CN103289946A - Establishment method of streptomyces diastatochromogenes expression system - Google Patents

Abstract

The invention discloses an establishment method of a streptomyces diastatochromogenes expression system. The establishment method comprises the following steps of: screening a necessary or efficient ingredient of the streptomyces diastatochromogenes expression system by virtue of a green fluorescent protein gene gfp at first, and then constructing the expression system of a target gene, wherein the necessary or efficient ingredient is erythromycin resistance genome constructive promoter permE*; the target gene promotes production of toyocamycin; and the target gene is a haemoglobin gene vgb. The construction processes are as follows: 1) constructing a carrier pIB139-gfp; and 2) integrating the expression carrier into a streptomyces diastatochromogenes chromosome by virtue of a conjugational transfer method, so as to obtain a recombinant bacterium. The invention provides an efficient screening method and application of the streptomyces diastatochromogenes expression system. Compared with the original strain, the recombinant bacterium has the advantages that the toyocamycin yield of the recombinant bacterium is increased by at least 21.2%, and the concentration of the recombinant bacterium is increased by 11.6%.

Description

The establishment method of streptomyces diastatochromogenes expression system

Technical field

The present invention relates to the establishment method of streptomyces diastatochromogenes expression system, belong to gene engineering technology field.

Background technology

Toyokamycin is a kind of novel nucleoside microbiotic, and molecular formula is C ₁₂H ₁₃N ₅O ₄, ribose C ₁The deazapurine ring that connects similar guanine, core texture are pyrroles's pyrimidine nucleoside analoys.The mechanism of action mainly is the growth that influences thalline of transcribing by the inhibition microorganism, and its bioactivity research report mainly concentrates on clinical medicine domain.Have in the recent period and discover that toyokamycin has good prevention effect to the various plants eqpidemic disease, life-time service can not cause environmental pollution, and plant-growth is also had certain regulating effect.Therefore, the application potential that has in agricultural plants disease control field of toyokamycin.With respect to chemical synthesis, the synthetic toyokamycin of biological process is raw material with the renewable resources, has the reaction conditions gentleness, pollutes less and advantage such as with low cost, and therefore, biological synthesis process is the both economical effective means of present toyokamycin suitability for industrialized production.

Toyokamycin belongs to the secondary metabolite of microorganism, and mycelial structure is dense during the fermentation, and the fermented liquid thickness causes dissolved oxygen level to be restricted, and causes the synthetic level of toyokamycin lower, and production energy consumption is bigger, is difficult to large-scale production.The existing way that addresses this problem generally all is by the traditional selection by mutation production bacterial strain as main means screening high yield and high quality, and the uncertain factor of efficient bacterial strain is many, the cycle is long but screen.Along with the development of Protocols in Molecular Biology, utilizing advanced meanses such as genetically engineered to carry out the metabolism breeding becomes a feasible technological line.Yet the research that utilizes molecular engineering to improve toyokamycin output does not appear in the newspapers.Secondary meta-bolites such as agricultural antibiotic is how synthetic by streptomycete, be exactly to set up the system that efficiently expresses that is applicable to it to the important prerequisite of streptomycete molecular modification, and the foundation of expression system depends on multiple factor.At present research is more thorough, be derive from red saccharopolyspora ( Saccharopolyspora erythraea) erythromycin resistant gene promoter-( PermE*), it be a kind of in streptomycete the strong promoter of constitutive expression.

Yet the metabolic process of streptomycete is the multi-level regulation process of very complicated, a mutual coupling, and therefore, the promotor of streptomyces gene is the ubiquity complicacy on transcriptional profile, therefore, PermE*Promotor does not possess versatility in streptomycete, some studies report PermE*The promoter transcription activity is lower even invalid.Especially at streptomyces diastatochromogenes, still there is not the effectively expressing system at present.

Summary of the invention

In order to overcome the deficiencies in the prior art, the invention provides a kind of establishment method of streptomyces diastatochromogenes expression system.

Streptomyces diastatochromogenes ( Streptomyces diastatochromogenes) 1628 are strain antagonism actinomycetes, its fermented liquid has stronger restraining effect to the various plants pathogenic fungi, through separation and Extraction, determines that its main effective constituent is toyokamycin, does not see the research report of the genetic expression system of streptomyces diastatochromogenes as yet.The present invention, has successfully made up and has contained as reporter gene with green fluorescence protein gene (gfp) PermE ^*The streptomyces diastatochromogenes recombinant expression vector pIB139-gfp of promotor studies the back to its promoter activity in streptomyces diastatochromogenes and finds, it is green that the reorganization bacterium mycelium that obtains is under fluorescent microscope, illustrates in streptomyces diastatochromogenes PermE ^*But the expression of promotor effectively start foreign gene gfp.

A kind of establishment method of streptomyces diastatochromogenes expression system at first utilizes green fluorescence protein gene GfpNecessary or efficient composition, the establishing target expression of gene system then of screening streptomyces diastatochromogenes expression system.

Described necessity or efficient composition are erythromycin resistance gene constitutive promoter permE*.

Described target gene promotes the production of toyokamycin.

Described target gene is hemoglobin gene Vgb

The process of described structure is as follows:

1) carrier construction pIB139-gfp;

2) utilize the conjugal transfer method that described expression vector is integrated into streptomyces diastatochromogenes karyomit(e), obtain the reorganization bacterium.

Described reorganization bacterium is compared with original strain, and reorganization bacterium toyokamycin output has improved 21.2% at least, and bacterium is dense has improved 11.6%.

Beneficial effect of the present invention:

The invention provides a kind of screening method and application of effective streptomyces diastatochromogenes expression system, at first utilize gfp to be reporter gene, survey the transcriptional activity of the neccessary composition permE* promotor in the expression system, set up the expression system that is applicable to streptomyces diastatochromogenes; And the expression system of using foundation has obtained the integration hemoglobin gene VgbStreptomyces diastatochromogenes, make the original bacterium of energy force rate of its synthetic toyokamycin improve 21.2%, bacterium is dense has improved 11.6%.

Description of drawings

Fig. 1 is the structure synoptic diagram of recombinant plasmid pIB139-gfp.

Fig. 2 is the restriction enzyme digestion and electrophoresis proof diagram of plasmid pET28-gfp.

Wherein, M ₁: λ DNA/ HinD III Marker, M ₂: DL2000 Marker, 1:pET28a- Gfp/ EcoR I+ HinD III double digestion; 2:pMD18-T- Gfp/ EcoR I+ HinD III double digestion; 3: GfpThe PCR product

Fig. 3 is plasmid pIB139- GfpThe restriction enzyme digestion and electrophoresis proof diagram.

Wherein, M:DL2000 Marker; 1:pIB139- Gfp/ XbaI+ NotThe I double digestion; 2:pIB139/ XbaI+ NotThe I double digestion;

Fig. 4 is the PCR electrophoresis proof diagram of reorganization bacterium 1628-GFP.

Wherein, 1-4: 1628-GFP is the template pcr amplification with the reorganization bacterium GfpGene; 5: be the template pcr amplification with 1628 Gfp; M:DL2000 Marker

Fig. 5 is promoter transcription determination of activity figure.

Wherein, the green fluorescence of starting strain 1628 and reorganization bacterium 1628-GFP detects: A. starting strain 1628; B. recombinant bacterial strain 1628-GFP

Fig. 6 is recombinant plasmid pIB139 -vgbThe structure synoptic diagram.

Fig. 7 is recombinant plasmid pET28a- VgbThe restriction enzyme digestion and electrophoresis proof diagram.

Wherein, 1:pET28a- Vgb/ EcoR I+ HinD III double digestion; 2:pET28a/ EcoR I+ HinD III double digestion; M:DL2000.

Fig. 8 is recombinant plasmid pIB139 -vgbThe restriction enzyme digestion and electrophoresis proof diagram.

M:DL2000; 1:pIB139/ NdeI+ NotThe I double digestion; 2:pIB139- Vgb/ NdeI+ NotThe I double digestion.

Fig. 9 is the PCR electrophoresis proof diagram of reorganization bacterium 1628-VHB.

Wherein, 1-4: 1628-VHB is the masterplate pcr amplification with the reorganization bacterium AprGene; 5: be the template pcr amplification with original bacterium 1628 AprGene; 6-9: 1628-VHB is the masterplate pcr amplification with the reorganization bacterium VgbGene; 10: be the template pcr amplification with original bacterium 1628 VgbGene; M:DL2000 Marker.

Figure 10 is the CO differential spectra figure of reorganization streptomyces diastatochromogenes 1628-VHB.

Embodiment

The present invention is described further below in conjunction with drawings and Examples.

Embodiment 1: with green fluorescence protein gene GfpStructure as the streptomyces diastatochromogenes genetic expression system of reporter gene:

Be template with plasmid pCAMBIA1302, with P GfpF EcoR I and P GfpR HinDIII is that primer PCR obtains to contain EcoR I with HinTwo restriction enzyme sites of dIII GfpFragment is connected with pMD18-T Vector, makes up cloning vector pMD18-T- Gfp( EcoR I+ HinDIII).With cloning vector pMD18-T- Gfp( EcoR I+ HinDIII) be converted into E. coliThe JM109 recipient bacterium is coated on the LB agar plate that contains Amp, IPTG, X-gal, after 37 ℃ of overnight incubation, grows many white colonies on the flat board.The white clone of picking enzyme is served sea living worker's biotechnology company limited order-checking usefulness after cutting evaluation at random EcoR I with HinThe dIII double digestion is contained EcoR I with HindTwo restriction enzyme sites of III GfpFragment, the pET28a that cuts with same enzyme is connected, and obtains transformant at Kan resistance LB plate screening behind the transformed into escherichia coli JM109, receives plasmid pET28a- GfpUse then XbaI with NotI double digestion pET28a- Gfp, be connected conversion with the pIB139 that same enzyme is cut, coating apramycin resistant panel, the picking transformant, enzyme is cut checking, namely obtains recombinant plasmid pIB139- Gfp(Fig. 1).

Green fluorescence protein gene according to the GenBank announcement GfpSequence, the design primer amplifies from plasmid pCAMBIA1302 by round pcr GfpSequence.Design of primers is as follows:

P gfpF? EcoR?I：5’-ACG TCTAGA ATGGTAGATCTGACTAG

P gfpR? HindIII：5’-ACG GCGGCCGC CCCGATCTAGTAAC

Shown in Fig. 2 and 3, be respectively recombinant plasmid pET28a- GfpAnd pIB139- GfpEnzyme is cut the checking result.Recombinant plasmid pET28a- GfpWarp EcoR I with HinObtain the dna fragmentation of about 1.1kb and 5.3kb behind the dIII double digestion, respectively with GfpBig or small consistent (Fig. 2) of gene fragment and plasmid pET28a.Recombinant plasmid pIB139- GfpWarp XbaI with NotObtain the dna fragmentation of about 1.1kb and 5.9kb behind the I double digestion, respectively with GfpBig or small consistent (Fig. 3) of gene fragment and plasmid pIB139.Above result proves recombinant plasmid pIB139- GfpMake up correct.

Extract plasmid pIB139- Gfp, produce the look streptomycete with conjugal transfer method transferred amylase.After the some bacterium colonies of picking are cultivated at random on the apramycin resistant panel, extract karyomit(e) in the CP substratum.The PCR experiment all can amplify GfpGene (Fig. 4) proves that reorganization streptomyces diastatochromogenes 1628-GFP successfully constructs.

Embodiment 2: with green fluorescence protein gene GfpPreliminary assessment as the streptomyces diastatochromogenes genetic expression system of reporter gene:

Picking is at the streptomyces diastatochromogenes colony inoculation CP substratum of the flat board growth that contains 50 μ g/mL apramycins, 28 ℃, 180 r/min shaken overnight, and then be inoculated in respectively with the inoculum size of 1:50 and be equipped with in the fresh CP substratum of 50 mL, be cultured to logarithmic growth mid-term, dry cell weight (DCW) is about 0.3 g/L, the centrifugal collection mycelium of 6000 r/min, behind PBS damping fluid washing mycelium 2 times, be resuspended among the 30 mL PBS.Get 5-10 μ L bacterium liquid smears, observe under Olympus BX50 fluorescent microscope, the Color Video Camera with SONY 3CCD takes pictures simultaneously.With wild bacterium in contrast, the selective exitation spectral filter is 485 nm, and the emission spectral filter is 520 nm, determines the transcriptional activity of promotor.

The result shows that reorganization bacterium 1628-GFP mycelia can send stable bright green fluorescence (Fig. 5), and the no fluorescence of contrast illustrates promotor in streptomyces diastatochromogenes 1628 PermE ^*Can the effectively start foreign gene GfpExpression.

Embodiment 3:Vitreoscilla hemoglobin gene VgbExpression in streptomyces diastatochromogenes:

Recombinant plasmid pIB139- VgbStructure as shown in Figure 6, step and recombinant plasmid pIB139- VgbConstruction process similar.

As shown in Figure 7 and Figure 8, be respectively recombinant plasmid pET28a- VgbAnd pIB139- VgbEnzyme is cut the checking result.Reorganization matter plasmid pET28a- VgbWarp EcoR I with HinObtain the dna fragmentation of about 0.5kb and 5.3kb behind the dIII double digestion, respectively with VgbGene fragment and plasmid pET28a's is big or small consistent.Recombinant plasmid pIB139- VgbWarp NdeI with NotObtain the dna fragmentation of about 0.6kb behind the I double digestion, with VgbThe size of gene fragment is consistent.Above result proves recombinant plasmid pIB139- VgbMake up correct.

Extract plasmid pIB139- Vgb, produce look streptomycete 1628 with conjugal transfer method transferred amylase.After the some bacterium colonies of picking are repeatedly cultivated at random on the apramycin resistant panel, extract karyomit(e) in the CP substratum.The PCR experiment all can amplify VgbGene and apramycin resistant gene Apr(Fig. 9), prove plasmid pIB139- VgbOn the karyomit(e) that is incorporated into streptomyces diastatochromogenes of success, and inheritance stability, reorganization streptomyces diastatochromogenes 1628-VHB successfully constructs.

Success is carried out the expression study of VHb to it, and has been measured the expression amount of VHb by CO differential spectra method after obtaining reorganization streptomyces diastatochromogenes 1628-VHB.

As shown in figure 10, do contrast with original streptomyces diastatochromogenes 1628, reorganization streptomyces diastatochromogenes 1628-VHB is combined the back and absorption peak is all occurred at the 419nm place with CO, prove VgbGene is host cell with the streptomyces diastatochromogenes, in promotor PermE ^*Effect can give expression to the oxyphorase with activity down.

The streptomyces diastatochromogenes 1628-VHB that will recombinate is inoculated in the fermention medium, in 28 ℃, and the 180r/min 96h that ferments.Control group is the streptomyces diastatochromogenes strain of setting out.After the fermentation ends, it is dense to have measured bacterium respectively, toyokamycin output.As shown in table 1 below, the plain level of the product of recombinant bacterial strain has improved 21.2% than starting strain, and bacterium is dense has improved 11.6%, and repeatability is good.The expression that VHb is described is conducive to the growth of streptomyces diastatochromogenes thalline and synthesizing of toyokamycin.

Table 1 VHb expresses 1628 thalli growths and the biosynthetic influence of toyokamycin

Thalline	Toyokamycin output (mg/L)	Dry cell weight (g/L)
			1628	134.97	0.43
1628-VHB	163.52	0.49

Claims (6)

1. the establishment method of a streptomyces diastatochromogenes expression system is characterized in that, at first utilizes green fluorescence protein gene GfpNecessary or efficient composition, the establishing target expression of gene system then of screening streptomyces diastatochromogenes expression system.

2. the establishment method of streptomyces diastatochromogenes expression system as claimed in claim 1 is characterized in that, described necessity or efficient composition are erythromycin resistance gene constitutive promoter permE*.

3. the establishment method of streptomyces diastatochromogenes expression system as claimed in claim 1 is characterized in that, described target gene promotes the production of toyokamycin.

4. the establishment method of streptomyces diastatochromogenes expression system as claimed in claim 1 is characterized in that, described target gene is hemoglobin gene Vgb

5. the establishment method of streptomyces diastatochromogenes expression system as claimed in claim 1 is characterized in that, the process of described structure is as follows:

1) carrier construction pIB139-gfp;

2) utilize the conjugal transfer method that described expression vector is integrated into streptomyces diastatochromogenes karyomit(e), obtain the reorganization bacterium.

6. the establishment method of streptomyces diastatochromogenes expression system as claimed in claim 5 is characterized in that, described reorganization bacterium is compared with original strain, and reorganization bacterium toyokamycin output has improved 21.2% at least, and bacterium is dense has improved 11.6%.

Images