CN104357539B - A kind of high-throughput screening method of organic acid superior strain - Google Patents
A kind of high-throughput screening method of organic acid superior strain Download PDFInfo
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Abstract
The invention discloses a kind of high-throughput screening method of organic acid superior strain, belong to microbial technology field.The present invention can be screened from a large amount of bacterial strains by pH indicator combination high flux screenings and obtain the of a relatively high bacterial strain of production of organic acids, be simplified whole screening process and improve screening efficiency.The high flux screening that pH indicator combines also is combined by the present invention with conventional breeding methods, obtains the superior strain of one plant of alpha Ketoglutarate, compared to original bacteria, the yield of the bacterial strain improves 51.8% in shaking flask, and 45.4% is improved on 3L fermentation tanks.
Description
Technical field
The present invention relates to a kind of high-throughput screening method of organic acid superior strain, belong to microbial technology field.
Background technology
α-ketoglutaric acid (α-KG) is a crucial mesostate in tricarboxylic acid cycle, is amino acid and protein
Main premise material, is widely used in the fields such as food, medicine, agricultural and cosmetics in metabolic process.But low production efficiency
This defect seriously limits the development and application of fermentation method production α-ketoglutaric acid industrially, therefore, to α -one penta 2
The screening of sour superior strain is extremely urgent.
At present, the detection of organic acid typically uses gas chromatography or HPLC methods, exists with the method come bacterium
The cumbersome, shortcoming such as efficiency is low.The also not no relevant report in terms of high flux screening α-ketoglutaric acid superior strain.
The present invention changes this characteristic by using pH in organic acid production process, with reference to pH indicator and high-throughput techniques
The method for establishing efficient, quick screening organic acid superior strain.
The content of the invention
The present invention provides a kind of high-throughput screening method of organic acid superior strain, is first by bacterial strain to be screened in containing change
Color range contains and primary dcreening operation is carried out in the culture medium of the pH indicator of any pH value in pH 2.6~5.2, choose become chromosphere compared with
After big bacterial strain ferments in high pass orifice, the pH that color change interval contains any pH value in pH 1.4~3.2 is added
Indicator carries out secondary screening, chooses the larger bacterial strain that changes colour and carries out fermentation checking.
Methods described, it is first to be carried out with treating bacterium containing bromocresol green or thymol blue or Congo red culture medium
Primary dcreening operation, select and become the larger bacterium colony of chromosphere, after being fermented in high pass orifice, take fermented liquid supernatant, add quinaldine red or orange
Yellow IV pH indicator, carries out secondary screening, chooses the larger bacterial strain that changes colour and carries out fermentation checking.
The organic acid can be it is following any one：Citric acid, isocitric acid, butanedioic acid, pyruvic acid, malic acid, α-
Ketoglutaric acid, phenylpyruvic acid, a- ketoisocaproates, fumaric acid, aliphatic acid, oxaloacetic acid.
The organic acid is α-ketoglutaric acid in one embodiment of the invention.
The bromocresol green or thymol blue are Congo red, and addition in the medium is conventional addition.
The compound method of the bromocresol green indicator：Bromocresol green 0.1g, add 0.05mol/L sodium hydroxide solutions
2.8ml makes dissolving, adds water and is diluted to 200ml, color change interval pH 3.6~5.2.
The bromocresol green, it is directly to add final concentration in the medium to exist in one embodiment of the invention
0.2-0.5g/L solid bromocresol green.In the culture medium of high yield organic acid bacterial strain, bromocresol green discoloration is obvious, and to thin
Born of the same parents do not have toxic action.
The primary dcreening operation can be by bacterial strain dibbling, coating or rule on flat board.
The high pass orifice can be that 96 deep-well plates, 96 shallow bore hole plates, 48 deep-well plates, 24 deep-well plates, 12 deep-well plates etc. are appointed
Anticipate it is a kind of with high flux screening effect culture vessel.
The compound method of the quinaldine red pH indicator：Quinaldine red 0.1g is taken, adds methanol 100ml to make dissolving, produces.
Color change interval pH1.4~3.2 (colourless → red).
The amount for adding quinaldine red pH indicator, is supernatant volume 1/25 to 1/100.
The amount for adding orange IV pH indicator, is supernatant volume 1/50 to 1/200.
After the addition quinaldine red or orange IV pH indicator, 5-60min is reacted.
The amount for adding quinaldine red pH indicator, it is the 1/ of supernatant volume in one embodiment of the invention
50。
The amount for adding orange IV pH indicator, it is the 1/ of supernatant volume in one embodiment of the invention
200。
The supernatant, in one embodiment of the invention, obtained after zymotic fluid is centrifuged.
The secondary screening, in one embodiment of the present invention, it can also be after adding quinaldine red pH indicator, measure
OD520, the relatively small bacterial strain that as secondary screening obtains of numerical value.
The secondary screening, in one embodiment of the present invention, it can also be after adding orange IV pH indicator, measure
OD520, the relatively large bacterial strain that as secondary screening obtains of numerical value.
The present invention also provides a kind of method that α-ketoglutaric acid superior strain is screened using the high-throughput screening method,
It is that bacterial strain to be screened is first subjected to primary dcreening operation in the culture medium containing bromocresol green, picking becomes the larger bacterium colony of chromosphere, then at height
After being fermented in flux orifice plate, fermented liquid supernatant is taken, adds quinaldine red pH indicator, the larger bacterial strain that changes colour is chosen and is obtained for secondary screening
The bacterial strain arrived, bacterial strain is obtained to secondary screening and carries out fermentation checking, that is, obtains α-ketoglutaric acid with respect to superior strain.
The bacterial strain is any one：The bacterial strain of organic acid can be produced.
The superior strain, it is the pH that can enable zymotic fluid during the fermentation in one embodiment of the invention
Enough reach less than 3.6 bacterial strain.
The bacterial strain, it is solution fat Asia Lip river yeast Yarrowia lipolytica in one embodiment of the invention.
The bacterial strain, it is Yarrowia lipolytica CCTCC NO in one embodiment of the invention：
M207143 and its bacterial strain obtained after ARTP mutagenesis.
The secondary screening, in one embodiment of the present invention, it can also be after adding quinaldine red pH indicator, measure
OD520, the relatively small bacterial strain that as secondary screening obtains of numerical value.
The volume for adding quinaldine red pH indicator is the 1/50 of supernatant volume, and the amount of α-ketoglutaric acid is in supernatant
During 0.6g/L-4.8g/L, after reacting 30min, OD520Linear equation be present with α-ketoglutaric acid amount：Y=0.02842X+
0.2039。
The secondary screening, those skilled in the art can be by adjusting the side such as fermentation time, dilution or concentrated broth
Method, the amount of α-ketoglutaric acid in supernatant is set to be suitable for being screened with quinaldine red pH indicator in a certain OK range.
The screening technique of the α-ketoglutaric acid superior strain, in one embodiment of the invention, it is specifically：To be more
Strain Yarrowia lipolytica are cultivated in the culture medium containing bromocresol green indicator, are chosen and are become chromosphere relatively
Big bacterial strain is the bacterial strain that primary dcreening operation obtains, and is fermented in high pass orifice, after zymotic fluid centrifugation, takes supernatant to add volume
For the quinaldine red pH indicator reaction 30min of supernatant 1/50, OD is determined520, choose OD520Relatively small bacterial strain is as secondary screening
Obtained bacterial strain, verified after being cultivated in fermentation medium.
Fermented in the high flux deep-well plates, in one embodiment of the invention, referred in fermentation medium,
In 28 DEG C, under 900rpm, ferment 96h.
The checking, refer to using α-ketoglutaric acid yield after high performance liquid chromatography measure strain fermentation, to determine secondary screening
Bacterial strain is relative superior strain.
The present invention establishes a kind of new screening technique, the method for combining the high flux screening of pH indicator, greatly
Simplify whole screening process.The inventive method can rapidly screening obtains the bacterial strain of high yield organic acid from a large amount of bacterial strains.
The present invention can also combine with other breeding methods (such as mutation breeding, metabolism breeding technique, metabolic engineering etc.)
Come, aimed strain is screened from a large amount of bacterial strains.In addition, the present invention has also combined constant temperature atmospheric plasma induced-mutation technique
(ARTP), pH indicator and High Throughput Screening Assay, improve the yield of α-ketoglutaric acid and reduce the yield of accessory substance pyruvic acid,
The present invention screens again by multiple batches of mutagenesis, has obtained the superior strain of one plant of α-ketoglutaric acid, has correspondingly reduced simultaneously
The yield of fermentation byproduct pyruvic acid and the production efficiency for improving whole fermentation process.
Brief description of the drawings
Fig. 1：OD520 and production of organic acids linear relationship；A is quinaldine red 1/25；B is quinaldine red 1/50；C is quinoline
Any pyridine red 1/100；D is orange IV 1/50；E is orange IV 1/100；F is orange IV 1/200.
Embodiment
The present invention bacterial strain to be screened, can be isolated from nature primary dcreening operation product or induced mutations or
The product of transformation.It is organic the present disclosure applies equally to quickly be screened from the higher or relatively low bacterial strain of organic acid output value level
The of a relatively high bacterial strain of acid yield, because the yield of organic acid is relatively low in high pass orifice first, it is not up to shaking flask or fermentation
The same Peak output in tank, in addition, for obtain the orifice plate culture technique for the purpose of target product be it is known in the art, according to
Target strain interested, related seed culture medium, fermentation medium, condition of culture and time etc. are referred to existing skill
Related guidance in art, adopted or adjusted with improving, these adjustment belong to the routine of those skilled in the art with improving
Technical ability, such as those skilled in the art can be according to strain properties, by adjusting fermentation time, dilution or concentrated broth
The methods of, organic acid content is accumulated in a certain OK range, to be adapted to this screening technique to be screened.The inventive method is applicable
In the bacterial strain of the bacterial strain, especially production α-ketoglutaric acid of various production organic acids.
The measure of dry cell weight：
A certain amount of bacteria suspension is drawn in 10mL volumetric flasks, add 2mL dissolving with hydrochloric acid suspensions in calcium carbonate, add from
Sub- water constant volume shakes up to 10mL, with the type visible spectrophotometers of UV 7500, OD values is determined at 570nm, use dry cell weight
Standard curve draws dry cell weight.
The measure of α-ketoglutaric acid, pyruvic acid and glycerine：High performance liquid chromatography (HPLC)
Instrument：The high performance liquid chromatographs of Agilent 1260 (match somebody with somebody UV-vis detector, differential refraction detector and work
Stand), chromatographic condition：
Chromatographic column：Aminex HPX-87H ion exchange column
Mobile phase：5mM H2SO4
Flow velocity：0.5mL/min
Column temperature：40℃
Sample size：10μL
UV-detector wavelength：210nm (detection α-ketoglutaric acid and pyruvic acid)
Differential refraction detector：Detect glycerine
Sample preparation：1mL zymotic fluids centrifuge 5min under 12,000rpm, take supernatant be put into 1.5mL centrifuge tubes survey α-
The content of ketoglutaric acid, pyruvic acid and glycerine.100 μ L of supernatant liquid are taken in 5mL volumetric flasks, ultra-pure water constant volume to graduation mark, warp
0.22 μ L membrane filtrations, filtrate supply efficient liquid phase chromatographic analysis.
The calculating of fatal rate：
Wherein A represents the CFU number on blank control flat board, and S is represented by mutagenic treatment rear plate
CFU number.
Required culture medium：
Seed culture medium (g/L)
Glucose 20g, soybean protein 10g, potassium dihydrogen phosphate 1.0g, epsom salt 0.5g, solid medium addition 20g
Agar.115 DEG C of sterilizing 15min.
Primary dcreening operation culture medium (g/L)
Glucose 20g, soybean protein 10g, potassium dihydrogen phosphate 1.0g, epsom salt 0.5g, bromocresol green 0.5g, block that
Mycin 1.0g, agar 20g.115 DEG C of sterilizing 15min.
Fermentation medium (i.e. secondary screening and checking culture medium) (g/L)
Glycerine 100g, ammonium sulfate 3g, potassium dihydrogen phosphate 3g, epsom salt 1.2g, sodium chloride 0.5g, dipotassium hydrogen phosphate
0.1g, thiamine hydrochloride 6 × 10-7G (filtration sterilization), calcium carbonate 10g (individually sterilizing).115 DEG C of sterilizing 15min.Verify culture medium
Middle addition 10g/L calcium carbonate.
Embodiment 1：ARTP mutagenesis
The Yarrowia lipolytica CCTCC NO that will be deposited in glycerol tube：M207143 strains are coated on eggplant shape
After 24h being activated on bottle inclined-plane, in the ring thalline of picking one access seed culture medium (500mL shaking flasks fill 50mL), 200r/min, 28 DEG C
17-18h is cultivated, takes 1mL bacteria suspensions in 1.5mL sterile centrifugation tubes, 4500r centrifugation 10min, after brine 2 times, then
Diluted in right amount with physiological saline and cell concentration is made 106~107Between bacteria suspension, the bacteria suspension of dilution is spread evenly across
Sterile slide surface, slide glass is placed on microscope carrier, is handled with ARTP mutation breedings system, is 100W, helium in incident power
Under the conditions of flow is 10SLM, 10s, 20s, 30s, 40s, 50s, 60s, 90s, 120s are irradiated respectively, sample treatment finishes, with nothing
Bacterium tweezers put slide glass into the EP pipes equipped with 1ml physiological saline, vibrate 1min, the microorganism being attached on fungus slide glass is washed
Take off in liquid, form new bacteria suspension, 10ul bacterium solutions are directly taken in control group into 1ml physiological saline.Will be all new
Bacteria suspension is diluted to 10-1、10-2、10-3Three gradients, 100 μ L10 are taken respectively-1、10-2、10-3The bacteria suspension applying solid of gradient
On flat board, every group do 3 groups it is parallel.After cultivating 24h in 28 DEG C of constant incubators, the CFU on every piece of flat board is counted
Number, then the fatal rate of each processing time is calculated respectively, and then it is 100W, helium gas flow 10SLM to obtain in incident power
Under the conditions of the research of the early stage such as destruction curve, Laroussi show that mutation effect of the fatal rate more than 90% is best, therefore,
30s is as the optimal mutagenic treatment time.
Embodiment 2：The selection of developer
According to the Variation Features of pH value in α-ketoglutaric acid fermentation process, BG, quinaldine red, orange IV, hole have chosen
Sparrow is green, thymol blue, Congo red, 7 kinds of developers of bromocresol green are used as pre-selection developer, has the preliminary experiment in 48 deep-well plates to survey again
Go out the yield of α-ketoglutaric acid and pyruvic acid in below 2g/L, therefore using fermentation medium as lysate, add α -one penta 2
The amount 0.6g/L-4.8g/L of acid gradient concentration, is dissolved in 200 μ L fermentation mediums, is adding the developer of gradient proportion
(1/400,1/200,1/100,1/50,1/25), normal-temperature reaction 30min, in the maximum visible absorption value of various developers
Determine light absorption value OD.As a result show that bromocresol green indicator just has obvious metachromasia under low acid concentration, and be not present
Sterilization and bacteriostasis effect, can be as the developer on primary dcreening operation flat board；Good line be present when being 1/50 in quinaldine red additional proportion
Sexual intercourse, OD520With α-ketoglutaric acid amount existing for linear equation be：Y=-0.0306X+0.214, quinaldine red is as secondary screening
Developer.
Embodiment 3：The screening of high yield α-ketoglutaric acid bacterial strain
Bacteria suspension after ARTP mutagenic treatments 30s is diluted to 10-1、10-2Two concentration gradients, it is equal that 100 μ L are drawn respectively
It is even to be coated on pre-sifted flat board, 48h is cultivated in 200r/min, 28 DEG C of constant incubators, the big change chromosphere of picking colony form is big
Colony lift into the seed culture medium in 96 deep-well plates, 900r/min, train in 28 DEG C of high flux deep-well plates constant temperature oscillators
48h is supported, is transferred to 10% inoculum concentration in 48 deep-well plates (dress 1mL fermentation mediums (volume after inoculation)), 900r/min, 28
DEG C fermented and cultured 96h, 48 deep-well plates 3500r/min are centrifuged into 10min, take 200 μ L of supernatant liquid in 96 orifice plates, add 4 μ L quinolines
After any pyridine red pH indicator reactions 30min, the light absorption value OD under 520nm visible rays is determined with ELIASA520.Select OD520Relatively
Less bacterial strain carries out secondary screening experiment.By after activation go out bacterium germination and mutagenic strain that primary dcreening operation obtains is respectively with 10% inoculum concentration
Access in fermentation medium (500mL shaking flasks dress 50mL (volume after inoculation)), 200r/min, 28 DEG C of culture 168h.Obtain one plant
The amount of production α-ketoglutaric acid improves 51.8% bacterial strain compared to starting strain, and yield is still stable after Secondary Culture several times.
In addition, being quantitative determined in screening process, while from HPLC to bacterial strain production α-ketoglutaric acid amount, find：
The big bacterial strain acid producing ability of change chromosphere, which is better than, during primary dcreening operation becomes the relatively small bacterial strain of chromosphere, and during secondary screening, discoloration is smaller
Or OD520Larger bacterial strain, its α-ketoglutaric acid yield are actually lower than the larger or OD that changes colour520Less bacterial strain, illustrates this
Screening technique is reliable.
Embodiment 4：The fermenting and producing checking of high yield α-ketoglutaric acid bacterial strain
The maximum bacterial strain improved of yield that embodiment 3 obtains is inoculated into seed culture medium with starting strain to (500mL shakes
Bottled 50mL), 17-18h is cultivated under the conditions of 200r/min, 28 DEG C of culture 17-18h, 3L hairs are linked into 10% inoculum concentration
In fermentation tank (dress 1.5L (volume after inoculation)), 600r/min, 28 DEG C, ferment 168h under the conditions of 1.5vvm.After fermentation 168h, with going out
Bacterium germination strain is compared, and the yield of α-ketoglutaric acid is 31.46g/L, and starting strain is 21.64g/L improves 45.4%, pyruvic acid
Amount be 30.45g/L, and starting strain is 39.23g/L, reduces 22.4%.
Embodiment 5：The screening of high yield pyruvic acid bacterial strain
Can fermentation production of acetone acid by Torulopsisglabrata CCTCC M202019.By Torulopsis
After glabrataWSH-Y carries out ultraviolet mutagenesis, mass mutation bacterial strain is obtained.By mutant strain and starting strain, dibbling in containing
On the flat board of 0.2g/L bromocresol green, negative mutant strain is rejected, then chooses and becomes the big bacterial strain of chromosphere in high pass orifice
Carry out secondary screening, after 72h fermentation ends, centrifugation, added in 200 μ L of supernatant liquid 3/100 quinaldine red, 10min is reacted, in enzyme
The detection for carrying out absorbance in instrument under 520nm wavelength to its reaction solution is marked, obtains OD520The 12 plant bacterium low compared with starting strain, will
This 12 plants of bacterium shake flask fermentation 72h, detect acetone acid content.As a result find in this 12 plants of bacterium, the OD of said determination520Lower production
Pyruvic acid ability is stronger.The bacterial strain that one plant of output of pyruvic acid is 36.6g/L is also obtained simultaneously, it is relative for starting strain 32.4g/
L improves 12.96%.Adopting said method, quickly and accurately screening has obtained one plant of production pyruvic acid relatively from a large amount of bacterial strains
High bacterial strain.
Embodiment 6：The selection of developer
Congo red, bromocresol green and thymol blue are added in the culture medium containing various concentrations organic acid, found
Color reaction can occur under low acid concentration, can all be used as primary dcreening operation indicator.As shown in figure 1, it is bromocresol green and thymol
Blue colouration is dismissed, and is as a result shown, the color of bromocresol green is changed into yellow from blueness, and color change is most obvious, and bromocresol green
Cytotoxic evil is acted on, it is best for primary dcreening operation indicator, effect.
By with fermented supernatant fluid volume ratio be 1/25,1/50,1/100 quinaldine red indicator, and 1/50,1/100,
1/200 orange IV indicator, OD is determined respectively after reacting 30min with fermentation supernatant520.Also measured were simultaneously in fermentation supernatant
Organic acid content, in order to show the reliability of experimental data, every group do respectively 5 it is parallel, as a result find OD values and organic acid
Certain linear relationship between containing all be present, such as Fig. 1 (A, quinaldine red 1/25；B, quinaldine red 1/50；C, quinaldine red 1/
100；D, orange IV 1/50；E, orange IV 1/100；F, orange IV 1/200) shown in, 1/50 quinaldine of addition supernatant volume
After red indicator, OD520It is best with the linear relationship of organic acid content, therefore quinaldine red indicator can be as the present invention's
One of preferred embodiment.
Embodiment 7：The screening of high yield organic acid bacterial strain
Obtained 52 saccharomycetes will be screened from natural environment, coating or dibbling in containing Congo red culture medium,
The 24h times are cultivated in 30 DEG C, selects and becomes the bacterial strain that arrives of the relatively large 24 plants of bacterium of chromosphere into primary dcreening operation, in containing seed culture medium
Fermented and cultured 72h, takes zymotic fluid to centrifuge in 24 deep-well plates, 1/200 orange IV indicator is added in 200 μ L of supernatant liquid, instead
60min is answered, the detection of absorbance is carried out under 520nm wavelength to its reaction solution in ELIASA, obtains OD520Of a relatively high 6
Strain bacterium, by this 6 plants of bacterium shake flask fermentation 144h, detect total organic acids content.As a result find in this 6 plants of bacterium, the OD of said determination520
Lower production organic acid ability is stronger, and one plant of obtained production of organic acids is 43.6g/L fractional yield highest bacterial strain.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (4)
1. a kind of high-throughput screening method of organic acid superior strain, it is first to contain bacterial strain to be screened in containing color change interval
Primary dcreening operation is carried out in the culture medium of the pH indicator of any pH value in pH2.6~5.2, chooses and becomes the larger bacterial strain of chromosphere in high pass
After being fermented in orifice, the pH indicator for adding any pH value that color change interval contains pH 1.4~3.2 carries out secondary screening, choosing
The larger bacterial strain that changes colour is taken to carry out fermentation checking；
Methods described is first to carry out primary dcreening operation with treating bacterium containing bromocresol green or thymol blue or Congo red culture medium,
Select and become the larger bacterium colony of chromosphere, after being fermented in high pass orifice, take fermented liquid supernatant, add quinaldine red indicator and carry out
Secondary screening, choose the larger bacterial strain that changes colour and carry out fermentation checking；
Wherein, the amount for adding quinaldine red pH indicator is the 1/50 of supernatant volume；The amount of α-ketoglutaric acid is 0.6g/ in supernatant
L-4.8g/L；
The organic acid is α-ketoglutaric acid, and the bacterial strain is solution fat Asia Lip river yeast.
2. according to the method for claim 1, it is characterised in that after the addition quinaldine red indicator, reaction 5 is arrived
60min。
3. according to the method for claim 1, it is characterised in that the bacterial strain is primary dcreening operation product or the people for being isolated from nature
After work mutagenesis bacterial strain or it is Metabolically engineered after bacterial strain.
4. according to the method for claim 1, it is characterised in that the secondary screening is after adding quinaldine red pH indicator, instead
30min is answered, determines OD520, the relatively small bacterial strain that as secondary screening obtains of numerical value.
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| CN107782885B (en) * | 2017-06-30 | 2020-01-21 | 江南大学 | High-flux screening system based on single mechanical arm |
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| CN109749956A (en) * | 2019-01-08 | 2019-05-14 | 甘肃农业大学 | A kind of Oenococcus Oeni bacterial strain high-throughput screening method based on 48 hole deep-well plates |
| CN111471740A (en) * | 2020-06-02 | 2020-07-31 | 驻马店华中正大有限公司 | Rapid screening method of clostridium butyricum |
| CN114456959B (en) * | 2022-01-26 | 2022-11-18 | 山东星光首创生物科技有限公司 | Hyperosmotic-resistant yarrowia lipolytica strain, breeding method and application |
| CN115232856A (en) * | 2022-07-27 | 2022-10-25 | 河南省健康元生物医药研究院有限公司 | High-throughput screening method for acremonium chrysogenum based on solid fermentation |
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| 柠檬酸发酵菌黑曲霉的诱变育种研究;黄旭初;《中国优秀博硕士学位论文全文数据库 (硕士) 基础科学辑》;20061215(第12期);第A006-215页 * |
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