CN105316246B - Beta carotene high-yield strains and its application - Google Patents
Beta carotene high-yield strains and its application Download PDFInfo
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Abstract
The invention discloses a kind of Yarrowia lipolytica (Yarrowia lipolytica), the deposit number of the strain is CGMCC No.8940.The Yarrowia lipolytica CGMCC No.8940 can utilize common carbon source and nitrogen source, and it is the strain excellent of high yield beta carotene that after fermented culture, beta carotene yield, which can reach 4.5g/L, have extensive prospects for commercial application.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of beta carotene high-yield strains and its application.
Background technique
So far, more than 600 kinds of carotenoid are had reported, comes derived bacterium, algae, yeast and plant and has concurrently, β-Hu
Radish element is one of them.Precursor one of of the beta carotene as vitamin A, is FAO (Food and Agriculture Organization of the United Nation) and world health group
Knit the A class wholefood hardening agent of food additives joint specialist committee identification, at the same by 52 countries in the world and
Area approval uses, and is recorded by U.S. Food Chemistry product additive model (FCC).In addition, beta carotene is as drug
By United States Pharmacopeia (USP) 1990 editions, 1995 editions, European Pharmacopoeia 1997 editions, British Pharmacopoeia (BP) 1998 editions is formally recorded, and China defends
Life portion issuing standard has also recorded this product, first list of OTC medicines that China National Drug Surveillance Authority announces also records
This product (analysis of S.J.Chem. beta carotene domestic market, finely and specialty chemicals, 2003,8:8-9).
Currently, the demand of whole world beta carotene is increased every year with 7%~9% speed, the demand in the U.S. is close
Year is increased with 10%~15% speed, annual requirement 1000t of global beta carotene or so.China β-Hu in 2000
40% for exporting in radish element yield, domestic consumption 60%, wherein 70% is used for food industry, 20% is used for health care product,
10% be used for pharmaceutical production, wherein about 75% be using fermentation method production (S.J.Chem. beta carotene domestic market analyze,
Finely and specialty chemicals, 2003,8:8-9).
The molecular formula of beta carotene is C40H56, molecular weight 536.88 has alltrans, 9- cis- and 15- cis- 3
Kind structure.The method of production beta carotene mainly has Nature inorganic bone method, chemical synthesis and microbe fermentation method at present.Sieve
Family name company produces beta carotene using chemical synthesis at first in 1954, and BASF AG also begins to use chemical synthesis within 1972
Method production, natural beta-carotin is trans- and cis- mix-configuration, and chemically synthesized is then alltrans configuration, and with
The time passage and research deeply, it has been found that using chemical method synthesis beta carotene due to that cannot be inhaled completely by human body
It receives, and a degree of toxic side effect is generated to human body, irreversible lesion can also be generated to human body by taking for a long time, therefore be changed
The beta carotene for learning synthesis has been prohibited to be used as food additives in western countries.In addition, beta carotene can be from Hu trailing plants
It is extracted in the natural plants such as fore-telling, sea-buckthorn, tomato, potato, fructus lycii and corn, but content beta-carotene is very in natural goods
Low, extraction process is complicated, and at high cost, product purity is low, and largely plantation needs to expend many land resource, crop cycle
It is longer.Comparatively, the shortcomings that biological fermentation process production beta carotene then overcomes both the above method, has production technology
Simply, the advantages that period is short, at low cost, good product quality.
The study found that such as photosynthetic bacteria (Rhodobacter sphaeroides), yeast such as phaffiafhodozyma in bacterium
(Xanthophyllomyces dendrorhous), rhodotorula mucilaginosa (Rhodotorula mucilaginosa), rhodotorula
(Rhodotorula glutinis), rhodothece rubra (Rhodotorula rubra) etc., mould such as three spore cloth Laplaces are mould
(Blakeslea trispro), phycomyces blakesleeanus (Phycomyces blakesleeanus) and volume branch Mucor (Mucor
) etc. circinelloides can synthesize and with algae such as Du Shi algae (Dunaliella) etc. in intracellular accumulation carotenoid
(rushing the present Research of equal different microorganisms production carotenoid, 2011,32 (2): 179-183), in total carotinoid
Containing the beta carotene of different proportion.
The fermentation production technology of Du Shi algae (Dunaliella) and three spore cloth Laplaces mould (Blakeslea trispro) compares
Maturation comes into industrialized production, wherein three spore cloth Laplaces mould (Blakeslea trispro) cultivate 5 days β-carrots
Plain yield may be up to 800~900mg/L, and Du Shi algae (Dunaliella) ferments content of the beta carotene in dry mycelium can be high
Up to 10%.But three spore cloth Laplaces are mould that character easily fails due to mode of reproduction of mould etc., algal culture condition
It is required that harsh, these deficiencies limit its development, so seeking strain excellent becomes particularly important.
Summary of the invention
In order to overcome the shortcomings in the prior art, the present invention is intended to provide a kind of high-yield strains of beta carotene.Firstly,
It, will be from the beta carotene route of synthesis of Mucor circinelloides ATCC90680 by gene package technique
2 genes carB, carRP, and solution is introduced from two the genes GGS1 and tHmg1 of Yarrowia lipolytica NRRL Y-1095
Rouge Ye Shi yeast ATCC MYA2613 is configured to produce the engineering bacteria of beta carotene.Then engineering fungi degradation building completed
Rouge Ye Shi yeast CIBTS-1177 bacterial strain carries out further mutagenic and breeding, is finally obtained the β-Hu Luo of high yield and stable yield
Bu Su producing strains Yarrowia lipolytica (different name: solution rouge Asia sieve yeast) (Yarrowia lipolytica) HCCB08563, in
On March 20th, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), culture presevation
Number be CGMCC No.8940.
Therefore, the first purpose of this invention is to provide a kind of Yarrowia lipolytica (Yarrowia lipolytica),
The deposit number of the strain is CGMCC No.8940.
Second object of the present invention is to provide the Yarrowia lipolytica CGMCC No.8940 for producing β-Hu
The application of radish element.
Third object of the present invention is to provide a kind of production method of beta carotene, passes through the fermentation solution rouge
Ye Shi yeast CGMCC No.8940 obtains beta carotene, and the condition of culture of the fermentation is as follows:
Slant medium are as follows: glucose 1.5~4%, peptone 0.5~3%, yeast extract powder 0.5~2%, agar
2%；
Seed culture medium are as follows: glucose 0.5~4%, peptone 0.2~3%, yeast extract powder 0.5~3.5%；
Fermentation medium are as follows: glucose 1~7%, peptone 0.5~3%, yeast extract powder 1~3.5%, biphosphate
Potassium 0.01~0.5%, dipotassium hydrogen phosphate 0.01~0.5%.
The condition of culture of preferred embodiment in accordance with the present invention, the fermentation is as follows:
Slant medium: glucose 2%；Tryptone 2%；Yeast extract powder 1%；Agar 2%；
Seed culture medium: glucose 2%；Peptone 2%；Yeast extract powder 1%；
Fermentation medium: glucose 3%；Peptone 2%；Yeast extract powder 1%；Potassium dihydrogen phosphate 0.08%；Phosphoric acid hydrogen
Dipotassium 0.08%.
Beneficial effects of the present invention: the present invention is using Yarrowia lipolytica ATCC MYA2613 as starting strain, through gene work
Journey building and mutagenesis screening, obtain one plant of Yarrowia lipolytica CGMCC No.8940, can utilize common carbon source and nitrogen source,
After fermented culture, beta carotene yield can reach 4.5g/L, be the strain excellent of high yield beta carotene.
The present invention provides a kind of new Yarrowia lipolyticas, can be used for high yield beta carotene, have extensive industry
Application prospect.
Detailed description of the invention
Fig. 1 is the integration assembling sequence schematic diagram of each netic module on chromosome.
Fig. 2 is the PCR qualification result that beta carotene produces bacterium.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.It should be understood that following embodiment is merely to illustrate this
The range of invention and is not intended to limit the present invention.
Yarrowia lipolytica (different name: solution rouge Asia sieve yeast) (Yarrowia lipolytica) HCCB08563 of the invention
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation are preserved on March 20th, 2014
Address is No. 3 Institute of Microorganism, Academia Sinica, institute of Chaoyang District Beijing North Star West Road 1, deposit number CGMCC No.8940.
Embodiment 1, engineering bacteria building
1.1, experimental program and design of primers
(ATCC, USA, bacterial strain information: MATA ura3-302leu2- are purchased from Yarrowia lipolytica ATCC MYA2613
Expression is from Mucor circinelloides in 270xpr2-322axp2-deltaNU49XPR2::SUC2)
Two gene carB (GenBank:AJ238028.1), carRP of the beta carotene route of synthesis of ATCC90680
(GenBank:AJ250827.1), and from Yarrowia lipolytica NRRL Y-1095 two genes GGS1 and tHmg1, and
URA3 gene is expressed as selection markers simultaneously, selects the site yeast chromosomal rDNA as integration site, each netic module exists
Integration assembling sequence on chromosome is as shown in Figure 1.Each promoter, terminator and gene are numbered respectively, as shown in table 1.
Table 1, each promoter, terminator and gene number
| Promoter number | Promoter title | Gene number | Gene Name | Terminator number | Terminator title |
| A | TEF1p | 1 | GGS1 | a | xpr2t |
| B | EXP1p | 2 | carB | b | mig1t |
| C | GPDp | 3 | carRP | c | lip2t |
| 4 | tHmg1 |
Construct following gene expression module: rDNAu-TEF1p-GGS1-xpr2t, EXP1p-carB-mig1t, GPDp-
CarRP-lip2t, EXP1p-tHmg1-xpr2t-URA3-rDNAd, i.e. A1aB2bC3cB4a combination.
For design primer sequence for expanding above each small fragment, primer sequence is as shown in table 2.
Table 2, primer sequence
1.2, the building of plasmid
It (is purchased from carrier pUC57 is cloned into from carB, carRP gene order of Mucor circinelloides
Nanjing Genscript Biotechnology Co., Ltd.), plasmid pUC57-carB is obtained, pUC57-carRP is transformed into E.coli bacterial strain DH5
α saves plasmid.
1.3, the extraction of template DNA
The extracting method of Yarrowia lipolytica chromosomal DNA is as follows: the full single colonie of picking is connected to 3ml YPD Liquid Culture
Base (glucose 2%, tryptone 2%, yeast extract powder 1%) test tube 30 DEG C, is incubated overnight about 18h or so, collects bacterium solution,
Extract chromosomal DNA (operating with reference to Shanghai Lai Feng Biotechnology Co., Ltd Yeast genome extraction agent box specification).
The extracting method of plasmid is as follows: DH5 α bacterium of the inoculation containing corresponding plasmid is in 3ml LB liquid medium, wherein containing
There is the final concentration of 100 μ g/ml of ampicillin, 37 DEG C, is incubated overnight about 15h or so, extracts plasmid pUC57-carB, pUC57-
CarRP (is operated) referring to Axygen plasmid extraction kit specification.
1.4, the building of the PCR amplification of small DNA fragmentation and gene expression module
The PCR amplification that each small fragment is carried out using primer described in table 2 is coagulated PCR product using the agarose of 1% concentration
Gel electrophoresis separation, rubber tapping, purification and recovery segment, and the molten concentration of each segment weight is made to keep quite, saving backup.
Each small DNA fragmentation obtained using PCR constructs each gene expression module rDNAu- as template, using Overlap PCR
TEF1p-GGS1-xpr2t、EXP1p-carB-mig1t、GPDp-carRP-lip2t、EXP1p-tHmg1-xpr2t-URA3-
rDNAd.Overlap PCR product is used to the agarose gel electrophoresis of 1% concentration, rubber tapping, purification and recovery segment saves backup
With conversion.
2.4, it converts
Using lithium acetate transformation method by gene expression module rDNAu-TEF1p-GGS1-xpr2t, EXP1p-carB-
Mig1t, GPDp-carRP-lip2t, EXP1p-tHmg1-xpr2t-URA3-rDNAd cotransformation Yarrowia lipolytica ATCC
MYA2613。
Operating method is as follows: picking single colonie connects YPD fluid nutrient medium, and in 30 DEG C, 200rpm/min overnight incubation is transferred
Fresh 25ml YPD fluid nutrient medium, 30 DEG C, 200rpm/min shaken cultivation, to bacterium solution OD600When reaching 0.8~1.0,4 DEG C,
Thalline were collected by centrifugation by 4000rpm, 5min, distinguishes washing thalline with ice bath sterile water and 100mM LiAc, then uses 100mM
Thallus is resuspended to cell concentration about 2 × 10 in LiAc8Competent cell is made in a/mL.Take 50 μ l competent cells, 4 DEG C,
Thalline were collected by centrifugation by 13000rpm, 30s, sequentially adds the PEG6000 of 240 μ l 50%, the LiAc of 36 μ l 1M, the 50 single-stranded fishes of μ l
Smart DNA, the 24 sterile ddH of μ l2The DNA of O and 10 μ l or so is vortexed and mixes, then 30 DEG C of water-baths 30min, 42 DEG C of water-bath 25min,
Thalline were collected by centrifugation by 13000rpm, 30s, and thallus is resuspended with 1ml YPD fluid nutrient medium, multiple in 30 DEG C of shaken cultivation recovery 6h
Thallus is resuspended in 13000rpm after Soviet Union, 30s centrifugation, 100 μ l sterile waters, is coated with SC-ura (the basic nitrogen source of glucose 20g/l, YNB
Each 50mg/l of 1.7g/l, Lys and Leu) solid medium, it is cultivated 3~4 days in 30 DEG C.
2.5, it identifies
The deeper bacterium colony of color in transformant is selected, is inoculated in 3ml YPD fluid nutrient medium test tube, 30 DEG C, 200rpm/
Min shaken cultivation about 18h takes bacterium solution, extracts genome, uses C1-F/R, C2-F/R, C3-F/R, the C4-F/R marked in table 2
Primer pair PCR verifies (C1:2457bp, C2:3649bp, C3:3477bp, C4:5487bp), by PCR product electrophoretic analysis, as a result
As shown in Fig. 2, obtaining one plant of PCR verifies the bright genotype positive colony of four bands, engineering bacteria is named as CIBTS-
1177。
Embodiment 2, induction mutation of bacterium screening and optimizing
2.1, strain culturing
It will solution Zhi Shi yeast (different name: solution rouge Asia sieve yeast) (Yarrowia lipolytica) CIBTS-1177 freezing
Pipe strain is separated through YPD solid medium (glucose 2%, tryptone 2%, yeast extract powder 1%, agar powder 2%) plate
It is inoculated in YPD culture medium slant after purification, 28 DEG C are cultivated 3~4 days.
2.2, natural separation
It takes the fresh inclined-plane of bacterial strain that appropriate amounts of sterilized water is added, slant pore is gently scraped with inoculation shovel, is poured into equipped with nothing
In the triangular flask of bacterium bead, shake well 30min is filtered with the sterile funnel for being plugged with absorbent cotton, obtains monospore suspension.It will
It is coated on after monospore suspension gradient dilution in YPD solid medium tablets, culture obtains single bacterium colony.
2.3, mutagenic treatment
(1) ultraviolet mutagenesis is handled
The continuous ultraviolet mutagenesis processing method of bacteria suspension: by monospore bacterial suspension inoculation to fluid nutrient medium containing 10mlYPD and
In the 50ml triangular flask of magnetic agitation rotor, it is placed under the ultraviolet lamp that power is 30W, wavelength 253.7nm, irradiation distance 33cm
Mutagenesis liquid is applied in YPD solid medium tablets by mutagenic treatment 40s.
(2) EMS (ethylmethane sulfonate) mutagenic treatment
Bacteria suspension 10000r/min is centrifuged 10min, abandons supernatant, thallus is washed with the phosphate fliud flushing of 0.05M pH7.0
Afterwards, it is suspended in the iodine flask of the phosphate buffer containing 10ml, 200 μ l EMS is added and are lured in oscillation treatment 20min on shaking table
10ml concentration is added after change processing and terminates reaction into 5% hypo solution.5ml treatment fluid is finally drawn in centrifuge tube
In, YPD solid medium is coated on twice, after gradient dilution with brine after being centrifuged 10min at 10000r/min
On plate.
2.4, shaking flask is screened
The single colonie handled through breeding is inoculated with YPD culture medium slant, is carried out after being cultivated 3~4 days in 28 DEG C
Shaking flask second order fermentation, and with HPLC detection beta carotene yield.
Using HPLC detection, (method, as foundation, finally obtains one by repeated screening referring to WO2009/126890A2) result
Strain Yarrowia lipolytica (Yarrowia lipolytica) superior strain HCCB08563, was preserved on March 20th, 2014
State's Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), culture presevation number are CGMCC No.8940.
2.5, fermentation medium is investigated
According to beta carotene superior strain Yarrowia lipolytica (Yarrowia lipolytica) CGMCC of use
The characteristic of No.8940 investigates beta carotene fermentation medium, fermentation condition.Confirm that the superior strain is available
Seed and fermentation medium type include carbon source: glucose, sucrose, soluble starch, starch, glycerol, soya-bean oil, rapeseed oil
Deng；Nitrogen source: peptone, yeast powder, soybean powder, ammonium sulfate etc.；And various phosphate, various metallic salts include magnesium salts, sodium
Salt, calcium salt, molysite, zinc salt, cobalt salt etc..
Better suited slant medium are as follows: glucose 1.5~4%, peptone 0.5~3%, yeast extract powder 0.5~
2%, agar 2%.
Appropriate seed culture medium are as follows: glucose 0.5~4%, peptone 0.2~3%, yeast extract powder 0.5~
3.5%.
Appropriate fermentation medium are as follows: glucose 1~7%, peptone 0.5~3%, yeast extract powder 1~3.5%,
Potassium dihydrogen phosphate 0.01~0.5%, dipotassium hydrogen phosphate 0.01~0.5%.
Embodiment 3, fermentation
3.1, shake flask fermentation
Yarrowia lipolytica (Yarrowia lipolytica) CGMCC No.8940 is inoculated in containing following inclined-plane culture
On the inclined-plane of base: glucose 2%；Tryptone 2%；Yeast extract powder 1%；Agar 2% after inclined plane inoculating, cultivates 3 in 28 DEG C
It.
Using dig block method by inclined plane inoculating in the 250ml triangle shake bottle of the seed culture fluid containing 30ml.Seed culture medium: Portugal
Grape sugar 2%；Peptone 2%；Yeast extract powder 1%；For 24 hours in 28 DEG C of cultures, shaking speed 220r/m.
By cultured seed liquor by 5~8% inoculum concentration transferred speciess in the 250ml triangle shake bottle of the fermentation culture containing 30ml
In, in 28 DEG C, shaking speed 220r/m, cultivate 5 days.Fermentation medium: glucose 4%；Peptone 2%；Yeast extract powder
1%.Terminal fermentation yield (grams of the every liter of fermentation liquid containing beta carotene) about 2.1g/L.4 batches of experimental results are as indicated at 3.
Table 3, shake flask fermentation result
3.2,7.5L glass jar ferments
Strain is CGMCC No.8940, inclined-plane and the seed culture side Yarrowia lipolytica (Yarrowia lipolytica)
Formula is the same as embodiment 3.1.Cultivation temperature: 28 DEG C；Shaking speed 220r/m；Incubation time: for 24 hours.
Seed is inoculated in the 7.5L glass fermentation tank of the fermentation medium containing 3.5L by transferred species amount 5~8%.Fermented and cultured
Base: glucose 3%；Peptone 2%；Yeast extract powder 1%；Potassium dihydrogen phosphate 0.08%；Dipotassium hydrogen phosphate 0.08%.
The fermentation of 7.5L glass jar: 28 DEG C of fermentation temperature, ventilatory capacity 1:1 (vol:vol), speed of agitator 550r/m fermented
50% glucose, rate 3ml/h.l are added in journey, fermentation period is 5 days, average product 4.5g/L.4 batches of fermentation results are such as
Shown in table 4.
Table 4, glass jar fermentation results
Claims (5)
1. a kind of Yarrowia lipolytica (Yarrowia lipolytica), which is characterized in that the deposit number of the strain is CGMCC
No.8940。
2. the application that Yarrowia lipolytica CGMCC No.8940 as described in claim 1 is used to produce beta carotene.
3. a kind of production method of beta carotene, which is characterized in that pass through the solution rouge Ye Shi ferment as described in claim 1 that ferments
Female CGMCC No.8940 obtains beta carotene.
4. production method as claimed in claim 3, which is characterized in that the condition of culture of the fermentation is as follows:
Slant medium are as follows: glucose 1.5~4%, peptone 0.5~3%, yeast extract powder 0.5~2%, agar 2%；
Seed culture medium are as follows: glucose 0.5~4%, peptone 0.2~3%, yeast extract powder 0.5~3.5%；
Fermentation medium are as follows: glucose 1~7%, peptone 0.5~3%, yeast extract powder 1~3.5%, potassium dihydrogen phosphate
0.01~0.5%, dipotassium hydrogen phosphate 0.01~0.5%.
5. production method as claimed in claim 4, which is characterized in that the condition of culture of the fermentation is as follows:
Slant medium: glucose 2%；Tryptone 2%；Yeast extract powder 1%；Agar 2%；
Seed culture medium: glucose 2%；Peptone 2%；Yeast extract powder 1%；
Fermentation medium: glucose 3%；Peptone 2%；Yeast extract powder 1%；Potassium dihydrogen phosphate 0.08%；Dipotassium hydrogen phosphate
0.08%.
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| CN106987550B (en) * | 2017-05-18 | 2020-10-13 | 陕西师范大学 | Recombinant bacterium for producing beta-carotene and construction method and application thereof |
| CN108949599A (en) * | 2018-06-29 | 2018-12-07 | 华南理工大学 | A kind of production alpha, beta-lonone genetic engineering bacterium and its construction method and application |
| CN113151340B (en) * | 2020-11-25 | 2023-03-24 | 广州智特奇生物科技股份有限公司 | Genetic engineering bacterium for increasing yield of beta-carotene and application thereof |
| CN112831427B (en) * | 2021-01-20 | 2022-08-23 | 山东大学 | Yarrowia lipolytica for high yield of beta-carotene and application thereof |
| CN115261244B (en) * | 2022-06-30 | 2024-02-23 | 山东微研生物科技有限公司 | Culture medium combination and fermentation process for high-yield canthaxanthin by utilizing yarrowia lipolytica |
| CN118064533A (en) * | 2023-07-04 | 2024-05-24 | 广东筑美生物医疗科技有限公司 | Method for producing beta-carotene by enhancing yarrowia lipolytica |
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