CN106591136A - High-glucose-tolerance crypthecodinium cohnii obtained from orient domestication, and preparation method and application thereof - Google Patents
High-glucose-tolerance crypthecodinium cohnii obtained from orient domestication, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a high-glucose-tolerance crypthecodinium cohnii domesticated strain, and a preparation method and an application thereof. The high-glucose-tolerance crypthecodinium cohnii strain is purchased from American Type Culture Collection ATCC, an algal strain with the code of ATCC 30556 is used as a starting strain, and crypthecodinium cohnii selects the strain from the ATCC (having the strain code of ATCC 30556) as the starting strain; the initial crypthecodinium cohnii ATCC 30556 is used as a processing object, and domestication culture is performed for multiple times in a by+ culture medium having glucose gradually added, and a processing object of each-time domestication culture is an algal strain obtained after last-time domestication culture; the glucose concentration is increased to 54 g/L after domestication is finished; the domestication process is performed totally for 650 days, and the total number of generations is 130; the obtained high-glucose-tolerance crypthecodinium cohnii domesticated strain is named as ALE3. The high-glucose-tolerance crypthecodinium cohnii domesticated strain has obvious economic advantages and industry prospects in fermentation production of docosahexaenoic acid (DHA).
Description
Technical field
The present invention discloses the Kou Shi Crypthecodinium cohnii domestication strain of a plant height glucose-tolerant, preparation method and application.Belong to biological
Technical field.
Background technology
Docosahexenoic acid (Docosahexaenoic acid, DHA), belongs to ω -3 class unsaturated fatty acids, is pole
For a kind of important fatty acid with high nutritive value, the normal of the normal development, adult brain's function to infant brain is sent out
Wave and play an important role.Traditionally DHA is extracted from bathypelagic fish oil, but gradually reducing with world fisheries resource, finds DHA
Other effort for substituting source are also increasingly subject to pay attention to.Wherein, it is considered using marine microalgae heterotrophism metabolism fermenting and producing DHA
One main alternative method.
Kou Shi Crypthecodinium cohnii has preferable DHA synthesis capabilities, and DHA accounts for more than the 30% of cytolipin.Kou Shi Crypthecodinium cohnii
(Crypthecodinium cohnii) fermenting and producing DHA, with series of advantages compared with traditional fish oil is originated, such as ferments week
Phase is short, and training method is simple, and growth of microorganism is fast, it is easy to large-scale culture；Content of polyunsaturated fatty acid is high, and product quality is steady
Fixed, preferably, unsaturated fatty acid composition is single, low without EPA or EPA content, it is easy to isolate and purify for oxidation stability；While gram
Having taken traditional DHA that obtains from fish oil is affected by many restrictions factor such as raw material, weather, the place of production, production cycle.
At present, glucose is the high-quality carbon source that Kou Shi Crypthecodinium cohnii produces DHA, but earlier studies have shown that high concentration glucose
Crypthecodinium cohnii growth and oil and fat accumulation can be suppressed.Therefore, the improvement of Crypthecodinium cohnii algae strain germplasm is for promotion Crypthecodinium cohnii production DHA products
The sustainable development of industry is highly important.Earlier studies have shown that, microorganism is placed in into specific ambient pressure by artificial
Under, tame through the orientation to microorganism of a period of time, can obtain has more resistance, adaptability or profit to the stress conditions
With the mutant strain of ability.Due to the biosynthesiss mechanism of the full-length genome and DHA of Kou Shi Crypthecodinium cohnii it is not yet clear and definite, genetic engineering
Transformation metabolic pathway difficulty is larger.The operating process of orientation domestication is simple can effectively to change strain performance again, so as to be felt
The phenotype of interest.Up to the present, the report of the Crypthecodinium cohnii algae strain of high glucose tolerance is not obtained using orientation domestication.
It is a discovery of the invention that the Kou Shi Crypthecodinium cohnii that high glucose tolerance can be obtained by orienting domestication tames strain, with the bacterium that sets out
Strain is compared, and the ability of the domestication strain tolerance glucose is significantly improved, and oil and fat accumulation also increased.Four core metabolic indicators
Raising occur in thing glycerol, glutamic acid, malonic acid and succinic acid, this and tame the raising of strain glucose tolerance and have and significantly associate.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, the Kou Shi Crypthecodinium cohnii for obtaining a plant height glucose-tolerant is tamed and dociled
Change strain, and the growth and metabolism of analysis domestication strain is special in terms of glucose tolerance, oil and fat accumulation and metabolite content change etc.
Levy.
For achieving the above object, the technical solution used in the present invention is：
It is starting strain from the Kou Shi Crypthecodinium cohnii (bacterial strain code ATCC 30556) from american strain collection ATCC.
The initial Kou Shi Crypthecodinium cohnii is incubated at into by+Culture medium, can reach exponential phase mid-term, OD to 36h490Can
Reach 0.8；The by+Culture medium is consisted of：Glucose 9g/L, yeast extract 2g/L, sea salt 25g/L, pH are 6.5.By institute
State initial Kou Shi Crypthecodinium cohnii and be incubated at the by for having augmented high concentration glucose+In culture medium, growth rate can be remarkably decreased；It is described
The by for having augmented glucose+In culture medium concentration of glucose be in state incremented by successively, yeast extract 2g/L, sea salt 25g/L；
When concentration of glucose is higher than 25g/L, the time that the initial Kou Shi Crypthecodinium cohnii reaches exponential phase mid-term may extend to
48～72h.
Using initial Kou Shi Crypthecodinium cohnii ATCC 30556 as process object, in the by for gradually augmenting glucose+In culture medium
Repeatedly domestication culture is carried out, the process object of the domestication culture is algae strain after last domestication culture every time；At the end of domestication
Concentration of glucose increases to 54g/L；Domestication process experiences 650 days altogether, altogether 130 generation；The Kou Shi of the high tolerance glucose for obtaining
Crypthecodinium cohnii domestication strain is named as ALE3.Detailed process is as follows：
During the domestication, initial Kou Shi Crypthecodinium cohnii is incubated in the conical flask of 250mL, the supplement equipped with 50mL in bottle
The by of glucose+Culture medium；Control initial inoculation OD490For 0.08, as the OD of Crypthecodinium cohnii algae solution490Meet or exceed 0.8 it
After passed on.
During the domestication, OD after domestication strain growth 36h490Can reach or during more than 0.8, improve concentration of glucose.
During the domestication, the concentration of glucose in culture medium is in state incremented by successively；Concentration of glucose is higher than
During 18g/L, the concentration of glucose is gradually increased with the amplification of 4g/L, when concentration of glucose is higher than 36g/L, concentration of glucose amplification
It is changed to 2g/L.
During the domestication, cell sample can be taken at set intervals and with Olympus BX43 fluorescence microscopes
The form of domestication strain, while and combining the method exclusion biological pollution phenomenon such as method detection of painting flat board.
During the domestication, domestication strain reached for 130 generations, and caking phenomenon occurs in cell, and domestication terminates.Now glucose is dense
Degree has been improved to 54g/L.
The domestication process is altogether after 650 days.The domestication strain obtained by the domestication process is named as ALE3.
The domestication strain ALE3 is incubated at by respectively that augmented glucose+In culture medium, concentration of glucose is 9g/L,
36g/L, 45g/L and 54g/L.Sent out using glucoseoxidase and determine remaining sugar concentration in culture medium, using BD companies
FACSCalibur flow cytometers and grease extraction determine fat content, using Agilent 7890A/5975C gas chromatography mass spectrometry
Instrument determines culture DHA content, tames strain ALE3 and the significant difference set out between algae strain using statistics t check analyses,
Cell metabolite is extracted from parallel culture simultaneously, the change feelings of intracellular metabolite is tested and analyzed using metabolism group method
Condition.
The metabolism group data of gained are weighted into related network analysis (WGCNA), are found out and domestication by module detection
The related module of strain (every kind of module is represented with a kind of color)；Then filter out significantly correlated confidence module；It is finally right respectively
Every kind of significantly correlated confidence module construction metabolism network topology diagram, finds out core compound.In WGCNA construction, if
One compound is connected with having more than 5 compounds, then it is assumed that this compound is core metabolic markers.
By analysis, the Kou Shi Crypthecodinium cohnii domestication strain of the high glucose tolerance for obtaining, it is characterised in that：(1) 36,45 and
In 54g/L high glucose concentration culture medium cultivate, relative to starting strain, specific growth rate be respectively increased 1.03,2.78 and
3.52 times, mean doubling time has been respectively shortened 23.5%, 31.9% and 6.4%, while glucose consumption rate is respectively increased
2.67,2.62 and 2.51 times；After 72h is cultivated at (2) 25 DEG C, oils and fatss are extracted, relative to starting strain, oil and fat accumulation increases respectively
6.8%, 14.2% and 16.3%, meanwhile, being dyeed using Nile red and flow cytometry analysis, cell interior complexity has
Increased；(3) by the metabolism group method and WGCNA methods of GC-MS, detect and analyze the changes of contents feelings of intracellular metabolite
Condition, relative to starting strain, 21 compounds are substantially raised, and are located at respectively and positively related blue, green two modules of algae strain
In；Wherein blue module identification goes out glycerol, glutamic acid, four core metabolic markers of malonic acid and succinic acid.Described core
Metabolism mark thing is verified relevant with anti-stress, such as glycerol, glutamic acid, but they and Crypthecodinium cohnii glucose tolerance improve phase
Association is found first.
Compared with the prior art the present invention has following distinguishing feature and good effect：One plant is obtained effectively by orienting domestication
The Kou Shi Crypthecodinium cohnii of high glucose tolerance.This domestication strain can not only enduring high-concentration glucose, and oil and fat accumulation also has substantially increasing
Plus, also disclose the metabolic markers related to domestication strain glucose-tolerant raising.
Description of the drawings
Fig. 1 was illustrated for the domestication time of Kou Shi Crypthecodinium cohnii ATCC 30556.
Fig. 2 is original algae strain and growth and the glucose consumption of domestication strain of setting out of Kou Shi Crypthecodinium cohnii under 9g/L concentration of glucose
Comparison diagram.
Fig. 3 be 36g/L concentration of glucose under Kou Shi Crypthecodinium cohnii it is original set out algae strain with tame strain growth and glucose disappear
Consumption comparison diagram.
Fig. 4 be 45g/L concentration of glucose under Kou Shi Crypthecodinium cohnii it is original set out algae strain with tame strain growth and glucose disappear
Consumption comparison diagram.
Fig. 5 be 54g/L concentration of glucose under Kou Shi Crypthecodinium cohnii it is original set out algae strain with tame strain growth and glucose disappear
Consumption comparison diagram.
Fig. 6 is original algae strain and the oil and fat accumulation and DHA relative amount comparison diagrams of domestication strain of setting out of Kou Shi Crypthecodinium cohnii.Statistics
Credit analysis p value shows to set out less than 0.05 and there is significant difference (* p between algae strain and domestication algae strain<0.05), p value is less, poor
It is different bigger.
Fig. 7 is that the original algae strain of setting out of Kou Shi Crypthecodinium cohnii is compared point with the flow cytometer of domestication strain under different concentration of glucose
Analysis.In figure, blue scatterplot represents starting strain, and red scatterplot represents domestication strain.In figure FL2 the and FL3 signal intensitys of red scatterplot compared with
Blue scatterplot shows by force to tame the intracellular neutral fat of strain and polarity fat content is high；Red scatterplot and blue scatterplot in figure
Fsc signal no significant difference shows to tame strain and starting strain cell size no significant difference；In figure the SSC signals of red scatterplot compared with
Blue scatterplot is strong, shows that taming strain cell interior complexity increases.
Fig. 8 is the thermal map of WGCNA analysis modules and domestication strain dependency.In figure, different colours represent disparate modules, do not have
Substantially the compound of dependency is attributed to Grey Simulation.Correlation coefficient r value is compound and domestication strain positive in positive representation module
Close, r values are that negative indication is negatively correlated；The absolute value of r shows that more than 0.5 the compound in module is obvious with the dependency of domestication strain；
Statistical analysis p value represents that less than 0.05 dependency is substantially thinkable.In figure red font represent filter out two with
The module of domestication strain height correlation.
Fig. 9 is the core metabolite and its associated metabolic network topology structure figure that WGCNA analyzes blue module.Section in figure
Point represents the metabolite with obvious relation between persistence, and gray line represents the interrelational form between each metabolite, the Connected degree of red font
Core metabolic markers are represented more than 5.
Specific embodiment
Below by embodiment and combine accompanying drawing the present invention is described in further detail.Used in following embodiments
Experimental technique if no special instructions, is conventional method.Material used, reagent etc. in following embodiments, if no special instructions,
Commercially obtain.
Embodiment is in order that those skilled in the art better understood when the present invention, but the present invention is not appointed
What limits.
Embodiment 1
The orientation of high concentration glucose tolerance is carried out to Crypthecodinium cohnii (Crypthecodinium cohnii) ATCC 30556
Domestication, comprises the steps：
1. Crypthecodinium cohnii is cultivated in the culture medium of sea salt 25g/L, control containing glucose 9g/L, yeast extract 2g/L
Initial inoculation OD490For 0.08, measure once per 12h, 3 Duplicate Samples are done per group, with Shimadzu UV-1750 spectrophotometric determination
Wavelength is the light absorption value under 490nm, after culture 36h, OD490Can reach or more than 0.8, draw block diagram, then passed on；
Simply the concentration of glucose in Secondary Culture base gradually increases as shown in Figure 1；Tianjin Ounuo Instrument Co., Ltd. HNY-
202B shaking table parameters are set to rotating speed 180rpm, 25 DEG C of temperature；
2. essentially identical with step 1, simply the generation time is 10 days, and the concentration of glucose in Secondary Culture base is increased to
22g/L, OD under the conditions of this490There is prolongation in the time that can reach 0.8.OD490After meeting or exceeding 0.8, training is passed in continuous passage
Foster base concentration of glucose maintains 22g/L, treats the OD of new algae solution490When can meet or exceed 0.8 after 36h is cultivated, carried
High glucose concentration is passed on.As shown in figure 1, concentration of glucose is gradually increased with the amplification of 4g/L；
3. essentially identical with step 2, simply the generation time is 200 days, and the concentration of glucose in Secondary Culture base is increased to
36g/L, concentration of glucose amplification are changed to 2g/L (Fig. 1).
4. essentially identical with step 3, simply the generation time is 650 days, and the concentration of glucose in Secondary Culture base is increased to
54g/L, substantially, domestication terminates Crypthecodinium cohnii cell caking phenomenon.
5. can detect that the algae solution that exclusion is passed on has microorganism pollution phenomenon at set intervals：1mL algae solutions are taken, it is appropriate to carry out
Dilution, takes 100 μ L and coats on the solid medium of corresponding concentration of glucose.Seen with Olympus BX43 fluorescence microscopies simultaneously
Examine, amplification is 400 times；The solid medium flat board of coating is positioned in constant incubator, 25 DEG C of temperature, dark condition
Lower culture.
6. Crypthecodinium cohnii tame strain specificity analysises, by Crypthecodinium cohnii set out algae strain and domestication algae strain carry out Heterotrophic culture, cultivate
Base, condition of culture are identical with example 1.3 Duplicate Samples are done per group, after cultivating 72 hours, Nile red dyeing, using BD companies
FACSCalibur flow cytometers determine intracellular fat content, while extracting oils and fatss, determine DHA content；From parallel culture
Middle extraction cell metabolite, tests and analyzes the change of intracellular metabolite using Agilent company 7890A/5975C gas chromatograph-mass spectrometers
Situation.
7. substantially the same with the situation of step 6, simply in culture medium, concentration of glucose is 36g/L.
8. substantially the same with the situation of step 6, simply in culture medium, concentration of glucose is 45g/L.
9. substantially the same with the situation of step 6, simply in culture medium, concentration of glucose is 54g/L.
Metabolism group data obtained by 10.WGCNA analytical procedures 6-9, by metabolite associated with each other according to its dependency
It is divided into different modules, and is represented with different colours, the metabolite for not having relatedness is attributed to into Grey Simulation；According to correlation coefficient
r>0.5, data statisticss confidence level p<0.05 is screening conditions, and red font represents filter out 2 and domestication strain height correlation
Module (Fig. 8).
The 11. metabolism network topological structure for building confidence module, find out core compound.In WGCNA analyses, if a change
Compound is connected with having more than 5 compounds, then it is assumed that this metabolite is core metabolite.
Find finally by compared with Kou Shi Crypthecodinium cohnii starting strains：Domestication strain glucose tolerance significantly improve (Fig. 3-
5), in 36,45 and 54g/L high glucose concentration culture medium, specific growth rate is significantly improved, and has been respectively increased 1.03,2.78
And 3.52 times, mean doubling time has been respectively shortened 23.5%, 31.9% and 6.4%, while glucose consumption rate is carried respectively
It is high 2.67,2.62 and 2.51 times；Domestication strain oil and fat accumulation substantially increases (Fig. 6), total fat content has been respectively increased 6.8%,
14.2% and 16.3%；Domestication strain cell interior complexity increased (Fig. 7)；Domestication strain have 21 metabolite it is notable on
Adjust, and be located at respectively and tame in the positively related blue and green module of strain (table 1), 4 are found in blue module with domestication strain
Glucose-tolerant improves related core compound, respectively glycerol, glutamic acid, malonic acid and succinic acid (Fig. 9), green module
Core compound of the Connected degree more than 5 is not found.
21 metabolite substantially raised in the domestication strain of table 1*
*：Red font represents the metabolite positioned at blue module, and black font represents the metabolite positioned at green module.
Gained high glucose domestication algae strain of the invention is compared with following features with algae strain of setting out：(1) 36,45 and
In 54g/L high glucose concentration culture medium, specific growth rate is significantly improved, and has been respectively increased 1.03,2.78 and 3.52 times, averagely
Doubling time has been respectively shortened 23.5%, 31.9% and 6.4%, while glucose consumption rate has been respectively increased 2.67,2.62
And 2.51 times；After 72h is cultivated at (two) 25 DEG C, oils and fatss are extracted, relative to starting strain, oil and fat accumulation substantially increases for domestication strain, point
6.8%, 14.2% and 16.3% is not increased, meanwhile, dyeed using Nile red and flow cytometry analysis, also further demonstrated that
Oil and fat accumulation increased；(3) by the metabolism group detection method and weighted association based on gas chromatograph-mass spectrometer (GC-MS)
The method of analysis of network (WGCNA), detects and analyzes the changes of contents situation of two kinds of algae strain intracellular metabolite, relative to setting out
Strain, 21 compounds substantially raise, wherein glycerol, glutamic acid, malonic acid and succinic acid be confirmed as core metabolic marker thing and
The raising of domestication strain glucose tolerance has significantly association.
The high glucose domestication strain of the Kou Shi Crypthecodinium cohnii of the invention has in docosahexenoic acid (DHA) of fermenting and producing
There are obvious economic advantages and industrial prospect.
Embodiment 2
Identical with the step of embodiment 1, simply concentration of glucose is gradually increased with the amplification of 4g/L, and the generation time is
200 days, compared with starting strain, glucose tolerance was significantly improved the naturalized strain for obtaining.
Embodiment 3
It is identical with the step of embodiment 1, simply the generation time be 400 days, the naturalized strain for obtaining compared with starting strain,
Glucose tolerance is significantly improved.
Claims (9)
1. the Kou Shi Crypthecodinium cohnii domestication strain of one plant of tolerance high glucose concentration, it is characterised in that：Kou Shi Crypthecodinium cohnii is from from U.S.
(the bacterial strain code ATCC 30556) of state strain collections ATCC is starting strain；
Using initial Kou Shi Crypthecodinium cohnii ATCC 30556 as process object, in the by for gradually augmenting glucose+Carry out in culture medium many
Secondary domestication culture, the process object that the domestication is cultivated every time are algae strain after last domestication culture；Glucose at the end of domestication
Concentration increases to 54g/L；Domestication process experiences 650 days altogether, altogether 130 generation；The Kou Shi Crypthecodinium cohnii of the high tolerance glucose for obtaining
Domestication strain is named as ALE3.
2. the Kou Shi Crypthecodinium cohnii domestication strain of one plant of tolerance high glucose concentration according to claim 1, it is characterised in that will
The initial Kou Shi Crypthecodinium cohnii is incubated at by+Culture medium, can reach exponential phase mid-term, OD to 35-36h490Can reach 0.8；
The by+Culture medium is consisted of：Glucose 9g/L, yeast extract 2g/L, sea salt 25g/L, pH are 6.5.
3. the Kou Shi Crypthecodinium cohnii domestication strain of one plant of tolerance high glucose concentration according to claim 1, it is characterised in that institute
That what is stated has augmented the by of glucose+In culture medium concentration of glucose be in state incremented by successively, yeast extract 2g/L, sea salt 25g/
L；When concentration of glucose is higher than 25g/L, the time that the initial Kou Shi Crypthecodinium cohnii reaches exponential phase mid-term may extend
To 48～72h.
4. the Kou Shi Crypthecodinium cohnii of one plant of tolerance high glucose concentration tames the preparation method of strain, it is characterised in that Kou Shi Crypthecodinium cohnii is selected
It is starting strain with (the bacterial strain code ATCC 30556) from american strain collection ATCC；Detailed process is as follows：
During the domestication, initial Kou Shi Crypthecodinium cohnii is incubated in the conical flask of 250mL, and what 50mL was housed in bottle has augmented Portugal
The by of grape sugar+Culture medium；Control initial inoculation OD490For 0.08, as the OD of Crypthecodinium cohnii algae solution490Meet or exceed 0.8 it is laggard
Row is passed on；
During the domestication, OD after domestication strain growth 35-36h490Can reach or during more than 0.8, improve concentration of glucose；
During the domestication, the concentration of glucose in culture medium is in state incremented by successively；Concentration of glucose is higher than 18g/L
When, the concentration of glucose is gradually increased with the amplification of 4g/L, when concentration of glucose is higher than 36g/L, concentration of glucose amplification is changed to
2g/L。
5. the Kou Shi Crypthecodinium cohnii of one plant of tolerance high glucose concentration according to claim 4 tames the preparation method of strain, its
During being characterised by the domestication, cell sample can be taken at set intervals and with Olympus BX43 fluorescence microscopes
The form of domestication strain, while and combining the method exclusion biological pollution phenomenon such as method detection of painting flat board.
6. the Kou Shi Crypthecodinium cohnii of one plant of tolerance high glucose concentration according to claim 4 tames the preparation method of strain, its
During being characterised by the domestication, domestication strain reached for 130 generations, and caking phenomenon occurs in cell, and domestication terminates；Now glucose is dense
Degree has been improved to 54g/L.
7. the Kou Shi Crypthecodinium cohnii of one plant of tolerance high glucose concentration according to claim 4 tames the preparation method of strain, its
It is characterised by the domestication process altogether after 650 days；The domestication strain obtained by the domestication process is named as ALE3.
8. a kind of Kou Shi Crypthecodinium cohnii of the high glucose tolerance described in claim 1 tames the application of strain, it is characterised in that：It is described
Application of the domestication strain of high tolerance glucose in algae oil of the fermenting and producing rich in DHA.
9. the Kou Shi Crypthecodinium cohnii as described in claim 8 tames the application of strain, it is characterised in that：Fructus Vitis viniferae in the fermentation medium
Sugared concentration can be 1～54g/L.
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| CN109913513A (en) * | 2018-11-29 | 2019-06-21 | 厦门大学 | A method of domestication schizochytrium limacinum Lipid-producing |
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| CN108753855A (en) * | 2018-05-28 | 2018-11-06 | 天津大学 | The method that WGCNA identifies D-ALPHA-Hydroxypropionic acid fermentation process notable module and Hubs metabolins |
| CN109136162A (en) * | 2018-06-07 | 2019-01-04 | 昆明藻能生物科技有限公司 | A kind of method that multifactor coevolution improves hidden dinoflagellate DHA yield |
| CN109913513A (en) * | 2018-11-29 | 2019-06-21 | 厦门大学 | A method of domestication schizochytrium limacinum Lipid-producing |
| JP2021166510A (en) * | 2020-04-10 | 2021-10-21 | スバル株式会社 | Euglena culture method |
| JP7286183B2 (en) | 2020-04-10 | 2023-06-05 | 株式会社ユーグリード | Euglena culture method |
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