Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The structure of embodiment 1, frr expression vector
One, the construction of recombinant plasmid that contains frr gene and himself promotor
1, contains the amplification of the frr gene of self promotor
The design primer, be used for pcr amplification and be positioned at the frr gene [NC_003155.4 (3224362...3223805)] that contains self promotor on the Avid kyowamycin karyomit(e), upstream primer PrimerF1 (CCGACATGACCGCGATCAC) is positioned at frr upstream from start codon 300bp place, downstream primer PrimerR1 (CG
GAATTCGTCATGCGCATCGTACGTGG, band underscore base is a restriction enzyme EcoRI recognition site) being positioned at 150bp place, frr terminator codon downstream, amplified production should be 976bp.
Total DNA with Avid kyowamycin wild type strain ACTT31267 is a template, and PrimerF1 and Primer R1 are primer, carry out pcr amplification, and amplification condition is 95 ℃, 4min; (95 ℃, 1min; 60 ℃, 1min; 72 ℃, 1min) * 25 circulation; 72 ℃, 10min.Amplified production is carried out agarose gel electrophoresis detect, a specific amplified band is arranged at about 1kb place.
2, the structure of recombinant plasmid pFR1-1139 and pFR1-152
Reclaim test kit with DNA and reclaim pcr amplification product, be connected with T carrier pMD18-T (TaKaRa company), show through the order-checking comparison, the fragment that is increased is really for containing the frr gene of self promotor.The T carrier that will contain the frr gene is cut through BamHI and HindIII enzyme, reclaim the frr fragment of 1008bp, this fragment is connected to pIJ2925 carrier (the Janssen G R that cuts through BamHI and HindIII enzyme, and Bibb M J.Derivatives of pUC18that have BglII sites flanking a multiple cloningsite and that retain ability to identify recombinant clones by visualscreening of Escherichia coli colonies.Gene, 1993,124:133-134), called after pFR1-2925, enzyme is cut checking.To verify that then correct pFR1-2925 cuts with BglII and EcoRI enzyme, reclaim the frr fragment of about 1020bp.With this fragment respectively with carrier pKC1139 (the Bierman M that cuts through BamHI and EcoRI enzyme, Logan R, O ' Brien K, etal.Plasmid cloning vectors for the conjugal transfer of DNA fromEscherichia coli to Streptomyces spp.Gene, 1992,116:43-49) link to each other, connect the product competent cell (TaKaRa company) of transformed into escherichia coli DH5 α respectively with the carrier pSET152 (the same pKC1139 of reference) that cuts through same enzyme.Extract from transformant that plasmid carries out PCR and enzyme is cut checking, with correct recombinant plasmid called after pFR1-1139 and pFR1-152 respectively.PFR1-1139 is the plasmid of multiple copied, and pFR1-152 is an integrative plasmid, and they all have frr gene and himself promotor.The plasmid map of pFR1-1139 and pFR1-152 as shown in Figure 1.
Two, the construction of recombinant plasmid that contains frr gene and erythromycin resistance gene strong promoter
1, the amplification of frr structure gene
The design primer, being used for increases to be positioned at contains ribosome bind site (ribosome binding site, frr structural gene sequence RBS), upstream primer Primer F4 (GA on the Avid kyowamycin karyomit(e)
AGATCTCCTACTCAAGACACGCAGGAG, band underscore base is a restriction enzyme BglII recognition site) be positioned at frr upstream from start codon 20bp place, downstream primer Primer R4 (CCC
AAGCTTGTCATGCGCATCGTACGTGG, band underscore base is a restriction enzyme HindIII recognition site) being positioned at 120bp place, frr terminator codon downstream, amplified production should be 700bp.
Total DNA with Avid kyowamycin wild type strain ACTT31267 is that template, PrimerF4 and Primer R4 are primer, carries out pcr amplification, and amplification condition is with " one " in the present embodiment.Amplified production is carried out electrophoresis detection, an amplified band is arranged at the 700bp place.
2, the structure of recombinant plasmid pFR4-1139 and pFR4-152
Reclaim amplified production, it is connected with T carrier pMD18-T (TaKaRa company), show that through the order-checking comparison fragment that is increased is not really for containing the frr structure gene that promotor contains ribosome bind site.Cut the T carrier that contains frr structure gene with BglII and HindIII enzyme, reclaim the frr structure gene fragment of 700bp, this fragment is connected on the pIJ117 carrier that contains erythromycin resistance gene strong promoter (ermE*p) that BamHI and HindIII enzyme are cut (the BglII fragment that will contain ermE*p is inserted into to make up on the corresponding restriction enzyme site of pIJ2925 and forms), get recombinant plasmid pFR4-117, enzyme is cut evaluation.Then correct pFR4-117 is cut with the BglII enzyme, reclaim the ermE*p+frr fragment of about 1kb, this fragment is linked to each other with the carrier pSET152 that cuts through same enzyme with the carrier pKC1139 that cuts through the BamHI enzyme respectively, connect product transformed into escherichia coli DH5 α respectively.Extract from transformant that plasmid carries out PCR and enzyme is cut checking, with correct recombinant plasmid called after pFR4-1139 and pFR4-152 respectively.PFR4-1139 is the plasmid of multiple copied, and pFR4-152 is an integrative plasmid, and the frr structure gene in these two plasmids all places under the erythromycin resistance gene strong promoter ermE*p.The plasmid map of pFR4-1139 and pFR4-152 as shown in Figure 2.
The conversion of embodiment 2, recombinant plasmid
Made up four kinds of frr expression of gene plasmid vectors altogether by embodiment 1, be respectively pFR1-1139, pFR1-152, pFR4-1139 and pFR4-152, original plasmid in contrast is pKC1139 and pSET152.Now the characteristics of these six kinds of plasmids are made brief description (table 1).
Table 1 contains the characteristics of the recombinant plasmid and the original plasmid of frr gene
Owing to there is very strong restriction modification effect in the Avid kyowamycin, directly transform Avid kyowamycin with the plasmid of carrying from E.coliDH5 α, transformation efficiency is extremely low, sometimes even can not get transformant.And use plasmid from the recipient bacterium E.coli ET12567 of modification without limits, its transformation efficiency obviously improves.Therefore, the recombinant plasmid and the control plasmid that build are transformed into earlier E.coli ET12567 (Kieser T respectively, Bibb M J, Buttner M J, etal.Practical Streptomyces Genetics, 2000, Norwich:The John InnesFoundation.) obtaining non-methylated DNA, and then transforms the protoplastis of Avid kyowamycin with non-methylated plasmid DNA in.
This example has selected for use three kinds of different Avid kyowamycin bacterial strains to do the bacterial strain that sets out: ATCC31267, GB-165 and 76-02-e.ATCC31267 is the Avid kyowamycin wild type strain, produces the grey spore; GB-165 (Cai Yujuan. produce the research of green spore Avid kyowamycin genetic modification and fermentation condition. master thesis, 2006, Beijing: the Avid kyowamycin bacterial strain that only produces Avrmectin B component that China Agricultural University) to be this laboratory obtain through mutagenesis and genetic engineering means, produce green spores; 76-02-e (Guo Weiqun. the seed selection of Avrmectin superior strain and Optimizing Conditions of Fermentation. master thesis, 2007, Beijing: be this laboratory strain Avrmectin superior strain that wheel mutagenesis obtains through how China Agricultural University), only produce a spot of spore.The protoplastis for preparing these three kinds of Avid kyowamycin bacterial strains, transform protoplastis with the plasmid that from E.coli ET12567, extracts, be applied on the RM14 flat board of the not added with antibiotic that has dried up, behind 28 ℃ of cultivation 16-20h, on flat board, be coated with the aqueous solution that 1mL contains 1000 μ g apramycins, continue to cultivate 7-10 days at 28 ℃, the bacterium colony that grows is transformant.Because the transformant of Avid kyowamycin does not produce spore on the RM14 regeneration culture medium,, cultivate for 28 ℃ and recovered to produce spore in 7 days so three transformants of each picking are inoculated on the YMS flat board that contains 10 μ g/mL apramycins.Transformant carries out next step fermentation research after plasmid extraction and PCR checking correctly.
The preparation of colibacillary conversion in the present embodiment, Avid kyowamycin protoplastis and method for transformation, RM14 and YMS culture medium preparation referring to the Master's thesis of Cai Yujuan (Cai Yujuan. produce the research of green spore Avid kyowamycin genetic modification and fermentation condition. master thesis, 2006, Beijing: China Agricultural University).
The fermentation research of embodiment 3, transformant
One, the fermentation research of ATCC31267 and transformant thereof
1. the shake flask fermentation of Avid kyowamycin
Seed culture medium: Zulkovsky starch 30g, malt extract 2g, soy peptone 2g, CoCl
26H
2O5mg, adding distil water transfer pH to 7.0-7.2 to 1L.
Fermention medium: Zulkovsky starch 50g, yeast powder 12g, MgSO
47H
2O0.5g, K
2HPO
43H
2O0.5g, KCl4g; CaCO
32g, CoCl
26H
2O5mg, adding distil water transfer pH to 7.0-7.2 to 1L.
Avid kyowamycin wild type strain ATCC31267 and different transformants thereof are inoculated in the seed culture medium (loading amount is the 100mL/500mL triangular flask) grow abundant spore on the YMS substratum after, 28 ℃ of shaking tables are cultivated 24 hours (rotating speed 180rpm, eccentricity 2.5cm).Be inoculated in the fermention medium (loading amount is the 50mL/300mL triangular flask) by 5% inoculum size, cultivate 10 days (rotating speed 250rpm, eccentricity 2.5cm) for 28 ℃, put bottle, with the fermentation unit of HPLC method mensuration Avrmectin, the result as shown in Figure 3.
2, the HPLC of tunning analyzes
1) sample preparation: get the 1.0mL fermented liquid, add 4.0mL methyl alcohol, soak more than 30 minutes, every vibration in 10 minutes once, centrifugal 10 minutes of 4000rpm gets the analysis of supernatant liquor sample introduction;
2) HPLC analysis condition: C
18Reversed-phase column, column length 150mm, column internal diameter 4.6mm, 40 ℃ of column temperatures, moving phase is methyl alcohol: water (85:15), flow velocity 1.0mL/min, sampling volume 10 μ L, wavelength is 246nm.
The fermentation result of Fig. 3 shows, the conversion of control plasmid pKC1139 and pSET152 is little to the yield effect of Avrmectin, and the abamectin fermented unit that contains the ATCC31267 transformant of frr expression vector compares all obviously increase with starting strain, two kinds of abamectin fermented units that contain high copy recombinant plasmid transformed have increased 3-3.7 doubly, and two kinds of abamectin fermented units that contain integrated recombinant plasmid transformed have increased 71%-86%.Can determine the dosage effect of frr gene in ATCC31267 thus.The increase that contains frr gene plasmid copy number causes the increase of frr expression amount, and the frr gene pairs Avrmectin synthetic promoter action of high copy is better than the frr gene of low copy.Relatively be used to express of the influence of two kinds of different promoters of frr gene to Avrmectin output, as can be seen, the abamectin fermented unit that contains two kinds of transformants of frr gene self promotor all is higher than two kinds of transformants that contain erythromycin resistance gene strong promoter (ermE*p), may be when expressing frr erythromycin resistance gene strong promoter (ermE*p) be not better than self promotor of frr gene.
Two, the fermentation research of GB-165 and transformant thereof
Shake flask fermentation and HPLC analytical procedure are with " one " in the present embodiment, and difference is that used fermention medium is fermention medium G: Zulkovsky starch 95g, peanut protein powder 25g, cottonseed protein 7g, maltose 3g, MgSO
47H
2O0.3g, CoCl
26H
2O7.5mg, (NH
4)
2SO
40.1g, CaCO
31g, K
2HPO
43H
2O0.3g, ZnSO
47H
2O2mg, adding distil water transfer pH to 7.0-7.2 to 1L.
From the fermentation result of Fig. 4 as can be seen, frr gene crossing in GB-165 expressed and can be promoted the synthetic of Avrmectin equally, and the frr gene pairs Avrmectin synthetic promoter action of high copy is better than the frr gene of low copy.
Equally, the abamectin fermented unit of two kinds of transformants that contains frr gene self promotor is all a little more than two kinds of transformants that contain erythromycin resistance gene strong promoter (ermE*p), shows that once more erythromycin resistance gene strong promoter (ermE*p) is not better than self promotor of frr gene when expressing frr.
Three, the fermentation research of 76-02-e and transformant thereof
Shake flask fermentation and HPLC analytical procedure are with " one " in the present embodiment, and difference is that the spore of 76-02-e is not abundant, and the seed culture time is 40-46 hour, used fermention medium is fermention medium E: W-Gum 120g, groundnut meal 26g, soybean cake powder 2g, yeast powder 8g, (NH
4)
2SO
40.25g, CoCl
26H
2O25mg, MnSO
44H
2O30mg adds tap water to 1L, the pH nature.
The fermentation result of Fig. 5 shows, in superior strain 76-02-e, expresses the frr gene and still can promote the synthetic of Avrmectin, and be the frr gene that the high frr gene pairs microbiotic synthetic promoter action that copies is better than low copy equally.
Different with the GB-165 transformant with ATCC31267 is, the output that contains Avrmectin in the 76-02-e transformant of erythromycin resistance gene strong promoter ermE*p may be because 76-02-e is different with ATCC31267 and GB-165 on the frr gene expression regulation a little more than the transformant that contains frr gene self promotor.
The study on the stability of embodiment 4, engineering strain
The engineering strain of constructed frr gene overexpression is transferred five times on the YMS inclined-plane continuously, find that this bacterial strain is identical on morphological specificity and cultural characteristic with starting strain, growth conditions is good, and proterties is stable.Engineering strain through transferring after five times carries out shake flask fermentation again, and considerable change does not all take place HPLC check Avrmectin output, illustrate that the engineering bacteria of structure is stable.
Embodiment 5, frr gene overexpression are to the influence of thalli growth
Present embodiment has compared the changing conditions of frr gene overexpression strains A TCC31267 (pFR1-1139) and starting strain ATCC31267 Avrmectin output and dry cell weight in the shake flask fermentation process.
The HPLC analytical procedure of shake flask fermentation and Avrmectin is with " one " among the embodiment 3, difference is to select for use the fermention medium II fermentation culture thalline of solubility, this is owing to contain insoluble components in the Avid kyowamycin fermention medium, is unfavorable for the accurate mensuration of dry cell weight.Different time is taken a sample, and measures the fermentation unit (Fig. 6) and the dry cell weight (Fig. 7) of Avrmectin respectively.
Fermention medium II: Zulkovsky starch 70g, yeast enzymolysis powder 16g, MgSO
47H
2O0.5g, K
2HPO
43H
2O0.5g, KCl4g, CoCl
26H
2O5mg, adding distil water transfer pH to 7.0-7.2 to 1L.
The mensuration of dry cell weight: with the 50ml filtering fermentation liquor, thalline is cleaned with distilled water, dries to constant weight, claims dry cell weight.
By Fig. 6 and Fig. 7 as can be known, Avrmectin output and the dry cell weight of ATCC31267 (pFR1-1139) are higher than ATCC31267 always, show that expression frr gene can promote thalli growth.As seen, in the frr gene overexpression bacterial strain raising reason of Avrmectin output some give the credit to the raising of thalline biomass in the fermented liquid, some reason may be the expression amount that the overexpression of frr gene has improved positive regulating gene aveR in the Avrmectin biological synthesis gene cluster and Avrmectin biosynthesizing relevant enzymes, but this supposition awaits further confirming by experiment.