CN103555755A - Poor oxygen resistance high density fermentation natamycin gene engineering strain constructed by using vgb gene, and application thereof - Google Patents
Poor oxygen resistance high density fermentation natamycin gene engineering strain constructed by using vgb gene, and application thereof Download PDFInfo
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Abstract
The invention relates to a poor oxygen resistance high density fermentation natamycin gene engineering strain (Streptomyces gilvosporeus sw-807, CGMCC No.6890) constructed by using vgb gene, and an application thereof. According to the present invention, vitreoscilla hemoglobin gene vgb is adopted to construct recombinant plasmid, a conjugal transfer manner is adopted to integrate the vgb gene into Streptomyces gilvosporeus genome to obtain the recombinant gene engineering strain capable of being directly used for natamycin fermentation production, and VHb expression in the recombinant strain can be provided for effectively solving oxygen supply and demand contradiction in natamycin fermentation production so as to substantially increase natamycin yield, reduce production cost and bring great economic benefits.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to utilize the brown yellow spore streptomycete engineering strain of the gene constructed restructuring of Vitreoscilla hemoglobin (vgb), by the expression of vgb gene, improve the utilization ratio of brown yellow spore streptomycete to oxygen of recombinating in tennecetin process of high-density fermentation, to improve the output of tennecetin.
Background technology
Tennecetin (Natamycin) is a kind of natural, wide spectrum, polyene macrocyclic lactones is antifungal agents based efficiently.Compare with other antimicrobial components, tennecetin is extremely low to the toxicity of mammalian cell, can be widely used in by fungus-caused disease, as tennecetin is used to the fungi infestation of anti-skin and mucous membrane with formulations such as suspension agent, emulsion, ointment and sheath shape tablets.In addition, the solubleness of tennecetin is low, can be used for the processing to food surfaces, extends the quality guaranteed period of food, does not but affect local flavor and the mouthfeel of food.In June nineteen eighty-two, U.S. FDA (Food and drug dministration) official approval tennecetin can be used as food preservatives, and is classified as the row of GRAS product, becomes at present unique in the world antimycotic microbiological antiseptic.Within 1997, China Ministry of Health official approval tennecetin is as food preservatives.Tennecetin has been widely used in production and the preservation of the food such as milk-product, meat product, fermented wine, beverage in more than 50 countries at present.
Along with tennecetin is the continually developing of medicine and food service industry, its demand constantly increases.But because tennecetin fermentation level is on the low side at present, cause production cost very high, seriously limited its application in each field.Wherein in fermenting process, dissolved oxygen deficiency is the major reason that causes tennecetin fermentation level low.The generation bacterium of tennecetin as brown yellow spore streptomycete be aerobic bacteria, along with fermentation time extends, cell density increases sharply, oxygen-consumption is very big, and every physical parameter of fermentor tank can not meet the supply of oxygen, and especially highdensity brown yellow spore streptomycete mycelium is wound around mutually, make local oxyty lower, result thalli growth slows down, and the synthetic key enzyme expression amount of tennecetin reduces, and directly causes tennecetin yield reducation.Therefore, how to improve during the fermentation tennecetin and produce bacterial strain utilization to oxygen under conditions of high density, to improving the output tool significance of producing cell density and tennecetin.
Vitreoscilla hemoglobin (Vitreoscilla Hemoglobin, VHb) is the most thorough, the most widely used prokaryotic organism oxyphorase of a class research.Research shows, VHb is combined and can be formed oxygenate state with oxygen, and gets involved some committed step or the approach tapping point in the cell pathways metabolism relevant with oxygen with which, thereby changes the original pathways metabolism of cell while limitting oxygen.Under same culture conditions, the expression of VHb can be accelerated the growth velocity of cell, improves cellular respiration intensity, reduces the critical dissolved oxygen concentration of cell, significantly in dissolved oxygen variation, keeping constant respiration rate, thereby making it under hypoxia condition, still there is certain growth advantage.At present more and more deep to the research of Vitreoscilla hemoglobin gene, except intestinal bacteria, also in the multiple-microorganisms such as false pseudomonas bacillus, Erwinia, Molds and yeasts, be studied, and be successfully applied in industrial production, improve amylase, penicillin phthalein enzyme, unwrapping wire rhodomycetin and etc. the output of multiple biochemical products.
In sum, VHb can strengthen the utilize ability of vgb gene recombination bacterium to oxygen from molecular level, therefore can be under oxygen deprivation condition Promote cell's growth and product synthetic, thereby increase substantially the output of tunning.The present invention is integrated into Vitreoscilla Hemoglobin gene in brown yellow spore streptomyces gene group, by the expression of VHb, effectively solve the oxygen disparities between supply and demand in tennecetin fermentative production, increase substantially the output of tennecetin, reduce production costs, increase economic benefit.
Summary of the invention
The object of the present invention is to provide the genetic engineering bacterium of the gene constructed anti-oxygen deprivation high density fermentation tennecetin of a kind of vgb of utilization.The successful expression of VHb in constructed engineering strain, guaranteed the oxygen uptake capacity of Host Strains in high density fermentation Hypoxic habitats, maintained the metabolism of cell in hypoxemia situation, thereby improved recombination engineering strain high density fermentation, produce the oxygen supply and demand problem in tennecetin process, greatly improved the fermentation level of engineering strain.
The present invention also aims to provide the construction process of described genetic engineering bacterium.
The object of the invention is to reach by following measures: by pcr amplification, obtain vgb gene, vgb gene is inserted to respective carrier, build integrated recombinant plasmid, then utilize the mode of conjugal transfer to transform brown yellow spore streptomycete, through antibiotics resistance screening, obtain the recombinant strain that is integrated with vgb gene, by shake flask fermentation experiment screening, obtain and express oxyphorase (VHb), and can be directly used in the recombinant bacterium of high yield tennecetin.
The promotor of described vgb gene is himself promotor.
Described recombinant plasmid is pSET152-vgb.
Wherein, plasmid also comprises other carriers in construction recombination plasmid pSET152-vgb process: p
bBR-MCS5-vgb and pSET152.P wherein
bBR-MCS5-vgb contains vgb gene complete sequence and promotor thereof; PSET152 contains plasmid and shifts required element and phage
intergrase and integration site; Recombinant plasmid pSET152-vgb is with p
bBR-MCS5-vgb carrier is template, and pcr amplification obtains vgb gene and promoter sequence thereof, inserts in pSET152 plasmid and build and form after double digestion.
The step of described conjugal transfer is: recombinant plasmid is imported in intestinal bacteria ET12567 (PUZ8002), recombination bacillus coli and brown yellow spore streptomycete are incubated to MS altogether (containing 10mM MgCl
2) on solid medium, cultivate 16～20h for 30 ℃, be then coated with 1mL sterilized water (containing 0.5mg nalidixic acid and 1mg apramycin) and cover dull and stereotypedly, 30 ℃ are continued to be cultured to and grow transformant.
The condition of described shake flask fermentation experiment is 28～30 ℃, rotating speed 200～300r/min, and 3～5d ferments.
Seed culture based component is: glucose 2%, soy peptone 0.6%, yeast powder 0.6%, NaCl0.5%, pH7.0～7.5,115 ℃, steam sterilizing 20min.
Fermentation culture based component is: peptone 2%, yeast powder 0.45%, NaCl0.2%, MgSO
4.7H
2o0.1%, 121 ℃, steam sterilizing 20min.
After fermentation ends, get 20mL fermented liquid and after ultrasonication, carry out CO in conjunction with differential spectra analysis, detect the activity of VHb.Separately get 1mL fermented liquid, add the methyl alcohol of 9mL, ultrasonic extraction 20min, centrifugal, supernatant liquor, after the membrane filtration of 0.22 μ m, is measured the output of tennecetin through HPLC.
The application in preparing tennecetin of described recombinant plasmid and engineering bacteria.
Tool of the present invention has the following advantages and effect:
The invention provides the engineering strain that utilizes the gene constructed anti-oxygen deprivation high density fermentation tennecetin of vgb.Construction process is vgb gene to be imported to tennecetin produce in bacterium, by the expression of VHb, guaranteed that engineering strain improves the oxygen uptake capacity of cell in the environment of high density fermentation oxygen deprivation, maintain the metabolism of cell in hypoxemia situation and meet tennecetin synthetic in demand to oxygen, thereby improved the problem of the oxygen supply deficiency in constructed engineering strain high density fermentation tennecetin process.Constructed engineering strain is applied to, in tennecetin production, can improve output 60%～80%, greatly reduces the production cost of tennecetin.
Accompanying drawing explanation
Fig. 1: recombinant bacterium PCR checking
Swimming lane M:1Kb DNA marker; Swimming lane 1 and 3: take original bacterium genome as increase the respectively PCR result of resistant gene (acc) and vgb gene of template; Swimming lane 2 and 4: take recombinant bacterium genome as template, the PCR result of increase respectively resistant gene (acc) and vgb gene.
Fig. 2: CO is in conjunction with differential spectra analysis
Embodiment
The structure of recombinant plasmid pSET152-vgb.
(1) sequence (NO.M30794.1) of the vgb gene of including according to GenBank and promotor thereof, designs respectively upstream and downstream primer:
Upstream primer vgbL:5 '-GGC
gAATTCaGTTTTAAGAGGCAAT-3 ' (underscore is denoted as EcoRI restriction enzyme site); Downstream primer vgbR:5 '-CT
tCTAGAtTATTCAACCGCTTGAGCGTAC-3 ' (underscore is denoted as XbaI enzyme cutting site).
With plasmid p
bBR-MCS5-vgb is template, and pcr amplification obtains vgb gene and promoter sequence thereof.
(2) PCR product and carrier pSET152 are used respectively to EcoRI/XbaI double digestion, cut after glue recovery, with the mixed in molar ratio of 3: 1～10: 1, add T4 ligase enzyme, 16 ℃ of connections are spent the night.By connecting fluid and CaCl
2the standby competent escherichia coli cell E.coli ET12567 of legal system mixes, and 42 ℃ of heat shock 90s cultivate after 1h for 37 ℃, and coating is containing the LB solid plate of kantlex, paraxin and apramycin resistance.Picking transformant, extraction plasmid carries out enzyme and cuts checking, obtains recombinant plasmid pSET152-vgb.
The structure of the brown yellow spore streptomycete of restructuring vgb gene, the mode of conjugal transfer between employing intestinal bacteria-streptomyces, concrete steps are as follows:
(1) picking is containing the mono-bacterium colony of intestinal bacteria E.coli ET12567 of recombinant plasmid pSET152-vgb in LB substratum (containing the kantlex of 25 μ g/mL, the apramycin of the paraxin of 25 μ g/mL and 50 μ g/mL), and 37 ℃ of shaking culture are spent the night.
(2) by 2% inoculum size, the restructuring E.coli ET12567 that activation is spent the night transfers in fresh LB substratum (containing kantlex, paraxin and apramycin), and 37 ℃ are cultured to OD
600=0.4～0.6, centrifugal, with isopyknic LB substratum washing thalline twice, be resuspended in the LB substratum of 0.1 times of volume;
(3), when processing restructuring E.coli ET12567, get approximately 10
8the spore of individual brown yellow spore streptomycete is cultivated and is concentrated in 500 μ l2 * YT, cooling after 50 ℃ of heat shock 10min; Or will on MS solid medium, cultivate the mycelium of 3～4d, and with 20% glycerine of 2-3mL, collect, standby;
(4) get spore suspension or the isopyknic mycelium that 500 μ l restructuring E.coli ET12567 and equal-volume heat shock process and mix, gently hang, remove after most of supernatant, the cell of mixing is resuspended in remaining liquid;
(5) cell mixing is coated containing 10mM to MgCl
2mS solid medium on, cultivate 16-20h for 30 ℃;
(6) coating 1mL sterilized water (containing 0.5mg nalidixic acid and 1mg apramycin) covers dull and stereotypedly, and 30 ℃ are continued to be cultured to and grow transformant;
(7) picking transformant (containing the nalidixic acid of 25 μ g/mL and the apramycin of 30 μ g/mL) on MS substratum carries out repeated screening;
In liquid nutrient medium, activation obtains each transformant, extract the genomic dna of each transformant, with primer vgbL and vgbR, pcr amplification vgb gene, with primer Ass (5 '-TCCCCGCGGGGTTCATGTGCAGCTCCATCA-3 ') and Aca (5 '-GGTAACTATTGCCGTCTCAGCCAATCGACTGGCGAGCGGCA-3 ') amplification apramycin resistance gene (aac), do further checking (Fig. 1).In Fig. 1, swimming lane 2 and swimming lane 4 arrow indications are respectively aac and the vgb gene band amplifying, and take the karyomit(e) of starting strain, all do not amplify respective strap (swimming lane 1 and 3) during as template, have confirmed the successful integration of recombinant plasmid pSET152-vgb.
The gained restructuring vgb brown brown yellow spore streptomycete of yellow spore streptomycete called after of gene (Streptomyces gilvosporeus) sw-807, in (address: Datun Road, Chaoyang District, Beijing City, in November, 2012 27 China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is brown yellow spore streptomycete (Streptomyces gilvosporeus), and preserving number is CGMCC No.6890.
The application of S.gilvosporeus sw-807 in tennecetin fermentation.
With the slant pore of the sterilized water brown yellow spore streptomycete S.gilvosporeus of washing and recombinant bacterium S.gilvosporeus sw-807, make spore suspension (approximately 10
8individual/mL); By 2%～5% inoculum size, be inoculated in the seed culture medium of 5mL 28～30 ℃ of shaking culture 48h; Be forwarded to again liquid amount and be in the triangular flask of 250mL of 30mL seed culture medium, cultivate 16～20h; Then with 2%～5% inoculum size, be forwarded in the triangular flask of the 250mL that 30mL and 50mL fermention medium are housed, 28～30 ℃, rotating speed 200～300r/min, 3～5d ferments.
After fermentation ends, measure respectively the activity of VHb and the output of tennecetin.
VHb determination of activity step is as follows:
(1) get 20mL fermented liquid centrifugal, precipitation, with after the washing once of equal-volume physiological saline, is resuspended in the phosphoric acid buffer (pH7.5) of 10mL0.1M, ultrasonication (300-400W, broken 10s, gap 10s, broken time 20min);
(2) 4 ℃, the centrifugal 15min of 1000r/min, gets supernatant 3mL, adds 3mL phosphoric acid buffer, and adds sodium sulfite to concentration 2.5mg/mL;
(3) sample through above-mentioned processing is divided into two parts, a logical CO, 3min;
(4) with ultraviolet-visible pectrophotometer, within the scope of 400～500nm, respectively to two duplicate samples scannings, the not sample of logical CO of take is contrast, and curve obtained is CO differential spectra figure (Fig. 2).
As shown in Figure 2, S.gilvosporeus compares with starting strain, and S.gilvosporeus sw-807, in the appearance of 420nm place absorption peak, has proved the successful expression of vgb gene.
Tennecetin determination of yield method, for getting 1mL fermented liquid, adds 9mL methyl alcohol, after ultrasonic extraction 20min, centrifugal (1000r/min, 10min), supernatant liquor carries out HPLC analysis after the membrane filtration of 0.22 μ m, detects the tennecetin output of starting strain and recombinant bacterial strain in ultraviolet 303nm.Result shows (table 1), different liquid amounts, and recombinant bacterial strain tennecetin output has all had and has significantly improved than starting strain, and when liquid amount is 30mL/250mL, recombinant bacterial strain tennecetin output can improve 60% left and right; When liquid amount is 50mL/250mL, recombinant bacterial strain tennecetin output improves more remarkable, can reach 80% left and right.
Table 1S.gilvosporeus and the comparison of recombinant bacterium S.gilvosporeus sw-807 tennecetin fermentation yield
Claims (6)
1. utilize Vitreoscilla hemoglobin gene (Vitreoscilla hemoglobin, vgb) the anti-oxygen deprivation high density fermentation tennecetin engineering strain and the application thereof that build, it is characterized in that: by pcr amplification, obtain vgb gene, vgb gene is inserted to respective carrier, build integrated recombinant plasmid, then utilize the mode of conjugal transfer to transform brown yellow spore streptomycete, through antibiotics resistance screening, obtain the recombinant strain that is integrated with vgb gene, by shake flask fermentation experiment screening, obtain the recombinant bacterial strain of expressing oxyphorase (VHb) and can be directly used in high yield tennecetin.
2. the gene constructed anti-oxygen deprivation high density fermentation tennecetin engineering strain of vgb that utilizes claimed in claim 1, the promotor that it is characterized in that described vgb gene is himself promotor.
3. the gene constructed anti-oxygen deprivation high density fermentation tennecetin engineering strain of vgb that utilizes claimed in claim 1, is characterized in that described recombinant plasmid is pSET152-vgb.
4. the gene constructed anti-oxygen deprivation high density fermentation tennecetin engineering strain of vgb that utilizes claimed in claim 1, it is characterized in that: the step of described conjugal transfer, for recombinant plasmid is imported in intestinal bacteria ET12567 (PUZ8002), is incubated at MS (containing 10mM MgCl altogether by recombination bacillus coli and brown yellow spore streptomycete
2) on solid medium, cultivate 16～20h for 30 ℃, be then coated with 1mL sterilized water (containing 0.5mg nalidixic acid and 1mg apramycin) and cover dull and stereotypedly, 30 ℃ are continued to be cultured to and grow transformant.
5. the gene constructed anti-oxygen deprivation high density fermentation tennecetin engineering strain of vgb that utilizes claimed in claim 1, is characterized in that, the condition of described shake flask fermentation experiment is 28～30 ℃, rotating speed 200～300r/min, and 3～5d ferments.
6. the recombinant plasmid described in claim 1～5 any one and the application of engineering bacteria in preparing tennecetin.
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| CN105907778A (en) * | 2015-11-30 | 2016-08-31 | 天津科技大学 | Streptomyces gilvosporeus recombinant expression plasmid, and engineering bacterium and application thereof |
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Application publication date: 20140205 |