CN105907778A - Streptomyces gilvosporeus recombinant expression plasmid, and engineering bacterium and application thereof - Google Patents

Streptomyces gilvosporeus recombinant expression plasmid, and engineering bacterium and application thereof Download PDF

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CN105907778A
CN105907778A CN201610203779.6A CN201610203779A CN105907778A CN 105907778 A CN105907778 A CN 105907778A CN 201610203779 A CN201610203779 A CN 201610203779A CN 105907778 A CN105907778 A CN 105907778A
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pime
vgb
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刘浩
孙春杰
何希宏
王海霞
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Tianjin University of Science and Technology
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Abstract

The invention relates to a Streptomyces gilvosporeus recombinant expression plasmid, and an engineering bacterium and application thereof. An expression vector pIMEP::pimE::pimM::vgb is successfully constructed by cloning two genes (pimM and pimE) with positive regulatory effect and in the natamycin biosynthesis gene cluster of Streptomyces gilvosporeus and an exogenous ferrohemoglobin gene vgb. A strain of the Streptomyces gilvosporeus engineering bacterium is obtained through a conjugative transfer experiment of Streptomyces gilvosporeus and Escherichia coli ETZ with the expression vector pIMEP::pimE::pimM::vgb. Through the superimposed effect of overexpression of the regulatory gene pimM and the cholesterol oxidase gene pimE in the natamycin biosynthesis gene cluster of the Streptomyces gilvosporeus engineering bacterium and dysoxia resistance of the ferrohemoglobin gene vgb, transcription of the natamycin biosynthesis gene cluster can be improved; and thus, the output of natamycin can be substantially improved.

Description

Brown yellow spore streptomycete recombinant expression plasmid and engineering bacteria and application
Technical field
The present invention relates to gene engineering technology field, the most brown yellow spore streptomycete recombinant expression plasmid and work Journey bacterium and application.
Background technology
Natamycin is a kind of polyene macrolides broad-spectrum antifungals.It can effectively suppress and kill Mycete, yeast, filamentous fungi, therefore can be effectively reduced strong pathogenic fungus element and invade the mankind Evil.Natamycin is extremely low to the toxicity of mammalian cell compared with other antimicrobial components, can extensively answer For fungus-caused disease.Owing to natamycin is nontoxic, the not spy such as teratogenesis, harmless noresidue Property, nineteen eighty-two, the food preservative purposes of U.S. FDA official approval natamycin.China's Ministry of Public Health in The food preservative qualification of also official approval natamycin in 1997.Nowadays, natamycin the most extensively quilt It is applied to food service industry, additionally, natamycin is also widely used in the fields such as medical treatment, feedstuff, grain storage.
At present, at streptomyces (Streptomyces), brevibacterium (Brevibacterium), false list The antibacterials such as born of the same parents Pseudomonas (Pseudomonas), Rhod (Schizopylium) are all found to have cholesterol Oxidase.In natamycin biological synthesis gene cluster, pimE encodes cholesterol oxidase.At present the most from Isolating cholesterol oxidase gene from rhodococcus equi in streptomyces, it is solid that the cholesterol oxidase of its coding belongs to acetyl gallbladder The member of alcohol oxidase family, can be catalyzed cholesterol and form 17-KS and hydrogen peroxide.2007 Marta et al. is found with complementation test by gene knockout, in Streptomyces natalensis, and pimE coding one Plant functional cholesterol oxidase, and this enzyme is relevant to the biosynthesis of natamycin.Natamycin Biosynthetic process is not required to the participation of cholesterol, but passes through in the engineered strain knocking out pimE, When adding the sterin oxidase of PimE albumen after purification or allos, all it is remarkably improved the product of natamycin Amount.Therefore Marta et al. speculates that cholesterol oxidase is that the one in natamycin biosynthesis is secreted into born of the same parents Outer signal protein, plays positive regulating and controlling effect to the biosynthesis of natamycin.Zhejiang University Sun Zhi person of outstanding talent discusses The article pointed out, cholesterol oxidase gene from rhodococcus equi also has similar effect in streptomyces chatanoogensis, and pushes away Survey cholesterol oxidase gene from rhodococcus equi and two controlling gene pimR and pimM may to natamycin biosynthesis There is coordinated regulation effect.
The controlling gene of natamycin biological synthesis gene cluster is pimR and pimM, is separately encoded two and transcribes The factor.In Streptomyces natalensis (Streptomyces natalensis), PimM gene is to receive that he is mould The downregulation factor in element biological synthesis gene cluster.PimM is as PAS modulin, it is possible to activate base In cause bunch, portion gene transcribes.In Streptomyces natalensis, pimM knock-out bacterial strain loses natamycin Synthesis capability, increase pimM gene copy can improve natamycin yield.Zhejiang University Sun Zhi person of outstanding talent Paper is pointed out, in streptomyces chatanoogensis (Streptomyces chattanoogensis), and receives him The controlling gene pimM functionally similar controlling gene ScnRII of mycin biological synthesis gene cluster is just similarly Controlling gene, and this gene is likely to also in conjunction with eight bases in natamycin biological synthesis gene cluster The promoter region of cause.In commercial production, by directly increasing the copy of approach specific regulatory control gene Number improves the method for antibiotic yield and extensively applies at structure high yield streptomyces strain.
Brown yellow spore streptomycete is aerobic bacteria, and along with longer fermentation times, cell density increases sharply.Fermentation During, dissolved oxygen can not make thalli growth slow completely, and natamycin synthesis key enzyme expression reduces, and enters And make natamycin yield reduce.Research finds, VHb expression under oxygen lean conditions, can remarkably promote big Enterobacteria and the growth of brewing yeast cell, improve protein synthesis capacity, increases the yield of purpose product. Additionally, vgb gene is also applied in the microorganisms such as bacillus cereus, Erwinia, product cephalosporium chrysogenum, become Merit improves amylase, vitamin C, the yield of the biogenetic products such as cephalosporin.Also have been reported that this base Also succeed because importing plant is expressed.Therefore this gene is imported to brown yellow spore streptomycete by the present invention equally In, make engineered strain obtain resistance to oxygen deprivation ability by the expression of this gene, and then improve natamycin Yield.
Additionally, according to what gorgeous tinkling of pieces of jade " fed-batch fennentation improves natamycin yield ", at the beginning of making Beginning concentration of glucose 40g/L, interval mends sugar makes concentration of glucose maintain 2%, can make natamycin yield 2.3g/L is brought up to from 1.5g/L.The happy report of Zhejiang University's beam scape " educate by natamycin superior strain space Kind, process optimization and industry amplify research ", by relatively more different control conditions, on 30L fermentation-scale Optimum control condition be: the middle pH value of sweat is 5.8-6.2, and omnidistance speed of agitator is 400rpm, Ventilation is 1.0vvm, and temperature is 28 DEG C.Brown yellow spore streptomycete is aerobic bacteria, therefore at its sweat In can by provide rotating speed improve dissolved oxygen meet the thalline requirement to dissolved oxygen.During the fermentation, how Control fermentation condition equally to the yield important in inhibiting improving natamycin.
Summary of the invention
The technical problem to be solved is to provide a kind of brown yellow spore streptomycete recombinant expression plasmid.
Another technical problem to be solved by this invention is to provide containing above-mentioned brown yellow spore streptomycete restructuring table Reach the engineering bacteria of plasmid.
Another technical problem to be solved by this invention is to provide the application of above-mentioned engineering bacteria.
For solving above-mentioned technical problem, the technical scheme is that
A kind of brown yellow spore streptomycete recombinant expression plasmid, is by controlling gene pimM, cholesterol oxidase base Because pimE and external source hemoglobin gene vgb inserts in the carrier pIMEP containing erythromycin promoter respectively Obtain recombinant expression carrier pIMEP::pimE::pimM::vgb, recombinant expression carrier The sequence of pIMEP::pimE::pimM::vgb is sequence shown in sequence table<400>17；Described recombinant expressed Plasmid is the expressing in series of three genes, all has erythromycin promoter, the core of described pimM before each gene Nucleotide sequence is sequence shown in sequence table<400>1, and the nucleotides sequence of pimE is classified as shown in sequence table<400>2 Sequence, the nucleotides sequence of vgb is classified as sequence shown in sequence table<400>3, pimE/pimM/vgb and promoter Sequence be sequence shown in sequence table<400>16；Wherein, described controlling gene pimM and the carrier that sets out PIMEP BamH I/EcoR I carries out enzyme action, pimE and the carrier pIMEP BamH I that sets out and carries out enzyme Cutting, vgb and the carrier pIMEP Kpn I that sets out carries out enzyme action；Containing erythromycin promoter and controlling gene (Perm::pimM) fragment and the carrier pIMEP::pimE EcoR I that sets out carry out enzyme action, containing red The fragment of mycin promoter and hemoglobin gene (Perm::vgb) and the carrier that sets out PIMEP::pimE::pimM Mun I/Spe I carries out enzyme action.
Preferably, above-mentioned brown yellow spore streptomycete recombinant expression plasmid, described controlling gene pimM, cholesterol The promoter of lysyloxidase gene pimE and external source hemoglobin gene vgb is erythromycin promoter ermE*(Perm)。
A kind of engineering bacteria (Streptomyces containing above-mentioned brown yellow spore streptomycete recombinant expression plasmid Gilvosporeus/pIMEP::pimE::pimM::vgb), preserving number is CGMCC No.11790.
The genome of above-mentioned engineering bacteria is integrated and has recombinant expression carrier pIMEP::pimE::pimM::vgb, Having the ability of high yield natamycin, the yield of fermentation 120h reaches 10.136 grams.
The construction method of above-mentioned engineering bacteria, specifically comprises the following steps that
Above-mentioned brown yellow spore streptomycete recombinant expression plasmid is first changed and proceeds to, in escherichia coli ETZ, utilize brown Huang Spore Streptoinyces-E. coli Conjugative tiansfer is tested, by brown yellow spore streptomycete spore suspension with containing restructuring table The recombination bacillus coli ETZ bacterium solution mixing reaching carrier pIMEP::pimE::pimM::vgb is applied to MS and (contains 5mM MgCl2) flat board, cultivate 16-20h in 28 DEG C of incubators, coating 1ml sterilized water is (containing naphthyridines afterwards Keto acid 0.4mg and A Pu mycin 1.5mg) screen；In 28 DEG C continue cultivations can obtain containing The brown yellow spore streptomycete engineered strain of recombinant expression carrier pIMEP::pimE::pimM::vgb.
Above-mentioned brown yellow spore streptomycete recombinant expression plasmid is the regulation and control containing natamycin biological synthesis gene cluster Gene pimM and cholesterol oxidase gene from rhodococcus equi pimE and the recombinant expression carrier of exogenous gene vgb pIMEP::pimE::pimM::vgb。
Preferably, the construction method of above-mentioned engineering bacteria, utilize brown yellow spore streptomycete engineered strain to contain A Pu Chloramphenicol resistance, screens transformant on the SS flat board containing A Pu chloramphenicol resistance, and in flat board, A Pu mycin is eventually Concentration is 15 μ g/ml；Flat board is verified, and correct transformant extracts genome, with PCR method checking weight Whether group expression vector pIMEP::pimE::pimM::vgb is successfully plugged into brown yellow spore streptomyces gene group In.
The application of above-mentioned engineering bacteria high yield natamycin.
Preferably, the application of above-mentioned engineering bacteria, the fermentation condition of described engineering bacteria is: prepare Fresh spores Suspension, is inoculated in seed culture medium by the inoculum concentration of 2%-5%, 28 DEG C, 200rpm cultivate 2d, seed Culture medium is transferred to 28 DEG C, 200rpm fermentation culture in fermentation medium by the inoculum concentration of 5%.
Preferably, the application of above-mentioned engineering bacteria, described seed culture medium is calculated as by g/L: glucose 20, Peptone 6,10, pH=7.0,121 DEG C of sterilizing 20min of yeast extract 6, NaCl.
Preferably, the application of above-mentioned engineering bacteria, described fermentation medium is calculated as by g/L: glucose 40, Soy peptone 15, yeast extract 5, beef extract powder 5, pH=7.5,121 DEG C sterilizing 20min, wherein glucose compound concentration is 50%, 115 DEG C of sterilizing 30min, fermentation culture bar Part: 28 DEG C, 200rpm fermentation culture in fermentation medium.
Preferably, the application of above-mentioned engineering bacteria, including supplementary carbon source, regulating and controlling ventilating amount and speed of agitator, In sweat, 30h-108h adds 50% glucose solution with the flow velocity stream of 2.5g/L 3g/L；Control Fermentation liquid pH is 6；Initial ventilation is that 4L/min, 7h rise to 6L/min；When corresponding difference Between some regulation rotating speed, no longer change after being adjusted to 650rpm to 58h.
The invention has the beneficial effects as follows:
The present invention utilizes technique for gene engineering to clone the controlling gene coming from brown yellow spore streptomycete itself PimE and pimM and external source hemoglobin vgb gene, and make its successful expression, it is thus achieved that natamycin produces Measure the brown yellow spore streptomycete engineered strain S.gilvosporeus/ that more brown yellow spore streptomycete starting strain is high pIMEP::pimE::pimM::vgb.This brown yellow spore streptomycete engineered strain is applied in natamycin production, In the case of consuming same substrate, brown yellow spore streptomycete engineered strain can produce more natamycin, reduces Production cost, and then improve enterprise's productivity effect.
Accompanying drawing explanation
Fig. 1 is the structure plasmid map of recombinant expression carrier pIMEP::pimE::pimM::vgb of the present invention.
Fig. 2 is that recombinant expression carrier pIMEP::pimE::pimM::vgb is proceeded to brown yellow spore strepto-by the present invention The transformant obtained of bacterium (Streptomyces gilvosporeus).
Fig. 3 is that recombinant expression carrier pIMEP::pimE::pimM::vgb of the present invention converts brown yellow spore streptomycete (Streptomyces gilvosporeus) obtains the PCR of recombinant bacterial strain afterwards and verifies electrophoretogram: random picking 3 transformants all can amplify the specific band that size is 3230bp, result shows to be the positive Transformant, wherein, wherein, 1 refers to negative control, does not adds template；2 is positive control, and its template is structure The recombinant expression carrier built；3 compare for wild strain, and its template is brown yellow spore streptomyces gene group；4,5, 6 is positive transformant, and its template is the genome of corresponding transformant.
Fig. 4 be brown yellow spore streptomycete engineered strain with wild-type strain after shake flask fermentation, its natamycin The ratio of yield and dry cell weight changes over figure: it can be seen that brown yellow spore streptomycete engineering The ability of bacterial strain synthesis natamycin is better than wild-type strain, and wherein, () refers to brown yellow spore streptomycete (S.gilvosporeus S139)；(▲) refers to brown yellow spore streptomycete engineered strain (S.gilvosporeus/pIMEP::pimE::pimM::vgb)。
Fig. 5 be brown yellow spore streptomycete engineered strain with wild-type strain shaker fermentation after, take the 4th day send out The Bactericidal test that ferment liquid does.Wherein, the left side is brown yellow spore streptomycete (S.gilvosporeus S139), The right is brown yellow spore streptomycete engineered strain (S.gilvosporeus/pIMEP::pimE::pimM::vgb). Indicator strain is yeast.Its result is consistent with Fig. 4 result.
Preservation information
Classification noun: brown yellow spore streptomycete Streptomyces gilvosporeus
Depositary institution's title: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on December 04th, 2015
Preserving number: CGMCC No.11790
The biomaterial (strain) of ginseng evidence: S280
Detailed description of the invention
In order to make those skilled in the art be better understood from technical scheme, below in conjunction with specifically Technical scheme of the present invention is described in further detail by embodiment.
One as it is shown in figure 1, build containing controlling gene pimM, cholesterol oxidase gene from rhodococcus equi pimE and blood The expression vector pIMEP::pimE::pimM::vgb of Lactoferrin gene vgb, primer sequence is sequence table <400>sequence shown in 4-<400>15；
Two, the brown yellow spore streptomycete engineering bacteria containing expression vector pIMEP::pimE::pimM::vgb is built Strain: this bacterial strain is by the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preservation Day: December in 2015 preserving number on the 04th is: CGMCC No.11790；
Three, the ferment control of brown yellow spore streptomycete engineered strain and the mensuration of natamycin content；
The present invention with brown yellow spore streptomycete (Streptomyces gilvosporeus) strain gene group is Template, by encoding regulator gene pimM, coding cholesterol oxidation in natamycin biological synthesis gene cluster Enzyme gene pimE and external source Vitreoscilla Hemoglobin gene are cloned into and set out on carrier pIMEP, Controlling gene pimM, cholesterol oxidase gene from rhodococcus equi pimE, hemoglobin gene vgb is made to be positioned at this carrier After erythromycin promoter Perm.After bacterium colony PCR checking and digestion verification, success construction expression carries Body pIMEP::pimM/pIMEP::pimE/pIMEP::vgb.Afterwards with expression vector pIMEP::pimE For the carrier that sets out, with expression vector pIMEP::pimM for template amplification gene Perm::pimM, use EcoR I single endonuclease digestion builds recombinant expression carrier pIMEP::pimE::pimM.Then with expression vector PIMEP::pimE::pimM is the carrier that sets out, with expression vector pIMEP::vgb for template amplification gene Perm::vgb, builds recombinant expression carrier with Mun I/Spe I single endonuclease digestion pIMEP::pimE::pimM::vgb.This supported is proceeded in escherichia coli ETZ competent cell.Profit With the escherichia coli ETZ containing recombinant expression carrier pIMEP::pimE::pimM::vgb and brown yellow spore chain The experiment of mycete Conjugative tiansfer can obtain brown yellow spore streptomycete engineered strain S.gilvosporeus /pIMEP::pimE::pimM::vgb.The brown yellow spore streptomycete engineered strain obtained is carried out shaker fermentation Test, and take fermentation liquid and mix with 1:9 ratio with methanol, it is suitable to dilute after ultrasonic echography 20min Multiple, measures the yield of brown yellow spore streptomycete engineered strain natamycin by HPLC method.
Embodiment 1
The structure of expression vector pIMEP::pimE::pimM::vgb
(1) structure of expression vector pIMEP::pimE
As template, table is utilized with brown yellow spore streptomycete (Streptomyces gilvosporeus) genome Cholesterol oxidase gene from rhodococcus equi pimE in the primer amplification natamycin biological synthesis gene cluster of 1, draws downstream Thing pimE-R is with his label.
The primer PCR amplification system utilizing table 1 is: 2 × PCR Buffer is (containing Mg2+) 10 μ l, dNTP (2.5 MM) each 1 μ l of 2 μ l, forward primer pimE-F and downstream primer pimE-R (10 μMs), template is (brown Yellow spore streptomyces gene group DNA) 1 μ l, KOD Fx DNA (purchased from TOYOBO company, article No. KFX-101) Polymerase 0.5 μ l, adding sterilized water to final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 2min, 98 DEG C of degeneration 15s, 58 DEG C of annealing 30s, 68 DEG C Extend 100s, react 35 circulations, after 68 DEG C, extend 10min.
Table 1 the primer sequence
Acquired DNA fragmentation pimE BamH I is carried out enzyme action, after recovery with as at restriction endonuclease The plasmid fragments pIMEP managed is attached.Product conversion e. coli jm109 competence will be connected thin Born of the same parents, and be spread evenly across on the LB flat board with A Pu chloramphenicol resistance (50 μ g/ml), cultivated for 37 DEG C Night, picking monoclonal, carry out bacterium colony PCR checking and digestion verification, expression vector can be obtained pIMEP::pimE。
(2) structure of expression vector pIMEP::pimM
Equally with brown yellow spore streptomycete (Streptomyces gilvosporeus) genome as template, profit With controlling gene pimM, downstream primer in the primer amplification natamycin biological synthesis gene cluster of table 2 PimM-R is with his label
PCR amplification system is: 2 × PCR Buffer is (containing Mg2+) 10 μ l, dNTP (2.5mM) 2 μ l, The each 1 μ l of forward primer pimM-F and downstream primer pimM-R (10 μMs), template (brown yellow spore streptomycete Genomic DNA) 1 μ l, KOD Fx DNA (purchased from TOYOBO company, article No. KFX-101) polymerase 0.5 μ l, adding sterilized water to final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 2min, 98 DEG C of degeneration 15s, 58 DEG C of annealing 30s, 68 DEG C extend 37s, react 35 circulations, extend 10min after 68 DEG C.
Table 2 the primer sequence
Equally acquired DNA fragmentation pimM BamH I/EcoR I is carried out enzyme action, with same after recovery The plasmid fragments pIMEP that sample restriction endonuclease processed is attached.Product will be connected and convert e. coli jm109 Competent cell, and be spread evenly across on the LB flat board with A Pu chloramphenicol resistance (50 μ g/ml), 37 DEG C overnight incubation, picking monoclonal, carry out bacterium colony PCR checking and digestion verification, it is thus achieved that expression vector pIMEP::pimM。
(3) structure of expression vector pIMEP::pimE::pimM
With the expression vector pIMEP::pimM that successfully constructs as template, carry with the expression successfully constructed simultaneously Body pIMEP::pimE is the carrier that sets out.With the primer amplification Perm::pimM gene of table 3.
PCR amplification system is: 2 × PCR Buffer is (containing Mg2+) 10 μ l, dNTP (2.5mM) 2 μ l, The each 1 μ l of forward primer Perm-F and downstream primer pimM-R (10 μMs), template (brown yellow spore streptomycete Genomic DNA) 1 μ l, KOD Fx DNA (purchased from TOYOBO company, article No. KFX-101) polymerase 0.5 μ l, adding sterilized water to final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 2min, 98 DEG C of degeneration 15s, 58 DEG C of annealing 30s, 68 DEG C extend 52s, react 35 circulations, extend 10min after 68 DEG C.
Table 3 the primer sequence
Acquired DNA fragmentation Perm::pimM EcoR I is carried out enzyme action, after recovery with as in Cut the plasmid fragments pIMEP::pimE that ferment treatment crosses to be attached.Product will be connected and convert escherichia coli JM109 competent cell, and be spread evenly across on the LB flat board with A Pu chloramphenicol resistance (50 μ g/ml), 37 DEG C of overnight incubation, picking monoclonal, carry out bacterium colony PCR checking and digestion verification, it is thus achieved that expression vector pIMEP::pimE::pimM。
(4) structure of expression vector pIMEP::vgb
With plasmid vector puc57::vgb (pLH217) as template, utilize the primer amplification vgb base of table 4 Cause, this gene carries his label.
PCR amplification system is: 2 × PCR Buffer is (containing Mg2+) 10 μ l, dNTP (2.5mM) 2 μ l, The each 1 μ l of forward primer vgb-F and downstream primer vgb-R (10 μMs), template (brown yellow spore streptomycete base Because of group DNA) 1 μ l, KOD Fx DNA (purchased from TOYOBO company, article No. KFX-101) polymerase 0.5 μ L, adding sterilized water to final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 2min, 98 DEG C of degeneration 15s, 58 DEG C of annealing 30s, 68 DEG C extend 29s, react 35 circulations, extend 10min after 68 DEG C.
Table 4 the primer sequence
Acquired DNA fragmentation vgb Kpn I is carried out enzyme action, after recovery with as restriction endonuclease process The plasmid fragments pIMEP crossed is attached.Product will be connected and convert escherichia coli jm109 competent cell, And be spread evenly across on the LB flat board with A Pu chloramphenicol resistance (50 μ g/ml), 37 DEG C of overnight incubation, Picking monoclonal, carries out bacterium colony PCR checking and digestion verification, can obtain expression vector pIMEP::vgb.
(5) structure of expression vector pIMEP::pimE::pimM::vgb
With the expression vector pIMEP::vgb that successfully constructs as template, simultaneously with the expression vector successfully constructed PIMEP::pimE::pimM is the carrier that sets out.With the primer amplification Perm::vgb gene of table 5.
PCR amplification system is: 2 × PCR Buffer is (containing Mg2+) 10 μ l, dNTP (2.5mM) 2 μ l, The each 1 μ l of forward primer Perm-F and downstream primer vgb-R (10 μMs), template (brown yellow spore streptomycete base Because of group DNA) 1 μ l, KOD Fx DNA (purchased from TOYOBO company, article No. KFX-101) polymerase 0.5 μ L, adding sterilized water to final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 2min, 98 DEG C of degeneration 15s, 58 DEG C of annealing 30s, 68 DEG C extend 43s, react 35 circulations, extend 10min after 68 DEG C.
Table 5 the primer sequence
Acquired DNA fragmentation Perm::vgb Mun I/Spe I is carried out enzyme action, with same after recovery The plasmid fragments pIMEP::pimE::pimM that sample restriction endonuclease processed is attached.Product will be connected convert Escherichia coli jm109 competent cell, and be spread evenly across with A Pu chloramphenicol resistance (50 μ g/ml) On LB flat board, 37 DEG C of overnight incubation, picking monoclonal, carry out bacterium colony PCR checking and digestion verification, obtain Obtain expression vector pIMEP::pimE::pimM::vgb.
LB culture medium:
Tryptone: 10.0g, yeast extract: 5.0g, NaCl:10.0g, deionized water dissolving After, it is settled to 1.0L, pH and is adjusted to 7.0-7.2, the agar powder that solid medium adds 1.5%.121℃ Sterilizing 20min.
Embodiment 2
The structure of the brown yellow spore streptomycete engineered strain containing expression vector pIMEP::pimE::pimM::vgb
1. the Conjugative tiansfer of escherichia coli-brown yellow spore streptomycete
(1) recombiant plasmid pIMEP::pimE::pimM::vgb correct for checking is converted escherichia coli ETZ In competent cell, cultivate after 45min in 37 DEG C of shaking tables, take 100 μ l fermentation liquids and be spread evenly across and contain There are kanamycin (final concentration of 50 μ g/ml), tetracycline (final concentration of 15 μ g/ml), chloromycetin On the LB flat board of (final concentration of 25 μ g/ml) and A Pu mycin (final concentration of 50 μ g/ml) resistance, After growing bacterium colony, picking monoclonal is connected in the LB liquid medium containing same four kinds of antibiotic, enters Row bacterium colony PCR checking and digestion verification.
(2) will verify that correct monoclonal is connected in the LB liquid medium containing identical four kinds of antibiotic, Cultivate to OD600Value is between 0.4 and 0.6.Collect thalline with 1.5ml EP pipe, cultivate with liquid LB After base washing thalline 2-3 time, standby with 0.5ml LB fluid medium suspension thalline in 1.5ml EP With.
(3) the brown yellow spore streptomycete spore that the cotton swab collection of use sterilizing is fresh is in 1.5ml EP pipe, uses In 0.5ml 2 × YT fluid medium, 50 DEG C of water-bath 10min, take out and are cooled to room temperature.
(4) mixed processing is good Bacillus coli cells and brown yellow spore streptomycete spore suspension, be coated with MS (containing 5mM MgCl2) flat board, cultivate 16-20h in 28 DEG C of incubators, coating 1ml sterilized water (contains afterwards Nalidixic acid 0.4mg and A Pu mycin 1.5mg).Continue to cultivate in 28 DEG C and have bacterium colony to long.
(5) each flat board about grows 50-200 transformant (Fig. 2).Transformant is transferred to A Pu On the SS flat board of chloramphenicol resistance.
2. the checking of transformant
The transformant of the brown yellow spore streptomycete engineered strain obtained is taken Fresh spores and is inoculated into seed culture In base, cultivate and be centrifuged bacterium solution extraction genome two days later.With the genome of transformant as template, wild type The genome of bacterial strain is comparison, and expression plasmid pIMEP::pimE::pimM::vgb is that positive control carries out PCR Checking.
PCR reaction system is: 10 × PCR Buffer 2 μ l, dNTP (10mM) 0.4 μ l, upstream Primer pimE-F and downstream primer pimM-R (10 μMs) is shown in Table 6 each 0.4 μ l, template 1 μ l, Taq archaeal dna polymerase (purchased from Fermentas company, article No. EP0405) 0.4 μ l, adds sterilized water extremely Final volume is 20 μ l.
PCR reaction condition is: 94 DEG C of denaturations 5min, 96 DEG C of degeneration 30s, 58 DEG C of annealing 30s, 72 DEG C extend 194s, react 35 circulations, and 72 DEG C re-extend 10min.
Table 6 the primer sequence
Result as it is shown on figure 3, compared with brown yellow spore streptomycete wild-type strain, recombinant expression carrier PIMEP::pimE::pimM::vgb, brown yellow spore streptomycete engineered strain can amplify the band of 3230bp, Illustrate that recombinant expression carrier pIMEP::pimE::pimM::vgb has been successfully plugged into brown yellow spore streptomycete base Because of in group.Wherein, 1 refers to negative control, does not adds template；2 is positive control, and its template is the weight built Group expression vector pIMEP::pimE::pimM::vgb；3 compare for wild strain, and its template is brown yellow spore chain Mycete genome；4,5,6 is positive transformant, and its template is the genome of corresponding transformant.
Culture medium prescription:
MS culture medium:
Mannitol: 20.0g；Analysis for soybean powder: 20.0g；Agar: 15.0g；After deionized water dissolving, It is settled to 1.0L.121 DEG C of sterilizing 20min.
Seed culture medium:
Glucose: 20.0g, peptone: 6.0g, yeast extract:6.0g, NaCl:10.0 G, after deionized water dissolving, is settled to 7.0,121 DEG C of sterilizing 20min of 1.0L, PH.
Embodiment 3
The ferment control of brown yellow spore streptomycete engineered strain and the mensuration of natamycin content
1, the shaker fermentation of brown yellow spore streptomycete engineered strain controls
Brown yellow spore streptomycete engineered strain (the Streptomyces gilvosporeus/ that will screen PIMEP::pimE::pimM::vgb) CGMCC No.11790 and wild-type strain (S.gilvosporeus S139) all line SS flat board, cultivate 8-12d at 28 DEG C.Fresh spores is taken, preparation with aseptic washing Fresh spores suspension.Spore suspension is inoculated into equipped with 20ml seed culture medium by the inoculum concentration of 2%-5% 50ml triangular flask in, 28 DEG C, 200rpm cultivate 2d.Then by seed culture medium by 5% connect Kind of amount is transferred to fill in the 250ml triangular flask of 50ml fermentation medium, 28 DEG C, 200rpm ferments training Support 5d.
2, brown yellow spore streptomycete engineered strain 5L ferment tank controls
The brown yellow spore streptomycete engineered strain (S.gilvosporeus that will screen / pIMEP::pimE::pimM::vgb) and wild-type strain (S.gilvosporeu S139) all line SS Flat board, cultivates 8-12d at 28 DEG C.Take Fresh spores with aseptic washing, prepare Fresh spores suspension. Spore suspension is inoculated into the triangle of 500ml equipped with 150ml seed culture medium by the inoculum concentration of 2%-5% In Ping, 28 DEG C, 200rpm cultivate 2d.Then seed culture medium is transferred to 5L by the inoculum concentration of 5% In fermentation tank, fermentation liquid cumulative volume is 3L.The initial ventilation that ferments is 4L/min, whole sweat Middle pH value controls about 6.0, and temperature is 28 DEG C.And when 30h (fermentation liquid residual sugar is less than 2%), Drip 50% glucose with the flow velocity of 2.5g-3.0g per hour, stop stream during 108h and add.At whole During ferment, fermentation processes is as follows:
(1) 0-7h rotating speed is 250rpm, and ventilation maintains 4L/min；
(2) 7-8h rotating speed is 250rpm, and ventilation maintains 6L/min；
(3) 8-12h rotating speed is 300rpm, and ventilation maintains 6L/min；
(4) 12-16h rotating speed is 350rpm, and ventilation maintains 6L/min；
(5) 16-20h rotating speed is to 400rpm, and ventilation maintains 6L/min；
(6) 20-24h rotating speed is 450rpm, and ventilation maintains 6L/min；
(7) 24-30h rotating speed is 500rpm, and ventilation maintains 6L/min；
(8) 30-38h rotating speed is 550rpm, and ventilation maintains 6L/min；
(9) 38-50h rotating speed is 600rpm, and ventilation maintains 6L/min；
(10) 50-58h rotating speed is 625rpm, and ventilation maintains 6L/min；
(11) 58-120h rotating speed is 650rpm, and ventilation maintains 6L/min.
Seed culture medium:
Glucose: 20.0g, peptone: 6.0g, yeast extract:6.0g, NaCl:10.0 G, after deionized water dissolving, is settled to 7.0,121 DEG C of sterilizing 20min of 1.0L, pH.
Fermentation medium:
Glucose: 40.0g, soy peptone: 15.0g, yeast extract:5.0g, beef Leaching powder: 5.0g, after deionized water dissolving, is settled to 7.5,121 DEG C of sterilizing 20min of 1.0L, pH, Wherein glucose compound concentration is 50%, 115 DEG C of sterilizing 30min.
SS culture medium:
Glucose: 15.0g；Peptone: 5.0g, yeast extract:3.0g, Fructus Hordei Germinatus leaching powder: 3.0g, agar powder: 15.0g, after deionized water dissolving, be settled to 1.0L, 121 DEG C of sterilizing 20min.
3, the mensuration of natamycin content in fermentation liquid
After fermentation ends, take 1ml fermentation liquid and 9ml methanol mixed, mix, ultrasonic 20min, it Rear 8000r/min is centrifuged 15min, then obtains testing sample with the organic membrane filter of 0.22 μm, With HPLC quantitative analysis natamycin.
HPLC analysis condition: flowing phase: methanol: water=70:30；Flow velocity: 0.700ml/min；Detection Wavelength is 303nm；Sample size is 10 μ l；Column temperature is 30 DEG C.
Shaking table data are as shown in Figure 4: brown yellow spore streptomycete engineered strain (S.gilvosporeus/ PIMEP::pimE::pimM::vgb) with starting strain (S.gilvosporeus S139) shaker fermentation after Natamycin yield and dry cell weight ratio change over situation.The most brown yellow spore Streptomycete engineered strain (S.gilvosporeus/pIMEP::pimE::pimM::vgb) natamycin produces The amount wild plant height of ratio, and this species diversity was the most notable when the 4th day.Result of calculation shows, brown yellow spore chain Mycete engineered strain (S.gilvosporeus/pIMEP::pimE::pimM::vgb) when the 4th day its The natamycin relatively starting strain that fermentation liquid unit thalline is produced improves 56%.Thus prove that his mycin is biological Controlling gene pimM and the synergistic effect of cholesterol oxidase gene from rhodococcus equi pimE and external source in synthetic gene bunch The expression of gene vgb can significantly improve the yield of natamycin in brown yellow spore streptomycete.Fig. 5 is for the 4th The Bactericidal test that it fermentation liquid is cooked, its indicator strain is yeast.This figure finds out brown Huang the most intuitively Spore streptomycete engineered strain (S.gilvosporeus/pIMEP::pimE::pimM::vgb) synthesis receives him The ability of mycin is higher than wild strain.Data such as table 7 after wild strain and engineered strain 5L fermentor cultivation Shown in, the synergistic effect expressed due to pimM and pimE process LAN and exogenous gene vgb, engineered strain After the fermentation of upper tank, natamycin yield increases, and its rate of increase was gradually lowered in the later stage；But when 120h, Its natamycin yield relatively wild strain yield still improves 15.6%, reaches 10.136g/L.It is visible, Due to the synergistic effect between three, natamycin generated time is made to be greatly shortened.
Table 7 starting strain and engineered strain natamycin Yield compari@
Above-mentioned with reference to detailed description of the invention to this brown yellow spore streptomycete recombinant expression plasmid and engineering bacteria and application The detailed description carried out, is illustrative rather than determinate, if can according to restriction scope list Dry embodiment, therefore changing and modifications under without departing from present general inventive concept, should belong to the present invention's Within protection domain.

Claims (4)

1. a brown yellow spore streptomycete recombinant expression plasmid, it is characterised in that: be by controlling gene pimM, Cholesterol oxidase gene from rhodococcus equi pimE and external source hemoglobin gene vgb inserts respectively containing erythromycin promoter Carrier pIMEP in obtain recombinant expression carrier pIMEP::pimE::pimM::vgb, recombinant expressed The sequence of carrier pIMEP::pimE::pimM::vgb is sequence shown in sequence table<400>17.
2. the engineering bacteria containing yellow spore streptomycete recombinant expression plasmid brown described in claim 1, it is special Levy and be: preserving number is CGMCC No.11790.
3. the application of engineering bacteria high yield natamycin described in claim 2.
The application of engineering bacteria the most according to claim 3, it is characterised in that: include supplementary carbon source, Regulating and controlling ventilating amount and speed of agitator, in sweat, 30h-108h is with the flow velocity of 2.5g/L 3g/L Stream adds 50% glucose solution；Controlling fermentation liquid pH is 6；Initial ventilation is that 4L/min, 7h rise to 6L/min；Regulate rotating speed in corresponding different time points, no longer change after being adjusted to 650rpm to 58h.
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