CN103088104B - Method for optimizing fermentation medium by improving rapamycin production by using metabolic profiling analysis - Google Patents
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Abstract
The invention provides a method for optimizing a fermentation medium by improving rapamycin production by using metabolic profiling analysis, which researches dynamic changes and differences of bacteria intracellular metabolites under the culture conditions of initial fermentation medium and optimized fermentation medium by using the methods of dynamic detection and metabolic profiling analysis. The analysis result of partial least squares indicates that the bacteria primary metabolite profile and the rapamycin production have correlation and a further dynamic adding measure is provided to the medium by analyzing the dynamic changes of the key compounds combining related processes. Due to the research of S.hygroscopicus U1-6E7 fermentation medium, the rapamycin production is improved from 196mg/L to 307mg/L and is improved by 56.6%, which indicates that rational guidance of optimization of fermentation mediums by dynamic detection combining the metabolite profile analysis method is capable of successfully improving fermentation level of antibiotic and providing a new idea for optimization of fermentation mediums.
Description
Technical field
The invention belongs to metabolic profiling analysis and instruct fermentation optimization technical field, particularly in metabolic profile method performance analysis born of the same parents, affect the synthetic crucial metabolite of rapamycin, rationality instructs the interpolation optimization of high yield rapamycin fermention medium.
Background technology
Rapamycin is that Streptomyces hygroscopicus is synthetic, a kind of important natural lactone compound; it has multiple biological activity, as antimycotic, immunosuppressive activity, anti-tumor activity, neuroprotective (Steiner et al.1997) and anti-ageing.The potent immunosuppressive activity that particularly rapamycin has, has been approved for the treatment that treating organs is transplanted anti-rejection and autoimmune disease clinically.In addition the semi-synthetic derivative temsirolimus of rapamycin and everolimus also go through to be used for the treatment of clinically advanced renal cell cancer.Due to important pharmaceutical use and the biological activity that rapamycin has, its biosynthetic pathway and regulatory mechanism be being furtherd investigate gradually also.
In wild-type streptomyces hygroscopicus, the output of rapamycin is lower, therefore, for suitability for industrialized production and the clinical application of rapamycin, the further raising that in streptomyces hygroscopicus, rapamycin is tired and the various derivatives of rapamycin synthetic imperative.Fermention medium optimization is a kind of effective ways that improve antibiotic fermentation level, wherein Lee et al. is optimized rear rapamycin production by single factor experiment to Streptomyces hygroscopicus C9 fermentation culture nitrogenous source and is increased to 125mg/L by 93mg/L, before optimizing, has improved more than 30%; Cheng et al. is by fermention medium, optimization makes rapamycin production bring up to 97mg/L by 18mg/L; The optimum producting condition that Sallam et al. produces bacterial strain Streptomyces hygroscopicus ATCC29253 by experiment of single factor method to rapamycin is optimized, and rapamycin production is brought up to 41.46mg/L from 10.66mg/L.These fermention medium optimizations that utilize single factor or response surface method to carry out lay particular emphasis on the change of medium component and its proportioning.And the variation of fermentation culture based component has complicated impact to thalline born of the same parents intracellular metabolite, and the change of the thalline born of the same parents intracellular metabolite behavior that causes of medium optimization is not also furtherd investigate.
In microorganism, primary metabolite level changes directly affects the supply of microbiotic precursor and antibiotic synthetic.As secondary metabolites, rapamycin has complicated biosynthetic pathway, its direct biosynthesizing precursor comprises (4R, 5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC), nipecotic acid, 7 malonyl coenzyme As and 7 methylmalonyl CoAs, and the biosynthesizing of these direct precursors relates to multiple primary metabolite approach, as carbohydrate metabolism, tricarboxylic acid cycle, fatty acid metabolism, shikimic acid metabolism, amino acid metabolism etc.It is most important that the born of the same parents' intracellular metabolite thing dynamic change that relates to these pathways metabolisms affects in the synthetic born of the same parents of rapamycin key factor for deep understanding.Metabolism group, can be measured and evaluate biosystem and responded by dynamic multiparameter metabolism in the born of the same parents after the external world or internal disturbance by metabolic information in comprehensive analysis of biological samples as a powerful instrument.(the Korneli C such as Korneli, Bolten CJ, Godard T, Franco-Lara E, Wittmann C (2012) Debottlenecking recombinant protein production in Bacillus megaterium under large-scale conditions-targeted precursor feeding designed from metabolomics.Biotechnol Bioeng109:1538 – 1550) utilize metabolism group means to study the bottleneck of bacillus megaterium production green fluorescent protein under extensive condition, by stream, adding amino acid precursor makes green fluorescent protein output improve 100%.(the Lu S such as Lu, Wang J, Niu Y, Yang J, Zhou J, Yuan Y (2012) Metabolic profiling reveals growth related FAME productivity and quality of Chlorella sorokiniana with different inoculum sizes.Biotechnol Bioeng109:1651 – 1662) utilize metabolic profiling analysis method research different vaccination amount chlorella to be produced to the impact of fatty acid methyl ester output and quality, find that light intensity is wherein to limit the key factor of fatty acid methyl ester output under high inoculum size condition.Yet, up to the present, adopt metabolic profiling analysis, detection of dynamic born of the same parents' intracellular metabolite thing changes, the synthetic impact of the change of investigation fermentation culture based component on Streptomyces hygroscopicus primary metabolite level and rapamycin, analyze crucial metabolite in the synthetic born of the same parents of restriction rapamycin, and then rationality instructs the research of rapamycin fermention medium optimization not also to be in the news.
Summary of the invention
The present invention's object is to utilize detection of dynamic and gas chromatograph-mass spectrometer (GC-MS) for basic metabolic profiling analysis method, by detection of dynamic streptomyces hygroscopicus born of the same parents intracellular metabolite thing, change, (fermention medium M1 is initial medium to investigate the change of fermentation culture based component, fermention medium M2 is M1, and through response surface, optimization obtains) born of the same parents' intracellular metabolite thing metaboilic level difference of causing, analyze the synthetic dependency of thalline born of the same parents' intracellular metabolite and rapamycin, identify and affect key compound in the biosynthetic born of the same parents of rapamycin, in conjunction with relational approach and rapamycin biosynthetic pathway, on affecting key compound dynamic change in the synthetic born of the same parents of rapamycin, analyze, for the synthetic key factor of restriction rapamycin, M2 substratum has been proposed to further dynamic appending measure, thereby obtain optimization substratum M3, further to improve rapamycin production.Proposed to utilize metabolic profiling analysis to improve the method for the optimization fermention medium of rapamycin production.
The object of the invention is to be achieved through the following technical solutions:
Setting fermention medium M1 is initial medium, is called for short M1; Fermention medium M2 is that the optimization of M1 process response surface obtains, and is called for short M2: step is as follows:
1) under M1, M2 culture condition, streptomyces hygroscopicus fermentation character changes: rapamycin production, thalline biomass, fermented liquid pH value and fermented liquid total sugar content under fermention medium M1, two kinds of culture condition of M2 are carried out to detection of dynamic; Rapamycin biosynthesizing curve according to streptomyces hygroscopicus under M1 and M2 culture medium culturing condition is determined the sampling spot of metabolic profile research, and the time period that rapamycin production has a significant difference under two kinds of conditions samples.
2) coenzyme A compounds in M1 and M2 culture condition hypothallus born of the same parents is extracted, adopt high performance liquid chromatography-electrospray ion source-mass spectrum/GC-MS (LC-EIS-MS/MS) to detect, compare with standard coenzyme A compounds ion fragment, coenzyme A compounds in M1 and M2 culture condition hypothallus born of the same parents is carried out quantitatively.
3) utilize gas chromatograph-mass spectrometer (GC-MS) to detect M1 and M2 culture condition hypothallus born of the same parents intracellular metabolite thing, utilize Masslynx software to identify and quantification mass spectra peak, by the mass spectrum fragment of every kind of compound and NIST mass spectral database are contrasted, every kind of metabolite is identified.
4) utilize SIMCA-P Demo software to detect in born of the same parents compound to GC-MS and rapamycin production data are carried out Partial Least Squares Method, the dependency that research M1 and M2 culture medium culturing condition hypothallus primary metabolite and rapamycin are synthetic, identify two kinds of condition hypothallus Difference of Metabolisms and rapamycin production are had to the compound of remarkable contribution (VIP>1), and determine the pathways metabolism that these compounds relate to.
5) to step 2) in born of the same parents coenzyme A compound and the definite compound dynamic change of step 4) be analyzed, under research M2 culture condition, rapamycin production is higher than key factor in the born of the same parents of M1, and then for key factor in these born of the same parents, rapamycin fermention medium is proposed to innovative approach, obtain optimization fermention medium M3, rationality instructs the optimization of rapamycin fermention medium.
According to technique scheme, at streptomyces hygroscopicus Streptomyces hygroscopicus U1-6E7, preservation China common micro-organisms culture presevation administrative center strain number: in CGMCC6988, initial fermention medium M1(40g/L glucose, 20g/L N.F,USP MANNITOL, 45g/L soybean cake powder, 0.3g/L ammonium sulfate, 0.3g/L K
2hPO
4), response surface is optimized fermention medium M2(30g/L glucose, 30g/L N.F,USP MANNITOL, 40g/L soybean cake powder, 0.5g/L ammonium sulfate, 0.1g/L K
2hPO
4) carried out rapamycin fermention medium optimization research under condition.Detailed process is as follows:
1), according to rapamycin biosynthesizing curve under M1 and M2 condition, determine that it is 48h, 72h, 96h and 120h that group is learned the sampling spot of research.；
2) LC-EIS-MS/MS detects coenzyme class category-A compound in M1 and M2 condition hypothallus born of the same parents, according to coenzyme class category-A compound standard substance color atlas, determines coenzyme class category-A compound concentration in thalline born of the same parents.
3) GC-MS detects M1 and M2 condition hypothallus born of the same parents intracellular metabolite thing, and detection by quantitative goes out 91 kinds of compounds altogether, wherein 79 kinds identified, comprise 34 kinds of organic acids, 20 seed amino acids, 13 kinds of carbohydrates, 4 kinds of amines, 5 kinds other, 12 kinds are not identified.
4) PLS analyzes and to show that bacterial metabolism synthesizes and has dependency with rapamycin, identified that 16 kinds affect key compound in the synthetic born of the same parents of rapamycin, these compounds relate generally to tricarboxylic acid cycle (TCA), fatty acid metabolism, shikimic acid metabolism and amino acid metabolism.
5) in conjunction with rapamycin biosynthetic pathway and TCA circulation, fatty acid metabolism, shikimic acid metabolism and amino acid metabolism, on affecting the dynamic change of key compound in the synthetic born of the same parents of rapamycin, to analyze, rationality instructs medium optimization.On the basis of M2 fermention medium, fermentation 0h adds 1.0g/L Witconol 2301,12h adds 1.0g/L Methionin, 24h adds 0.5g/L shikimic acid, 36h adds 0.5g/L sodium succinate, 0.1g/L phenylalanine, 0.1g/L tryptophane and 0.1g/L tyrosine, the final optimization substratum M3 that obtains, rapamycin production has reached 307mg/L, than M2, has improved 56.6%.
The present invention utilizes detection of dynamic and metabolic profiling analysis method, be used for instructing the optimization of high yield rapamycin fermention medium, by investigating small molecules metabolite metaboilic level difference in the born of the same parents that cause under two kinds of fermention medium conditions, analyze the synthetic dependency of two kinds of condition lower eyelid intracellular metabolites and rapamycin, in conjunction with associated metabolic approach, small molecules metabolite dynamic change in two kinds of culture condition lower eyelids is analyzed, disclose the synthetic small molecules metabolite of restriction rapamycin, and analyze its deficiency, rationality proposes further dynamic appending measure, for improving rapamycin production, provide a new thinking.
Accompanying drawing explanation
Under Fig. 1: M2, M1 culture condition, rapamycin production, thalline biomass, pH value move and fermented liquid total reducing sugar state variation diagram.
Fig. 2: the bacterial metabolism profile based under M2, M1 culture condition and the PLS model of rapamycin production, model parameter R2 (X) is 0.603, R2 (Y) is 0.987, Q2 is 0.978.A:PLS shot chart t[1]/u[1] disclosed the otherness of two kinds of culture condition hypothalluses metabolism and the dependency between metabolite profile (t1) and rapamycin production (u1).B: the PLS model VIP being drawn by metabolite data schemes.
Fig. 3: the additives additive effect of different concns.
Fig. 4: substratum adds the impact of measure on rapamycin production and fermentation byproduct.
Embodiment
The method according to this invention below, the present invention will be further described in conjunction with specific embodiments:
Step 1:
We adopt streptomyces hygroscopicus be Streptomyces hygroscopicus U1-6E7.Preservation China common micro-organisms culture presevation administrative center strain number: CGMCC6988.Composition and the content of the fermention medium M1 selecting are: 40g/L glucose, 20g/L N.F,USP MANNITOL, 45g/L soybean cake powder, 0.3g/L ammonium sulfate, 0.3g/LK
2hPO
4; Utilize response surface method to be optimized initial fermention medium M1, obtained optimization fermention medium M2.The composition of fermention medium M2 and content are: 30g/L glucose, 30g/L N.F,USP MANNITOL, 40g/L soybean cake powder, 0.5g/L ammonium sulfate, 0.1g/L K
2hPO
4.Rapamycin resultant quantity, thalline biomass, fermentating liquid PH value and fermented liquid total sugar content under M2, two kinds of culture condition of M1 are carried out to detection of dynamic, and its result is as Fig. 1. as shown in.S.hygroscopicus U1-6E7 has obvious fermentation advantage in fermention medium M2, and rapamycin production has improved 39%, reaches 196mg/L.Wherein the synthesis rate of the rapamycin under M2 condition is obviously greater than M1, according to rapamycin resultant curve under M1, M2 culture condition, under two kinds of conditions, rapamycin is synthetic starts to produce notable difference (>15mg/L) and rapamycin a large amount of synthesis phases and carries out metabolic profiling analysis research and sample, and sampling spot is made as 48h, 72h, 96h and 120h.
Step 2:
In thalline, first the extraction of coenzyme A compounds, extract the thalline 15%(w/v of extraction to thalline in fermented liquid with containing silicone oil (AR200:DC200,4:1, δ=1.010)) trichoroacetic acid(TCA) suspension, then 20000 leave heart 15min.60% thalline is extracted in trichoroacetic acid(TCA) solution, utilize OASIS HLB SPE test kit to carry out vacuum filtration to trichoroacetic acid(TCA) thalline extraction liquid, utilize afterwards 0.15% trichoroacetic acid(TCA) solution and normal hexane washing reagent box, finally use methyl alcohol ammonium hydroxide (99:1, v/v) dissolve and extract coenzyme A compounds in thalline, obtain internal cell coenzyme category-A compound solution.Get 80 μ L coenzyme A compounds solution and carry out high performance liquid chromatography-electrospray ion source-mass spectrum/GC-MS (LC-EIS-MS/MS, Waters/Micromass, Manchester, UK).By high performance liquid chromatography-electrospray ion source-mass spectrum/mass spectrum (LC-EIS-MS/MS) figure comparison of coenzyme A standard substance; in detection chromatogram fragment, coenzyme A compound parent ion is to the conversion (m/zparent>m/z daughter) of daughter ion; result is: acetyl-CoA, 810>303; Propionyl coenzyme A, 824>317; Isobutyryl coenzyme A, 841>334; Malonyl coenzyme A, 854>347; Methylmalonyl CoA and succinyl-coenzyme A, 868>361.With standard substance coenzyme A compound ions fragment, in detected born of the same parents, the concentration of coenzyme A compound is as shown in table 1.
Table 1
Step 3:
The thalline sample sampling of metabolic profile research with cancellation method is: getting 5mL fermented liquid is that the cellulose acetate film of 0.8um carries out fast filtering with aperture is housed, and then with 15ml, 4 ℃, 0.9% sodium chloride solution, washs fast thalline three times.Thalline after washing is transferred to rapidly and is equipped with in 2.5mL ,-40 ℃, the centrifuge tube of the 50% cold methanol aqueous solution, and be stored in-80 ℃.Sample is carried out to thawing with freezing of three-wheel, vortex vibration 1min ,-20 degree, the centrifugal 15min of 10000g, quantitative collection supernatant is also stored in-80 ℃.Get 100ul born of the same parents' intracellular metabolite thing extracting solution in 1.5mL centrifuge tube, and add internal standard substance, carry out vacuum lyophilization.Dry sample is carried out to derivatization treatment.The analysis of born of the same parents' intracellular metabolite thing adopts GC-MS system, comprise gas-chromatography Agilent6890N, mass spectrum Agilent5975C MSD, automatic sampler Agilent7683B and chromatographic column DB-5MS capillary column (30m * 0.25mm, 0.25 μ m, Agilent Technologies).By gas chromatograph-mass spectrometer (GC-MS) altogether detection by quantitative go out 91 kinds of compounds (as shown in table 2), wherein 79 kinds identified, comprise 34 kinds of organic acids, 20 seed amino acids, 13 kinds of carbohydrates, 4 kinds of amines, 5 kinds other, 12 kinds are not identified.These materials have related to tricarboxylic acid cycle (TCA) in S.hygroscopicus U1-6E7 born of the same parents, carbohydrate metabolism, fatty acid metabolism, amino acid metabolism, nucleotide metabolism, shikimic acid pathway etc., have effectively reflected born of the same parents' intracellular metabolite state of S.hygroscopicus U1-6E7 fermentative production rapamycin.
Table 2
Step 4:
GC-MS is detected in born of the same parents to compound and rapamycin production data are carried out partial least square method (PLS) (PLS) analysis, PLS shot chart t[1]/u[1] (figure .2a) result shows that bacterial metabolism there are differences under M2 and two kinds of conditions of M1, i.e. the change of medium component causes the variation of thalline born of the same parents intracellular metabolite; Under same fermentation condition also there is significant difference in different time points bacterial metabolism; And PLS analytical proof bacterial metabolism and rapamycin there is very strong dependency between synthetic.Variable drop index (VIP) figure (figure .2b) has represented every kind of compound identifying contribution degree to two kinds of culture condition hypothallus Difference of Metabolisms.Wherein, VIP score is greater than 1 compound and shows that its Clustering on sample in PLS model has significant impact, and VIP score is higher shows that this compound is larger to the contribution degree of the Clustering of sample in PLS model.The VIP value that has 16 kinds of compounds is greater than 1(figure .2b), be palmitinic acid, α-ketoglutaric acid, nipecotic acid, lauric acid, citric acid, shikimic acid, stearic acid, oleic acid, Methionin, tryptophane, tyrosine, phenylalanine and 4 kinds of unknown compounds, these compounds have related to TCA cyclic metabolism, fatty acid metabolism, amino acid metabolism and the shikimic acid pathway of streptomyces hygroscopicus.
Step 5:
According to TCA circulation compound and coenzyme A compounds Dynamic Variation Analysis, in cell, rapamycin precursor methylmalonyl CoA metaboilic level is to affect a synthetic important factor of rapamycin.In M2 substratum, add sodium succinate, the succinate that external source is added enters TCA circulation and is converted into fumaric acid, oxysuccinic acid etc., reduce succinyl-coenzyme A to the conversion of succsinic acid, sodium succinate interpolation concentration and interpolation time are optimized, result is as shown in figure .3a.When the 36h in fermentation adds after the sodium succinate of 0.5g/L, rapamycin production is increased to 238mg/L by 196mg/L.
According to lipid acid in born of the same parents and coenzyme A compound Dynamic Variation Analysis, external source is added oily substance can slow down the synthetic Competition synthetic with rapamycin of lipid acid in cell on the one hand, makes on the other hand more lipid acid enter beta-oxidation approach and generates acetyl-CoA.In M2 substratum, to add respectively soya-bean oil and Witconol 2301, and its interpolation concentration and interpolation time are optimized, result is as shown in figure .3bc, and after the 12h of fermentation adds 0.5g/L soya-bean oil, rapamycin production is increased to 241mg/L by 196mg/L.And rapamycin production is increased to 268mg/L by 196mg/L after 0h adds 1.0g/L Witconol 2301, improved 36.7%.
According to shikimic acid pathway metabolite Dynamic Variation Analysis, (4R in cell, 5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid(DHCHC) metaboilic level is to affect a synthetic important factor of rapamycin, in order to strengthen chorismic acid in shikimic acid pathway, to DHCHC, transform, in M2 substratum, add respectively shikimic acid, and its interpolation concentration and interpolation time are optimized, its effect, as shown in figure .3d, is increased to 223mg/L to rapamycin production after adding 0.5g/L shikimic acid in M2 by 196mg/L at the 24h of fermentation.In addition, on the basis of shikimic acid, add phenylalanine, tryptophane and tyrosine mixing mother liquor simultaneously, and it is added to concentration and the time is optimized, result (figure .3e) shows that on the basis of adding shikimic acid rapamycin production has been brought up to 247mg/L by 196mg/L after the 36h of fermentation adds phenylalanine, tryptophane and the tyrosine mother liquor of 0.1g/L simultaneously.
According to the analysis to Methionin metabolite metaboilic level, in strengthening S. hygroscopicus U1-6E7, nipecotic acid is to the conversion of rapamycin heterocycle unit, in fermention medium, add Methionin, utilize Methionin to carry out making more Methionin enter catabolic pathway on the basis of growth metabolism to be converted into nipecotic acid meeting thalline.In M2, add Methionin, and its interpolation concentration and time are optimized, its effect is as shown in figure .3f, and after the 12h in fermentation adds the Methionin of 1.0g/L, rapamycin production is increased to 244mg/L by 196mg/L.
According to born of the same parents' intracellular metabolite thing Dynamic Variation Analysis, on M2 basis, in fermentation, 0h adds 1g/L Witconol 2301, during 12h, add 1g/L Methionin, 24h adds 0.5g/L shikimic acid, 36h adds 0.5g/L sodium succinate and concentration is tryptophane, tyrosine and the phenylalanine of 0.1g/L, and finally obtains substratum M3.Above-mentioned interpolation measure on the impact of rapamycin production and fermentation byproduct as figure .4 as shown in, when fermentation ends (120h), under M3 culture condition, rapamycin production is increased to 307mg/L by 196mg/L, than the rapamycin production under M2 culture condition, has improved 56.6% (figure .4).And when fermentation ends (120h), compare with M2, M3 condition bottom fermentation by product elaiophylin and polyetherin A output are reduced to 37mg/L and 81mg/L by 51mg/L and 137mg/L respectively, compare with M2 and reduced by 35% and 52% (figure .4).
Claims (1)
1. utilize metabolic profiling analysis to improve the method for the optimization fermention medium of rapamycin production, setting fermention medium M1 is initial medium, is called for short M1; Fermention medium M2 is that the optimization of M1 process response surface obtains, and is called for short M2: it is characterized in that step is as follows:
1) under M1, M2 culture condition, streptomyces hygroscopicus fermentation character changes: rapamycin production, thalline biomass, fermented liquid pH value and fermented liquid total sugar content under fermention medium M1, two kinds of culture condition of M2 are carried out to detection of dynamic; Rapamycin biosynthesizing curve according to streptomyces hygroscopicus under M1 and M2 culture medium culturing condition is determined the sampling spot of metabolism group research, and the time period that rapamycin production has a significant difference under two kinds of conditions samples;
2) coenzyme A compounds in M1 and M2 culture condition hypothallus born of the same parents is extracted, adopt high performance liquid chromatography-electrospray ion source-mass spectrum/GC-MS to detect, compare with standard coenzyme A compounds ion fragment, coenzyme A compounds in M1 and M2 culture condition hypothallus born of the same parents is carried out quantitatively;
3) utilize gas chromatograph-mass spectrometer to detect M1 and M2 culture condition hypothallus born of the same parents intracellular metabolite thing, utilize Masslynx software to identify and quantification mass spectra peak, by the mass spectrum fragment of every kind of compound and NIST mass spectral database are contrasted, every kind of metabolite is identified;
4) utilize SIMCA-P Demo software to detect in born of the same parents compound to gas chromatograph-mass spectrometer and rapamycin production data are carried out Partial Least Squares Method, the dependency that research M1 and M2 culture medium culturing condition hypothallus primary metabolite and rapamycin are synthetic, identify two kinds of condition hypothallus Difference of Metabolisms and rapamycin production are had to the compound of remarkable contribution, and determine the pathways metabolism that these compounds relate to;
5) to step 2) in born of the same parents coenzyme A compound and the definite compound dynamic change of step 4) be analyzed, under research M2 culture condition, rapamycin production is higher than key factor in the born of the same parents of M1, and then for key factor in these born of the same parents, rapamycin fermention medium is proposed to innovative approach, obtain optimization fermention medium M3, rationality instructs the optimization of rapamycin fermention medium;
Described fermentation culture rapamycin bacterial strain used is streptomyces hygroscopicus CGMCC6988;
Described initial fermention medium M1 forms and content is: glucose 40g/L, N.F,USP MANNITOL 20g/L, soybean cake powder 45g/L, ammonium sulfate 0.3g/L, K
2hPO
40.3g/L;
Described fermention medium M2 forms and content is: glucose 30g/L, N.F,USP MANNITOL 30g/L, soybean cake powder 40g/L, ammonium sulfate 0.5g/L, K
2hPO
40.1g/L;
Described fermention medium M3 is on M2 basis, and in fermentation, 0h adds 1g/L Witconol 2301, adds 1g/L Methionin during 12h, and 24h adds 0.5g/L shikimic acid, and 36h interpolation 0.5g/L sodium succinate and concentration are tryptophane, tyrosine and the phenylalanine of 0.1g/L.
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