CN105779348A - Method for producing Rakicidins compounds by virtue of marine micromonospora fermentation - Google Patents

Method for producing Rakicidins compounds by virtue of marine micromonospora fermentation Download PDF

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CN105779348A
CN105779348A CN201610197210.3A CN201610197210A CN105779348A CN 105779348 A CN105779348 A CN 105779348A CN 201610197210 A CN201610197210 A CN 201610197210A CN 105779348 A CN105779348 A CN 105779348A
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周剑
林风
江红
连云阳
江宏磊
方东升
陈丽
赵薇
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Fujian Institute of Microbiology
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Abstract

The invention relates to the technical field of industrial microorganisms and the field of biological medicines, and in particular relates to a method for producing Rakicidins compounds by virtue of marine micromonospora fermentation. The method is characterized by comprising the following steps: performing solid slant culture on micromonospora which is collected in the China General Microbiological Culture Collection Center (CGMCC) and has a collection number of CGMCC NO. 12132, performing shake flask seed culture, seeding tank culture and fermentation tank fermentation culture, and performing after-treatment to obtain the compounds, wherein amino acid is replenished in the fermentation initial stage of a fermentation tank. The fermentation method provided by the invention is simple in operation and low in cost and can be used for realizing industrial large-scale production, and the highest total fermentation titer reaches about 600mg/L.

Description

A kind of method of sea micromonoad fermenting and producing Rakicidins compounds
Technical field
The present invention relates to industrial microbial technology field and field of biological medicine, a kind of method being specifically related to sea micromonoad fermenting and producing Rakicidins compounds.
Background technology
Grow at top speed in recent years in antitumor drug market, anticarcinogen market global marketing volume 100,000,000,000 dollars in 2014；By 2018, anticarcinogen market was up to 147,000,000,000 dollars, compound growth rate 11.6%, and research and development anticarcinogen will be made a profit huge.Anoxia is one of feature of malignant entity tumor, and anoxia is closely related with the angiogenesis of tumor, Invasion and Metastasis, chemicotherapy opposing and prognosis mala etc..Hypoxic inducing factor-1 (hypoxia-induciblefactor1, HIF-1) is the transcriptional regulatory factor the most key in the regulation and control of anoxia effect.HIF-1 selectivity in solid tumor tissue continues high expressed, and the generation development of downstream key controlling gene and tumor is closely related, as promoted angiogenesis, cell survival, suppression apoptosis of tumor cells, metabolism to reinvent and the adjustment of pH stable state.Just because of the difference of oxygen content in the environment residing for cancer cell under different space-times, the signal path of the promotion tumor growth thus activated is typically not to be induced in the normal tissue, so weary oxygen signal path becomes potential therapy target.Also the emphasis of tumor hypoxia effect regulating medicine research and development is become with the HIF-1 specific small molecule inhibitor being target spot.
RakicidinA and B is because containing 1 rare rare 4-amino-2 in its 15 yuan of cyclic lipopeptide structures, 4-pentadienoic acid monooctyl ester also has Anti-tumor angiogenesis and the [McBrienKD that receives publicity, BerryRL, LoweSEetal.Rakicidins, NewCytotoxicLipopeptidesfromMicromonosporasp.Fermentatio n, IsolationandCharacterization [J] .JAntibiot, 1995,48:1446].Yamazaki (2007) research finds that RakicidinA has the weary oxygen selective Anti-tumor angiogenesis of brilliance, under weary oxygen condition, inhibitor against colon carcinoma cells HCT-8 cytoactive is 17.5 times of [YamazakiY under normal oxygen condition, KunimotoS, IkedaD.RakicidinA:ahypoxia-selectivecytotoxin [J] .BiolPharmBull.2007,30 (2): 261-5.].Apoptosis [the TakeuchiM of marrow chronic leukemia stem cell can be induced under the weary oxygen condition of Takeuchi (2011) reported first RakicidinA, AshiharaE, YamazakiY, etal.RakicidinAeffectivelyinducesapoptosisinhypoxiaadapt edBcr-Ablpositiveleukemiccells [J] .CancerSci.2011,102 (3): 591-6.].Though the mechanism of action of the anti-hypoxic tumor cells of this compound and anti-CSC is unclear, but RakicidinA has been thought an anti-hypoxic tumor cells being rich in DEVELOPMENT PROSPECT and anti-CSC medicine by many colleagues.
First inventor seminar reports and is separated to RakicidinA and B from microbial metabolic products for 2006 at home, and the relevant biologic activity of RakicidinB (FW523-3) has been carried out preliminary study (Jiang Hong, Lin Ru, Zheng Wei, Deng. the separation discriminating of the lipopeptide compound FW523 that ocean Micromonospora chalcea FIM02-523 produces and biologic activity [J]. China's antibiotic magazine, 2006,31 (5): 267-270；Jiang Hong, Lin Ru, Cheng Yuanrong. the anti tumor activity in vitro of the brisk rhzomorph BFW523-3 in sea micromonoad source. China's antibiotic magazine, 2008,33 (9): 531；XieJJ, ZhouF, LiEM, JiangH, DuZP, LinR, FangDS, XuLY.FW523-3, anovellipopeptidecompound, inducesapoptosisincancercells.MolMedRep.2011,4 (4): 759-63).
Report is studied at present less about the fermentation manufacturing technique of Rakicidins class chemical combination.KimberlyD.Mcbrien etc. report the fermentation of Rakicidins compounds and separate purification, but it only provides basic flask process and fermentation period longer (8 days), technique [the McBrienKD of applicable industrialized production is not provided, BerryRL, LoweSEetal.Rakicidins, NewCytotoxicLipopeptidesfromMicromonosporasp.Fermentatio n, IsolationandCharacterization [J] .JAntibiot, 1995,48:1446-52].Domestic only inventor river exoerythrocytic stage is reported in the process screening new immunosuppressant, from the Micromonospora chalcea Micromonosporachalceasp.FIM02-523 fermentation liquid of ocean, extract Rakicidins compounds include RakicidinsA and RakicidinsB (Jiang Hong, Lin Ru, Zheng Wei, the separation discriminating of the lipopeptide compound FW523 that Cheng Yuanrong, ocean Micromonospora chalcea FIM02-523 produce and biologic activity.China's antibiotic magazine, 2006,31 (5): 267-270).But described is only basic fermentation medium and the cultural method of shaking flask, and for the accumulation of sample, its fermentation level is low, it is impossible to be applied to large scale fermentation and produce.
Summary of the invention
The technical problem to be solved is to overcome Rakicidins compounds fermentation titer in prior art low, it is impossible to reach the defect that the technology of industrialized production requires.Disclose the fermentable production technology of a kind of Rakicidins compounds, it is provided that a kind of simple to operate, with low cost, can industrial-scale production, the highest total fermentation titer reaches about 600mg/L.And that can pass through fermentation condition optimizes the effective ratio controlling each component in fermentation liquid and content.
The present invention will be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the micromonospora that deposit number is CGMCCNO.12132 carries out solid slant culture, shake-flask seed cultivation, seed tank culture, ferment tank cultivation, post processing and get final product, just adds aminoacid in the ferment tank incipient stage.One or more in valine, glycine, methionine of the described aminoacid added.Adding the concentration preferably 0.05～0.3%, 0.05～0.3%, 0.05～0.3% of valine, glycine, methionine, percentage ratio of the present invention is all weight percentage.
The invention discloses a micromonospora bacterial strain, be called for short: FIM02-523, Classification And Nomenclature: Micromonosporasp.FIM02-523.Its preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCCNo.12132.Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.Preservation day: on February 18th, 2016.Obtain from the marine clay of Putian, Fujian Province.
The compound of the preferred following any structure of described Rakicidins compounds:
Wherein RakicidinA and B is known compound, and RakicidinB1 is noval chemical compound.
Preferred preparation method includes:
A. solid slant culture: the micromonospora that deposit number is CGMCCNO.12132 is inoculated in solid slope, after in 30-37 DEG C constant temperature culture 10-15 days, rear preservation under room temperature；
B. shake-flask seed is cultivated: load seed culture medium 80ml in 500ml triangular flask, sterilizes 30 minutes for 121 DEG C, inoculates spore suspension, cultivate 48 hours in shaking table 230rpm, 28-35 DEG C after cooling；
C. seed tank culture: seed good for shake-flask culture is merged, is inoculated in seed tank culture base with the 1-5% of seed tank liquid amount, 200-300rpm, ventilation 1:1vvm, cultivates 48 hours by 28 DEG C；
D. ferment tank is cultivated: be inoculated in fermentation tank by seed good for seed tank culture with the inoculum concentration of 5-10%, incubation time is 72-100 hour, fermentation rotating speed controls 200-400rpm, ventilation controls 0.08-1.2VVM, adds one or more in valine, glycine, methionine during the fermentation as the precursor fermented.
E. post processing, to obtain final product.
Preferably adding amino acid whose concentration is: valine is 0.05-0.3%, glycine be 0.05-0.3%, methionine is 0.05-0.3%.The interpolation time is preferably 0-36 hour that fermentation starts, gradation or disposable add.Sweat adds 0.05～0.3% valine can improve the constituent content of RakicidinA；Sweat adds 0.1～0.3% glycine can improve the constituent content of RakicidinB；Sweat adds 0.1～0.3% methionine can improve the constituent content of RakicidinB1 and B.
Wherein the component of solid medium and the percentage composition of each component are preferred: soluble starch 2, L-asparagine 0.05, KNO30.1、K2HPO4·3H2O0.05、NaCl0.05、MgSO4·7H2O0.05、CaCO30.1, agar 1.5, pH7.5.
In shake-flask seed incubation step, the component of seed culture medium and the weight percentage of each component are preferably: soluble starch 1～3, glucose 0.5～1.0, yeast powder 1～2, peptone 0.5～2.0, MgSO4·7H2O0.05、FeSO4·7H2O0.005、CuSO4·5H2O0.005、CoCl2·6H2O0.0005、CaCO30.1～0.3, tap water prepare, pH, 7.0～7.5.
The component of fermentation medium and the weight percentage of each component are preferably: soluble starch 3～6, sucrose 0.5～2.0, soybean cake powder 1.0～3.0, yeast powder 1.0～3.0, MgSO4·7H2O0.04、FeSO4·7H2O0.005、CuSO4·5H2O0.005、CoCl2·6H2O0.0005、CaCO30.3～0.6, tap water is prepared, pH7.5.
The preferred component of each culture medium and percentage composition be:
Seed culture medium: soluble starch 2.0, glucose 1.0, yeast powder 2.0, peptone 1.0, MgSO4·7H2O0.05、FeSO4·7H2O0.005、CuSO4·5H2O0.005、CoCl2·6H2O0.0005、CaCO30.1～0.3, tap water is prepared, pH, and 7.0～7.5.Temperature preferably 28～32 DEG C.
Fermentation medium: soluble starch 5.0, sucrose 1.0, soybean cake powder 3.0, yeast powder 1.0, MgSO4·7H2O0.04、FeSO4·7H2O0.005、CuSO4·5H2O0.005、CoCl2·6H2O0.0005、CaCO30.5, tap water is prepared, pH7.5.Temperature preferably 32 DEG C.
The present invention passes through the optimization of culture medium and the control of fermentative process conditions, particularly add at fermentation the amino acid whose of incipient stage, in the fermenting experiment of 20-1000L fermentation tank, sea micromonoad (Micromonosporasp.FIM02-523) fermentation produces the total titer of Rakicidins compounds and reaches about 600mg/L, be conducive to the extraction work of Rakicidins compounds, it is possible to meet industrialization demand.
From table 1, embodiment and reference examples fermentation results are it can be seen that the present invention is by significantly improving the titer of product after adding aminoacid.
The impact on each component yield of Rakicidins compounds of the table 1 different aminoacids adding conditional
Reference examples 1
Bacterial suspension inoculation is made in the cultivation of shaking flask first order seed after being scraped by sea micromonoad (Micromonosporasp.FIM02-523) spore of solid slope preservation, at 28 DEG C, 220rpm, cultivates 48 hours.
Seed culture based formulas (%): soluble starch 2.0, glucose 1.0, yeast powder 2.0, peptone 1.0, MgSO4·7H2O0.05, FeSO4·7H2O0.005,CuSO4·5H2O0.005,CoCl2·6H2O0.0005, CaCO30.1～0.3, tap water is prepared, pH, and 7.0～7.5；28～32 DEG C.
Being inoculated in 20L fermentation tank (actual liquid amount 15L) according to the inoculum concentration of 5% afterwards and ferment, 28 DEG C of cultivations, initial rotating speed is 200rpm, is adjusted to 350rpm and is about 96-120 hour to fermentation ends after 48 hours.Tank pressure 0.03-0.05Mpa, ventilation is 1:1vvm.
Fermentation medium is: soluble starch 5.0, sucrose 1.0, soybean cake powder 3.0, yeast powder 1.0, MgSO4·7H2O0.04, FeSO4·7H2O0.005,CuSO4·5H2O0.005,CoCl2·6H2O0.0005, CaCO30.5, tap water is prepared, pH7.5；32℃.Sweat detects the content of Rakicidins compounds in fermentation liquid by HPLC, and final fermentation titer is 50mg/L, and wherein RakicidinA content is 15mg, RakicidinB1 content be 10mg, RakicidinB content is 35mg.
Detailed description of the invention
Embodiment 1
Bacterial suspension inoculation is made in the cultivation of shaking flask first order seed after being scraped by sea micromonoad (Micromonosporasp.FIM02-523) spore of solid slope preservation, at 28 DEG C, 220rpm, cultivates 48 hours.
Seed culture based formulas (%): soluble starch 2.0, glucose 1.0, yeast powder 2.0, peptone 1.0, MgSO4·7H2O0.05, FeSO4·7H2O0.005,CuSO4·5H2O0.005,CoCl2·6H2O0.0005, CaCO30.1～0.3, tap water is prepared, pH, and 7.0～7.5；28～32 DEG C.
Being inoculated in 20L fermentation tank (actual liquid amount 15L) according to the inoculum concentration of 5% afterwards and ferment, 28 DEG C of cultivations, initial rotating speed is 200rpm, is adjusted to 350rpm and is about 96-120 hour to fermentation ends after 48 hours.Tank pressure 0.03-0.05Mpa, ventilation is 1:1vvm.
Fermentation medium is: soluble starch 5.0, sucrose 1.0, soybean cake powder 3.0, yeast powder 1.0, MgSO4·7H2O0.04, FeSO4·7H2O0.005,CuSO4·5H2O0.005,CoCl2·6H2O0.0005, CaCO30.5, tap water is prepared, pH7.5；32℃.Fermentation is disposable after starting 24 hours adds fermentating liquid volume content 0.1% valine.Sweat detects the content of Rakicidins compounds in fermentation liquid by HPLC, and final fermentation titer is 190mg/L, and wherein RakicidinA content is 120mg/L, RakicidinB1 content be 20mg/L, RakicidinB content is 50mg/L.
Embodiment 2
Sweat basal conditions, with embodiment 1, is only distinguished and is started disposable after 24 hours to add fermentating liquid volume content 0.2% methionine in fermentation.Sweat detects the content of Rakicidins compounds in fermentation liquid by HPLC, and final fermentation titer is 270mg/L, and wherein RakicidinA content is 50mg/L, RakicidinB1 content be 120mg/L, RakicidinB content is 110mg/L.
Embodiment 3
Sweat basal conditions, with embodiment 1, is only distinguished and is started disposable after 24 hours to add fermentating liquid volume content 0.3% glycine in fermentation.Sweat detects the content of Rakicidins compounds in fermentation liquid by HPLC, and final fermentation titer is 570mg/L, and wherein RakicidinA content is 140mg/L, RakicidinB1 content be 80mg/L, RakicidinB content is 350mg/L.
Embodiment 4
Sweat basal conditions, with embodiment 1, is only distinguished and is started disposable after 24 hours to add fermentating liquid volume content 0.1% methionine and 0.2% glycine in fermentation.Sweat detects the content of Rakicidins compounds in fermentation liquid by HPLC, and final fermentation titer is 600mg/L, and wherein RakicidinA content is 100mg/L, RakicidinB1 content be 180mg/L, RakicidinB content is 320mg/L.
Embodiment 5
It is inoculated in shaking flask primary-seed medium after making bacteria suspension after being scraped by sea micromonoad (Micromonosporasp.FIM02-523) spore of solid slope preservation, at 28 DEG C, 220rpm, cultivates 48 hours.It is inoculated in 20L seed tank (actual liquid amount 15L) according to the inoculum concentration of 5% afterwards and carries out second order fermentation, 28 DEG C of cultivations, rotating speed is 300rpm, tank pressure 0.03～0.05Mpa, ventilation is 1:1vvm, cultivates 48 hours.
Shake-flask seed and seed tank formula: soluble starch 2.0, glucose 1.0, yeast powder 2.0, peptone 1.0, MgSO4·7H2O0.05, FeSO4·7H2O0.005,CuSO4·5H2O0.005,CoCl2·6H2O0.0005, CaCO30.1～0.3, tap water is prepared, pH, and 7.0～7.5；28～32 DEG C.
Being inoculated in 100L fermentation tank (actual liquid amount is 75L) according to 8% inoculum concentration, ventilation is 1:1vvm, and initial rotating speed is 200rpm, and declining according to dissolved oxygen after 24 hours steps up rotating speed to 400rpm.Start disposable after 24 hours to add fermentating liquid volume content 0.3% glycine in fermentation.
Fermentation medium is: soluble starch 5.0, sucrose 1.0, soybean cake powder 3.0, yeast powder 1.0, MgSO4·7H2O0.04, FeSO4·7H2O0.005,CuSO4·5H2O0.005,CoCl2·6H2O0.0005, CaCO30.5, tap water is prepared, pH7.5；32℃；Percent in described nutrient media components is mass fraction.Sweat detects the content of Rakicidins compounds in fermentation liquid by HPLC, and final fermentation titer is 540mg/L, and wherein RakicidinA content is 130mg/L, RakicidinB1 content be 90mg/L, RakicidinB content is 330mg/L.
Embodiment 6
It is inoculated in shaking flask primary-seed medium after making bacteria suspension after being scraped by sea micromonoad (Micromonosporasp.FIM02-523) spore of solid slope preservation, at 28 DEG C, 220rpm, cultivates 48 hours.
It is inoculated in 50L seed tank (actual liquid amount 35L) according to the inoculum concentration of 5% afterwards and carries out second order fermentation, 28 DEG C of cultivations, rotating speed is 300rpm, tank pressure 0.03～0.05Mpa, ventilation is 1:1vvm, cultivates 48 hours.
Shake-flask seed and seed tank formula: soluble starch 2.0, glucose 1.0, yeast powder 2.0, peptone 1.0, MgSO4·7H2O0.05, FeSO4·7H2O0.005,CuSO4·5H2O0.005,CoCl2·6H2O0.0005, CaCO30.1～0.3, tap water is prepared, pH, and 7.0～7.5；28～32 DEG C.
Being inoculated in 1000L fermentation tank (actual liquid amount is 720L) according to 5% inoculum concentration, ventilation is 1:1vvm, and initial rotating speed is 120rpm, and declining according to dissolved oxygen after 24 hours steps up rotating speed to 250rpm.Start disposable after 24 hours to add fermentating liquid volume content 0.3% glycine in fermentation.
Fermentation medium is: soluble starch 5.0, sucrose 1.0, soybean cake powder 3.0, yeast powder 1.0, MgSO4·7H2O0.04, FeSO4·7H2O0.005,CuSO4·5H2O0.005,CoCl2·6H2O0.0005, CaCO30.5, tap water is prepared, pH7.5；32℃；Percent in described nutrient media components is mass fraction.Sweat detects the content of Rakicidins compounds in fermentation liquid by HPLC, and final fermentation titer is 570mg/L, and wherein RakicidinA content is 110mg/L, RakicidinB1 content be 80mg/L, RakicidinB content is 380mg/L.
Embodiment 7
The preparation of RakicidinB1
Step one: the fermentation culture conditions reference literature (Jiang Hong of micromonospora bacterial strain FIM02-523, Lin Ru, Zheng Wei, Deng. the separation discriminating of the lipopeptide compound FW523 that ocean Micromonospora chalcea FIM02-523 produces and biologic activity [J]. China's antibiotic magazine, 2006,31 (5): 267-270).
Step 2: by the FIM02-523 fermentation liquid of step one gained by after the centrifugal 15min of 4500rpm, obtain mycelia slag, the mycelia slag obtained overnight is soaked 2 times with absolute methanol or the ethanol of 2 times of volumes, containing spirituous mycelia slag again to be merged by supernatant after the centrifugal 15min of 4500rpm, fermented extracted liquid will be obtained.
Step 3: HP20 macroporous resin adsorption column chromatography (blade diameter length ratio is 1:5～1:10, fills column volume 1.5-2.5L): the fermented extracted liquid (40-60L) of employing is with the alcohol concentration of 50%-55%, and flow velocity is that 40ml/min carries out upper prop absorption；After absorption completely, carry out gradient elution with the ethanol of concentration 60%-80%, detect eluent 1 with HPLC, merge the same composition (30L) containing RakicidinB1.
Step 4: step 3 eluent carries out NM200 resin absorption column chromatography (blade diameter length ratio is 1:2～1:10, fill column volume 1-2L): by the step 3 eluent alcohol concentration with 50%-55%, flow velocity is 40ml/min, carries out upper prop absorption；After absorption completely, carry out gradient elution with the ethanol of concentration 55%-80%, detect eluent 2 with HPLC, merge the same composition (25L) containing RakicidinB1.
Step 5: step 4 eluent 2, after monoploid hydrops dilutes, with equal-volume extraction into ethyl acetate 2 times, concentrating under reduced pressure, obtains crude product.
Step 6: crude product step 5 obtained, dissolves with methanol, carries out the anti-phase C18 column chromatography of middle pressure (blade diameter length ratio 1:3～1:10), flow velocity 30ml/min, with concentration for 60%-90% acetonitrile-water gradient, fraction collection, collect liquid with HPLC detection, merge same composition.
Described micromonospora FIM02-523 hid and China Committee for Culture Collection of Microorganisms's common micro-organisms center in February, 2016, and deposit number is CGMCCNo.12132.
Compound R akicidinB1: white amorphous powder.It is dissolved in chloroform, methanol, DMSO, water insoluble.High resolution mass spectrum (HR-ESI-MS) shows its molecular ion peak [M+H]+be 621.1175, infers that its molecular formula is C33H56N4O7, degree of unsaturation is 8.13CNMR and DEPT135 shows that this molecule contains 33 carbon signals, including 5 quaternary carbon (δC172.6,172.4,169.2,167.6,165.9), 4 double key carbons [comprise a sp2Mesomethylene carbon, two sp2Methine carbon (δC138.4,118.8)], 6 sp3Methine carbon [comprises two even oxygen carbon atom (δC78.0,72.4)], 13 sp3Mesomethylene carbon and 5 methine carbon atom (δC36.5,19.1,15.4,13.2,11.2)。1HNMR shows that this compound has 4 double bond proton [δH6.87 (1H, d, J=15.0Hz), 6.16 (1H, d, J=15.0Hz), 5.44 (1H, s), 5.32 (1H, s)], 5 exchangeable protons [δH8.88 (1H, s), 8.05 (1H, d, J=9.9Hz), 7.31 (1H, s), 7.28 (1H, s), 5.66 (1H, d, J=6.0Hz)], 5 methyl proton [δH2.95 (3H, s), 1.05 (3H, d, J=7.0Hz), 0.93 (3H, d, J=6.9Hz), 0.83 (3H, t, J=7.0Hz), 0.81 (3H, d, J=6.7Hz)].All of Hydrogen Proton passes through hsqc spectrum1H–13C is relevant to be pointed out.1H–1The coupling constant of HCOSY Correlated Spectroscopy and proton shows that this compound contains 4 independent spin coupling systems: NH-2 C-2 C-3 OH-3, C-9 C-10, C-31 C-14 C-15 C-16 (C-32) C-17 C-18 and C-26 C-27 C-28 (C-33) C-29 C-30.In conjunction with1H–1HCOSY and HMBC is it can be seen that H-2 and C-1/C-5 is correlated with, and H-3 and C-4 is correlated with, and OH-3 and C-2/C-3 is correlated with, and Ha-6 and C-5/C-7 is correlated with, H3-7 is relevant to C-6/C-8, and H-9 and C-8/C-11 is correlated with, and H-10 and C-8/C-11 is correlated with, and Hb-12 and C-10/C-11 is correlated with, and H-15 and C-1 is correlated with, H3-30 is relevant to C-28/C-29, H3-31 is relevant to C-13/C-14/C-15, H3-32 is relevant to C-15/C-16/C-17, and H3-33 is relevant to C-27/C-28/C-29, and the chemical shift of hydrogen and carbon is listed in the table below:

Claims (9)

1. the method for a fermenting and producing Rakicidins compounds, including: China Committee for Culture Collection of Microorganisms's common micro-organisms center will be preserved in, the micromonospora that deposit number is CGMCCNO.12132 carries out solid slant culture, shake-flask seed cultivation, seed tank culture, ferment tank cultivation, post processing and get final product, is characterized in that: just add aminoacid in the ferment tank incipient stage.
2. the process of claim 1 wherein that Rakicidins compounds is selected from the compound of following any structure:
3. one or more in valine, glycine, methionine of the aminoacid added described in the process of claim 1 wherein.
4. the method for claim 3, wherein add valine, glycine, methionine concentration respectively to 0.05～0.3%, 0.05～0.3%, 0.05～0.3%, be all weight percentage.
5. the method for claim 1, including:
A. solid slant culture: the micromonospora that deposit number is CGMCCNO.12132 is inoculated in solid slope, after in 30-37 DEG C constant temperature culture 10-15 days, rear preservation under room temperature；
B. shake-flask seed is cultivated: load seed culture medium 80ml in 500ml triangular flask, sterilizes 30 minutes for 121 DEG C, inoculates spore suspension, cultivate 48 hours in shaking table 230rpm, 28-35 DEG C after cooling；
C. seed tank culture: seed good for shake-flask culture is merged, is inoculated in seed tank culture base with the 1-5% of seed tank liquid amount, 200-300rpm, ventilation 1:1vvm, cultivates 48 hours by 28 DEG C；
D. ferment tank is cultivated: be inoculated in fermentation tank by seed good for seed tank culture with the inoculum concentration of 5-10%, incubation time is 72-100 hour, fermentation rotating speed controls 200-400rpm, ventilation controls 0.08-1.2vvm, adds one or more in valine, glycine, methionine during the fermentation as the precursor fermented.
E. post processing, to obtain final product.
6. the method for claim 5, wherein valine concentration to be 0.05-0.3%, glycine concentration be 0.05-0.3%, methionine concentration are 0.05-0.3%.
7. the method for claim 5, wherein in shake-flask seed incubation step, the component of seed culture medium and the weight percentage of each component are: soluble starch 1～3, glucose 0.5～1.0, yeast powder 1～2, peptone 0.5～2.0, MgSO4·7H2O0.05、FeSO4·7H2O0.005、CuSO4·5H2O0.005、CoCl2·6H2O0.0005、CaCO30.1～0.3, tap water is prepared, pH, and 7.0～7.5.
8. the method for claim 5, wherein the component of fermentation medium and the weight percentage of each component are: soluble starch 3～6, sucrose 0.5～2.0, soybean cake powder 1.0～3.0, yeast powder 1.0～3.0, MgSO4·7H2O0.04、FeSO4·7H2O0.005、CuSO4·5H2O0.005、CoCl2·6H2O0.0005、CaCO30.3～0.6, tap water is prepared, pH7.5.
9. the method for claim 5, wherein post-processing approach includes: taking fermentation liquid and add methanol mixed uniformly, after ultrasonic Treatment, 10000rpm is centrifuged, and takes clear liquid after 0.45um membrane filtration for measuring each component titer of fermentation liquid.
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* Cited by examiner, † Cited by third party
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CN108130284A (en) * 2017-11-24 2018-06-08 福建省微生物研究所 A kind of the sea micromonoad strain and its application of the production Rakicidin A that ferment
CN108300672A (en) * 2017-11-24 2018-07-20 福建省微生物研究所 A kind of the sea micromonoad strain and its application of fermentation production Rakicidin B1
CN108300672B (en) * 2017-11-24 2019-02-15 福建省微生物研究所 A kind of fermentation produces the sea micromonoad strain and its application of Rakicidin B1
CN110698537A (en) * 2019-08-12 2020-01-17 福建省微生物研究所 Natural Rakicidins compound Rakicidin B1-2 and fermentation extraction method thereof
CN110698537B (en) * 2019-08-12 2023-05-12 福建省微生物研究所 Natural Rakicidins compound Rakicidin B1-2 and fermentation extraction method thereof
CN112608952A (en) * 2020-09-11 2021-04-06 福建省微生物研究所 Method for producing macrolide compound FW05328-1 through high-efficiency fermentation
CN112608952B (en) * 2020-09-11 2021-08-03 福建省微生物研究所 Method for producing macrolide compound FW05328-1 through high-efficiency fermentation

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