CN108753855A - The method that WGCNA identifies D-ALPHA-Hydroxypropionic acid fermentation process notable module and Hubs metabolins - Google Patents
The method that WGCNA identifies D-ALPHA-Hydroxypropionic acid fermentation process notable module and Hubs metabolins Download PDFInfo
- Publication number
- CN108753855A CN108753855A CN201810524408.7A CN201810524408A CN108753855A CN 108753855 A CN108753855 A CN 108753855A CN 201810524408 A CN201810524408 A CN 201810524408A CN 108753855 A CN108753855 A CN 108753855A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- alpha
- hydroxypropionic acid
- intracellular
- wgcna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of methods that WGCNA identifies D-ALPHA-Hydroxypropionic acid fermentation process notable module and Hubs metabolins；D-ALPHA-Hydroxypropionic acid fermentation technology optimization research is carried out, including logical nitrogen maintains anaerobic fermentation environment, regulation and control fermentation pH value, replaces neutralizer, replaces cheap nitrogen source, and above-mentioned fermentation condition is combined optimization, obtains the optimal zymotechnique route of D-ALPHA-Hydroxypropionic acid；Dynamic detection is carried out to the intracellular metabolin under various technological condition for fermentation, D-ALPHA-Hydroxypropionic acid fermentation strain intracellular is obtained and is metabolized dynamic change characterization curve；Statistics parsing is carried out to the intracellular metabolites characteristic measured in step 2) using the mathematical model of WGCNA, is obtained and the highly relevant notable metabolism module of each fermentation condition and Hubs metabolins.The present invention discloses Lactobacillus delbrueckii under the conditions of different fermentations for the first time by systems biology means, and the intracellular significant difference metabolism module and core metabolin of the D-ALPHA-Hydroxypropionic acid that ferments instruct follow-up strain promotion and fermentation optimization.
Description
Technical field
The invention belongs to lactobacillus system biology fields, are related to a kind of Lactobacillus delbrueckii (Lactobacillus
Delbrueckii, Laboratories Accession are bought in China General Microbiological culture presevation administrative center, bacterium numbering
CGMCC1.2624) under a variety of technological condition for fermentation, using weighted association network analysis (Weighted correlation
Network analysis, referred to as WGCNA) carry out metabolism group data parsing, to identify significant difference metabolism module and
The method of core (Hubs) metabolin.
Background technology
D-ALPHA-Hydroxypropionic acid molecular formula CH3CHOHCOOH, relative molecular weight 90.08, No. CAS：10326-41-7, be it is a kind of the most
Common metabolite, being widely used in the industries such as food, chemical industry, agricultural and medicine is general, is three great tradition organic acids
One of.D-ALPHA-Hydroxypropionic acid is the precursor of a variety of chiral materials as a chiral centre, is widely used in pharmacy, higher effective and lower toxic pesticide
And the chiral synthesis in the fields such as herbicide, cosmetics.Another even more important purposes of D-ALPHA-Hydroxypropionic acid is as synthesizing polylactic acid
(PLA) starting monomer of material.A kind of renewable, recyclable emerging plastics that polylactic acid receives significant attention in recent years are
The potential substitute of petroleum-based plastics.
Currently, the main method of production D-ALPHA-Hydroxypropionic acid is microbe fermentation method.Since biological fermentation process production D-ALPHA-Hydroxypropionic acid rises
Time do not grow, at present to both at home and abroad to D-ALPHA-Hydroxypropionic acid fermentation research rest on extracellular conceptual phase mostly, as strain evolve, training
Base improvement and fermentation operation condition optimizing etc. are supported, report yet there are no to the system research of the intracellular metabolic mechanism of D-ALPHA-Hydroxypropionic acid fermentation process
Road.
As the important component of systems biology, metabolism group is another after genomics and proteomics
Door new branch of science.For industrial microorganism, during strain transformation and fermentation behavior manipulation, multiple metabolism of cell
Functional areas or metabolic pathway can change simultaneously, all have limitation to the independent analysis of any single point, can not manage comprehensively
Solve the physiological metabolism change mechanism of thalline.Only from the angle analysis of system, the metabolism row of microorganism could be more clearly understood
It is characterized, illustrates its metabolism mechanism.Therefore, it can be determined and be synthesized with target product with height by metabonomic analysis
The key metabolites or Key Metabolic approach of relevance, to provide and really may be used for strain transformation, process optimization and process adjustment
Capable theoretical direction.Metabonomic technology is more and more extensive in agricultural, environmental science, human health etc. application, relevant
Patent of invention is also more and more.It is killed for example, the Liu Pengfei et al. of China Agricultural University has invented a kind of disclosed using metabolism group
Method a kind of (fungicide study on mechanism method based on metabolism group of microbial inoculum mechanism of action.Publication number：
CN107796884A), based on GC-MS technologies to carrying out generation containing fungicide and without the pathogen mycelia cultivated in medicine control medium
It thanks to a group detection, difference metabolin is found by comparing metabolism group organon, exclude the shared metabolin of different role mechanism, obtain
The specific biomarkers for obtaining target fungicide mechanism of action, are realized using the variation of biomarker content to fungicide
The identification of mechanism of action and high throughput analysis.The biomarker that the invention also discloses succinate dehydrogenase inhibitors is amber
Acid, the screening fungicide mechanism of action biomarker method provided using the invention is quick, reliable, the biomarker of acquisition
High specificity.It may be implemented to the quick identification of active material mechanism of action and high throughput using biomarker specific variations
Analysis.Jia Xiaoqiang of University Of Tianjin et al. has invented a kind of utilization metabolism group method raising Rhodococcus sp degradation cycloaromatics pyrene ability
Method (method of the Rhodococcus sp degradation condition to improve polycyclic aromatic hydrocarbon pyrene degradation rate is improved based on metabolism group.Publication number：
CN107796906A), Rhodococcus sp degrading polycyclic aromatic hydrocarbons pyrene is disclosed using metabolism group means and combination difference significance analysis
Metabolin difference is further illustrated and the relevant metabolic alterations mechanism of polycyclic aromatic hydrocarbon pyrene degradation.By to Rhodococcus sp in different lifes
Degradation pyrene ability and metaboilic level under long environment are analyzed, and find and degrade the relevant metabolin of pyrene, to optimize its drop
Solution condition provides direction for the raising of polycyclic aromatic hydrocarbon pyrene degradation rate, and this method is alternatively other degrading polycyclic aromatic hydrocarbons microorganism drops
The optimizing research of solution condition provides new idea and method.The Honkonen et al. of Cleaning corporation of the U.S. has invented a kind of using generation
Thank to a group method (Metabonomic methods to assess health of for method analysis human skin health status
Skin.Patent No.7,761,242), using metabolism group means, over the course for the treatment of from different skin parts or it is different when
Between the sample that acquires for diagnosing skin or the various skin treating effects of evaluation, identify and skin health highlights correlations
Biomarker.Application aspect about metabolism group in D-ALPHA-Hydroxypropionic acid production process at present, yet there are no patent of invention.
Complicated metabolism group data are parsed, in addition to traditional non-supervisory method and measure of supervision, are newly risen in recent years
A kind of method of entitled weighted association network analysis (WGCNA) is also applied in metabolism group data analysis.As mass spectrum is examined
The development of survey technology, metabonomic analysis generate a large amount of detection data, metabolism group are carried out using traditional statistical analysis technique
Data are faced with a huge challenge when parsing, that is, how to be converted to the group data of large amount of complex and anticipate with biology
The information of justice.First, traditional statistical analysis technique is difficult the metabolin for identifying that variation multiple is small, causes analysis result
Deviation；Secondly, traditional analysis is more suitable for sample analyze two-by-two comparing, can not be simultaneously to a variety of source samples
High-throughput data carry out systematic comprehensive analysis.And WGCNA is one kind excavation module (module) letter from high-throughput data
The algorithm of breath breaches above-mentioned both sides constraint.WGCNA is to be used for transcript profile data analysis at the beginning, in the method mould
Block is defined as one group of gene with similar express spectra, if certain genes in a physiology course or different tissues always
Change with similar expression, then we it is reasonable that these genes were functionally correlated, can determine them
Justice is a module.This seems to be somewhat similarly to obtained by carrying out clustering as a result, unlike but, and the cluster of WGCNA is accurate
Then there is the result that biological significance, and unconventional clustering method, therefore this method are obtained to have higher confidence level.Mesh
Before, about applications of the WGCNA in metabolism group data analysis, it yet there are no patent of invention.
Therefore, the method that the present invention uses metabolism group combination weighted association network analysis model (WGCNA) for the first time, identification
L.delbrueckii is under a variety of technological condition for fermentation, the notable metabolism module and Hubs metabolins of D-ALPHA-Hydroxypropionic acid process of fermenting, with
Instruct that subsequent strain is promoted and technological condition for fermentation advanced optimizes.
Invention content
Weighted association network analysis (WGCNA) is applied to metabolism group data solution the object of the present invention is to provide a kind of
Analysis, the method to identify significant difference metabolism module and core (Hubs) metabolin；It is as follows：
1) carry out D-ALPHA-Hydroxypropionic acid fermentation technology optimization research, including logical nitrogen maintain anaerobic fermentation environment, regulation and control fermentation pH value,
It replaces neutralizer, replace cheap nitrogen source, and above-mentioned fermentation condition is combined optimization, obtain the optimal zymotechnique road of D-ALPHA-Hydroxypropionic acid
Line；
2) dynamic detection is carried out to the intracellular metabolin under various technological condition for fermentation in step 1), obtains D-ALPHA-Hydroxypropionic acid fermentation
Bacterial strain intracellular is metabolized dynamic change characterization curve；
3) it uses the mathematical model of WGCNA to carry out statistics parsing to the intracellular metabolites characteristic measured in step 2), obtains
Obtain the notable metabolism module and Hubs metabolin highly relevant with each fermentation condition.
In the step 1), (purchase is in China General Microbiological culture presevation by progress L.delbrueckii on 7.5L tanks
Administrative center, bacterium numbering CGMCC1.2624) production D-ALPHA-Hydroxypropionic acid optimization of fermentation condition, be passed through sterile nitrogen 0.5h, arrange
Except the initial dissolution oxygen in fermentation medium；Neutralizer is replaced with to the calcium hydroxide and hydrogen of control fermentation pH respectively by calcium carbonate
Sodium oxide molybdena, and it is 5.9 to optimize fermentation pH；Nitrogen source is changed to the compound nitrogen source of peptone and yeast powder by beef extract.
Using GC-MS and LC-MS/MS technologies to the fermentation under different fermentations process conditions in step 1) in the step 2)
Liquid carries out Dynamic sampling and detects intracellular metabolin, and carries out processing and multi-variate statistical analysis.
It is described as follows：
1) D-ALPHA-Hydroxypropionic acid fermentation technology optimization research is carried out, including is continuously passed through sterile nitrogen 0.5h and maintains anaerobic fermentation ring
Border, regulation and control fermentation pH value are 5.9, replacement neutralizer are changed to calcium hydroxide or sodium hydroxide, are replaced expensive nitrogen source beef extract
Cheap nitrogen source protein peptone and yeast powder compound nitrogen source are changed, and above-mentioned fermentation condition is combined optimization, it is optimal to obtain D-ALPHA-Hydroxypropionic acid
Zymotechnique route.Detailed technological condition for fermentation illustrated in table 1；
2) gas chromatography mass spectrometry (GC-MS) and LC-MS (LC-MS/MS) technology platform are used, to various zymotechniques in 1)
Under the conditions of intracellular metabolin carry out dynamic detection, obtain D-ALPHA-Hydroxypropionic acid fermentation strain intracellular be metabolized dynamic change characterization curve；
3) use weighted association network analysis (WGCNA) mathematical model pair 2) in measure intracellular metabolites characteristic carry out
Statistics parses, and obtains and the highly relevant notable metabolism module of each fermentation condition and Hubs metabolins.
1 technological condition for fermentation explanation of table
The present invention relates to weighted association network analysis (Weighted correlation network analysis, abbreviations
For WGCNA) metabolism group data parsing is carried out, to identify that significant difference is metabolized the side of module and core (Hubs) metabolin
Method.The present invention discloses Lactobacillus delbrueckii (Lactobacillus delbrueckii, experiment for the first time by systems biology means
Room preservation is bought in China General Microbiological culture presevation administrative center, bacterium numbering CGMCC1.2624) in different fermentations work
Under the conditions of skill, the intracellular significant difference metabolism module and core (Hubs) metabolin of high-efficiency fermenting D-ALPHA-Hydroxypropionic acid, after reaching guidance
Continuous strain is promoted and the purpose of further optimization of fermentation condition.
Description of the drawings
Under the optimal technological condition for fermentation of Fig. 1, the apparent kinetics indicatrix of D-ALPHA-Hydroxypropionic acid fermentation；
Hubs metabolins under the optimal technological condition for fermentation of Fig. 2 and its metabolism association analysis figure.
Specific implementation mode
In order to make the purpose of the present invention, technical solution and effect be more clear, the present invention is with reference to the drawings and specific embodiments
It illustrates：
As the preferred embodiment of the present invention, one kind is provided and utilizes metabolism group combination network point in L.delbrueckii
The method for being significantly metabolized module and Hubs metabolins in analysis identification D-ALPHA-Hydroxypropionic acid fermentation process.
The first step carries out the optimization of fermentation condition of L.delbrueckii production D-ALPHA-Hydroxypropionic acids on 7.5L tanks, optimizes item
Part includes：It is passed through sterile nitrogen 0.5h, the initial dissolution oxygen in fermentation medium is excluded, builds the yeasting of anaerobism；Will in
The calcium hydroxide and sodium hydroxide of fermentation pH can be accurately controlled by being replaced with respectively by calcium carbonate with agent, and is optimized fermentation pH and be
5.9；Nitrogen source is changed to the compound nitrogen source of peptone and yeast powder by beef extract, substantially reduces fermentation raw material cost；Will more than
Technological condition for fermentation is combined optimization, obtains best D-ALPHA-Hydroxypropionic acid zymotechnique route.Second step, using GC-MS and LC-MS/
MS technologies carry out Dynamic sampling to the zymotic fluid under different fermentations process conditions in the first step and detect intracellular metabolin, and are located
Reason and multi-variate statistical analysis (PCA)；Third walks, and carries out WGCNA analyses to second step treated metabolism group data, identification is notable
It is metabolized module and Hubs metabolins.
Embodiment
1, L.delbrueckii produces the optimization of fermentation condition of D-ALPHA-Hydroxypropionic acid
Lactobacillus delbrueckii (buy in China General Microbiological by Lactobacillus delbrueckii, Laboratories Accession
Strain
Preservation administrative center, bacterium numbering CGMCC1.2624)
First, the ripe L.delbrueckii shake-flask seeds of culture are inoculated into the ratio of 10% (v/v) equipped with 4.5L
(Tween 80 1g/L, peptone 22.5g/L, yeast extract 7.5g/L, hydrogen citrate two in the 7.5L fermentation tanks of fermentation medium
Ammonium 2g/L, dipotassium hydrogen phosphate 2g/L, anhydrous sodium acetate 3g/L, anhydrous magnesium sulfate 0.2g/L, manganese sulfate 0.1g/L, glucose 80g/
L, pH=5.9).42 DEG C, 150rpm fermentations 48h.Remaining fermentation condition is as shown in table 1.Fermentation results are shown, in combined fermentation work
Under the conditions of skill, D-ALPHA-Hydroxypropionic acid fermentation yield is up to 133g/L (Fig. 1).
2, the preparation of intracellular metabolin sample
(1) intracellular metabolin extracts
1) 20mL fermentation broth samples are taken, the 60% cold methanol-aqueous solution of isometric -40 DEG C of precoolings is rapidly joined, vortex shakes
2s is swung, is placed in -20 DEG C, 5min is quenched to cell, terminates intracellular metabolic response, at 4 DEG C, 5000rpm centrifuges 10min, abandons
Clearly, bacterial sediment is with 4 DEG C, and three times, in 4 DEG C after washing, 5000rpm centrifuges 5min, abandons supernatant for 0.9% NaCl solution washing,
Retain thalline, removes nutrient media components.
2) liquid nitrogen grinding, fracturing cell walls is used to weigh about 200mg in the mortar that the thalline after washing is pre-chilled at -20 DEG C
Thalline is in 1.5mL EP pipes, addition 1mL extracting solutions (50% (v/v), -40 DEG C of methanol solutions), anti-with liquid nitrogen after vortex mixing
Multiple freeze thawing 3 times, each 1min.
3) by above-mentioned crude extract under the conditions of 4 DEG C, 8000rpm centrifuges 5min, takes in supernatant to new EP pipes, lower layer bacterium
Body precipitation uses 0.5mL extracting solutions (50% (v/v), -40 DEG C of methanol solutions) to extract again, and under the conditions of 4 DEG C, 8000rpm, from
Supernatant is taken after heart 5min, after merging supernatant twice, again under the conditions of 4 DEG C, 8000rpm centrifuges 5min, takes supernatant.
4) it to same sample, takes 100 μ L cell extracts in two clean EP pipes respectively, is separately added into 10 μ L
Succinic d4acid (0.1mg/mL) and 10 μ L D-sorbitol-13C6 (0.1mg/mL) are used as GC-MS and LC-MS/MS
Detection internal standard, after mixing well, vacuum refrigeration (- 48 DEG C) is until sample is completely dried.
5) low-temperature preservation is spare in -80 DEG C of ultra low temperature freezers or directly carries out analyte derivative for the sample after being freeze-dried
Change.
(2) derivatization of sample
The methoxyl group ammonium salt hydrochlorate pyridine solution for preparing 20mg/mL, takes in the sample that 50 μ L are added to after being completely dried, whirlpool
Rotation concussion mixing, and 90min is fully reacted under the conditions of 40 DEG C in metal bath, add 80 μ L trimethylsilyl trifluoroacetyls
Amine, mixing fully react 30min in metal bath under the conditions of 40 DEG C.By 0.22 μm of oil film of the sample after abundant derivatization
Filtering, takes 150 μ L in sample injection bottle, in case GC-MS and LC-MS/MS analyses.
3, intracellular is metabolized analyte detection
(1) GC-MS is detected
GC-MS systems：Gas-chromatography (Agilent 6890N), mass spectrum (Agilent 9575C MSD) and autosampler
(Agilent 5183-2085).Chromatographic column using DB-5MS capillary columns (30mm × 0.25mm, 0.25 μm, Agilent
Technologies).Chromatographic condition is as follows：1 μ L of sample size, split ratio 1:1, injector temperature and interface temperature are 280 DEG C, are carried
Gas (helium, constant pressure), flow velocity 1.0mL/min.Column temperature temperature program：70 DEG C of holding 2min；5 DEG C/min is warming up to 290 DEG C, protects
Hold 3min.Mass spectrometry parameters：Using electron impact ion source, electron bombardment energy is 70eV, and ion source temperature is 250 DEG C, electric current
For 40 μ A, scanning range 50-650m/z.
(2) intracellular metabolin LC-MS/MS is detected
In GC-MS detections, because of the part intracellular in pentose phosphate pathway (PP approach) and glycolytic pathway (EMP Embden Meyerbof Parnas pathway)
Sugar phosphate metabolin can not detect, such as glucose 6-phosphate (G6P), fructose-1, 6-diphosphate (F6P), fructose 1,6-diphosphate (F-
1,6-P), glyceraldehyde 3-phosphate (3PG), phosphoenolpyruvate (PEP), ribulose 5-phosphate (RiBP), ribose 5-phosphate
(R5P), X 5P (X5P), sedoheptulose 7-phosphate (S7P) and erythrose-4-phosphate (E4P).Therefore, for upper
It states and is detected using LC-MS/MS systems approaches in the detection of metabolin this research, liquid chromatogram (Perkin- is respectively adopted
Elmer series 200pump) and Symmetry C18 columns (3.9mm × 150mm, 5 μm, Waters Chromatography
BV, Etten Leu, The Netherlands) it detaches and sprays source (Turbo Ion Spray) equipped with electronic and ionic
API-3000 tandem mass spectrums (PE-Sciex) carry out Mass Spectrometric Identification, and wherein source parameters is：Ion spray voltage -2500V, from
400 DEG C of source temperature, atomising air and impinging air flows are respectively set as 10 and 4.
4, metabolin data processing and multi-variate statistical analysis
(1) mass spectrogram of GC-MS detections uses AMDIS (Version3.2, National Institute of
Standards and Technology, Gaithers burg, MD, USA), NIST (National Institute of
Standards and Technology mass spectral library, 2005) and GMD (Golm Metabolome
) etc. Database Agilent software kits deconvoluted first, the identification of noise reduction, integrating peak areas and metabolin, then to each
Identification metabolin carries out derivative group processing to get to each metabolin data.It is identified altogether using GC-MS and sxemiquantitative obtains
To 80 intracellular metabolins.
(2) LC-MS/MS detection datas then carry out quantitative/qualitative analysis using corresponding metabolin standard items determination data,
And each metabolin and internal standard compound are analyzed by Window NT software (Ver.1.3.1), it is final to obtain each
The concentration of metabolin.It is identified altogether using LC-MS/MS and sxemiquantitative obtains 10 intracellular metabolins.
(3) all metabolins that GC-MS is detected pass through metabolin peak area divided by sample dry weight and corresponding internal standard compound peak
Area is standardized, and the absolute concentration for the metabolin that LC-MS/MS is detected carries out standard using identical method in GC-MS
Change, will two groups of data merge after be normalized with it is sized, using SIMCA-P package (Ver 11.5；Umetrics,
Umea, Sweden) carry out principal component analysis.Each sample is implemented 5 independent biochemicals and is repeated, and data are accurate using average value mark-on
Difference indicates.The results show that under different fermentations process conditions, intracellular metabolism shows huge difference for principal component analysis.
5, the structure of WGCNA methods
According to WGCNA standard operation programs, GC-MS and LC-MS/MS are detected and the metabolism group after being normalized
Data construct correlation networks analysis model.Detailed process is as follows：Weighting Pearson's phase of metabolism group data is calculated first
Relationship matrix number is then converted into metabolin bonding strength matrix, while using topology overlapping (topological
Overlap, TO) correlation degree between metabolin is calculated, metabolin similitude related network is obtained, and carries out generation based on TO
It thanks to object distinctiveness ratio hierarchical clustering, establishes hierarchical cluster tree, will have high relevance metabolin to gather using dynamic shearing tree algorithm
To together, eventually finding, 9 significant difference metabolism modules with biological significance are as shown in table 2.Hub metabolites
(Hubs) identification is then that it is further that conspicuousness is metabolized module data in the determining conspicuousness module basis of WGCNA analyses
It is input to VisANT and Cytoscape softwares and carries out visualization processing, screened after being ranked up by the Connected degree to metabolin
Go out 8 metabolins with high Connected degree, as Hub metabolites (Fig. 2).
Table 2：Significance difference anomalous mode
6, seed culture condition
The bacterial strain for taking 2mL glycerol tube preservations is inoculated in 200mL seed culture mediums ((Tween 80 1g/L, peptone 10g/
L, yeast extract 5g/L, diammonium hydrogen citrate 2g/L, dipotassium hydrogen phosphate 2g/L, anhydrous sodium acetate 3g/L, anhydrous magnesium sulfate 0.2g/
L, manganese sulfate 0.1g/L, glucose 20g/L, pH=5.9), 42 DEG C of stationary cultures to exponential phase, generally 11~13h, bacterium
Body just starts to precipitate.Layering between thalline and supernatant is not obvious, and when microscopy, most of thalline were rod-short, also there is part
Elongated rod shape in splitting status.At this time between thalline OD600=1.5~2.1.
7, the measurement of D-ALPHA-Hydroxypropionic acid yield and chiral purity
(1) D-ALPHA-Hydroxypropionic acid yield is detected using high performance liquid chromatography (Agilent 1200, USA).Concrete operations are as follows：
1) 1mL zymotic fluids, 10000rpm is taken to centrifuge 2min.
2) 100 μ L of supernatant are taken, are added in 900 μ L 0.1M dilution heat of sulfuric acid, 10000rpm centrifuges 5min after mixing.
3) the water system filter membrane that supernatant crosses 0.22 μm is taken, efficient liquid phase chromatographic analysis is used for：A：Chromatographic column HPX-87H
(250mm × 4.6mm, BioRad, America), mobile phase：2.5mM sulfuric acid solutions, flow velocity 0.4ml/min, 65 DEG C of column temperature, profit
It is detected with parallax monitor, 20 μ L of sample size；B：Chromatographic column ZOBAX SB-C18 (250mm × 4.6mm, Agilent,
America), mobile phase：5mM sulfuric acid solutions, flow velocity 0.5mL/min, 30 DEG C of column temperature, ultraviolet detection wavelength 210nm, sample size 20
μL。
(2) D-ALPHA-Hydroxypropionic acid chiral purity carries out separation determination using high performance liquid chromatography.Concrete operations are as follows：
1) 1mL zymotic fluids, 10000rpm is taken to centrifuge 2min.
2) 100 μ L of supernatant are taken, are added in 900 μ L water, 10000rpm centrifuges 5min after mixing.
3) the water system filter membrane that supernatant crosses 0.22 μm is taken, efficient liquid phase chromatographic analysis is used for：Chromatographic column Chiral
Separation columns CRS10W (50mm × 4.6mm, MCI GEL, Japan), mobile phase：2mM copper-baths, stream
Fast 0.5ml/min, 25 DEG C of column temperature, ultraviolet detection wavelength 254nm, 20 μ L of sample size.
What the present invention was disclosed and proposed is significantly metabolized module using weighted association network analysis identification D-ALPHA-Hydroxypropionic acid fermentation process
With the method for Hubs metabolins, those skilled in the art can be appropriate to change the links such as condition route reality by using for reference present disclosure
Existing, although the methods and techniques of the present invention are described by preferred embodiment, related technical personnel obviously can be not
Methods and techniques described herein route is modified or is reconfigured in disengaging the content of present invention, spirit and scope, is come real
Now final technology of preparing.In particular, it should be pointed out that all similar replacements and change are for a person skilled in the art
It is it will be apparent that they are considered as being included in spirit of that invention, range and content.
Claims (3)
- The method that 1.WGCNA identifies D-ALPHA-Hydroxypropionic acid fermentation process notable module and Hubs metabolins；It is characterized in that steps are as follows：1) D-ALPHA-Hydroxypropionic acid fermentation technology optimization research is carried out, including logical nitrogen maintains anaerobic fermentation environment, regulation and control fermentation pH value, replaces Neutralizer replaces cheap nitrogen source, and above-mentioned fermentation condition is combined optimization, obtains the optimal zymotechnique route of D-ALPHA-Hydroxypropionic acid；2) dynamic detection is carried out to the intracellular metabolin under various technological condition for fermentation in step 1), obtains D-ALPHA-Hydroxypropionic acid fermentation strain Intracellular is metabolized dynamic change characterization curve；3) mathematical model of WGCNA is used to carry out statistics parsing to the intracellular metabolites characteristic measured in step 2), obtain with The highly relevant notable metabolism module of each fermentation condition and Hubs metabolins.
- 2. the method as described in claim 1 carries out L.delbrueckii productions it is characterized in that in step 1) on 7.5L tanks The optimization of fermentation condition of D-ALPHA-Hydroxypropionic acid is passed through sterile nitrogen 0.5h, excludes the initial dissolution oxygen in fermentation medium；It will neutralize Agent is replaced with the calcium hydroxide and sodium hydroxide of control fermentation pH by calcium carbonate respectively, and it is 5.9 to optimize fermentation pH；By nitrogen source by Beef extract is changed to the compound nitrogen source of peptone and yeast powder.
- 3. the method as described in claim 1, it is characterized in that using GC-MS and LC-MS/MS technologies in step 1) in step 2) Zymotic fluid under different fermentations process conditions carries out Dynamic sampling and detects intracellular metabolin, and carries out processing and multivariate statistics point Analysis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810524408.7A CN108753855A (en) | 2018-05-28 | 2018-05-28 | The method that WGCNA identifies D-ALPHA-Hydroxypropionic acid fermentation process notable module and Hubs metabolins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810524408.7A CN108753855A (en) | 2018-05-28 | 2018-05-28 | The method that WGCNA identifies D-ALPHA-Hydroxypropionic acid fermentation process notable module and Hubs metabolins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108753855A true CN108753855A (en) | 2018-11-06 |
Family
ID=64002761
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810524408.7A Pending CN108753855A (en) | 2018-05-28 | 2018-05-28 | The method that WGCNA identifies D-ALPHA-Hydroxypropionic acid fermentation process notable module and Hubs metabolins |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108753855A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952164A (en) * | 2006-11-10 | 2007-04-25 | 南京工业大学 | Combined fermentation process for producing D-lactic acid |
| CN102286551A (en) * | 2011-07-01 | 2011-12-21 | 南京工业大学 | Fermentation process regulation and control method of fumaric acid radical-producing mould |
| CN103088104A (en) * | 2013-01-12 | 2013-05-08 | 天津大学 | Method for optimizing fermentation medium by improving rapamycin production by using metabolic profiling analysis |
| CN104419647A (en) * | 2013-09-06 | 2015-03-18 | 中国医学科学院药用植物研究所 | Muscodor malipoensis with biocontrol effect |
| CN104450834A (en) * | 2014-12-04 | 2015-03-25 | 天津大学 | Method of increasing yield of spinosad by improving fermentation condition of saccharopolyspora spinosa based on metabonomics |
| CN104704364A (en) * | 2012-06-15 | 2015-06-10 | 韦恩州立大学 | Biomarker test for prediction or early detection of preeclampsia and/or hellp syndrome |
| CN106591136A (en) * | 2016-12-28 | 2017-04-26 | 昆明藻能生物科技有限公司 | High-glucose-tolerance crypthecodinium cohnii obtained from orient domestication, and preparation method and application thereof |
| WO2018049268A1 (en) * | 2016-09-08 | 2018-03-15 | Duke University | Biomarkers for the diagnosis and characterization of alzheimer's disease |
-
2018
- 2018-05-28 CN CN201810524408.7A patent/CN108753855A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952164A (en) * | 2006-11-10 | 2007-04-25 | 南京工业大学 | Combined fermentation process for producing D-lactic acid |
| CN102286551A (en) * | 2011-07-01 | 2011-12-21 | 南京工业大学 | Fermentation process regulation and control method of fumaric acid radical-producing mould |
| CN104704364A (en) * | 2012-06-15 | 2015-06-10 | 韦恩州立大学 | Biomarker test for prediction or early detection of preeclampsia and/or hellp syndrome |
| CN103088104A (en) * | 2013-01-12 | 2013-05-08 | 天津大学 | Method for optimizing fermentation medium by improving rapamycin production by using metabolic profiling analysis |
| CN104419647A (en) * | 2013-09-06 | 2015-03-18 | 中国医学科学院药用植物研究所 | Muscodor malipoensis with biocontrol effect |
| CN104450834A (en) * | 2014-12-04 | 2015-03-25 | 天津大学 | Method of increasing yield of spinosad by improving fermentation condition of saccharopolyspora spinosa based on metabonomics |
| WO2018049268A1 (en) * | 2016-09-08 | 2018-03-15 | Duke University | Biomarkers for the diagnosis and characterization of alzheimer's disease |
| CN106591136A (en) * | 2016-12-28 | 2017-04-26 | 昆明藻能生物科技有限公司 | High-glucose-tolerance crypthecodinium cohnii obtained from orient domestication, and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| BIN ZHANG等: "A general framework for weighted gene co-expression network analysis", 《STAT APPL GENET MOL BIOL》 * |
| JIANGXIN WANG等: "Global Metabolomic and Network analysis of Escherichia coli Responses to Exogenous Biofuels", 《JOURNAL OF PROTEOME RESEARCH》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shao et al. | Tradeoffs among microbial life history strategies influence the fate of microbial residues in subtropical forest soils | |
| Treves et al. | Carbon flux through photosynthesis and central carbon metabolism show distinct patterns between algae, C3 and C4 plants | |
| Ellis et al. | Metabolomics-assisted synthetic biology | |
| Duportet et al. | The biological interpretation of metabolomic data can be misled by the extraction method used | |
| Mareya et al. | Untargeted metabolomics reveal defensome-related metabolic reprogramming in Sorghum bicolor against infection by Burkholderia andropogonis | |
| Antoniewicz | Tandem mass spectrometry for measuring stable-isotope labeling | |
| Sanford et al. | Genomics to fluxomics and physiomics—pathway engineering | |
| Romano et al. | Proton transfer reaction–mass spectrometry: online and rapid determination of volatile organic compounds of microbial origin | |
| Heux et al. | A novel platform for automated high-throughput fluxome profiling of metabolic variants | |
| Liebeke et al. | Staphylococcus aureus metabolic response to changing environmental conditions–a metabolomics perspective | |
| Spinelli et al. | Emission of volatile compounds by Erwinia amylovora: biological activity in vitro and possible exploitation for bacterial identification | |
| Hollywood et al. | Metabolomics tools for the synthetic biology of natural products | |
| Muhamadali et al. | Metabolomics investigation of recombinant mTNFα production in Streptomyces lividans | |
| Ezra et al. | Proton transfer reaction-mass spectrometry as a technique to measure volatile emissions of Muscodor albus | |
| CN110320303A (en) | A kind of Efficiency for Soil Aquifer Treatment metabonomic analysis methods based on UPLC-MS | |
| Li et al. | Insights on acetate-ethanol fermentation by hydrogen-producing Ethanoligenens under acetic acid accumulation based on quantitative proteomics | |
| Holland et al. | The in‐line measurement of plant cell biomass using radio frequency impedance spectroscopy as a component of process analytical technology | |
| Shen et al. | Quantitative metabolic network profiling of Escherichia coli: An overview of analytical methods for measurement of intracellular metabolites | |
| Ganie et al. | Metabolite profiling and network analysis reveal coordinated changes in low-N tolerant and low-N sensitive maize genotypes under nitrogen deficiency and restoration conditions | |
| Aryal et al. | Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light | |
| Kluger et al. | Metabolomics and secondary metabolite profiling of filamentous fungi | |
| Bachinger et al. | Searching for process information in the aroma of cell cultures | |
| Li et al. | Dynamic metabolic response of Aspergillus niger to glucose perturbation: Evidence of regulatory mechanism for reduced glucoamylase production | |
| Zhao et al. | Bioconversion of lutein to form aroma compounds by Pantoea dispersa | |
| Daly et al. | Towards a mechanistic understanding of microalgae–bacteria interactions: integration of metabolomic analysis and computational models |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181106 |