccgcat cgaaattttc ctgaaggaca acctgccggt cgatccggcg tagcgctttg 1200

aatttacgca gttctttcga agaattgctc aatggaggca agc 1243

<210> 3

<211> 1514

<212> DNA

<213> Sinorhizobium meliloti

<400> 3

agataggcgc gctcgacggc gccgaaggca gccgagcggg cgctttcggc agcaaaggcg 60

ttgtcgaatg ccggggcgta aggcgtggcg acgcgcgtcg cttcggccgg tgccggttcg 120

atatcctctt ccgatttcag ccgctcgagc accgactgaa agactgacat tccaaacatg 180

acgtctccga tcgcggcgca ccatggcagg cggagattat cccgggctga tcagactgat 240

catgcccggg ctgatcaggc gcggccctgc ttgaggatca ggtcgtagag cagcttggcg 300

ctcggcggca gcgcgcgtcc ttcggcagtg atcagcccat agggtttgat tcggatcggg 360

atttcggtct tcaggatgcg gatttcgccg gccggcgtac cgttgccggc aatcaggttg 420

gcgacgtcga gtgccacagg tgcaattgcg ttggtgttgc gcaccatcga cagcgtcagg 480

atgatcgagg aggtgttgat caccgtcgcc ggcagtctca ccccggccgc cacgaagctg 540

tcttcgaccg cgcgtctcag aagggtgccg ggcggctgga atacccagtc ataggagggc 600

aagtcctcgg cgacgaccat gggcttgtcg agcagcggat ggccgtcgcg cacgatcagg 660

caaacctcct cgtagccgat ctccaccatg ttgaagagcc gcggcggctg gtcgtcgggg 720

atgcggccga tgatgaaatc gtggcgggcg gcgagcagtt cccttgccag gacgttgctg 780

ttttcgatct ggacgttgat ctcgatgccg ggataggcgg tcatcacctg acggatggcg 840

ggcacggcga gattgagcgc cgggcctgtg accgagccga gcgaaaccga gccgccgctg 900

ccggtcttca actcgttgat ctcgcgcgat gcctcccgga gttccagaaa gatgctgcgc 960

gcgcgtcgcg ccagcgcttc gccatagggc gtcaacagga caccgcgggc aacgcgttcg 1020

cagaccggtg ccttcagcat ggtctcgatt tccgagagca tgcgcgacgc cgccggttgc 1080

gagatgccga gggcgtcagc ggcggcgctg atgcggcggt gatcgtcgat cgcgagaatc 1140

agccggaggt ggctgatctt caggcccgag cggaaaagcg tcgaatcgaa gagttcgccg 1200

gcggagctcg gtttctgatc gcgcatttca acctctgcgt ttttttcttt ggttttcagg 1260

gattttcaaa aatcacactg tttgcgctcg atatatcatt tttggtatgt attcaaagca 1320

atattgtatt tgacagttat atggtttcat tccagttttt gcttatgtgc cagcgcatgt 1380

aggagcatgc gaggcgggga ggcctctcct tcaccttgat ccgctgccgg agacgaagat 1440

ctcgggcggc ggattgctgc gcatagacgg aaaggctctc gcctgccggc ttcaactttt 1500

ttgggagaga actg 1514

<210> 4

<211> 987

<212> DNA

<213> Sinorhizobium meliloti

<400> 4

cgacaaggtc gagcgcgcca tcgaagcgct cggctatgtg cgcgacgtcg ccgccgccaa 60

cctcgccaag agccggatct acccgctggt cttcatcctg ccggagggcg acaacacctt 120

catgcgcggt cttgcggccg aggtggaggc ggcgaaggcg cgctcggcag tcgagcgcac 180

ggcgatcacg gtcttgtccg tccgggcctt cgacccggaa gcgttagctg cagcgcttca 240

taaagcgcgg gcgcttgacc cggccggcgt cgcggtcgtt gcgatcgatg cgcccgaggt 300

cgtcagtgcg gtagatgcgc tgcgtgaaga cggcgtcgcc gtcgtgacgt tggtttccga 360

cctgccgggg tcgtcgcgcg atcatttcgc cggtgtcgac aacgttgccg ccggccggac 420

cgccggcacc ctgatggggc gtttcctcgg tggccgaaac ggcccggtgg cgatcgtagc 480

cggctcgatg ctggtgcgcg accatcgcga ccgtctcgaa ggctttggcg cggccatggc 540

agaaagtgcg cctggccgcc ggctgctgcc cgtcgtcgaa ggccatgacg atccggctga 600

ggtcgatcgc ctcgtctcca accttctcgc cgatcaaccg gagcttgccg gcatctacag 660

tctcggcgcc ggcaatcgcg gcctgatcgc ggcactggaa aaggcaggtc gcgaaaaggc 720

tgtctgcgcc atcgctcatg aactgacgcc gcacagccgc gcggcacttc tgtccggcac 780

gctcgacgca ctcctcaatc aggacggcgg ccatgaggtg cgcagcgcca tccgggtgct 840

gaaggcccgc gccgacggcc tttcggtgat cggggcgcag gagcgcatcc gcatcgaaat 900

tttcctgaag gacaacctgc cggtcgatcc ggcgtagcgc tttgaattta cgcagttctt 960

tcgaagaatt gctcaatgga ggcaagc 987

<210> 5

<211> 687

<212> DNA

<213> Sinorhizobium meliloti

<400> 5

cgtcagtgcg gtagatgcgc tgcgtgaaga cggcgtcgcc gtcgtgacgt tggtttccga 60

cctgccgggg tcgtcgcgcg atcatttcgc cggtgtcgac aacgttgccg ccggccggac 120

cgccggcacc ctgatggggc gtttcctcgg tggccgaaac ggcccggtgg cgatcgtagc 180

cggctcgatg ctggtgcgcg accatcgcga ccgtctcgaa ggctttggcg cggccatggc 240

agaaagtgcg cctggccgcc ggctgctgcc cgtcgtcgaa ggccatgacg atccggctga 300

ggtcgatcgc ctcgtctcca accttctcgc cgatcaaccg gagcttgccg gcatctacag 360

tctcggcgcc ggcaatcgcg gcctgatcgc ggcactggaa aaggcaggtc gcgaaaaggc 420

tgtctgcgcc atcgctcatg aactgacgcc gcacagccgc gcggcacttc tgtccggcac 480

gctcgacgca ctcctcaatc aggacggcgg ccatgaggtg cgcagcgcca tccgggtgct 540

gaaggcccgc gccgacggcc tttcggtgat cggggcgcag gagcgcatcc gcatcgaaat 600

tttcctgaag gacaacctgc cggtcgatcc ggcgtagcgc tttgaattta cgcagttctt 660

tcgaagaatt gctcaatgga ggcaagc 687

<210> 6

<211> 583

<212> DNA

<213> Sinorhizobium meliloti

<400> 6

ttgccgccgg ccggaccgcc ggcaccctga tggggcgttt cctcggtggc cgaaacggcc 60

cggtggcgat cgtagccggc tcgatgctgg tgcgcgacca tcgcgaccgt ctcgaaggct 120

ttggcgcggc catggcagaa agtgcgcctg gccgccggct gctgcccgtc gtcgaaggcc 180

atgacgatcc ggctgaggtc gatcgcctcg tctccaacct tctcgccgat caaccggagc 240

ttgccggcat ctacagtctc ggcgccggca atcgcggcct gatcgcggca ctggaaaagg 300

caggtcgcga aaaggctgtc tgcgccatcg ctcatgaact gacgccgcac agccgcgcgg 360

cacttctgtc cggcacgctc gacgcactcc tcaatcagga cggcggccat gaggtgcgca 420

gcgccatccg ggtgctgaag gcccgcgccg acggcctttc ggtgatcggg gcgcaggagc 480

gcatccgcat cgaaattttc ctgaaggaca acctgccggt cgatccggcg tagcgctttg 540

aatttacgca gttctttcga agaattgctc aatggaggca agc 583

<210> 7

<211> 537

<212> DNA

<213> Sinorhizobium meliloti

<400> 7

tggccgaaac ggcccggtgg cgatcgtagc cggctcgatg ctggtgcgcg accatcgcga 60

ccgtctcgaa ggctttggcg cggccatggc agaaagtgcg cctggccgcc ggctgctgcc 120

cgtcgtcgaa ggccatgacg atccggctga ggtcgatcgc ctcgtctcca accttctcgc 180

cgatcaaccg gagcttgccg gcatctacag tctcggcgcc ggcaatcgcg gcctgatcgc 240

ggcactggaa aaggcaggtc gcgaaaaggc tgtctgcgcc atcgctcatg aactgacgcc 300

gcacagccgc gcggcacttc tgtccggcac gctcgacgca ctcctcaatc aggacggcgg 360

ccatgaggtg cgcagcgcca tccgggtgct gaaggcccgc gccgacggcc tttcggtgat 420

cggggcgcag gagcgcatcc gcatcgaaat tttcctgaag gacaacctgc cggtcgatcc 480

ggcgtagcgc tttgaattta cgcagttctt tcgaagaatt gctcaatgga ggcaagc 537

<210> 8

<211> 1088

<212> DNA

<213> Sinorhizobium meliloti

<400> 8

atgcgcccga cagttcacga tatcgctgct agagccgggg tcagcctcgc gaccgtcgac 60

cgggttctga acaaccggcc gggcgtgcgt ggtgtcaccc gcgacaaggt cgagcgcgcc 120

atcgaagcgc tcggctatgt gcgcgacgtc gccgccgcca acctcgccaa gagccggatc 180

tacccgctgg tcttcatcct gccggagggc gacaacacct tcatgcgcgg tcttgcggcc 240

gaggtggagg cggcgaaggc gcgctcggca gtcgagcgca cggcgatcac ggtcttgtcc 300

gtccgggcct tcgacccgga agcgttagct gcagcgcttc ataaagcgcg ggcgcttgac 360

ccggccggcg tcgcggtcgt tgcgatcgat gcgcccgagg tcgtcagtgc ggtagatgcg 420

ctgcgtgaag acggcgtcgc cgtcgtgacg ttggtttccg acctgccggg gtcgtcgcgc 480

gatcatttcg ccggtgtcga caacgttgcc gccggccgga ccgccggcac cctgatgggg 540

cgtttcctcg gtggccgaaa cggcccggtg gcgatcgtag ccggctcgat gctggtgcgc 600

gaccatcgcg accgtctcga aggctttggc gcggccatgg cagaaagtgc gcctggccgc 660

cggctgctgc ccgtcgtcga aggccatgac gatccggctg aggtcgatcg cctcgtctcc 720

aaccttctcg ccgatcaacc ggagcttgcc ggcatctaca gtctcggcgc cggcaatcgc 780

ggcctgatcg cggcactgga aaaggcaggt cgcgaaaagg ctgtctgcgc catcgctcat 840

gaactgacgc cgcacagccg cgcggcactt ctgtccggca cgctcgacgc actcctcaat 900

caggacggcg gccatgaggt gcgcagcgcc atccgggtgc tgaaggcccg cgccgacggc 960

ctttcggtga tcggggcgca ggagcgcatc cgcatcgaaa ttttcctgaa ggacaacctg 1020

ccggtcgatc cggcgtagcg ctttgaattt acgcagttct ttcgaagaat tgctcaatgg 1080

aggcaagc 1088

<210> 9

<211> 540

<212> DNA

<213> Sinorhizobium meliloti

<400> 9

tacggcgccc cctgtctcga tccgccgccg cgtgctcagc gggccggaac cttgtcgagg 60

aaaccggtga ccgtcttcag gcgaccatct tcgatctggc agacatcggt gccttcgatg 120

acagagggaa cgccgtccgg accgagtgcc caggaaaagc ggatcgtgtc ggcgtaaccg 180

tcggcggcac ccttcagcgt gaagcggaag ccggggaact gtccgtgcac gccggcaatg 240

agcgcatcca cgccggcatg gccgtcgccc tgcatgatcg ggtcgcgata ggcgacgtcg 300

gcggtgaagg ccttcgcgat ctggccggcg cggcttgcgg cgtcggtcgt gttccaggcg 360

gcgatgtagg ctctagcgat agcgttgaag tcgtgcatcg aagtgttcct ttcgtttggc 420

gtttcgatgc ttccggtatc gccggacgag ggaatgaaac caattacctt ccacgtaatg 480

gccgcgccta tcgcatgggc gacgccggcc gacaccactc ttgaaagagg agcaagaccc 540

<210> 10

<211> 1995

<212> DNA

<213> Sinorhizobium meliloti

<400> 10

atgtatctcg gactggatct cggcacctcg ggcgtcaagg caatgttgat cgatggcggc 60

cagcgcatcg tcggttcggc ctccggcgcg ctcgacgtct cgcggccgca tcccggctgg 120

tcggaacagg acccggctga ttgggtgcgc gccaccgaag aggcaattgc cgggctgaag 180

gcggcacacc cggcggagct ttcggcggtg cgcggtatcg gcctttccgg ccagatgcac 240

ggtgcgacgc tgcttgatgc cgacgaccgg gtgcttcggc cctgcatcct ctggaacgac 300

acccgcagct tccgagaggc ggcagcgctc gatgcggacc cgcagttccg cgccatcacc 360

ggcaacatcg tcttccccgg cttcaccgca ccgaagcttg cctgggtgcg ggccaacgag 420

cccgacatct tcgccaaggt gcgctgggtg ctgctgccga aggattatct gcgcctgtgg 480

ctgaccggcg agcacatgtc ggaaatgtcc gacagcgccg gcactgcctg gctcgatacc 540

ggcaagcggc agtggtcgga aagcctgctt gccgccaccg acatggacga aaagcagatg 600

ccgagcctgg tcgagggaac cgatgcggcc gggaagttgc gttcggaact ggccgcgcgc 660

tggggcattt ccggtgacgt cgtggttgcc ggcggtgcag gcgacaatgc ggcttctgcg 720

tgtggcatgg gcacggtcgg cgagggcaat gccttcgtct cgctcggtac ctcgggtgtt 780

ctcttcgcgg cgaatgcaag ctacttgccc aacccggaaa gcgccgtgca tgccttctgc 840

cacgcgctgc cgaatacctg gcaccagatg ggcgtgatcc tgtctgcgac cgatgcgttg 900

aactggtact ccggcatcgc cggccgcagt gctgccgacc tgagcgccga actcggcgat 960

catctgaagg cgccttcggg cgtcactttc cttccctatc tgtcgggcga gcgtacgccg 1020

cacaatgacg cggcgatccg tggcgccttt gtcggacttg gccatgagag cggccgggtg 1080

gtgctgaccc aggcggtgat ggagggcgtg tccttcgcca ttcgcgacag cctggaagcc 1140

ttgcgcgccg ccggcacgac gctttctcgg gtgacggcga tcggtggtgg ttcgcgctcg 1200

cgctactggc tcaaatcgat cgccacggcg ctcaacctgc cggtcgatct gcccgccgac 1260

ggcgatttcg gcgctgcctt cggggcggcc cgccttggcc tgatcgccgc gaccggtgcc 1320

gatcccgttg tcacctgcta cgccccacag acggccgaga ccattgcgcc ggaggcgtcg 1380

ctggtttcgg cctatgaggc gagctaccag cgctaccgcc ggctctatcc ggcgatcaag 1440

ggcgccgtgt cgtgatacgg cgccccctgt ctcgatccgc cgccgcgtgc tcagcgggcc 1500

ggaaccttgt cgaggaaacc ggtgaccgtc ttcaggcgac catcttcgat ctggcagaca 1560

tcggtgcctt cgatgacaga gggaacgccg tccggaccga gtgcccagga aaagcggatc 1620

gtgtcggcgt aaccgtcggc ggcacccttc agcgtgaagc ggaagccggg gaactgtccg 1680

tgcacgccgg caatgagcgc atccacgccg gcatggccgt cgccctgcat gatcgggtcg 1740

cgataggcga cgtcggcggt gaaggccttc gcgatctggc cggcgcggct tgcggcgtcg 1800

gtcgtgttcc aggcggcgat gtaggctcta gcgatagcgt tgaagtcgtg catcgaagtg 1860

ttcctttcgt ttggcgtttc gatgcttccg gtatcgccgg acgagggaat gaaaccaatt 1920

accttccacg taatggccgc gcctatcgca tgggcgacgc cggccgacac cactcttgaa 1980

agaggagcaa gaccc 1995

<210> 11

<211> 155

<212> DNA

<213> Sinorhizobium meliloti

<400> 11

cctttcctag gtcgaaaaat ctatctgaat caaatattta aggtgtcgaa tcccctgtct 60

ccggacagcc gtgcggcgct ccgttttcca cgtctcgcgg atttttgatt tacgtacatc 120

agaaattcct ctaaaggcag aacccggagg aaaac 155

<210> 12

<211> 1218

<212> DNA

<213> Sinorhizobium meliloti

<400> 12

atgatggatt tcgagaattt ctttaagaac gagctggacg ggctgcatca ggaaggccgc 60

taccgggttt tcgcagacct cgcccgtcat cgcggccaat tcccgaaggc cgcgcgccat 120

acggctgaag gcgttcagga agtcaccgtc tggtgttcga acgactatct cggcatgggc 180

cagcattctg tcgtcaccga ggcgatgaag cgcgccatcg acgaatgcgg cgccggcgcc 240

ggcggcaccc gcaacatttc cggtaccaac cattaccacg tcctgcttga gcgcgagctc 300

gcggacctgc atggcaagga atcggcgctg ctctttactt cgggctacgt gtccaactgg 360

gccgcgctcg gaacgctctg ttccaagatt cccggtgtta tcgtcttctc ggacgccggg 420

aatcacgctt cgatgatcga ggggatccgt cactccaagt gcgaacgcgt catcttcaag 480

cataattcgg tcgctgacct cgaggccaag ctcgctgccg ccgatccgcg tgcgccgaag 540

ctcatcgctt tcgagtccgt ctattcgatg gatggcgaca tcgcgccgat caaggaattc 600

tgcgacctcg ccgacaagta cggcgccatg acctatctcg acgaagtgca tgcggtcggc 660

atgtacggtc cgcgcggcgg cggcattgcc gagcgcgaag gcctgatgca ccgcctgacg 720

gtgatcgaag gcacgctcgg caaggctttc ggcgtgatgg gcggctacat caccggctca 780

gccgcactct gcgacttcat ccgctcgttt gcctccggct tcatcttcac gacggcgctg 840

ccgccgacgc ttgccgccgg tgcgctcgcc tcgatccgcc acctgaagga aagccaggtc 900

gaacgtttcg cgcaccagga gcgtgtgcgt cgtctgcgct cgctgctcga ccagcgcggc 960

attccgcaca tgccgaaccc gagccatatc gtgccggtca tggttggcga cgctgccaag 1020

tgcaagtgga tctcggatct cctgctcgac aatttcggcg tctacgtgca gccgatcaac 1080

tatccgacgg tgccgaagaa gaccgagcgc ctgcgcatca ccccgacgcc gctccattcg 1140

gatgccgaca tcgaccatct cgtcggcgcg ctgcattcgc tgtggtcgcg ctgtgcgctg 1200

gcccgcgctg tcgcgtaa 1218

Claims (14)

1. A xylose-inducible promoter is characterized in that the nucleotide sequence of the xylose-inducible promoter is shown as a sequence table SEQ ID NO.1 or a sequence table SEQ ID NO. 2.

2. A plasmid vector comprising the xylose-inducible 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, which is characterized in thatCharacterized in that the episomal vector comprises replicons recognizable by gram-negative bacteria andmoba broad host shuttle plasmid vector for the gene; the integration vector is a homologous recombination vector carrying a homologous recombination arm with 500-4000 bp flanking target genes.

5. The plasmid vector according to claim 4, wherein the episomal vector is pBBR1MCS 2; the integrative vector is pUC series plasmid.

6. The plasmid vector of claim 2, further comprising a nucleic acid sequence encoding a polypeptide of interest.

7. The plasmid vector of claim 6 wherein the nucleic acid sequence encoding the polypeptide of interest comprises a fluorescent protein.

8. The plasmid vector of claim 7 wherein the fluorescent protein is green fluorescent protein.

9. The plasmid vector of any one of claims 2 to 8, wherein the xylose is induced to have a xylose concentration of 0.1 to 4%.

10. The plasmid vector according to claim 9, wherein the concentration of xylose is 0.5 to 2%.

11. A host cell comprising the plasmid vector of claim 2.

12. The host cell of claim 11, wherein the host cell is Sinorhizobium meliloti (R.)Sinorhizobium meliloti) Zymomonas mobilis (A)Zymomonas mobilis) Bacillus crescentus (A), (B), (C)Caulobacter crescentus) Pseudomonas denitrificans (B)Pseudomonas denitrificans) Agrobacterium tumefaciens (II)Agrobacterium tumefaciens) Brucella abortus: (Brucella abortus) Pseudomonas fluorescens (A)Pseudomonas fluorescens) Rhizobium leguminosarum (A), (B), (C)Rhizobium leguminosarum) Or with Rhizobium sorokinii (A), (B)Sinorhizobium adhaerens) One kind of (1).

13. The host cell of claim 12, wherein the host cell is Sinorhizobium meliloti (R.)Sinorhizobium meliloti）。

14. Use of the xylose inducible promoter of claim 1 or the plasmid vector of any one of claims 2 to 10 or the host cell of any one of claims 11 to 13 to induce the initiation of expression of a gene of interest under xylose inducing conditions.

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