CN102250764A - Micro holographic biological sensing reactor system - Google Patents
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Abstract
The invention relates to a micro holographic biological sensing reactor system, in particular to a micro holographic biological sensing reactor system for metabolic flux analysis and applications of the micro holographic biological sensing reactor system. The device comprises a micro reactor for microbial fermentation, a data acquisition device for collecting parameters, a control system for adjusting oxygen supply and feed supplement, an exhaust spectrometer for online determination, and a gas chromatograph-mass spectrometer for determining amino acid marked by 13C in the fermentation system. The micro holographic biological sensing reactor system disclosed in the invention can determine intracellular microbial metabolic flux in the fermentation accurately, timely and cheaply.
Description
Technical field
The present invention relates to the fermentation field, relate more specifically to a kind of miniature holographic formula bio-sensing reactor assembly and application thereof that metabolic flux is analyzed that can be used for.
Background technology
Produce for biological product, want to improve and improve production process, a detailed understanding at first must be arranged the metabolism state in the extracellular microbial, the strongest instrument of this respect research is to utilize metabolism network flux distribution method, comes the physiological status parameter of pair cell and metabotic change situation to measure.
Metabolic flux (metabolic flux) is a basic determinative of stechiology, also is most important parameter in the pathways metabolism.Metabolic flux is analyzed (metabolic flux analysis, MFA) be to determine a kind of method that metabolic flux distributes in the whole metabolic reaction network, in metabolic engineering, occupy critical role according to measured data in each quantitative relation that reacts and the experiment in the metabolic pathway.Distribute by the metabolic flux that calculates under different approaches or the different condition, metabolic capacity that can characterize cells is seen clearly the influence of genetic modification pair cell metabolism state, thus for further more reasonably genetic modification theoretical foundation is provided.
MFA successful Application has realized comprising the raising of multiple purpose product productive rates such as organic acid, VITAMIN, ethanol in the multiple biological metabolism approach optimization.MFA experiences the fast development in more than 20 year, no matter at experimental technique, measurement means, still aspect data analysis and the assessment, related correlation technique is gradually improved, and has become the diagnostic tool of a kind of standard in the metabolic engineering and widespread use.
Traditional MFA is based on the conservation of matter, utilize the stoichiometry model and the material balance (black-box model) in the born of the same parents of main chemical reactions in the born of the same parents to calculate the interior metabolic flux of born of the same parents, therefore also be referred to as metrology MFA (Vallino, J.J.and Stephanopoulos, G., 1993).The measuring parameter of metrology MFA mainly comprises the composition and the CO of substrate uptake rate, product generating rate, biomass
2(vanGulik, 1995 such as release; Jin, S., 1997).Yet this metrology MFA demonstrates a lot of limitation (Zupke when handling practical problems, 1994): at first this limitation shows on the Energy Balance Analysis, Energy Balance Analysis need determine that all relate to cofactor (NADH or NADPH etc.) and the generation of ATP and the reaction of consumption in the cell, owing to determine very difficulty of the reaction relevant, comprehensively so some flux that calculates thus is inaccurate with energy; Secondly, have a large amount of invalid circulations in prokaryotic organism, this handles the energy balance problem to metrology MFA and has brought bigger difficulty; Once more, metrology MFA can not determine the net flux in a large amount of reversible reaction, two-way reaction, anaplerotic reaction and parallel reactions that exist in the cell.And the size of the existence of these approach and flux plays important effect (Wolfgang Wiechert, 2001) in the building-up process of product.
In recent years, based on the above-mentioned limitation of metrology MFA, developed with
13The C labelling experiment (
13Clabeling experiments is basic CLEs)
13The analysis of C metabolic flux (
13C-metabolic fluxanalysis,
13C-MFA), this method utilizes the substrates such as glucose, glycerine, amino acid and methyl alcohol of mark to carry out mark metabolism experiment, biochemical route metabotic change according to atom in the metabolic process, set up the mapping map network matrix between material carbon atom and the carbon atom, and according to the quality demeanour segment information of the mark carbon atom that obtains, the metabolism circulation is calculated (Schmidt, K., 1997; Christensen, B., and Nielsen, J., 1999; Stephanopoulos, G.1999).
13The most typical application of C-MFA is to make metabolism reach quasi-stable state in culture of continuous cultivation, utilizes the label information of tropina composition original acid at this moment, comes the center metabolic flux is calculated.Yet, the main drawback of this method is: because these former albumen generate in whole process, so can not represent the metabolism rheologyization in a certain moment in the metabolic process, thereby can not carry out determination and analysis to instantaneous metabolism stream, can only carry out the abundance measurement of former Argine Monohydrochloride for the quasi-stable state process that is in logarithmic phase and cultured continuously, calculate the metabolic flux of this process.
In recent years, some achievements in research show, utilize free amino acid information in the born of the same parents, rather than the information of former Argine Monohydrochloride to carry out the metabolic flux analysis be a kind of efficient ways.At the non-growing period of somatic cells, the renewal rate of the former Argine Monohydrochloride of thalline is very slow, but the rate of change in total free aminoacids pond is very fast in the born of the same parents, so the mark abundance information of total free aminoacids can be used for the distributed intelligence of instantaneous metabolic flux in the born of the same parents.Kromer and Wittmann, C. etc. utilize the metabolic flux of analyzing Corynebacterium glutamicum of GC-MS by total free aminoacids in the born of the same parents that measure the different moment in batch culturing process; Wahl (2004) utilizes NMR to analyze L-Phe and produces metabotic change in the bacterium building-up process; Lys building-up process flux distribution that Drysch utilized the total free aminoacids information research in 2004.
Van Winden proposed in 2005
13The novel method that C-MFA analyzes, this method is based on the quick inactivating of quick sampling and cell.The abundance information of utilizing LC-MS to analyze total free aminoacids in the born of the same parents in culture of continuous cultivation is used for the calculating of metabolism stream; Iwatani S. etc. has proposed in the metabolic flux analysis process, has increased the correction to already present intermediate metabolites influence inside and outside the born of the same parents.Influencing each other between precursor substance, total free aminoacids and the thalline amino acid also carried out discussing analysis.
The real purpose of isotopic labeling experiment will be used to instruct production process, because the price of labeled substrate is very expensive, therefore limited the isotopic labeling test 25 liters, 50 liters or the fermentor tank of more volume or the direct application in the analysis of reactor metabolic flux.So, need utilize microreactor to go to follow the tracks of and produce fermentor tank, thereby by in microreactor, carrying out the analysis that mark test carries out metabolic flux.
In sum, this area still lacks accurately, in time, the technology of microbial metabolism flux in the not expensive understanding fermenting process, therefore press for exploitation new can measure microbial metabolism flux method in the fermenting process accurate, timely, inexpensively.
Summary of the invention
Purpose of the present invention just provides a kind of method and relevant device of measuring microbial metabolism flux in the fermenting process accurate, timely, inexpensively.
In a first aspect of the present invention, a kind of microreactor apparatus that isotope-labeled metabolic flux is analyzed that is used for is provided, described device comprises:
1) microreactor that is used for microbial fermentation, described microreactor is furnished with dissolved oxygen sensor, pH transmitter and the temperature sensor of measuring fermentation system;
And described microreactor is furnished with inlet pipe, vapor pipe, feed supplement pipe and stopple coupon;
And the volume of described microreactor is 30-500ml;
2) data collector that is used to gather fermentation parameter, described data set links to each other with temperature sensor with described dissolved oxygen sensor, pH transmitter;
3) Controlling System that is used to regulate oxygen supply level and feed supplement, described Controlling System comprise the micro mass flow meter of control air air inlet, the magnetic stirring apparatus that control is stirred and the peristaltic pump of controlling feed supplement;
4) being used for deflated molecular weight to microreactor is 45
13C mark carbonic acid gas and molecular weight are the exhaust gas mass spectrograph that 44 cold carbon dioxide component is carried out on-line determination; And
5) be used for measuring fermentation system
13Amino acid whose gas phase-mass spectrometry the instrument system (GC-MS system) of C mark, described GC-MS system carries out the amino acid of (a) thalline hydrolysis and/or (b) the amino acid whose mensuration of free in the born of the same parents to the sampling sample of fermenting process, thereby obtains being used for the mark abundance data that metabolic flux is analyzed.
In another preference, in described device, by micro mass flow meter and magnetic stirring apparatus, according to fermentation system to the demand of oxygen supply and regulate charge flow rate and mixing speed automatically; And described device is regulated the rotating speed of peristaltic pump automatically according to the reading of the electronic scale of the setting of feed rate and the described microreactor weight of weighing, thereby whether and feed supplement speed the control feed supplement.
In another preference, described device comprises that also the air inlet molecular weight that is used for reactor is 45
13C mark carbonic acid gas and molecular weight are the air inlet gas mass spectrograph that 44 cold carbon dioxide component is carried out on-line determination.
More preferably, described air inlet gas mass spectrograph and exhaust gas mass spectrograph are same gas mass spectrograph.
In another preference, the volume of described microreactor is 50-250ml, and more preferably, the volume of described microreactor is 70-100ml.
In another preference, described device comprises that also one produces reactor, and the volume of wherein said production reactor is (as the 50-10000 liter) more than 50 liters;
And the exhaust gas mass spectrograph that is used for the deflated carbon dioxide component of producing reactor is carried out on-line determination.
In another preference, being used to measure the deflated exhaust gas mass spectrograph of producing reactor and microreactor is same gas mass spectrograph.
In another preference, the Controlling System of described microreactor is according to the determination data of exhaust mass spectrograph to the exhaust components of described production reactor and microreactor, described microreactor is implemented feedback control, thereby make the formation of the exhaust components of producing reactor and microreactor consistent as far as possible.When the formation same or similar (consistent as far as possible) of the exhaust components of producing reactor and microreactor, can think that the bacterial metabolism in the microreactor is similar or identical with the metabolism of the interior thalline of production reactor.
In another preference, described temperature electrode double as is a baffle plate.
In another preference, described data collector carries out online acquisition with temperature, dissolved oxygen and pH signal by AD converter (analog-digital converter).
In another preference, described data collector is also gathered the feed supplement data, and the feed supplement signal is carried out online acquisition by AD converter.
In another preference, described device also comprises the air inlet pretreatment unit that is used for removing the air inlet carbonic acid gas.
In another preference, described pretreatment unit is the container that the NaOH solution of 1M is housed.
In another preference, the blade diameter length ratio of described microreactor is 1: 1.8-2.2.
In another preference, the magnetic force rotor stirs control to described magnetic stirring apparatus in the microreactor by driving.
In a second aspect of the present invention, the purposes of the microreactor apparatus described in the first aspect present invention is provided, it is used to
13The isotope-labeled metabolic flux of C is measured.
In a third aspect of the present invention, provide a kind of
13The isotope-labeled metabolic flux measuring method of C comprises step:
1) in the microreactor in the microreactor apparatus described in the first aspect present invention,
13The carbon source of C mark exists down, carries out microbial fermentation and cultivates;
2) in the fermentation culture process, from the fermentation system of microreactor, take a sample, obtain sample to be analyzed;
3) by the gas phase in the described microreactor apparatus-mass spectrometry instrument system, the sampling sample is carried out the amino acid of (a) thalline hydrolysis and/or (b) the amino acid whose mensuration of free in the born of the same parents, obtain being used for the mark abundance information that metabolic flux is analyzed, thereby obtain the measurement result of metabolic flux.
In another preference, described method also comprises:
Produce in the reactor one, do not having
13The carbon source of C mark exists down, carries out microbial fermentation and cultivates, and wherein, the volume of described production reactor is (as the 50-10000 liter) more than 50 liters;
And in the fermentation culture process, to measuring respectively of the exhaust components of described production reactor and microreactor, and described microreactor implemented feedback control, thereby make the formation consistent as far as possible (same or similar) of formation and the exhaust components of production reactor of the exhaust components of microreactor.
In another preference, described feedback control is carried out in the following manner:
By micro mass flow meter and magnetic stirring apparatus, according to fermentation system to the demand of oxygen supply and regulate charge flow rate and mixing speed automatically; And/or,
Described device is regulated the rotating speed of peristaltic pump automatically according to the reading of the electronic scale of the setting of feed rate and the described microreactor weight of weighing, thereby whether and feed supplement speed the control feed supplement.
In another preference, described method also comprises:
13Two kinds of the C mark or two or more carbon source exist down, carry out microbial fermentation and cultivate; And
To molecular weight in the exhaust of the fermentation system of microreactor is 45
13C mark carbonic acid gas and molecular weight are that 44 cold carbon dioxide component is carried out on-line determination, and calculate metabolic flux according to observed value.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and specifically described in below (eg embodiment) each technical characterictic can make up mutually, thereby constitute new or optimized technical scheme.As space is limited, this tired no longer one by one stating.
Description of drawings
Fig. 1 has shown the holographic formula sensing response system in example of the present invention.
Fig. 2 has shown the Controlling System of the tracking production process in example of the present invention.
Fig. 3 has shown the change curve (OUR, CER and RQ) of the process in 50 liters of fermentor tanks.
Fig. 4 has shown the tracking fermenting process change curve in the micro sensing reactor.
Fig. 5 has shown in two parallel reactors (50 liters reactor and microreactor) amino acid and organic acid change in concentration outside cell concentration (by dry cell weight (DCW)), residual sugar content and the born of the same parents of the fermentation of vitamin B12.
Fig. 6 has shown that the microreactor system criticizes cultivation and investigates the performance graph that trimethyl-glycine utilizes process.
Fig. 7 has shown that 50L produces a jar fermenting process graphic representation.
Fig. 8 has shown the isotopic labeling exhaust information in the sensing response device and the real-time collection of macroscopical Fermentation Process of Parameter.
Fig. 9 has shown in the exhaust that the mark abundance of total free aminoacids changes in the carbonic acid gas and born of the same parents.
Figure 10 has shown the dynamic change of amino acid whose quality isotropic substance fragment in the born of the same parents.
Embodiment
The inventor has developed a kind of can be used for first through extensive and deep research
13The miniature holographic formula reactive system (being also referred to as microreactor apparatus) of C mark metabolic flux analysis experiment.This system comprises: one is used for the microreactor of microbial fermentation; One is used to gather the data collector of fermentation parameter; One is used to regulate the Controlling System of oxygen supply level and feed supplement; The exhaust mass spectrograph that is used for on-line determination; Measure in the fermentation system
13Amino acid whose gas phase-mass spectrometry the instrument system of C mark.Miniature holographic formula bio-sensing reactor assembly of the present invention can be measured the microbial metabolism flux in the born of the same parents in the fermenting process accurate, timely, inexpensively.
Miniature holographic formula reactor assembly
As used herein, term " miniature holographic formula reactor assembly ", " microreactor apparatus " and " sensing response device " are used interchangeably, and all refer to the device described in the first aspect present invention.
The miniature holographic formula reactor assembly that can be used for isotope-labeled metabolic flux analysis of the present invention comprises:
1) microreactor that is used for microbial fermentation, described microreactor is furnished with dissolved oxygen sensor, pH transmitter and the temperature sensor of measuring fermentation system;
And described microreactor is furnished with inlet pipe, vapor pipe, feed supplement pipe and stopple coupon;
And the volume of described microreactor is 30-500ml;
2) data collector that is used to gather fermentation parameter, described data set links to each other with temperature sensor with described dissolved oxygen sensor, pH transmitter;
3) Controlling System that is used to regulate oxygen supply level and feed supplement, described Controlling System comprise the micro mass flow meter of control air air inlet, the magnetic stirring apparatus that control is stirred and the peristaltic pump of controlling feed supplement;
4) being used for deflated molecular weight to microreactor is 45
13C mark carbonic acid gas and molecular weight are the exhaust gas mass spectrograph that 44 cold carbon dioxide component is carried out on-line determination; And
5) be used for measuring fermentation system
13Amino acid whose gas phase-mass spectrometry the instrument system (GC-MS system) of C mark, described GC-MS system carries out the amino acid of (a) thalline hydrolysis and/or (b) the amino acid whose mensuration of free in the born of the same parents to the sampling sample of fermenting process, thereby obtains being used for the mark abundance data that metabolic flux is analyzed.
A kind of preferred reactor assembly specifically comprises as shown in Figure 1:
1) microreactor: blade diameter length ratio 1: 2, volume 75-80ml has dissolved oxygen, pH and temperature electrode interface (doing baffle plate simultaneously uses), and inlet pipe, vapor pipe, feed supplement pipe and stopple coupon are arranged.Wherein, inlet pipe links to each other with micro flowmeter.As shown in the figure, microreactor should be furnished with magnetic stirring apparatus and AD transfer equipment.
2) data collecting system and 3) Controlling System: air is regulated and control by the micro mass flow meter, and the magnetic force rotor that drives in the tank body by magnetic stirring apparatus stirs control.The signal of temperature, dissolved oxygen, pH and feed supplement device carries out online acquisition and control by AD converter.
4) gas mass spectrograph: air inlet and deflated component to reactor are measured, especially the information of carbonic acid gas (
13C mark molecular weight of carbon dioxide 45, natural molecular weight of carbon dioxide are 44), can accurately measure.
5) GC-MS system: free amino acid and part organic acid in the amino acid of the thalline hydrolysis that obtains in the labelling experiment process or the born of the same parents are measured, and the mark abundance information that obtains is used for the calculating of metabolism stream.
The micro sensing reactor comes the monitoring of (comprising mixing speed, flow, pH, dissolved oxygen, temperature etc.) of the various parameters of realization response device by data collecting system, the information that Controlling System obtains according to the gas mass spectrograph is simultaneously carried out the feedback control of reactor, and the metabolism of thalline is similar in realizing the sensing response device and producing jar; Reach under the similar situation of metabolism at the two then, sampling is carried out the analysis of thalline amino acid flag state by gas phase-mass spectrograph fast, thereby understands the physiological metabolism situation of producing thalline in the jar.
In a preference, this reactor volume is about 80-120ml, the long-pending 35-50ml of dress liquid, and dissolved oxygen is adjusted dissolved oxygen by stirring and air flow quantity; To the influence of mark carbonic acid gas in the fermentation exhaust, air inlet is with the NaOH pre-treatment of 1M for fear of carbon dioxide in air, and pH adds 10% ammoniacal liquor by stream and carries out auto-control; Adopt the magnetic agitation drive system, maximum speed 1800rpm.
Follow the tracks of industries process control system
Miniature holographic formula reactor assembly of the present invention also can be used for following the tracks of production process and controlling.
Tracking industries process control system of the present invention is except comprising above-mentioned miniature holographic formula reactor assembly, comprise that also (i) is used for the production reactor of fermentative production, the volume of wherein said production reactor is not particularly limited, and is generally (as the 50-10000 liter) more than 50 liters; (ii) be used for the deflated carbon dioxide component of producing reactor is carried out the exhaust gas mass spectrograph of on-line determination.
A kind of sensing response device experimental installation of the present invention and mark flow instance are as shown in Figure 2.Sensing response device and scale operation reactor are to link to each other with flowing mode.When the labelling experiment investigation is carried out in metabolism to a certain fermentation stage process, to produce in the reactor in the aseptic importing sensing response of the nutrient solution device, come rapid adjustment micro sensing reactor parameter by process parameter and exhaust measuring analytical system, making has consistent running status with the production reactor.
For example, and in the fermentation culture process, to measuring respectively of the exhaust components of described production reactor and microreactor, and described microreactor implemented feedback control, thereby make the formation consistent as far as possible (same or similar) of formation and the exhaust components of production reactor of the exhaust components of microreactor.
Usually, described feedback control is carried out in the following manner:
By micro mass flow meter and magnetic stirring apparatus, according to fermentation system to the demand of oxygen supply and regulate charge flow rate and mixing speed automatically; And/or,
Described device is regulated the rotating speed of peristaltic pump automatically according to the reading of the electronic scale of the setting of feed rate and the described microreactor weight of weighing, thereby whether and feed supplement speed the control feed supplement.
In the present invention, because the metabolic flux basically identical of microorganism in microreactor and the production reactor, therefore only need in microreactor, to add a small amount of (usually only 1% or less amount) isotope-labeled substrate, and continuously fast sampling is handled and is measured total free aminoacids or organic acid label information in the born of the same parents, thereby measures and obtain the microreactor this state under and the metabolic flux distributed intelligence of production reactor.Therefore, the inventive method can reduce the cost that isotope-labeled metabolic flux is analyzed significantly.
Exhaust detects
In labelling experiment, the label information of carbonic acid gas also has very big effect in the exhaust in the metabolism flowmeter is calculated, the variation of pathways metabolism flux makes that the ratio of the mark carbonic acid gas that consumption specific markers substrate generates is different, and for great majority batch culturing process, even the Argine Monohydrochloride of thalline does not change, the replacement of the total free aminoacids in the born of the same parents is also very little, but as long as the consumption of underlined substrate, just have the generation of carbonic acid gas, therefore can come the pathways metabolism flux is calculated by the abundance information of carbonic acid gas, can carry out rapid detection.
In a preference of the present invention, use
13Two kinds of the C mark or two or more carbon source are (for example
13The different compounds of C mark sign (as fructose and glucose), or
13The carbon source of the same race that the C mark position is different (for example glucose)), carrying out microbial fermentation cultivates; And to molecular weight in the exhaust of the fermentation system of microreactor is 45
13C mark carbonic acid gas and molecular weight are that 44 cold carbon dioxide component is carried out on-line determination, thereby calculate metabolic flux according to observed value.
Major advantage of the present invention is:
(a) this microreactor system can follow the tracks of and simulate the microbial metabolism state in the large-scale reactor of microbial fermentation production process, dwindling and amplifying research technology platform is provided for production process.
(b) volume of microreactor is little, therefore in the pathways metabolism variations of flux process of utilizing isotopic labeling substrate research microorganism, can reduce the consumption of labeled substrate significantly, reduces cost.
(c) than traditional metabolic flux method compare can the more detailed description organism metabolism state.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Unless otherwise indicated, otherwise per-cent and umber by weight.
Materials and methods
Biosystem and culture condition
Bacterial classification: denitrified pseudomonas (Pseudomonas denitrificans): available from the flourish pharmaceutical Co. Ltd of China.
The bacteria suspension preparation: wash cultured inclined-plane with sterilized water, making bacterium dense is 10
8The bacteria suspension that individual cell is every milliliter.
Female bottle seed culture: with the bacterial suspension inoculation 2ml that makes in female flask culture base, loading amount 100ml/500ml, 32 ℃, rotating speed 260rpm cultivated 20-22 hour.
The 50L fermentor cultivation: will cultivate the aseptic female bottle seed liquor 1250ml of good back microscopy, the flame protection is inoculated in the fermentor tank that 25 substratum are housed, and culture condition is the secondary stirring arm, and 32 ℃, air flow 20L/min.
Complex medium: (g/L) sucrose 80, corn steep liquor 45, trimethyl-glycine 14, (NH
4)
2SO
41, KH
2PO
40.75, CoCL6H
2O 0.075, and MgO 0.5, and DMBI 0.05, ZnSO
47H
2O 0.08, CaCO
31, pH7.2-7.4.
Synthetic medium: (g/L) glucose 30, (NH
4)
2 HPO
410, KCl 0.2, MgSO
47H
2O 1.4, (NH
4)
2SO
45,5,6-dimethylbenzimidazole 0.0065, mixing solutions (TES) 100ml, pH7.2-7.4.TES (g/l): MnSO
4H
2O 0.2, ZnSO
47H
2O 0.2, CoCl
26H
2O 0.025, FeSO
47H
2O 0.03, Na
2MO
40.02.
Begin to add continuously glucose and trimethyl-glycine feed liquid substratum according to the thalli growth situation in the fermenting process,
Supplemented medium 1:(g/L) glucose 300, and DMBI 0.15, CoCL6H
2O 0.15
Supplemented medium 1:(g/L) trimethyl-glycine 30, and DMBI 0.4, CoCL6H
2O 0.3
Measuring method
Biomass is measured:
Light absorption value is measured in bacterium liquid dilution back in wavelength 700nm place, be contrast with the deionized water.Thalline optical density value (OD
700)=OD reading * extension rate has good linear relationship, DCW=OD between dry cell weight (DCW) and the thalline optical density(OD)
700* 0.78, dry cell weight calculates according to the optical density value that records.
Glucose assays:
Adopt conventional " the DNS method of improvement " to measure.
Determined amino acid:
Sample is measured chromatographic column model EclipseAAA post after deriving with o-phthalaldehyde(OPA) with HPLC (Aglient1100).
Organic acid is measured:
The Agilent1100 chromatographic system, chromatographic column, the C8 of AquaSep. company (4.6mm * 25cm, 5 μ m).
The vitamin B12 Determination on content:
Get the 10ml fermented liquid, add each 2.5ml of 8% sodium nitrite solution and Glacial acetic acid, shake up, in 95-100 ℃ of water-bath 30min; The water-bath postcooling adds deionized water and is settled to 50ml to room temperature, filters; Detect with the Agilent1100 chromatographic system, chromatographic column is Beckman (4.6mm * 250mm, 5 μ m); The detection wavelength is 361nm; Sample size 20 μ l; Flow velocity is 1.0ml/min.
GC-MS evaluation of markers amino acid:
Sample collection: get bacterial culture fluid 1ml in the EP pipe, the centrifugal 5min of 12000rpm cryogenic freezing collects thalline, after the distilled water washing, adds among the 6M HCl, and 95 ℃, acid hydrolysis 24h.To use air stream drying behind the filtering with microporous membrane of hydrolyzed solution with 0.22 μ m, the tropina hydrolysis amino acid sample that obtains is put into 50 ℃ of dried overnight of vacuum drying oven, guarantees the adiabatic drying of amino acid sample.
Analyte derivative: because the amino acid gasification point is higher, major part can not directly be carried out GC-MS and be measured mensuration after need deriving with silylating reagent.With of pyridine (chromatographically pure) dissolving of absolute anhydrous amino acid solid with 300 μ l, add 100 μ l derivating agent N-(tertiary butyl dimethyl-silicon)-N-methyl trifluoro ethanamides (MBDSTFA) again, with behind the oscillator concussion 10min mixing in 60 ℃, 30min derives, and filters the back and analyzes with GC-MS.
GC-MS condition: GC separation condition: the HP5MX post, 100 ℃ of initial column temperatures keep 3min, rise to 220 ℃ with 10 ℃/min again, and the back rises to 280 ℃ with 10 ℃/min, keeps 5min; 280 ℃ of injector temperatures; Carrier gas is high-purity helium, constant current, flow rate of carrier gas 1.0mL/min, splitting ratio 1: 10.MS condition: EI ionization mode, interface temperature, ion source temperature 200; The full scan pattern, quality of scanning scope 100-600.
The comparative analysis of producing jar, microreactor apparatus and shaking bottle
Present embodiment adopts 50 liters of fermentation reactors as producing jar, adopts microreactor apparatus shown in Figure 1 as the sensing response device.
The consistency analysis of 1 macroscopical metabolizing parameters
In order to investigate the feasibility of this minisize reaction system, the inventor has carried out producing the leavening property investigation of jar and sensing response device; In 50 liters of fermentation reactors, carry out vitamins B with complex medium
12Fermentation test, when fermenting process proceeds to certain phase, sterile sampling divides respectively and installs to microreactor neutralization and shake in the bottle from 50 liters of fermentation reactors, with carbon dioxide content, OUR and CER in the exhaust is controlled variable, by stirring and the aeration condition of regulating little reactor, guarantee the consistence of thalline physiological status in two reactors.
Process change curve in 50l reactor and the microreactor as shown in Figure 3 and Figure 4.From two reactors in the change curve of bacterial metabolism as can be seen, the physiology of respiration metabolic characteristic of thalline is consistent down with working condition in the micro sensing reactor, with the exhaust gas monitoring system is guidance, variation is adjusted to the thalline physiological characteristic in the sensing response device, can accomplish to respond quickly, thereby make the sensing response device can better represent the metabolism state under the working condition.
The investigation of metabolizing parameters in the 2 pairs of fermenting processs
For the feasibility of the tracking working condition of more in depth understanding the micro sensing reactor, two parallel reactor systems are carried out the while sampling analysis, measure its thalli growth situation, outer amino acid of the variation of sugar consumption and born of the same parents and organic acid variation.
The results are shown in Figure 5, from the result of sampling analysis as can be seen, thalli growth under sensing response device and the working condition and sugar consumption change very high similarity, and the degree of correlation is more than 98%, and the outer amino acid of the born of the same parents in while two reactor assemblies is also very consistent with the organic acid change in concentration.
On this similarity degree shows that not only two material concentrations in the reactor change, and shown on the rate of change of material, table 1 has been analyzed the wear rate of two culturing process 25-30 hour corresponding glucose, the specific growth rate of thalline and the generation of metabolite and wear rate, these materials are very approaching to the rate of change of unit thalline, the metabolic characteristic that these data prove absolutely thalline in the micro sensing reactor is consistent with metabolic characteristic under the corresponding working condition, and this sensing response device system can follow the tracks of and produce jar and carry out the real-time follow-up metabolic analysis.
Follow the tracks of the glucose consumption speed of cultivating 25-30 hour stage in two different reactors of table 1,
Carbonic acid gas rate of release and organic acid and amino acid whose generating rate
The application of microreactor in batch cultivation isotopic labeling experiment
In order better to study the utilize situation of trimethyl-glycine in the thalli growth stage, the glucose that utilizes mark is as substrate, in microreactor, carry out a batch cultivation, mark abundance information and the amino acid whose label information of tropina according to carbonic acid gas in the exhaust, the detailed analysis trimethyl-glycine utilize situation, the feasibility of checking minisize reaction system.
Present embodiment adopts 50 liters of fermentation reactors as producing jar, adopts microreactor apparatus shown in Figure 1 as the sensing response device.
1. labeled substrate and cultural method
Choosing of labeled substrate: select for use generally labelled glucose (5g/l) and natural trimethyl-glycine (3g/l) as unique carbonaceous material.
Culturing process: will cultivate the seed at all mark glucose synthetic medium, and after washing with sterilized water, get 0.5ml and be inoculated in the microreactor, the 1.5ml that at every turn takes a sample measures residual sugar, the dense and amino acid whose mark situation of tropina of bacterium, and exhaust system is gathered in real time.
2. labelling experiment is analyzed the situation of utilizing of trimethyl-glycine
Gather the label information of carbonic acid gas in the exhaust in the process in real time, and fermenting process is carried out the timing sampling analysis, Fig. 6 has shown the concentration of substrate in the fermenting process, the information of thalli growth and exhaust carbonic acid gas.
In culturing process, air inlet is controlled 30ml/min with mass flowmeter, and air is handled the CO that removes wherein through the NaOH of 1M
2, to avoid airborne CO
2To amino acid label information and exhaust CO
2The interference of mark abundance.From Fig. 6 b as can be seen, the pH of fermenting process in this system, dissolved oxygen, temperature can both gather and control well, and the Air quality control accuracy is higher, oxygen uptake rate OUR and CO in the fermenting process
2Generating rate CER can accurately be measured; The gas mass spectrograph can accurately be measured the mark situation of exhaust components.
Along with thalli growth enters exponential growth phase, sugar consumption speed increases sharply, and the concentration of trimethyl-glycine also reduces rapidly; Cultivate the fermented liquid component to be measured in 25 hours and do not find to have vitamins B
12Accumulation with its synthetic precursor substance 5-amino-laevulic acid.CO from exhaust
2Mark abundance information as can be seen, the CO of mark
2Account for to such an extent that ratio reaches minimum about 10 hours, because substrate uses is generally labelled glucose, the therefore CO that generates
2The mark abundance should be more than 98%, therefore the carbon source material beyond the glucose be arranged certainly, promptly trimethyl-glycine has participated in CO
2The generation metabolism.
Argine Monohydrochloride to the thalline in the culturing process is measured with GC-MS, and its measurement result sees Table 2.
Mark abundance information after Argine Monohydrochloride is proofreaied and correct after the table 2 fermentation thalline hydrolysis in 24 hours
Annotate: FL: the mark abundance of fragment; Mi: molecular weight is the fragment component of m+i.GC-MS data in the table are proofreaied and correct, and have removed the influence of natural isotopic.
The amino acid whose mark abundance of the tropina information that obtains from table 2 as can be seen, the mark abundance value FL of glycine and Serine is all less than 10%, illustrate that the glycine that generates after a large amount of trimethyl-glycines decomposes has participated in the thalline anabolism, illustrates also that simultaneously the Serine that generates tropina mainly is by glycine reverse synthetic under the effect of serine hydroxymethylase.
By pathways metabolism as can be known, the carbon atom of methionine(Met) 1-4 position is directed to the carbon atom of the 1-4 position of aspartic acid, and the 5th carbon atom derives from the transmethylation of folic acid; The label information of methionine(Met) shows, FLmet1-5 is 58.0% well below FLasp1-4, illustrates that the 5th carbon atom of methionine(Met) is mainly derived from the trimethyl-glycine that does not have mark.The mark abundance of the amino acid whose fragment of the overwhelming majority is 76 ± 3%, illustrates after trimethyl-glycine decomposes to have entered the center metabolism, has generated the required amino acid of thalli growth.22 ± 1.3% unmarked CO is arranged in the exhaust
2Exist, illustrate that the trimethyl-glycine of significant proportion has entered center metabolism generation CO
2, or under the effect of glycine synthetic enzyme, generate the CO that does not have mark by its degradation production glycine
2
In sum, miniature holographic formula reactive system of the present invention can well be used in a batch fermenting process, and can be with the isotopic abundance information in the exhaust, and the amino acid label information of tropina combines the physiological metabolism characteristic of thalline is accurately analyzed.Because this reactor is less, therefore the labeled substrate amount that needs is less, thereby greatly reduces the experimental implementation cost simultaneously.
Follow the tracks of the isotopic tracing analysis of production process
Investigation for the metabolic flux that labelling experiment is applied to fermenting process thalli growth stationary phase, real-time tracking fermentating metabolism process, just must overcome the restriction that the isotopic labeling experiment will reach isotopic distribution information stable state, reduce simultaneously the consumption of isotopic labeling substrate to greatest extent, so just require using miniature sensing response device to follow the tracks of metabolism state in the production process, add labeled substrate and carry out metabolic analysis; Yet because biomass growth rate is very low, the amino acid overwhelming majority of thalline constitutive protein generates before all being, their mark abundance information is not represented the metabolism state of this moment; Therefore should select for use metabolic pool to change faster intermediate product usually, change the calculating of carrying out carbon center's metabolic flux, total free aminoacids information in Here it is the born of the same parents according to their abundance; The interior total free aminoacids pond of born of the same parents is very fast at the renewal rate of thalli growth stationary phase, is easy to reach the steady state of isotopic distribution, can accurately be used for the calculating of metabolic flux stationary phase.
Present embodiment adopts 50 liters of fermentation reactors as producing jar, adopts microreactor apparatus shown in Figure 1 as the sensing response device.
With the aseptic taking-up of the nutrient solution that grows into stationary phase, be inoculated in the microreactor and cultivate, the 50ml liquid amount, air flow is 1vvm, by adjusting the rotating speed adjustment, make that the oxygen uptake rate of thalline is consistent with the production process on-line parameter with the carbonic acid gas generating rate in the microreactor, and add the glucose of mark, make final U-according to the content of the residual sugar that records
13C glucose and natural glucose sugar ratio are 20: 80.
Fig. 7 is the production process curve in 50 liters of fermentor tanks, 60 hours thalline grown into stationary phase, this moment the product vitamins B
12Beginning is a large amount of synthetic, and the quick aseptic taking-up of nutrient solution at this moment is inoculated in the micro sensing reactor, makes by the oxygen supply adjustment and keeps and produce jar more consistent online physiological parameter is arranged.Fig. 8 has reflected that the cellular process parameter in the sensing response device changes, and is consistent with production process parameters.
After adding the labelled glucose substrate, respectively at 30min, 60min, 90min, 120min, 180min and 360min take a sample fast, in 12000rpm, and-4 ℃ of centrifugal 1min, then with the liquid nitrogen that adds-80 ℃ in the thalline that obtains, carry out fragmentation with ultrasonic, the centrifuging and taking supernatant liquor carries out vacuum lyophilization, and amino acid whose label information in the born of the same parents is measured with GC-MS in the back of deriving.
The result of label information such as Fig. 9 are shown in 10.CO from exhaust
2The variation of mark abundance can find out that it is 18.9% that 80min just reaches balance, that is to say that thalline has at this moment reached balance to the utilization of labeled substrate.The mark abundance of total free aminoacids just reaches behind the amino acid whose isotopic labeling balance 120-150min in the born of the same parents, as can be seen than CO in the exhaust in the born of the same parents that record
2Balancing information evening 60min; Though visible CO
2Get the stability that the isotropic substance stable state has reflected base consumption, amino acid metabolite reaches the isotropic substance stable state in the born of the same parents but can not represent, because after the original amino acid of amino acid is fully replaced by newly-generated labeled amino acid in the born of the same parents, just can reach isotopic equilibrium, amino acid mark abundance information at this moment could be used for the calculating of metabolic flux effectively.For different microorganisms, add the labelled glucose substrate during the fermentation after, it is also different that born of the same parents' intracellular metabolite thing reaches time of response of isotopic equilibrium.Katharina etc. are in the unstable state metabolic process of utilizing labelled glucose substrate research E.coli K12, and the time of response of the interior total free aminoacids isotropic substance stable state of discovery born of the same parents is very short to be 20-40sec; Research such as Maciek R E.coli cultivates for K12 batch and produces 1, and in the ammediol process, the time of response that reaches the isotropic substance stable state after the labeled substrate consumption is 5min; Zheng Zhao etc. are when research penicillium chrysogenum fermentation unstable state labelling experiment, and the isotopic abundance information of born of the same parents' intracellular metabolite thing reaches balance and wants 50min.As seen all there is the generating rate of different born of the same parents' intracellular metabolite things in different microorganism and with the different physiological stages of a kind of microorganism, thereby it is also different to make when carrying out labelling experiment that the metabolite isotopic distribution reaches balance time length.
Above experimental result confirms, when utilizing the isotopic labeling substrate to investigate astable fermenting process, the steady-state response time of carbonic acid gas is starkly lower than the amino acid whose steady-state response time in the born of the same parents in the exhaust, and therefore in labelling experiment, the time of sampling and measuring born of the same parents' intracellular metabolite thing should be arranged in CO in the exhaust
2After the mark abundance stable state.For favourable directive function has been played in the setting of the sampling spot of labelling experiment.
Discuss
The experimental data of the above embodiment of the present invention confirms that this miniature holographic formula reactor assembly can well be applied to fermentation culture and the metabolic isotropic substance of production process is followed the tracks of investigation, and the major advantage of this system is summarized as follows:
1) this reactive system can carry out the tracking isotopic labeling The effects of batch fermentation culture and production process, by the on-line parameter acquisition system physiological status of thalline in the fermenting process is analyzed, can well be accomplished the process physiological status parameter more consistent with production process.
2) this reactor volume is little, volume 75-80ml, and liquid amount can be controlled in 35-50ml, and in carrying out the test of isotopic labeling The effects metabolic process, the consumption of labeled substrate is few, can significantly reduce the base consumption cost.
3) this reactor can be produced and the different periods of process are carried out isotopic labeling investigate, and this Controlling System can be controlled 13-15 platform microreactor simultaneously, and the substrate of available not isolabeling is followed the tracks of investigation to the fermenting process metabolic flux simultaneously.
4) the gas mass spectrograph can be analyzed the microreactor exhaust exactly and forms and to analyze, simultaneously CO in the exhaust in labelling experiment
2The mark abundant information also can accurately measure CO fast
2The judgement of the expending equilibrium that is changed to the process isotope substrate of mark abundance, and the stability of born of the same parents' intracellular metabolite thing isotopic labeling abundance provides directive function effectively.
5) this system can realize the quick sampling analysis to process at the trial, free amino acid and organic acid isotopic labeling information can accurately be measured by enough GC-MS in tropina composition amino acid, the born of the same parents, are used for the calculating of production process bacterial metabolism flux.
6) this reactor assembly also can be used for the fermenting process of other biological product.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Reference
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2) Drysch, A. wait the people, 2004.Serial flux mapping of Corynebacteriumglutamicum during fed-batch l-lysine production using the sensorreactor approach.Biotechnol.Bioeng.85 (5), 497-505.
3) Wahl, people such as A., 2004.Serial 13C-based flux analysis of anl-phenylalanine-producing E.coli strain using the sensor reactor.Biotechnol.Prog.20 (3), 706-714.
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8) people such as Zheng Zhao, 2008.Isotopic non-stationary 13C gluconate tracermethod for accurate determination of the pentose phosphate pathwaysplit-ratio in Penicillium chrysogenum.Metabolic Engineering.10,178-186.
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Claims (10)
1. one kind is used for the microreactor apparatus that isotope-labeled metabolic flux is analyzed, and it is characterized in that described device comprises:
1) microreactor that is used for microbial fermentation, described microreactor is furnished with dissolved oxygen sensor, pH transmitter and the temperature sensor of measuring fermentation system;
And described microreactor is furnished with inlet pipe, vapor pipe, feed supplement pipe and stopple coupon;
And the volume of described microreactor is 30-500ml;
2) data collector that is used to gather fermentation parameter, described data set links to each other with temperature sensor with described dissolved oxygen sensor, pH transmitter;
3) Controlling System that is used to regulate oxygen supply level and feed supplement, described Controlling System comprise the micro mass flow meter of control air air inlet, the magnetic stirring apparatus that control is stirred and the peristaltic pump of controlling feed supplement;
4) being used for deflated molecular weight to microreactor is 45
13C mark carbonic acid gas and molecular weight are the exhaust gas mass spectrograph that 44 cold carbon dioxide component is carried out on-line determination; And
5) be used for measuring fermentation system
13Amino acid whose gas phase-mass spectrometry the instrument system (GC-MS system) of C mark, described GC-MS system carries out the amino acid of (a) thalline hydrolysis and/or (b) the amino acid whose mensuration of free in the born of the same parents to the sampling sample of fermenting process, thereby obtains being used for the mark abundance data that metabolic flux is analyzed.
2. device as claimed in claim 1 is characterized in that, the volume of described microreactor is 50-250ml, and more preferably, the volume of described microreactor is 70-100ml.
3. device as claimed in claim 1 is characterized in that, described device comprises that also one produces reactor, and the volume of wherein said production reactor is (as the 50-10000 liter) more than 50 liters;
And the exhaust gas mass spectrograph that is used for the deflated carbon dioxide component of producing reactor is carried out on-line determination.
4. device as claimed in claim 1 is characterized in that, described temperature electrode double as is a baffle plate.
5. device as claimed in claim 1 is characterized in that, described data collector carries out online acquisition with temperature, dissolved oxygen and pH signal by AD converter (analog-digital converter).
6. device as claimed in claim 1 is characterized in that, described device also comprises the air inlet pretreatment unit that is used for removing the air inlet carbonic acid gas.
7. the purposes of microreactor apparatus as claimed in claim 1 is characterized in that, is used for
13The isotope-labeled metabolic flux of C is measured.
8. one kind
13The isotope-labeled metabolic flux measuring method of C is characterized in that, comprises step:
1) in the microreactor in the described microreactor apparatus of claim 1,
13The carbon source of C mark exists down, carries out microbial fermentation and cultivates;
2) in the fermentation culture process, from the fermentation system of microreactor, take a sample, obtain sample to be analyzed;
3) by the gas phase-mass spectrometry instrument system in the described microreactor apparatus of claim 1, the sampling sample is carried out the amino acid of (a) thalline hydrolysis and/or (b) the amino acid whose mensuration of free in the born of the same parents, obtain being used for the mark abundance information that metabolic flux is analyzed, thereby obtain the measurement result of metabolic flux.
9. method as claimed in claim 8 is characterized in that, described method also comprises:
Produce in the reactor one, do not having
13The carbon source of C mark exists down, carries out microbial fermentation and cultivates, and wherein, the volume of described production reactor is (as the 50-10000 liter) more than 50 liters;
And in the fermentation culture process, to measuring respectively of the exhaust components of described production reactor and microreactor, and described microreactor implemented feedback control, thereby make the formation consistent as far as possible (same or similar) of formation and the exhaust components of production reactor of the exhaust components of microreactor.
10. method as claimed in claim 9 is characterized in that, described feedback control is carried out in the following manner:
By micro mass flow meter and magnetic stirring apparatus, according to fermentation system to the demand of oxygen supply and regulate charge flow rate and mixing speed automatically; And/or,
Described device is regulated the rotating speed of peristaltic pump automatically according to the reading of the electronic scale of the setting of feed rate and the described microreactor weight of weighing, thereby whether and feed supplement speed the control feed supplement.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102703313A (en) * | 2012-05-14 | 2012-10-03 | 上海交通大学 | Batch type bioreactor |
| CN102703313B (en) * | 2012-05-14 | 2013-11-13 | 上海交通大学 | Batch type bioreactor |
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| CN108624516A (en) * | 2017-03-20 | 2018-10-09 | 华东理工大学 | A kind of metabolite amount in raising fermented cells and the method for preparing IDMS standard items |
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| CN113403193B (en) * | 2021-06-18 | 2022-11-25 | 华东理工大学 | Multistage continuous fermentation system |
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