CN103233034A - Method for establishing genetic engineering strain for producing single component tetracycline - Google Patents

Method for establishing genetic engineering strain for producing single component tetracycline Download PDF

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CN103233034A
CN103233034A CN2013101432169A CN201310143216A CN103233034A CN 103233034 A CN103233034 A CN 103233034A CN 2013101432169 A CN2013101432169 A CN 2013101432169A CN 201310143216 A CN201310143216 A CN 201310143216A CN 103233034 A CN103233034 A CN 103233034A
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tsiklomitsin
gene
engineering strain
plasmid
ctcp
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CN103233034B (en
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由德林
朱涛
邓子新
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Shanghai Jiaotong University
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Abstract

The invention relates to a method for establishing a genetic engineering strain for producing a single component tetracycline in the technical field of biological medicine. The method comprises a step of performing gene disruption of the related genes involved in aureomycin synthesis and chlorination in aureomycin producing bacteria so as to obtain a genetic engineering strain only producing tetracycline. According to the method provided by the invention, a homologous exchange plasmid is established, and DNA (deoxyribonucleic acid) homologous recombination is performed in the aureomycin producing bacteria; and the chlorination enzyme gene ctcP in charge of the chloro step is replaced by a spectinomycin resistance gene in an aureomycin synthesis process so as to disrupt the chloro step and obtain the genetic engineering strain only producing tetracycline. The strain can be applied to industrial production and has the obvious effects that (I) the strain ZTO4 obtained by genetic engineering reform only produces a single component tetracycline, and the output is as high as 18.9g/L; and (II) compared with the current fermentation technology, potassium bromide is not added as an inhibitor in a fermentation process, thus the production cost is saved, the technological flow is simplified, and industrial pollution caused by potassium bromide is avoided.

Description

Produce the construction process of the engineering strain of tsiklomitsin one-component
Technical field
The present invention relates to a kind of construction process that produces the engineering strain of tsiklomitsin one-component, belong to the biological medicine technology field.
Background technology
Tetracycline antibiotics is the synthetic large class Broad spectrum antibiotics of anti-bacteria albumen, all effective to multiple gram-positive and negative bacterium, rickettsiae, Mycoplasma and spirochete etc.Comprise first-generation tetracycline antibiotics tsiklomitsin, duomycin and terramycin, they are respectively by the streptomyces aureofaciens (Streptomyces aureofacines) of main pan mycin (chlortetracycline) and streptomyces rimosus (Streptomyces rimosus) the fermentation generation of producing terramycin (oxytetracycline).20 century 70s start to apply semi-synthetic technology tsiklomitsin and terramycin have been carried out to chemically modified, obtained the MINOCYCLINE HCL that s-generation tetracycline antibiotics is parent nucleus as 7-chloro-6-demecycline, doxycycline and the terramycin of take, these semi-synthetic derivatives that improve the property of medicine have promoted the development of tetracycline antibiotics.FDA (Food and Drug Adminstration) (FDA) approval Tigecycline listing in 2005, indicate the birth of third generation tsiklomitsin as the antibiotic listing of glycylcycline class of representative.Wherein first-generation tetracycline antibiotics tsiklomitsin, duomycin and terramycin are widely used in medicine, herding and water industry because of wide spectrum, easy to use, economic dispatch characteristics, and the world market demand is huge.At present, on industrial production, with the streptomyces aureofaciens that produces duomycin, produce tsiklomitsin, in substratum, add certain density Potassium Bromide, suppress the generation of duomycin and produce tsiklomitsin.The method of this interpolation Potassium Bromide fermentative production tsiklomitsin, not only increased technical process and production cost, and brought serious pollution problem.
Going deep into along with molecular genetics and chemicobiology research in recent years, can utilize metabolic engineering (metabolic engineering), the biosynthetic pathway of genetic modification natural product obtains the gene recombination bacterial strain specifically, thereby direct fermentation produces needed microbiotic and analogue (Keasling JD.Manufacturing molecules through metabolic engineering.Science.2010,330:1355-1358.) thereof.With chemosynthesis, compare, the purpose meta-bolites can be produced in a large number by the recombinant bacterial strain fermentation obtained, thereby can reduce production costs and environmental contamination reduction.Due to streptomyces aureofaciens genetic manipulation difficulty, biosynthetic research to tetracycline antibiotics can only be confined to the random mutation strain, so greatly limited tetracyclines biosynthesizing research and utilized metabolic engineering to carry out specifically the work of genetic modification aspect.
Through to the prior art literature search, do not find to change by knocking out the chlorination genes involved correlation technique report of bacterial classification secondary metabolites component and output.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of construction process that produces the engineering strain of tsiklomitsin one-component is provided.Method kind of the present invention has been carried out gene knockout by the chlorination genes involved, thereby only produce the method for the bacterial classification of tsiklomitsin while obtaining fermentation, not only tsiklomitsin output is high to adopt such strain fermentation, and need not add during the fermentation Potassium Bromide as inhibitor, saved production cost, simplify technical process, avoided industrial pollution.
The present invention is achieved by the following technical solutions, and the construction process of the engineering strain of the generation tsiklomitsin one-component the present invention relates to comprises the steps:
Duomycin is produced to the synthesizing chlorinated genes involved of participation duomycin in bacterium and carried out the gene interruption, and then obtain only producing the engineering strain of tsiklomitsin.
Preferably, described duomycin generation bacterium is streptomyces aureus (Streptomyces aureofaciens) F3CCTCCNO:M2013080.
Preferably, the synthesizing chlorinated genes involved of described participation duomycin is chlorination enzyme ctcP gene.
Preferably, described method comprises the steps:
Step 1, adopt spectinomycin resistance gene to build the homology exchange plasmid that chlorination enzyme gene ctcP interrupts;
Step 2, proceed to described homology exchange plasmid in streptomyces aureus, carries out the DNA homology restructuring, and then obtain producing the engineering strain of tsiklomitsin one-component.
Preferably, in step 1, the coemid pJTU4301 that described homology exchange plasmid is the ctcP sudden change.
Preferably, in step 1, described proceed to comprise the steps: by the homology of structure exchange plasmid by between intestinal bacteria and streptomyces aureus parents, carry out conjugal transfer, and then homology exchanged to plasmid proceed to streptomyces aureus.
Preferably, described intestinal bacteria are E.coli ET12567, and it carries plasmid pUZ8002.
Preferably, described construction process comprises the steps:
Step 1, adopt spectinomycin resistance gene to build the homology exchange plasmid that chlorination enzyme ctcP gene interrupts;
Utilize CopyControl tMfosmid Library Production test kit builds Streptomyces aureofaciens F3 genomic library, determine that according to chlB4 gene design probe coemid 11D1 comprises complete CTC synthetic gene bunch, by coemid 11D1, bacillus coli gene restructuring by λ-Red mediation, chlorination enzyme gene ctcP is suddenlyd change, obtain plasmid pJTU4301;
By plasmid pIJ778 double digestion, reclaim the 1.5kb fragment that comprises oriT+addA with EcoRI/HindIII, this fragment transforms intestinal bacteria DH10B, can not grow single bacterium colony on the spectinomycin resistant panel, is qualified;
Using the fragment of recovery as template, with primer targPF/targPR amplification, reclaim the PCR product of 1.5kb;
Transform 11D1 is imported to E.coli BW25113 competent cell by electricity, and preparation E.coli BW25113/11D1 Electroporation-competent cells, the electricity consumption of 1.5kb fragment is transformed and imports wherein, by homologous fragment and the 11D1 restructuring in 39bp on the PCR primer pair and the ctcP gene outside, the CTC synthetic gene bunch that the coemid pJTU4301 after double exchange comprises the ctcP sudden change;
Step 2, proceed to described homology exchange plasmid in streptomyces aureus, carries out the DNA homology restructuring, and then obtain producing the engineering strain of tsiklomitsin one-component;
Coemid pJTU4301, by the conjugal transfer between intestinal bacteria and streptomycete parents, is proceeded to streptomyces aureus by constructed plasmid;
The coemid pJTU4301 of ctcP sudden change must could import in acceptor streptomyces aureofaciens cell by conjugal transfer under the assistance of helper plasmid pUZ8002;
PJTU4301 is Transformed E .coli ET12567 at first, and incubated overnight under paraxin, spectinomycin and kantlex exist, cultivate 2 hours according to 1/10 inoculum size switching, collects thalline, washs thalline 3 times with fresh LB substratum standby; Streptomycete spore as acceptor need be processed through heat shock and pre-the sprouting;
The streptomycete spore is suspended in the TES damping fluid, and heat shock 10min in 50 ℃ of water-baths, add the pre-germination medium of equal-volume 2 * spore after being cooled to room temperature, and 37 ℃ of shaking tables are cultivated 2h, and centrifugal collection spore also evenly is suspended in appropriate LB substratum again, by 10 8: 10 8with after the Bacillus coli cells balanced mix, be coated on culture plate, carry out bacterium parents conjugal transfer;
Cover flat board with the 1ml sterilized water containing nalidixic acid and spectinomycin after 16 hours, put 30 ℃ of cultivations and can see transconjugant after 7～10 days; Finally obtain only producing the engineering strain of tsiklomitsin.
The present invention has following beneficial effect: the present invention is by building homology exchange plasmid, proceeding to streptomyces aureus F3(Streptomyces aureofaciens) F3 carries out the DNA homology restructuring, the chlorination enzyme ctcP gene of being responsible for the chloro step in the duomycin building-up process is substituted with spectinomycin resistance gene, thereby interrupt the chloro step, method of the present invention has successfully been carried out chlorination enzyme gene knockout, obtain only producing the genetic engineering bacterium of tsiklomitsin, this bacterial strain can be used in industrial production, and there is significant effect: first, this genetic engineering modified resulting bacterial strain ZT04 only produces the tsiklomitsin one-component, its output is up to 18.9g/L, secondly, during the fermentation, need not add Potassium Bromide as inhibitor, save production cost, simplify technical process, avoid the Potassium Bromide industrial pollution.
The streptomyces aureus F3(Streptomyces aureofaciens the present invention relates to) F3 was preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University on March 13rd, 2013; Postcode: 430072; Deposit number is CCTCC NO:M2013080.
The accompanying drawing explanation
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
The chemical structure of Fig. 1 tsiklomitsin and duomycin;
Fig. 2 chlorination enzyme gene interrupts the PCR checking of flow process and mutant strain ZT04;
Fig. 3 mutant strain ZT04 and wild type strain F3 fermentation production HPLC detected result;
Fig. 4 mutant strain ZT04 and wild type strain F3 tunning MS detected result, a left side is wild-type, the right side is mutant strain.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
In following examples, described intestinal bacteria E.coli ET12567(pUZ8002) be published in " Paget, M.S., Chamberlin, L., Atrih, A., Paget, M.S.et.al.Evidence that the extracytoplasmic function sigma factor σ eis required for normal cell wall structure in Streptomyces coelicolor A3 (2) .J.Bacteriol, 1999,181:204-211 ";
The plasmid used in following examples and reagent all can obtain by disclosed commercially available channel.
the structure of embodiment 1, homology exchange plasmid
For import the host and and karyomit(e) generation homologous recombination so as the acquisition targeted mutagenesis, be specially and utilize CopyControl tMfosmid Library Production test kit builds Streptomyces aureofaciens F3 genomic library, determine that according to chlB4 gene design probe coemid 11D1 comprises complete CTC synthetic gene bunch, by coemid 11D1, bacillus coli gene restructuring by λ-Red mediation, chlorination enzyme gene ctcP is suddenlyd change, obtain plasmid pJTU4301.At first, with EcoRI/HindIII, by plasmid pIJ778 double digestion, reclaim the 1.5kb fragment that comprises oriT+addA, this fragment transforms intestinal bacteria DH10B, can not grow single bacterium colony on the spectinomycin resistant panel, is qualified.Using the fragment of recovery as template, with primer targPF/targPR amplification, reclaim the PCR product of 1.5kb left and right.Next, transform 11D1 is imported to E.coli BW25113 competent cell by electricity, and preparation E.coliBW25113/11D1 Electroporation-competent cells, the electricity consumption of 1.5kb fragment is transformed and imports wherein, by homologous fragment and the 11D1 restructuring in 39bp on the PCR primer pair and the ctcP gene outside, the CTC synthetic gene bunch that the coemid pJTU4301 after double exchange comprises the ctcP sudden change.
targPF,5'-CGTCAAGTCGGTCTGAAGGAAAAGGAGTTCGACATGATTCCGGGGATCCGTCGA-3'（SEQ?IDNO.1）
targPR,5'-CCGTCCCGCGGACGCTGCTCGCCGTTGCCCCTCGCGCTATGTAGGCTGGAGCTGCTTC-3'（SEQ?ID?NO.2）
embodiment 2, for and the plasmid of karyomit(e) generation homologous recombination import the wild-type host
The plasmid of structure, by the conjugal transfer between intestinal bacteria and streptomycete parents, is proceeded to streptomyces aureus (streptomyces aureus F3(Streptomyces aureofaciens) F3 by constructed plasmid).The coemid pJTU4301 of ctcP sudden change must could import in acceptor streptomyces aureofaciens cell by conjugal transfer under the assistance of helper plasmid pUZ8002.PJTU4301 at first Transformed E .coli ET12567(carries pUZ8002), incubated overnight under paraxin, spectinomycin and kantlex exist, cultivate 2 hours according to 1/10 inoculum size switching, collects thalline, washs thalline 3 times with fresh LB substratum standby.Streptomycete spore as acceptor need be processed through heat shock and pre-the sprouting.The streptomycete spore is suspended in TES damping fluid (5ml0.05mol/L, pH8.0), and heat shock 10min in 50 ℃ of water-baths, add the pre-germination medium of equal-volume 2 * spore (Difco yeast powder 1%, Difco casamino acids 1%, CaCl after being cooled to room temperature 20.01mol/L), 37 ℃ of shaking tables (250rpm) are cultivated 2h, and centrifugal collection spore also evenly is suspended in appropriate LB substratum again, by 10 8: 10 8go up with being coated in culture plate (2% agarose, 2% N.F,USP MANNITOL, 2% soybean cake powder, pH7.2～7.5) after the Bacillus coli cells balanced mix, carry out bacterium parents conjugal transfer.Cover dull and stereotyped (final concentration: nalidixic acid 50ng/mL with the 1ml sterilized water containing nalidixic acid (suppressing colibacillary growth) and spectinomycin (importing plasmid pJTU4301 with this resistance) after 16 hours; Spectinomycin 50ng/mL), put 30 ℃ of cultivations and can see transconjugant after 7-10 days.
the screening of embodiment 3, mutant strain and checking
Select single conjugal transfer from wrapper plate and be inoculated into further confirmation resistance on resistance (spectinomycin) flat board, and amplify dull and stereotyped the cultivation.Prepare against the total DNA of roguing as pcr template; Primer targPCF and TargPCR are for the screening of mutant strain, and original starting strain DNA in contrast.Mutant strain ctcP gene complete encoding sequence is replaced by the spectinomycin resistance gene sequence, and PCR product size is 1601bp, and starting strain PCR product size is 1905bp, thereby has finally proved conclusively the exactness of targeted mutagenesis.
targPCF,5'-ACCACTTCGCCATCGGCGTCAAGTC-3'；（SEQ?ID?NO.3）
targPCR,5'-CGAGGTGCCCAGCCCGCTGATGTAC-3'；（SEQ?ID?NO.4）
the fermentation culture of embodiment 4, mutant strain, antibiotic separation and purification and product detect
Described streptomyces aureus is preserved bacterial classification inoculation oat flat board with 20% glycerine, the dull and stereotyped concrete formula of oat: 3.4% (w/v), MgSO 40.005%, KH 2pO 40.01%, (NH4) 2hPO 40.015%, all add the spectinomycin that corresponding final concentration is 30 μ g/mL in described mutant strain oat medium.Within 5-8 days, collect spore with 20% glycerine afterwards, get appropriate inoculation 30mL seed culture medium, container is the triangular flask of 250mL with spring, and 30 ℃ of shaking tables are cultivated after 24 hours by 5% inoculum size inoculation 100mL fermention medium, container is the triangular flask of 500mL with spring, and 30 ℃ of shaking tables are cultivated 4-5 days.
The seed culture based formulas is (w/v): W-Gum 4.0%, soybean cake powder 2.0%, yeast powder 0.5%, peptone 0.5%, calcium carbonate 0.4%, ammonium sulfate 0.3%, sodium-chlor 0.2%, sal epsom 0.025%, potassium primary phosphate 0.025%, autoclaving.
Fermentative medium formula is (w/v): W-Gum 8.0%, soybean cake powder 4.0%, yeast powder 0.1%, peptone 1.4%, corn steep liquor 0.8%, calcium carbonate 0.7%, ammonium sulfate 0.35%, sodium-chlor 0.25%, sal epsom 0.025%, add soya-bean oil by the 1.5mL/30-35mL volume ratio, autoclaving after packing.
Fermented liquid oxalic acid acidifying, regulate pH to 1.5-2.0, and the centrifugal 10min of 5000rpm/min collects supernatant liquor frozen in-20 ℃ of lucifuges, in order to next step detection.
The HPLC isolation identification of duomycin and tsiklomitsin is carried out on the Aglient1100series of Agilent company, and separating pillar is the reverse post of glient TC C18 (5.0 μ m, 4.6mm * 250mm); Mobile phase A, 0.02M oxalic acid/0.01M triethylamine (TEA, pH2.0), B, acetonitrile.Sample is loading after 0.45 μ m membrane filtration, and with under 20% acetonitrile Isocratic clution, flow velocity is 1.0mL/min, column temperature keeps room temperature, detects wavelength 360nm, and duomycin and tsiklomitsin retention time are respectively 6.9 minutes, 15 minutes, the two can finely separate, as shown in Figure 2.
The ZT04 mutant strain of starting strain streptomyces aureus and acquisition is fermented simultaneously, the separation and purification fermented liquid, same batch is carried out the HPLC detection.Shown in accompanying drawing 3, fermentation results shows, streptomyces aureus mutant strain ZT04, and the pan mycin, still do not accumulate a large amount of tsiklomitsins, and its output is up to 18.9g/L.
High pressure liquid chromatography-mass spectrometry (LC/MS) is to carry out on the Agilent1100series of Agilent company LC/MSD Trap system.Separating pillar is the reverse post of glient TC C18 (5.0 μ m, 4.6mm * 250mm); Mobile phase A, 0.02M oxalic acid/0.01M triethylamine/1% formic acid (TEA, pH2.0), B, acetonitrile.Sample is loading after 0.45 μ m membrane filtration, and with under 30% acetonitrile Isocratic clution, flow velocity is 0.3mL/min, and column temperature keeps room temperature, detects wavelength 360nm.
Mass spectrum working conditions: negative ion mode; Drying air stream: 10L/min; Atomizer pressure: 50psi; Dry temperature: 350 ℃; Bombarding voltage: 1.0-1.8V.
By LC-MS, tunning is checked, the product of further having proved conclusively accumulation is tsiklomitsin, the results are shown in Figure 4, and a left side is wild-type, and the right side is mutant strain.
Obviously, fermentation process of the present invention, during the fermentation, need not add Potassium Bromide as inhibitor, saved production cost, simplified technical process, avoided the Potassium Bromide industrial pollution.
In sum, the present invention is by building homology exchange plasmid, proceeding to streptomyces aureus F3(Streptomycesaureofaciens) F3 carries out the DNA homology restructuring, the chlorination enzyme ctcP gene of being responsible for the chloro step in the duomycin building-up process is substituted with spectinomycin resistance gene, thereby interrupt the chloro step, method of the present invention has successfully been carried out chlorination enzyme gene knockout, obtains only producing the genetic engineering bacterium of tsiklomitsin, this bacterial strain can be used in industrial production, and has significant effect.
Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.
Figure IDA00003090934900021

Claims (8)

1. a construction process that produces the engineering strain of tsiklomitsin one-component, it is characterized in that, comprise the steps: that duomycin is produced to the synthesizing chlorinated genes involved of participation duomycin in bacterium has carried out the gene interruption, and then obtain only producing the engineering strain of tsiklomitsin.
2. the construction process of the engineering strain of generation tsiklomitsin one-component as claimed in claim 1, is characterized in that, it is streptomyces aureus (Streptomyces aureofaciens) F3CCTCC NO:M2013080 that described duomycin produces bacterium.
3. the construction process of the engineering strain of generation tsiklomitsin one-component as claimed in claim 2, is characterized in that, the synthesizing chlorinated genes involved of described participation duomycin is chlorination enzyme ctcP gene.
4. the construction process of the engineering strain of generation tsiklomitsin one-component as claimed in claim 3, is characterized in that, described method comprises the steps:
Step 1, adopt spectinomycin resistance gene to build the homology exchange plasmid that chlorination enzyme ctcP gene interrupts;
Step 2, proceed to described homology exchange plasmid in streptomyces aureus, carries out the DNA homology restructuring, and then obtain producing the engineering strain of tsiklomitsin one-component.
5. the construction process of the engineering strain of generation tsiklomitsin one-component as claimed in claim 4, is characterized in that, in step 1, and the coemid pJTU4301 that described homology exchange plasmid is the ctcP sudden change.
6. the construction process of the engineering strain of generation tsiklomitsin one-component as claimed in claim 4, it is characterized in that, in step 1, described proceed to comprise the steps: by the homology of structure exchange plasmid by between intestinal bacteria and streptomyces aureus parents, carry out conjugal transfer, and then homology exchanged to plasmid proceed to streptomyces aureus.
7. the construction process of the engineering strain of generation tsiklomitsin one-component as claimed in claim 6, is characterized in that, described intestinal bacteria are E.coli ET12567, and it carries plasmid pUZ8002.
8. the construction process of the engineering strain of generation tsiklomitsin one-component as claimed in claim 6, is characterized in that, described construction process comprises the steps:
Step 1, adopt spectinomycin resistance gene to build the homology exchange plasmid that chlorination enzyme ctcP gene interrupts;
Utilize CopyControl tMfosmid Library Production test kit builds Streptomyces aureofaciens F3 genomic library, determine that according to chlB4 gene design probe coemid 11D1 comprises complete CTC synthetic gene bunch, by coemid 11D1, bacillus coli gene restructuring by λ-Red mediation, chlorination enzyme gene ctcP is suddenlyd change, obtain plasmid pJTU4301;
By plasmid pIJ778 double digestion, reclaim the 1.5kb fragment that comprises oriT+addA with EcoRI/HindIII, this fragment transforms intestinal bacteria DH10B, can not grow single bacterium colony on the spectinomycin resistant panel, is qualified;
Using the fragment of recovery as template, with primer targPF/targPR amplification, reclaim the PCR product of 1.5kb;
Transform 11D1 is imported to E.coli BW25113 competent cell by electricity, and preparation E.coli BW25113/11D1 Electroporation-competent cells, the electricity consumption of 1.5kb fragment is transformed and imports wherein, by homologous fragment and the 11D1 restructuring in 39bp on the PCR primer pair and the ctcP gene outside, the CTC synthetic gene bunch that the coemid pJTU4301 after double exchange comprises the ctcP sudden change;
Step 2, proceed to described homology exchange plasmid in streptomyces aureus, carries out the DNA homology restructuring, and then obtain producing the engineering strain of tsiklomitsin one-component;
Coemid pJTU4301, by the conjugal transfer between intestinal bacteria and streptomycete parents, is proceeded to streptomyces aureus by constructed plasmid;
The coemid pJTU4301 of ctcP sudden change must could import in acceptor streptomyces aureofaciens cell by conjugal transfer under the assistance of helper plasmid pUZ8002;
PJTU4301 is Transformed E .coli ET12567 at first, and incubated overnight under paraxin, spectinomycin and kantlex exist, cultivate 2 hours according to 1/10 inoculum size switching, collects thalline, washs thalline 3 times with fresh LB substratum standby; Streptomycete spore as acceptor need be processed through heat shock and pre-the sprouting;
The streptomycete spore is suspended in the TES damping fluid, and heat shock 10min in 50 ℃ of water-baths, add the pre-germination medium of equal-volume 2 * spore after being cooled to room temperature, and 37 ℃ of shaking tables are cultivated 2h, and centrifugal collection spore also evenly is suspended in appropriate LB substratum again, by 10 8: 10 8with after the Bacillus coli cells balanced mix, be coated on culture plate, carry out bacterium parents conjugal transfer;
Cover flat board with the 1ml sterilized water containing nalidixic acid and spectinomycin after 16 hours, put 30 ℃ of cultivations and can see transconjugant after 7～10 days; Finally obtain only producing the engineering strain of tsiklomitsin.
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CN110129244A (en) * 2019-01-23 2019-08-16 河北圣雪大成制药有限责任公司 Streptomycete chassis bacterial strain and its construction method, the application in heterogenous expression research
CN110129244B (en) * 2019-01-23 2023-09-15 河北圣雪大成制药有限责任公司 Streptomyces chassis strain, construction method thereof and application thereof in heterologous expression research
CN111647544A (en) * 2020-06-04 2020-09-11 上海交通大学 Genetic engineering strain for producing demethylated aureomycin and demethylated tetracycline and construction method thereof

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