CN1061375C

CN1061375C - Using allogeneic promoter to express transparent Tremellineae haemoglobin in streptomycete

Info

Publication number: CN1061375C
Application number: CN96116433A
Authority: CN
Inventors: 崔凤文; 杨胜利
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date: 1996-07-19
Filing date: 1996-07-19
Publication date: 2001-01-31
Anticipated expiration: 2016-07-19
Also published as: CN1171444A

Abstract

The present invention structures a plasmid pFW201 for expressing Vitreoscilla hemoglobin (VHb) in streptomycete after no action of a promoter of a Vitreoscilla hemoglobin gene (vgb) in the streptomycete is proved. After gene (vgb) is removed, the promoter of the Vitreoscilla hemoglobin gene (vgb) is inserted in the down stream of an ORF438 promoter on a plasmid pIJ702 of the streptomycete, and a DNA fragment with ATG between the ORF438 promoter and the Vitreoscilla hemoglobin gene is removed. Then, the chimeric gene is cloned to pIJ699 to obtain the plasmid pFW201 which can effectively express the Vitreoscilla hemoglobin and can be induced by tryptone.

Description

Utilize allogeneic promoter in streptomycete, to express Vitreoscilla hemoglobin

The present invention relates to a class can express Vitreoscilla hemoglobin (Vitreoscilla haemoglobin, plasmid VHb) belongs to bioengineering field in streptomycete.

Streptomycete and close bacterium are used for producing many kinds of microbiotic, as tsiklomitsin, erythromycin etc.The high viscosity characteristic of high-density streptomycete fermentation thing makes the oxygen supply in the production of antibiotics process become a subject matter.As secondary metabolite, antibiotic synthetic very responsive again to the supply of oxygen usually.Main optimization, the logical oxygen-rich air by reactor and add the oxygen consumption that some oxygen solubility promoter satisfies microorganism in the past.

1988, (Vitreoscilla haemoglobin gene vgb), and expressed in intestinal bacteria, has studied the characteristic of its hypoxia inducible to be cloned into Vitreoscilla hemoglobin gene by the Vitreoscilla (Vitreoscilla spp.) of protokaryon.Proof imports the efficient that the vgb gene can improve oxygen supply and raising generation ATP at cell levels in intestinal bacteria and fungal systems.Magnolo etc. have expressed VHb in sky blue streptomycete and paleness streptomycete, the result shows vgb introduced can play in the aerobic industrial microorganism and more effectively utilizes oxygen, cuts down the consumption of energy, and promotes growth, increases the beneficial effect of product production.But the expression plasmid pWLD5 of structures such as Magnolo and pWLD10 can not illustrate the key issue that vgb expresses definitely: can the vgb gene promoter work in streptomycete, the expression of VHb can not contain too much inessential heterologous gene fragment and also reduce its practicality by inducing the level that reaches higher on the plasmid.Proved that with plasmid pIJ4083-pro (the present invention's structure) the natural promotor of vgb is inoperative in paleness streptomycete (Streptomyces lividans) TK24 among the present invention.

The present invention aim to provide a kind of can be in streptomycete the genetically engineered plasmid of high level expression oxyphorase.The object of the present invention is achieved like this: the natural promoter of vgb gene (doctor D.A.Webster is so kind as to give) is removed, obtain the plasmid of energy effective expression VHb behind the insertion allogeneic promoter downstream.The vgb gene clone that specifically will remove natural promoter is gone up Sph I-Bgl II site in the ORF438 (open reading frame 438) to plasmid pIJ702 (Mr. Jiao Ruishen is so kind as to give), with ORF438 in the same way, obtain pIJ702-vgb '.ATG in the Sph I site just in time is that the beginning password is opened in the translation of ORF438, further removes the dna fragmentation that contains ATG between ORF438 promotor and the vgb ORF (hemoglobin gene open reading frame), and the two is linked to be mosaic gene, obtains plasmid pFW2.Again this mosaic gene is cloned into streptomycete plasmid pIJ699 (Mr. Jiao Ruishen is so kind as to give) and obtains plasmid pFW201.On the plasmid pFW201 that the present invention makes up, pIJ699 with terminator eliminated on the plasmid other gene fragments to the influence of ORF438 promoter expression.This promotor can be induced with Tryptones, and pFW201 should reach higher VHb expression level when needed.These several VHb expression plasmids all do not contain the non-streptomycete dna fragmentation except that vgb, have removed the unnecessary fragment of vgb5 ' and 3 ' end simultaneously, thereby have reduced the possibility that is limited by the host bacterium.This plasmid is introduced the streptomycete that is used for production of antibiotics, can improve microbiotic output by a relatively large margin, cut down the consumption of energy.

In following examples, invention is described in further detail:

Fig. 1 is the structure synoptic diagram of plasmid pIJ4083-pro.

Fig. 2 is the structure synoptic diagram of plasmid pOK12-vgb '.

Fig. 3 is the structure synoptic diagram of plasmid pFW201.

Fig. 4 expresses vHb vigor figure among the paleness streptomycete TK24.Wherein a.TK24/pIJ702 is as negative control, b.TK24/pFW201

Among the figure: Amp is an ampicillin resistance gene, and tsr is the thiostrepton resistant gene, and xy1E is a pyrocatechol 2, and 3-dioxygenase gene, Km are kalamycin resistance gene, and mel is a tyrosinase cdna, P _ORF438Be open reading frame 438 promotors, ter is a terminator, and Aat II, Afl II, BamH I, Bcl I, Bgl II, EcoR I, EcoRV, Hind III, Sph I etc. are restriction enzyme, and OD is an optical density(OD), and nm is a nanometer.

Embodiment 1, the structure of plasmid pIJ4083-pro.

Referring to Fig. 1.The Vitreoscilla dna fragmentation that contains the vgb gene on the pUC21-vgb (preserve in this laboratory) is about 1.4kb, and direction is promotor for the Hind III arrives the BamH I between Hind III and the Afl II, is ORF between Afl II and the Aat II.PUC21-vgb being gone up Hind III-BamH I fragment of 1.4kb downcuts, be connected with the T4 dna ligase with the promoter probe plasmid pIJ4083 (Mr. Deng Zixin is so kind as to give) that same enzyme is cut, obtain pIJ4083-vgb after transforming paleness streptomycete TK24 (Mr. Zhu Baoquan is so kind as to give), BamH I and Afl II double digestion pIJ4083-vgb, the Klenow enzyme is mended flat, freeze-thaw method reclaims big fragment, and the T4 dna ligase connects back conversion TK24 and obtains pIJ4083-pro.

Embodiment 2, and the vgb promotor does not have function in TK24.

To TK24/pIJ4083-pro at R ₂The bacterium colony that grows on the YE flat board drips not displaing yellow of 0.2mol/L pyrocatechol, does not promptly have the pyrocatechol 2 of xy1E genes encoding, 3-dioxygenase vigor; Cultivate TK24/pIJ4083-pro and to mid-log phase, seal the realization hypoxia inducible with preservative film, the final vacuum suction filtration was collected thalline in 12 hours, was suspended in the phosphoric acid buffer of 100mmol/L, pH7.5, in 4 ℃ of ultrasonications, get supernatant after centrifugal 10 minutes for 10,000 rev/mins.Collect thalline and make cell-free extract.Get 3ml cell-free extract, add 50 μ l 0.2mol/L pyrocatechol solution, lucifuge was in 30 ℃ of insulations 10 minutes, measure the photoabsorption at 375nm place, fail to measure pyrocatechol 2,3-dioxygenase enzyme is lived, show that the vgb promotor can not drive the xy1E gene transcription in TK24, promptly do not have function.

Embodiment 3, the structure of plasmid pOK12-vgb '.

Referring to Fig. 2.Cut pUC21-vgb with Afl II enzyme, the Klenow enzyme is mended flat, cut with BamH I enzyme again, the vgb ' (the vgb gene that removes natural promoter is called vgb ') of 1.3kb is reclaimed in freeze thawing, with is connected with the T4 dna ligase through the pWSK129 of BamH I and EcoR V double digestion (preserve in this laboratory), obtain pWSK129-vgb ' behind the transformed into escherichia coli DH5a (this laboratory preservation).Hind III and Aat II double digestion pWSK129-vgb ', the vgb ' of 800bp is reclaimed in freeze thawing, and is connected with the T4 dna ligase through the pOK12 of Hind III and Aat II double digestion (preserve in this laboratory), obtains pOK12-vgb ' behind the transformed into escherichia coli DH5a.

Embodiment 4, the structure of plasmid pFW201.

Referring to Fig. 3.Sph I and BamH I double digestion pOK12-vgb ', freeze thawing is reclaimed vgb ' fragment of 800bp and is connected with the T4 dna ligase with the pIJ702 of Sph I with Bgl II double digestion, obtain pIJ702-vgb ' after transforming TK24, Hind III and Sph I double digestion pIJ702-vgb ', T4 DNA polymerase is cut flat and is mended flat big fragment two ends, the T4 dna ligase connects back conversion TK24 and obtains pFW2, Bcl I enzyme is cut pFW2, the fragment that contains ORF438 promotor and vgb ' of 2.19kb is reclaimed in freeze thawing, EcoR I and Bgl II double digestion pIJ699, Bgl II-Bgl II segment of 5kb is reclaimed in freeze thawing, and the two is connected with the T4 dna ligase, obtains pFW201 behind the conversion TK24.(S.lividans TK24/pFW201 deposits the CCTCC M96007 China Wuhan Wuhan University Chinese in the school typical culture collection center that is labeled as).

Embodiment 5, contain the mensuration of VHb vigor among the TK24 of oxyphorase expression plasmid.

Referring to Fig. 4.The same system cell-free extract, with the vigor of VHb in the difference spectrum method mensuration thalline, the person expresses for VHb is arranged to have the charateristic avsorption band through spectral scan at the 420nm place.As seen TK24/pFW201 has absorption peak near 420nm, can detect the VHb vigor, and contrast TK24/pIJ702 does not have.

Claims

1. an energy is expressed the plasmid pFW201 of Vitreoscilla hemoglobin (VHb) in streptomycete, it is characterized in that it is included among the paleness streptomycete CCTCC No.M96007.