CN101649325B - Gene series technology for increasing main component content of gene engineering isovaleryl selectomycin - Google Patents

Abstract

The present invention relates to the application of genetic engineering technology in improving antibiotic components, specifically, it relates to a gene concatenation technology for increasing the main component content of genetically engineered isovalerylspiramycin, which is the gene of piramycin The ist gene closely related to the isovalerylation of spiramycin in engineering bacteria is connected in series, and the isovalerylation ability of the producing bacteria is improved by enhancing its gene dosage; at the same time, the promoter erythromycin resistance gene ermE with strong promoter activity is also used The gene promoter sequence replaces the original ist gene promoter sequence to enhance the expression of the tandem ist gene and increase the ratio of the main component of the isovalerylspiramycin produced by the genetically engineered bacteria from the source.

Description

A gene concatenation technology for increasing the main component content of genetically engineered isovalerylspiramycin

Technical field:

The invention relates to the application of genetic engineering technology in improving antibiotic components.

Background technique:

Bitespiramycin (formerly known as Shengjimycin) [patent number: ZL97104440.6] is a spiramycin derivative developed by our laboratory using genetic engineering technology. Spiramycin is a macrolide antibiotic. Its main core is a 16-membered ring consisting of a lactone ring that is homogeneous to Pratt lactone. Mycose, and the main component of spiramycin is a derivative of spiramycin mycose 4' hydroxyl group esterified with isovaleryl. Since the R hydroxyl group at the 3-position of the spiramycin lactone ring can be esterified by acetyl or propionyl to form the corresponding spiramycin I, II, and III components, so the spiramycin contains at least isovaleryl spiramycin I , II, III three components, the chemical structure of the main component of Bitespiramycin is as follows:

Phalosamine Pratt lactone Mycaminosamine Mycaminose

Bitespiramycin structure

Isovalerylspiramycin III R=COCH ₂ CH ₃ R'=COCH ₂ CH(CH ₃ ) ₂

Isovalerylspiramycin II R=COCH ₃ R'=COCH ₂ CH(CH ₃ ) ₂

Isovalerylspiramycin I R=HR'=COCH ₂ CH(CH ₃ ) ₂

Bitspiramycin has strong activity against Gram-positive bacteria, especially against mycoplasma and chlamydia, and against erythromycin and β-lactam antibiotic-resistant bacteria, influenza bacillus, Legionella, and perfringens Clostridium has antibacterial activity, and there is no complete cross-resistance with similar drugs. Bitspiramycin has high lipophilicity, rapid oral absorption, strong tissue permeability, wide distribution, long maintenance time in vivo, and good post-antibiotic effect. The completed phase II clinical trial of bitspiramycin with genetic engineering shows that the total effective rate of bitspiramycin in the treatment of upper respiratory tract infection is 93%, which is no different from azithromycin, which is the best control drug of the same kind currently used clinically. It has the potential application value of anti-urinary tract infection. Bitspiramycin, as a national first-class new drug, has completed the third phase of clinical trials, showing good efficacy and safety.

Bitspiramycin is a fermentation product of genetically engineered bacteria, and its formation requires the following elements and steps:

First, spiramycin is produced by fermentation of spiramycin-producing bacteria;

Secondly, clone the carbamycin 4'-position hydroxyl isovaleryl (ist) transferase gene from carbamycin-producing bacteria;

Again, transfer the ist gene into the spiramycin-producing bacteria to obtain genetically engineered bacteria;

Finally, the expression product (enzyme) of the ist gene can catalyze the 4'-position hydroxyl of mycaminose in the spiramycin molecule in the presence of a fatty acyl (isovaleryl) group substrate in the spiramycin-producing bacteria to carry out The O-acylation reaction results in the esterification of the 4'-hydroxyl isovaleryl of mycaminol, and the spiramycin in which the 4'-isovaleryl of mycaminol is esterified is called bitspiramycin. Due to the existence of various fatty acyl metabolites in the microbial fermentation process, such as acetyl, propionyl, butyryl, etc., although the isovaleryl (ist) transferase gene mainly recognizes isovaleryl, it has a certain ability to recognize other fatty acyl groups. Therefore, during the fermentation process, the 4' hydroxyl group of spiramycin mycaminose may also be acetylated, propionylated and butyrylated to form components such as acetyl, propionyl and butyryl spiramycin. In order to make the isovaleryl in the finished product of bitspiramycin become its main component (the new drug application quality standard requires that its isovalerylspiramycin content should not be less than 65%), it is usually necessary to increase the isovaleryl in the fermentation broth extraction process The number of times of washing the extract with water, however, each washing with water will reduce the extraction yield and increase the production cost. Therefore, the multi-component problem of antibiotic fermentation process products brings many difficulties and challenges to the post-extraction process and quality standard control in production. If the ability of strains to synthesize main components can be improved from the source, it will have significant economic and social benefits for simplifying the extraction process, reducing production costs, improving environmental protection and optimizing quality standard control.

Spiramycin is produced by S. spiramycetisus or S. ambofaciens. The spiramycin biosynthetic gene cluster (about 90kb in length) has been cloned [F Karray et al Microbiology2007, 153: 4111-4122], and its macrolide core system is mediated by about 40kb of polyketide synthase type I gene product lead synthesis, including 5 reading frames (SrmG I, II, III, IV, V), which utilize 4 malonyl CoA, 1 ethylmalonyl CoA, 1 methylmalonyl CoA and a formazan Oxymalonyl CoA constitutes a macrolide; five genes (orf5 ^* , orf22c, orf23c, orf24c, and orf25c) are associated with methoxymalonyl carrier proteins; one gene encoding p450 (orf1) is associated with C20 hydroxylation Related; a gene encoding an acyltransferase (orf6 ^* ) is related to the acylation of the hydroxyl group of the lactone ring to form spiramycin II and III components; orf31 encodes a ketoreductase related to the reduction of the C9 keto group of the lactone ring, and the C9 keto group After being reduced to a hydroxyl group, it is then glycosylated to form folosamine lactone. In the spiramycin biosynthesis gene cluster, 15 genes including orf1 ^* c, orf2-8, orf9c, orf12, orf15c, orf19, orf20, orf21c and orf27 are related to the synthesis of three sugar groups in spiramycin molecules; orf3 ^* c, orf2 ^* c, orf16-18, and orf26 encode glycosyltransferases; in addition, orf7 ^* c, orf11c (srmB) and orf29 are related to resistance; orf10 (srmR), orf13c, orf28c, and orf32c are helices Regulatory genes for mycin biosynthesis. In addition to producing spiramycin, it is known that spiramycin-producing bacteria also produce a kind of polyketide compound, and its biosynthesis is mediated by the PKS II enzyme gene [PangX etal Antimicrob.Agents Chemother.2004, 48 (2): 575 -588].

Isovalerylspiramycin is produced by isovalerylation of spiramycin mediated by isovaleryltransferase. Therefore, the output of isovalerylspiramycin depends on the output of spiramycin, and at the same time, the activity of isovaleryltransferase directly affects the conversion rate of spiramycin and the productive rate of isovalerylspiramycin, and isovalerylspiramycin The activity of acyltransferase, in turn, is regulated by gene dosage, gene promoter strength and environmental factors (ie, fermentation conditions).

Gene dosage is the gene copy number, which has an important impact on the level of gene expression. In general, the high copy number of the plasmid vector often corresponds to the dosage of the gene of interest. Many reports have shown that the increase in the number of plasmid copies can increase the production of the target protein [Coronado C et al Plasmid 1994, 32: 336-341, Shen SH et al PNAS1984, 1: 4627-4631]; The atrial natriuretic peptide gene increases the secretory expression of atrial natriuretic peptide in Escherichia coli [Zhou Chunyan and other biochemical journals, 1992, 8 (1): 26-32]; some people have also constructed an atrial natriuretic peptide containing 8 tandem repeats. Gene expression vector of atrial natriuretic peptide can increase the production of atrial natriuretic peptide [Lennick et al Gene 1987, 61: 103-112]. The gene copy number has a significant effect on the expression of tumor necrosis factor TNF, tetanus toxin and mouse epidermal growth factor in recombinant Pichia pastoris. Sreekrishna et al. integrated 1, 10 and 20 copies of the TNF gene into the chromosome of Pichia pastoris Above, the expression levels of TNF were 0.05, 4.0 and 10g/L, respectively, and the expression level of 20 copies of TNF recombinant was 200 times that of a single copy. The expression level of the 14 tetanus toxin genes tandem integrated in the Pichia yeast chromosome was 6 times higher than that of a single copy [Clare J J et al Biotechnol.1991, 9: 455-460]. Integrating 19 copies of mouse epidermal growth factor increased the expression level of recombinant Pichia pastoris by 13 times [Clare J J et al Gene1991, 105:205-212]. It has been reported that in Streptomyces clavuligerus (S. clavuligerus B71-14), the clavulanic acid biosynthesis positive regulatory gene CcaR was integrated in the chromosome to increase a copy, resulting in a 1.54-fold increase in clavulanic acid production [Bai Xiaojia et al. Food Research and Development 2008 , 29(8):].

The promoter is a DNA sequence located upstream of the 5' end of the structural gene, which can guide the RNA polymerase holoenzyme to correctly bind to the DNA template, activate the RNA polymerase, and initiate gene transcription. The region where RNA polymerase binds to the promoter is called the promoter region. In prokaryotes, it is generally -10 and -35 regions. The difference in the structure of the gene promoter, including the difference in the number of nucleotides between the -10 region and the -35 region of the promoter, will affect the activity of the promoter. The use of different promoters can affect gene transcription activity, thereby affecting gene expression. At present, it is believed that in Saccharopolyspora erythraea, the resistance gene promoter ermEp has a strong activation activity. Schmitt John T et al. compared several promoters with different structures, including chemically synthesized genes similar to E. coli Conserved sequence promoters, neomycin resistance gene apb I promoter, melanin gene melC promoter, thiostrepton-induced promoters tipA and ermEp, etc. are secreted and expressed amylase aprotinin in Streptomyces lividans (tendamistat), the results show that 500mg/ml amylase aprotinin can be obtained by adopting the ermEp promoter, the chemical synthesis is similar to the Escherichia coli promoter and can obtain the output of 10mg/ml, the melC promoter is 200mg/ml, and the tipA promoter is 0.5-40mg/ml yield [Schmitt-John T

et al. Appl Microbial. Biotechnol. 1992, 36(4): 493-8].

The erythromycin resistance gene ermE has two promoters, ermEp1 and ermEp2. The transcription initiation point of the former is close to the base G in front of the GTG start codon of ermE. The sequence of the -10 region is TAGGAT, and the sequence of the -35 region is TGGACA The distance between the two is 14bp; the transcription start point of ermEp2 is located 72bp upstream of the start codon of ermE, the sequence of the -10 region is GAGGAT, and the sequence of the -35 region is TTGACG, and the distance between the two is 18bp. The direction of transcription is the same for both promoters. The TGG codon in the ermEp1-35 region was deleted and mutated to a GGCACA sequence to obtain a stronger promoter ermE ^* p [Schmitt JT Applied Microbiology Biotechnology, 1992, 36: 493-498, Bibb MJ Molecular and General Genetics, 1986 , 203: 468-478]. The ist gene promoter is 36bp upstream of the ATG start codon of the reading frame, TAGACA in the -10 region and 61bp TTGCCC upstream of the -35 region, and the ribosome site is 6bp GAGG upstream [NCBI Gene Bank D3182].

The object of the present invention is to adopt the gene integration technology to connect the ist gene closely related to spiramycin isovalerylation in the spiramycin genetically engineered bacteria, so that the ist gene can be increased in the spiramycin-producing bacteria. Copy number, improve the isovalerylation ability of the producing bacteria by enhancing its gene dosage; at the same time, replace the original ist gene promoter sequence with a strong promoter such as the erythromycin resistance gene ermE promoter sequence to enhance the ist gene Expression, from the source to increase the ratio of the main component of isovalerylspiramycin produced by genetically engineered bacteria. The gene concatenation technology for increasing the content of the main component of genetically engineered isovalerylspiramycin mentioned in the present invention has not been reported at home and abroad so far.

Invention content:

The tandem of the double-copy ist gene provided by the present invention can adopt the repeated tandem mode, and two ist gene 1 and 2 fragments with their own promoters can be connected in series; the first ist gene with its own promoter can also be connected in series. Tandem with the second ist gene without a promoter to enable continuous transcription; in order to implement the above scheme, the upstream primers of the ist gene can be included or excluded according to the start codon ATG upstream of the reading frame of the ist gene The promoter region is designed; the ist gene downstream primer design can adopt two schemes with transcription terminator and without transcription terminator sequence. In the case of continuous transcription of two ist genes, the downstream primer design of the first ist gene should be in Near the stop codon, does not contain the transcription termination sequence. Using the pSW4 plasmid containing the ist gene [Shang Guandong et al The J ofantibiotics 200154(1):66-73] as a template, PCR was performed using the designed primers to obtain two ist genes, and the two ist genes were digested Points are connected to carry out its tandem connection, and a selectable marker gene is added at the same time to screen the gene transformants.

The integration of the double-copy ist gene provided by the present invention and the chromosome of the host bacterium can adopt homologous gene recombination double exchange or directly use an integrated plasmid vector. In the former scheme, in order to integrate the tandem ist exogenous gene into the chromosome of the spiramycin-producing bacteria, two fragments homologous to the chromosomal DNA can be added on both sides. Homologous recombination should be carried out with gene fragments that are not related to spiramycin biosynthesis, so as to ensure that the formation of spiramycin will not be affected during homologous gene recombination. The present invention adopts the PKS II gene cloned from the spiramycin-producing bacteria in the early stage as a gene fragment for homologous recombination with the chromosomal DNA of the host bacteria, designs two pairs of primers according to the PKS II gene sequence, and adds and ist at both ends of the primer nucleotide sequence. Corresponding restriction sites for linking gene fragments and restriction sites for cloning into vectors. Two gene fragments of about 1 kb were amplified, respectively connected to both sides of the tandem ist gene fragment. The PKS II gene is derived from the clone pCG4 in our laboratory [Tang Li et al., Acta Biological Engineering, 1991, 7(1): 24], and the gene sequence has been submitted to the NCBI Gene Bank for inclusion, with the number AY779544.

Another solution provided by the present invention is to use an integrative plasmid vector to directly integrate the double-copy ist gene on the chromosome. In principle, a variety of Streptomyces integrative vectors can be used. The present invention uses the pSET152 vector [Bierman Metal Gene 1992, 116: 43-49], which is a conjugative transfer plasmid vector in Streptomyces/Escherichia coli, containing Escherichia coli The pUC18 replication point does not contain the replication point of Streptomyces, so it cannot exist in a free form, but contains the ΦC31 integrase gene, which is integrated into the chromosome of the host bacterium through the Streptomyces phage ΦC31 attachment site for replication. Cloning the tandem ist gene into the Streptomyces integrative vector, transforming it into a spiramycin-producing strain, and confirming the integration of the tandem ist gene in the chromosome according to the selection marker and PCR verification.

The present invention also adopts a strong streptomyces promoter such as the promoter ermEp of the erythromycin resistance gene ermE to improve the expression of the tandem ist gene in the host bacterium, and then improve the synthetic isovalerylspiramycin main component of the genetically engineered bacteria yield.

Invention effect:

In the present invention, the isovaleryltransferase gene is serially expressed in the spiramycin-producing bacteria, which can increase the ratio of the main component isovalerylspiramycin produced by the bitspiramycin-producing bacteria from the source, thereby greatly simplifying the extraction process, Reduce production costs, improve environmental protection and optimize the control of quality standards, resulting in obvious economic and social benefits.

Description of drawings:

Figure 1. Schematic diagram of the construction and gene integration of the homologous recombination double exchange ist gene double-copy plasmid (pKC1139-5)

Among them: 1-chromosome of the original strain of spiramycin producing bacteria; 2-integration of double copy of ist gene in homologous recombination double exchange in chromosome; E, K, S, H, B, X and P are -EcoRI, KpnI, SphI respectively , HindIII, BamHI, XbaI and PstI restriction sites.

Figure 2. Construction of the integrated double-copy ist gene recombinant plasmid pSET-4

Among them: E, K, S, Sa, B, X, H and EV are EcoRI, KpnI, SphI, SalI, BamHI, XbaI and HindIII restriction sites, respectively.

Fig. 3. HPLC analysis of isovalerylspiramycin produced by transformant transformed with integrated double-copy ist gene in Streptomyces lividans to transform spiramycin.

Among them: A-bitspiramycin standard; B-spiramycin standard; C-pSET152-2 (ist single-copy plasmid) in the transformant of Streptomyces lividans TK24, the HPLC of isovalerylation of spiramycin Analysis; D-pSET152-4 (ist double-copy plasmid) in the transformant of Streptomyces lividans TK24, HPLC analysis of spiramycin isovalerylation; 1,2,3- is isovalerylspiramycin I , II, III; 1', 2', 3' are spiramycin I, II, III.

Figure 4. PCR identification of double-copy ist gene by homologous recombination double exchange.

Among them: M-λDNA/HindIII enzyme-cut marker; 1-spiramycin producing strain;

2-6-Newly constructed double-copy ist gene engineering strain containing homologous recombination double exchange.

Fig. 5. HPLC analysis of homologous recombination tandem ist gene in spiramycin-producing bacteria to increase the fraction of isovalerylspiramycin.

Among them: pSW4-control; pKC11395-homologous recombination double-copy ist gene engineering bacteria.

implementation plan:

The examples given below are only to help those skilled in the art better understand the present invention, but do not limit the present invention in any way.

<Example 1>: The concatenation of two double-copy ist genes with their own promoters

For the tandem of double-copy ist genes 1 and 2, use the repeated tandem method including its own promoter, and design P1-P2 and P3-P4 primers at the ist gene start codon ATG upstream - 129kb and terminator TAG downstream - 41 (Table 1), using the pSW4 plasmid containing the ist gene as a template, two about 1.3kb restriction sites with different restriction sites were amplified by PCR, namely the ist1 gene (KpnI-BamH I) and the ist 2 gene (BamHI- Xba I) fragment, the same BamH I restriction site can be used to carry out the double copy with its own promoter ist gene in series.

<Example 2>: Construction of double-copy ist gene tandem transcription

Design P5-P6 primers at the ist gene start codon ATG upstream -111bp and terminator TAG downstream -24bp (excluding the transcription termination sequence), and design P7 at the ATG upstream -105bp and terminator downstream -80bp (including the transcription termination sequence) - P8 primer (Table 1), the design site of the double-copy ist gene tandem primer is shown in Figure 1. Using the pSW4 plasmid containing the ist gene as a template, the ist 3 gene (including BamHI-XbaI site) and ist 4 gene (containing XbaI-SalI site) fragments were respectively obtained by PCR, and the same XbaI restriction site was used to perform double A tandem of copies of the ist gene.

<Example 3>: construction of homologous recombination double exchange ist gene double copy plasmid (pKC1139-5)

Using P9-P10 primers and pCG4 plasmid as a template, the homologous fragment H ₁ of about 1.1kb was amplified by PCR, and the homologous fragment H of about 1.1kb was amplified by PCR using the pCG4 plasmid as a template using P11-P12 primers ₂ , to construct a fragment of double crossover of homologous recombination on both sides of the ist gene. Digest the pWS1 plasmid with Xba I-Pst I [Shang Guandong et al The J of antibiotics 2001 54(1):66-73] to obtain a 1.3kb thiostrepton resistance gene (Tsr ^r ) fragment, as the same Screening markers for source recombination double crossovers. The H ₁ fragment (EcoR I-Kpn I), ist1 gene (Kpn I-BamH I), ist 2 gene (BamHI-Xba I) and Tsr ^r gene (Xba I-PstI) described in Example 1 and H ₂ The (PstI-Hind III) fragment was directly connected to the thermosensitive plasmid vector pKC1139 plasmid [Bierman M, et al.Gene, 1992, 116: 43-49] on the EcoR I-Hind III site, and transformed into Escherichia coli DH5α [Takara Company ], through plasmid extraction and sequencing, the recombinant plasmid pKC1139-5 (Fig. 1) of double-copy ist gene capable of homologous recombination double exchange was obtained.

Table 1 The sequences of the primers used in Examples 1-3

<Example 4>: PCR reaction in tandem ist gene construction

The main technical parameters of the PCR reaction are reaction volume 20 μl, using TakaRa company LATaq enzyme and 2×GC buffer I; take 50-100 ng of template gene DNA; 30 cycles, extended at 72°C for 5 min, and the reaction was terminated.

<Example 5>: Construction of a double-copy ist gene recombination plasmid with an integrated strong promoter

From the plasmid pGH113 [Journal of Mohongbo Bioengineering 2004, 20 (5): 662-666], the erythromycin resistance gene promoter region [Bibb MJ et al Gene, 1985, 38: 215-226] was subjected to KpnI and BamHI double digestion, obtain 279bp fragment, connect with ist 3 gene (BamHI-XbaI) and ist 4 gene (XbaI-SalI) fragment described in embodiment 2, and clone in pUC18 plasmid (Promega company) KpnI-SalI enzyme Cut the site, then digest the foreign fragment with EcoRI-SphI, and then perform semi-blunt ligation with the pSET152 plasmid [Bierman M et al Gene 1992, 116:43-49] EcoRI-EcoRV fragment to obtain pSET152-4 Recombinant plasmid (Figure 2). In a similar manner, the ist single gene was cloned into the pSET152 vector to obtain the pSET152-2 recombinant plasmid as a control.

<Example 6>: Confirmation of heterologous expression of double-copy ist gene with integrated strong promoter to improve spiramycin isovalerylation activity

The ist single-copy pSET152-2 control and the constructed pSET152-4 recombinant plasmid were transformed into Streptomyces universal host strain Streptomyces lividans TK24 [T Kieser et al Practical Streptomyces Genetics The John Innes Foundation, Norwich , 2000, p425].

Inoculate fresh slant spores of Streptomyces lividans into R2YE medium [10.3% sucrose, 1.0% glucose, 0.4% net yeast liquid, 0.2% tryptone, 0.4% peptone, 0.4% casein hydrolyzed amino acid, _{K2SO 4} 0.025%, anhydrous CaCl ₂ 0.216%, KH ₂ PO ₄ 0.005%, MgCl ₂ .6H ₂ O 1.012%, add NaOH (1M) 0.5ml to 100ml medium, Tris-HCl (0.25M) pH7.210ml, Trace element solution 0.2ml (ZnCl ₂ 0.004%, FeCl ₃ .6H ₂ O 0.02%, CuCl ₂ .2H ₂ O 0.001%, MnCl ₂ .4H ₂ O 0.001%, Na ₂ B ₄ O ₇ .10H ₂ O 0.001% , (NH ₄ ) ₆ Mo ₇ O ₂ .4H ₂ O 0.001%)], prepared pH 6.5 with distilled water, cultured in shaking flask at 37°C for 48 hours, and transferred the culture to freshly supplemented In the R2YE medium supplemented with 2% glycine, shake culture at 28°C for 20h. Take 10ml of bacterial liquid in a centrifuge tube, centrifuge at 3000r/min to collect mycelium, and use P buffer [1mol/LTris-HCl (pH8.0) 0.31ml, CaCl ₂ .2H ₂ O 0.368%, MgCl ₂ .6H for precipitation ₂ O 0.204%, sucrose 10.3%, glucose 0.1%, trace elements 0.2ml/100ml], pH 7.6, washed 3 times. Add 10ml of P buffer solution containing 2mg/ml lysozyme, mix well, incubate in a 37°C water bath for 1-2h, and shake once every 10-15min to promote the release of protoplasts. After filtering through absorbent cotton, the filtrate was centrifuged and washed twice with P buffer. Finally, suspend the protoplasts with 1ml P buffer.

Take 100 μl of protoplasts, add 10 μl of plasmid DNA solution, flick the tube wall to mix, quickly add 400 μl of P buffer containing 25% PEG-1000, blow and aspirate to mix, leave at room temperature for 1 min, take 200 μl and spread it on a dry R2YE plate On the top, after culturing at 28°C for a slight bacterial lawn (about 16-35 hours), cover with 50 μg/ml apramycin solution, continue to invert culture at 28°C for 3-5 days, and pick transformants. Inoculate the slant culture of the transformants on the transformation medium [starch 2.0%, glucose 2.0%, (NH ₄ ) ₂ SO ₄ 0.3%, yeast powder 0.5%, soybean cake powder 1.0%, KH ₂ PO4 0.05%, MgSO4.7H ₂ O 0.02%, CaCO ₃ 0.5%], cultured at 28°C for 30 hours, added 200 μg/ml spiramycin, and cultured for another 2 days. After the culture solution was filtered, the pH was adjusted to 8.0, extracted with ethyl acetate, concentrated and dissolved Carry out HPLC analysis in methanol, adopt Shimadzu LC-10ATvp liquid chromatograph, chromatographic column is Kromasil C184.5 * 150mm, mobile phase is methanol/1% sodium dihydrogen phosphate solution (55/45), flow velocity is 1ml/min , The detection wavelength is 231nm. According to the ratio of transformants converting spiramycin to isovalerylspiramycin, the constructed ist tandem gene was verified to express the activity of isovalerylase in Streptomyces lividans. Using the retention time (RT) of isovalerylated spiramycin in spiramycin and bitspiramycin as a standard, calculate the amount of isovalerylated spiramycin and the amount of spiramycin residue in the transformant according to the ratio of the UV absorption peak The ratio represents the activity of isovalerylase, and the results are shown in Table II. A representative HPLC analysis is shown in Figure 3.

Table 2. The ratio of the amount of isovalerylspiramycin formed and the amount of spiramycin residue in the Streptomyces lividans transformant

plasmid Spiramycin residues (peak %) Formation of isovalerylspiramycin (peak %) The ratio of the amount of isovalerylspiramycin formed to the amount of spiramycin residue pSET152-2 34.568 3.84 11.0 pSET152-4 15.046 12.564 53.6%

It can be seen from the results in the table that the tandem expression of the ist gene using the strong ermEp promoter can significantly increase the isovalerylation activity of spiramycin, and increase the ratio of the amount of isovalerylspiramycin formed to the amount of spiramycin residue by 138%.

<Example 7>: Verification that homologous recombination tandem ist gene increases isovalerylspiramycin component in spiramycin-producing bacteria.

Respectively, the recombinant vectors pKC1139-5 and pSET152-4 were introduced into the spiramycin-producing strain S.spiramyceticus [CGMCC4.118 of China General Microorganism Culture Collection and Management Center] by a method similar to the protoplast transformation in Example 6 to obtain anti- sex transformants. The pKC1139-5 transformant was cultured ^and grown on Tsr ^r plate [soybean cake flour-starch medium] at 28°C, and passaged on a non-resistant plate at 37°C after isolation. For the strains that do not grow on the Am ^r plate, obtain the engineered bacteria in which the ist gene is integrated into the host chromosome. See Figure 1 for the homologous recombination double crossover between the pKC1139-5 plasmid and the host bacterial chromosome. With the original strain of S.spiramyceticus and the total genome DNA of the newly constructed engineering bacteria as templates, the PCR with P1-P2 as primers is used to detect the ist gene; ) and P12 (downstream primer of H ₂ homologous fragment) were combined primers to amplify the full-length 5-segment gene product to verify the integration of the double-copy ist gene in the host chromosome (Figure 4). Five newly constructed double-copy ist gene strains and the original bitspiramycin genetically engineered bacteria were cultured by seeds [glucose 1-3%, starch 2-4%, soybean meal 1-3%, NaCl 0.4%, CaCO ₃ 0.3-0.8%] and fermentation culture [starch 2-6%, fish meal 1-3%, corn steep liquor 0.5-1.5%, NH ₄ NO ₃ 0.6%, KH ₂ PO ₄ 0.01-0.08%, MgSO ₄ 0.1-0.2% , NaCl 1-2%, CaCO ₃ 0.3-0.7%], the fermentation broth was extracted with ethyl acetate and detected by HPLC, and the proportion of the isovalerylspiramycin component in the fermentation product was calculated. The results are shown in Table 3. A representative HPLC analysis is shown in Figure 5.

Table 3, the proportion of isovalerylspiramycin components produced by fermentation of double-copy ist genetic engineering bacteria

Note: The results in the table are the average value of HPLC detection of three fermentations

As can be seen from the results in the table, the main component of isovalerylspiramycin produced by the homologous recombination double-copy ist gene engineering bacteria has increased by 62%.

sequence listing

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<213> Artificial sequence

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Claims (7)

1. gene polyphone technology that can improve gene engineering isovaleryl Spiramycin Base major constituent content is characterized in that said polyphone technology may further comprise the steps successively:

The polyphone of a, two copy ist genes;

B, employing streptomycete plasmid vector reach and the incoherent gene fragment of Spiramycin Base biosynthesizing makes up the two ist of copy dna homolog recombinant plasmids as homologous gene, or employing streptomycete integrative plasmid and the two copy of streptomycete strong promoter structure ist gene recombination plasmids;

C, change two copy ist dna homolog recombinant plasmids over to spiramycin-producing strain, or change the two copy of the integrated strong promoter of streptomycete ist gene recombination plasmids over to the allos muta lead mycillin;

D, two copy ist dna homolog recombinant plasmid improve isovaleryl Spiramycin Base major constituent content in spiramycin-producing strain, or the evaluation that heterogenous expression improves Spiramycin Base isoamyl acidylate in muta lead mycillin of the two copy of integrated strong promoter ist gene recombination plasmid.

2. the described gene of claim 1 polyphone technology, it is characterized in that, one of scheme of step a is two polyphones with the two copy of self promotor ist gene, frame initiator codon and self promotor upstream and terminator codon downstream sequence design primer are read according to the ist gene by system, and in design of primers, add corresponding restriction enzyme site sequence, with the plasmid that contains the ist gene is template, through pcr amplification, obtain two ist gene fragments, utilize corresponding restriction enzyme site, implement with the polyphone between the two copy of self promotor ist gene.

3. the described gene of claim 1 polyphone technology, it is characterized in that, two of the scheme of step a is pair structure that copy ist genes polyphone is transcribed, be first ist gene downstream primer design, be positioned at the terminator codon downstream but do not comprise its transcription termination sequence, second ist gene downstream primer design comprises transcription termination sequence, and in design of primers, add corresponding restriction enzyme site, with the plasmid that contains the ist gene is template, through pcr amplification, obtain two ist gene fragments, utilize corresponding restriction enzyme site, make second ist gene and first ist gene implement polyphone and consecutive transcription.

4. the described gene of claim 1 polyphone technology, it is characterized in that, one of scheme of step b is the structure of homologous recombination double exchange ist Gene Double copy plasmid, adopting design of primers and PCR method is template with spiramycin-producing strain karyomit(e), two and the incoherent gene fragment of Spiramycin Base biosynthesizing increase, and in design of primers, add corresponding restriction enzyme site, be connected to the both sides of the described polyphone of claim 2 ist gene fragment, simultaneously, add selected marker thiostrepton resistant gene, above-mentioned five fragment clonings to the streptomycete plasmid vector, are obtained can be used for homologous recombination double exchange ist Gene Double copy plasmid.

5. the described gene of claim 1 polyphone technology, it is characterized in that, two of the scheme of step b is the structure of the two copy of integrated strong promoter ist gene recombination plasmid, it utilizes streptomycete strong promoter fragment, transcribing fragment by the described two copy ist gene polyphones of corresponding restriction enzyme site and claim 3 is connected, and be cloned into streptomycete integrative plasmid carrier, obtain the two copy of integrated strong promoter ist gene recombination plasmid.

6. the described gene of claim 1 polyphone technology; it is characterized in that; one of scheme of step c and d is to change the two copy of the described integrated strong promoter of claim 5 ist gene recombination plasmid over to the allos streptomycete; analyze transformant with HPLC the Spiramycin Base that external source adds is converted into isovaleryl Spiramycin Base formation amount, improve the transformation efficiency of Spiramycin Base isoamyl acidylate to confirm the two copy of integrated strong promoter ist gene heterogenous expression.

7. the described gene of claim 1 polyphone technology, it is characterized in that, two of the scheme of step c and d is to change the described homologous recombination double exchange of claim 4 ist Gene Double copy plasmid over to spiramycin-producing strain, obtain transformant, after separating, on the non-resistant flat board, go down to posterity, obtain the engineering bacteria of ist gene integration to the host chromosome, through fermentation culture, extraction and product is carried out HPLC analyze, according to isovaleryl Spiramycin Base component concentration, identify the ratio of its raising.

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