CN103088104A - 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 the fermentation optimization technical field, particularly affect the synthetic crucial metabolite of rapamycin in metabolic profile method performance analysis born of the same parents, 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.Particularly the potent immunosuppressive activity that has of rapamycin, be approved for the treatment that treating organs is transplanted anti-rejection and autoimmune disease clinically.The semi-synthetic derivative temsirolimus of rapamycin and everolimus also go through to be used for the treatment of clinically advanced renal cell cancer in addition.Due to important pharmaceutical use and the biological activity that rapamycin has, its biosynthetic pathway and regulatory mechanism also gradually quilt are furtherd investigate.
In the 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 the 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, has improved more than 30% before optimizing; Cheng et al. is by optimization makes rapamycin production bring up to 97mg/L by 18mg/L to fermention medium; The optimum producting condition that Sallam et al. produces bacterial strain Streptomyces hygroscopicus ATCC29253 by the 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, the primary metabolite level changes supply and antibiotic the synthesizing that directly affects the microbiotic precursor.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.By metabolic information in the analysis-by-synthesis biological sample, can measuring and estimate in the born of the same parents after biosystem is subjected to the external world or internal disturbance dynamically, the multiparameter metabolism responds metabolism group 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 the bottleneck of bacillus megaterium production green fluorescent protein under the extensive condition of metabolism group means research, adding amino acid precursor by stream 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 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, investigate the change of fermentation culture based component to the synthetic impact of 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 also not to be in the news.
Summary of the invention
the present invention's purpose is to utilize detection of dynamic and gas chromatograph-mass spectrometer (GC-MS) to be basic metabolic profiling analysis method, change by detection of dynamic streptomyces hygroscopicus born of the same parents intracellular metabolite thing, (fermention medium M1 is initial medium to investigate the change of fermentation culture based component, fermention medium M2 is M1, and optimization obtains through response surface) 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, identifying affects key compound in the biosynthetic born of the same parents of rapamycin, analyze affecting in the synthetic born of the same parents of rapamycin the key compound dynamic change in conjunction with relational approach and rapamycin biosynthetic pathway, for the synthetic key factor of restriction rapamycin, further dynamic appending measure has been proposed the M2 substratum, thereby obtain optimization substratum M3, with further raising rapamycin production.Proposed to utilize metabolic profiling analysis to improve the method for the optimization fermention medium of rapamycin production.
The objective 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 be called for short M2: step is as follows:
1) under M1, M2 culture condition, the 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 detection of dynamic; Determine the sampling spot of metabolic profile research according to the rapamycin biosynthesizing curve of streptomyces hygroscopicus under M1 and M2 culture medium culturing condition, the time period that rapamycin production has a significant difference under two kinds of conditions takes a sample.
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 that mass spectra peak is identified and quantification, by the mass spectrum fragment of every kind of compound and NIST mass spectral database are compared, 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 the 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 the compound that two kinds of condition hypothallus Difference of Metabolisms and rapamycin production is had remarkable contribution (VIP〉1), and determine the pathways metabolism that these compounds relate to.
5) to step 2) coenzyme A compound and step 4) are determined in born of the same parents compound dynamic change is 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 to rapamycin fermention medium improvement measures, 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 the 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 the analysis showed that bacterial metabolism and rapamycin are synthetic and has a dependency, 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, the dynamic change that affects key compound in the synthetic born of the same parents of rapamycin to be analyzed, rationality instructs medium optimization.On the basis of M2 fermention medium, fermentation 0h adds the 1.0g/L Witconol 2301,12h adds 1.0g/L Methionin, 24h adds the 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, has improved 56.6% than M2.
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 the 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, provide a new thinking for improving rapamycin production.
Description of drawings
Under Fig. 1: M2, M1 culture condition, rapamycin production, thalline biomass, pH value move and fermented liquid total reducing sugar attitude variation diagram.
Fig. 2: based on the PLS model of the bacterial metabolism profile under M2, M1 culture condition and 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 that is drawn by metabolite data schemes.
Fig. 3: the additives additive effect of different concns.
Fig. 4: substratum adds measure to the impact of rapamycin production and fermentation byproduct.
Embodiment
Following the method according to this invention, 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 that selects are: 40g/L glucose, 20g/L N.F,USP MANNITOL, 45g/L soybean cake powder, 0.3g/L ammonium sulfate, 0.3g/LK
2HPO
4Utilize the response surface method that initial fermention medium M1 is optimized, 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
4Rapamycin 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 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, rapamycin production has improved 39%, reaches 196mg/L.Wherein the synthesis rate of the rapamycin under the M2 condition is obviously greater than M1, according to rapamycin resultant curve under M1, M2 culture condition, rapamycin is synthetic under two kinds of conditions begins to produce notable difference (〉 15mg/L) and rapamycin a large amount of synthesis phases carry out metabolic profiling analysis research and take a sample, sampling spot is made as 48h, 72h, 96h and 120h.
Step 2:
In thalline, at first the extraction of coenzyme A compounds, namely extract the thalline 15%(w/v of extraction to thalline in fermented liquid with containing silicone oil (AR200:DC200,4:1, δ=1.010)) the 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, use at last methyl alcohol ammonium hydroxide (99:1, v/v) coenzyme A compounds in thalline is extracted in dissolving, obtains 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).High performance liquid chromatography-electrospray ion source by the coenzyme A standard substance-mass spectrum/mass spectrum (LC-EIS-MS/MS) figure comparison, detect coenzyme A compound parent ion in the chromatogram fragment to the conversion of daughter ion (m/zparent〉m/z daughter), result is: acetyl-CoA, 810〉303; Propionyl coenzyme A, 824〉317; The 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 the cancellation method is: getting the 5mL fermented liquid is that the cellulose acetate film of 0.8um carries out fast filtering with the aperture is housed, and then washs fast thalline three times with 15ml, 4 ℃, 0.9% sodium chloride solution.Thalline after washing is transferred to rapidly 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 thawing with freezing of three-wheel, vortex vibration 1min ,-20 degree, the centrifugal 15min of 10000g, the quantitative collection supernatant also is stored in-80 ℃.Get 100ul born of the same parents' intracellular metabolite thing extracting solution in the 1.5mL centrifuge tube, and add internal standard substance, carry out vacuum lyophilization.Dry sample is carried out derivatization treatment.The GC-MS system is adopted in the analysis of born of the same parents' intracellular metabolite thing, 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 compound and the 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 the same fermentation condition, also there is significant difference in the different time points bacterial metabolism; And the PLS analytical proof bacterial metabolism and rapamycin have very strong dependency between synthetic.Variable drop index (VIP) figure (figure .2b) has represented that every kind of compound identifying is to the contribution degree of two kinds of culture condition hypothallus Difference of Metabolisms.Wherein, the VIP score shows that greater than 1 compound its Clustering on sample in the PLS model has significant impact, and the VIP score is higher shows that this compound is larger to the contribution degree of the Clustering of sample in the PLS model.Have the VIP value of 16 kinds of compounds 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.Add sodium succinate in the M2 substratum, the succinate that external source is added enters the 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.After the 36h in fermentation added the sodium succinate of 0.5g/L, rapamycin production was 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 the beta-oxidation approach and generates acetyl-CoA.In the 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 rapamycin production is increased to 241mg/L by 196mg/L after the 12h of fermentation adds 0.5g/L soya-bean oil.And rapamycin production is increased to 268mg/L by 196mg/L after 0h adds the 1.0g/L Witconol 2301, has 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 shikimic acid pathway, chorismic acid transforms to DHCHC in order to strengthen, in the M2 substratum, add respectively shikimic acid, and its interpolation concentration and interpolation time are optimized, its effect is as shown in figure .3d, and rapamycin production is increased to 223mg/L by 196mg/L after the 24h of fermentation adds the 0.5g/L shikimic acid in the M2.In addition, add simultaneously phenylalanine, tryptophane and tyrosine mixing mother liquor on the basis of shikimic acid, and its interpolation concentration and time are optimized, result (figure .3e) shows that rapamycin production has been brought up to 247mg/L by 196mg/L after the 36h that is fermenting on the basis of adding shikimic acid adds phenylalanine, tryptophane and the tyrosine mother liquor of 0.1g/L simultaneously.
According to the analysis to Methionin metabolite metaboilic level, in reinforcement S. hygroscopicus U1-6E7, nipecotic acid is to the conversion of rapamycin heterocycle unit, add Methionin in fermention medium, utilize Methionin to carry out making more Methionin enter catabolic pathway on the basis of growth metabolism to be converted into nipecotic acid satisfying thalline.Add Methionin in M2, and its interpolation concentration and time are optimized, its effect is as shown in figure .3f, when rapamycin production after the Methionin of the 12h of fermentation interpolation 1.0g/L is increased to 244mg/L by 196mg/L.
According to born of the same parents' intracellular metabolite thing Dynamic Variation Analysis, on the M2 basis, 0h adds the 1g/L Witconol 2301 in fermentation, add 1g/L Methionin during 12h, 24h adds the 0.5g/L shikimic acid, 36h adds the 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 shown in figure .4, when fermentation ends (120h), under the M3 culture condition, rapamycin production is increased to 307mg/L by 196mg/L, has improved 56.6% (figure .4) than the rapamycin production under M2 culture condition.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 have 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, the 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 detection of dynamic; Determine the sampling spot of metabolism group research according to the rapamycin biosynthesizing curve of streptomyces hygroscopicus under M1 and M2 culture medium culturing condition, the time period that rapamycin production has a significant difference under two kinds of conditions takes a sample.
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 Masslynxsoftware that mass spectra peak is identified and quantification, by the mass spectrum fragment of every kind of compound and NIST mass spectral database are compared, 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 the 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 the compound that two kinds of condition hypothallus Difference of Metabolisms and rapamycin production is had remarkable contribution, and determine the pathways metabolism that these compounds relate to.
5) to step 2) coenzyme A compound and step 4) are determined in born of the same parents compound dynamic change is 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 to rapamycin fermention medium improvement measures, obtain optimization fermention medium M3, rationality instructs the optimization of rapamycin fermention medium.
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| CN113249401A (en) * | 2020-02-10 | 2021-08-13 | 中国科学院分子植物科学卓越创新中心 | Method for improving streptomyces rapamycin yield |
| CN113249401B (en) * | 2020-02-10 | 2022-11-04 | 中国科学院分子植物科学卓越创新中心 | Method for improving streptomyces rapamycin yield |
| CN113627640A (en) * | 2020-05-08 | 2021-11-09 | 中国石油化工股份有限公司 | Productivity well testing prediction method and system for fracture-cavity type oil reservoir oil and gas well |
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