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

Abstract

The present invention has constructed a plasmid pFW201 capable of expressing Vitiligo hemoglobin (VHb) in Streptomyces after proving that the Vitiligo hemoglobin gene (vgb) promoter has no effect in Streptomyces. ) promoter was removed and then inserted into the downstream of the ORF438 promoter on Streptomyces plasmid pIJ702, the ATG-containing DNA fragment between the ORF438 promoter and the Vitiligo hyaline hemoglobin gene was removed, and the chimeric gene was cloned into pIJ699 to obtain an effective expression of transparent vitriol hemoglobin, and tryptone-inducible plasmid pFW201.

Description

Expression of Vitiligo hyaline hemoglobin in Streptomyces using a heterologous promoter

The invention relates to a kind of plasmid capable of expressing Vitreoscilla haemoglobin (VHb) in Streptomyces, belonging to the field of bioengineering.

Streptomyces and related bacteria are used to produce many kinds of antibiotics, such as tetracycline, erythromycin, etc. The highly viscous nature of Streptomyces hyperdensity ferments makes oxygen supply a major concern during antibiotic production. As secondary metabolites, the synthesis of antibiotics is usually very sensitive to the supply of oxygen. In the past, the oxygen consumption of microorganisms was mainly met by optimizing the reactor, passing oxygen-enriched air, and adding some oxygen co-solvents.

In 1988, the Vitreoscilla hemoglobin gene (Vitreoscilla haemoglobin gene, vgb) was cloned from the prokaryotic Vitreoscilla spp. and expressed in Escherichia coli to study its hypoxia-induced characteristics. In Escherichia coli and fungal systems, it has been proved that the introduction of vgb gene can improve oxygen supply and increase the efficiency of ATP production at the cellular level. Magnolo et al. expressed VHb in Streptomyces coelicolor and Streptomyces lividans, and the results showed that the introduction of vgb into aerobic industrial microorganisms can play a more effective role in utilizing oxygen, reducing energy consumption, promoting growth, and increasing product yield. However, the expression plasmids pWLD5 and pWLD10 constructed by Magnolo et al. cannot exactly explain the key issues of vgb expression: whether the vgb gene promoter can function in Streptomyces, the expression of VHb cannot be induced to a higher level, and the plasmid contains too much Non-essential heterologous gene segments also reduce their usefulness. In the present invention, the plasmid pIJ4083-pro (constructed by the present invention) is used to prove that the natural promoter of vgb does not work in Streptomyces lividans TK24.

The invention aims to provide a genetically engineered plasmid capable of high-level expression of hemoglobin in streptomyces. The purpose of the present invention is achieved as follows: the natural promoter of the vgb gene (gifted by Dr. D.A. Webster) is removed, and a plasmid capable of effectively expressing VHb is obtained after inserting the downstream of the heterologous promoter. Specifically, the vgb gene with the natural promoter removed was cloned into the SphI-BglII site in ORF438 (open reading frame 438) on the plasmid pIJ702 (gifted by Mr. Jiao Ruishen), in the same direction as ORF438, to obtain pIJ702-vgb'. The ATG in the SphⅠ site is just the translation initiation code of ORF438, further remove the ATG-containing DNA fragment between the ORF438 promoter and the vgb ORF (hemoglobin gene open reading frame), connect the two into a chimeric gene, and obtain the plasmid pFW2 . The chimeric gene was then cloned into Streptomyces plasmid pIJ699 (gifted by Mr. Jiao Ruishen) to obtain plasmid pFW201. On the plasmid pFW201 constructed in the present invention, the terminator carried by pIJ699 eliminates the influence of other gene fragments on the plasmid on the expression of the ORF438 promoter. This promoter is tryptone inducible and pFW201 should allow higher VHb expression levels if desired. These several VHb expression plasmids do not contain non-streptomyces DNA fragments except vgb, and at the same time, redundant fragments at the 5' and 3' ends of vgb are removed, thereby reducing the possibility of being restricted by host bacteria. The introduction of the plasmid into the Streptomyces used for antibiotic production can greatly increase the output of antibiotics and reduce energy consumption.

The invention is described in further detail in the following examples:

Figure 1 is a schematic diagram of the construction of plasmid pIJ4083-pro.

Fig. 2 is a schematic diagram of the construction of plasmid pOK12-vgb'.

Fig. 3 is a schematic diagram of the construction of plasmid pFW201.

Fig. 4 is a graph showing the activity of vHb expressed in Streptomyces lividans TK24. Where a.TK24/pIJ702 is used as negative control, b.TK24/pFW201

In the figure: Amp is the ampicillin resistance gene, tsr is the thiostrepton resistance gene, xy1E is the catechol 2,3-dioxygenase gene, Km is the kanamycin resistance gene, mel is Tyrosinase gene, P _oRF438 is the open reading frame 438 promoter, ter is the terminator, AatⅡ, AflⅡ, BamHI, BclⅠ, BglⅡ, EcoRI, EcoRV, HindⅢ, SphⅠ, etc. are restriction enzymes, OD is optical density , nm is nanometer.

Example 1, construction of plasmid pIJ4083-pro.

See Figure 1. The DNA fragment of Vitiligo hyaline containing the vgb gene on pUC21-vgb (preserved in our laboratory) is about 1.4kb in length, the direction is from HindⅢ to BamHI, the promoter is between HindⅢ and AflⅡ, and the ORF is between AflⅡ and AatⅡ. Cut out the 1.4kb HindⅢ-BamHI fragment on pUC21-vgb, connect it with the promoter probe plasmid pIJ4083 (gifted by Mr. Deng Zixin) with the same restriction enzyme digestion, and use T4 DNA ligase to transform Streptomyces lividans TK24 (gifted by Mr. Zhu Baoquan) To obtain pIJ4083-vgb, pIJ4083-vgb was digested with BamHI and AflⅡ, filled with Klenow enzyme, and the large fragment was recovered by freeze-thaw method. After ligation with T4 DNA ligase, TK24 was transformed to obtain pIJ4083-pro.

Example 2, the vgb promoter is not functional in TK24.

Adding 0.2mol/L catechol dropwise to the colony grown on the R ₂ YE plate by TK24/pIJ4083-pro does not show yellow color, that is, there is no catechol 2,3-dioxygenase activity encoded by the xy1E gene; Cultivate TK24/pIJ4083-pro to the mid-logarithmic phase and seal with plastic wrap to induce hypoxia. After 12 hours, collect the bacteria by vacuum filtration, suspend in 100mmol/L, pH7.5 phosphate buffer, and ultrasonically break at 4°C , centrifuge at 10,000 rpm for 10 minutes and take the supernatant. The bacteria were collected to make a cell-free extract. Take 3ml of cell-free extract, add 50μl 0.2mol/L catechol solution, keep it in the dark at 30°C for 10 minutes, measure the light absorption at 375nm, no catechol 2,3-bisaddition can be detected Oxygenase activity indicates that the vgb promoter cannot drive the transcription of the xy1E gene in TK24, that is, it has no function.

Example 3, construction of plasmid pOK12-vgb'.

See Figure 2. pUC21-vgb was digested with AflⅡ, filled with Klenow enzyme, then digested with BamH Ⅰ, and the 1.3kb vgb' (the vgb gene with the natural promoter removed is called vgb') was recovered by freezing and thawing, and combined with BamH Ⅰ and EcoR V double Digested pWSK129 (preserved in our laboratory) was ligated with T4 DNA ligase, and transformed into Escherichia coli DH5a (preserved in our laboratory) to obtain pWSK129-vgb'. HindⅢ and AatⅡ double enzyme digestion of pWSK129-vgb′, freeze-thaw recovery of 800bp vgb′, and HindⅢ and AatⅡ double enzyme digestion pOK12 (preserved in our laboratory) was ligated with T4 DNA ligase, transformed into Escherichia coli DH5a to obtain pOK12- vgb'.

Example 4, construction of plasmid pFW201.

See Figure 3. SphⅠ and BamHI double digested pOK12-vgb′, freeze-thawed 800bp vgb′ fragment was recovered and ligated with SphⅠ and BglⅡ double digested pIJ702 with T4 DNA ligase, transformed into TK24 to obtain pIJ702-vgb′, HindⅢ and SphⅠ double enzyme Cut pIJ702-vgb′, T4 DNA polymerase cuts and fills both ends of the large fragment, T4 DNA ligase ligates and transforms TK24 to obtain pFW2, digests pFW2 with BclⅠ, recovers a 2.19kb fragment containing ORF438 promoter and vgb′ by freezing and thawing , EcoRI and BglII double-digested pIJ699, freeze-thawed to recover the 5kb BglII-BglII fragment, connected the two with T4 DNA ligase, and transformed TK24 to obtain pFW201. (S.lividans TK24/pFW201, stored under the symbol CCTCC M96007 China·Wuhan·Wuhan University Campus·China Center for Type Culture Collection).

Example 5, Determination of VHb activity in TK24 containing hemoglobin expression plasmid.

See Figure 4. The cell-free extract was the same as that prepared before, and the activity of VHb in the bacteria was measured by differential spectroscopy, and those with characteristic absorption peaks at 420nm by spectral scanning were considered to have VHb expression. It can be seen that TK24/pFW201 has an absorption peak near 420nm, that is, the VHb activity can be detected, but the control TK24/pIJ702 does not.

Claims (1)

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.

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