CN105316246A - Beta-carotene high-yield strain and use thereof - Google Patents

Abstract

The invention discloses Yarrowia lipolytica. The Yarrowia lipolytica has an accession number of CGMCC No. 8940. The Yarrowia lipolytica with the accession number of CGMCC No. 8940 can utilize a common carbon source and a nitrogen source. Through fermentation culture of the Yarrowia lipolytica, a beta-carotene yield is 4.5g/L. The Yarrowia lipolytica is a high beta-carotene yield good strain and has a wide industrial application prospect.

Description

β-carotene high-yield strains and application thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of β-carotene high-yield strains and application thereof.

Background technology

Up to now, reported 600 multiple types carotene, come derived bacterium, algae, yeast and plant and have concurrently, β-carotene is one of them.β-carotene is as one of the precursor of vitamin A, it is the category-A wholefood reinforcer that Food and Argriculture OrganizationFAO and the foodstuff additive joint specialist council of the World Health Organization are assert, simultaneously by 52 countries and regions approval uses in the world, and recorded by U.S. food chemical additives model (FCC).In addition, as medicine, β-carotene by American Pharmacopeia (USP) 1990 editions, 1995 editions, European Pharmacopoeia 1997 editions, British Pharmacopoeia (BP) 1998 editions formally records, and ministry of Health of China issuing standard also records this product, first list of OTC medicines that China National Drug Surveillance Authority announces also records this product, and (S.J.Chem. β-carotene domestic market is analyzed, meticulous and specialty chemicals, 2003,8:8-9).

At present, the demand of whole world β-carotene every year with 7% ~ 9% speed increment, the demand of the U.S. in recent years with 10% ~ 15% speed increment, about the annual requirement 1000t of global β-carotene.In China's β-carotene output in 2000,40% for outlet, home consumption 60%, wherein 70% for foodstuffs industry, 20% for healthcare products, 10% for pharmaceutical production, and wherein about 75% is utilize fermentative Production (analysis of S.J.Chem. β-carotene domestic market, meticulous and specialty chemicals, 2003,8:8-9).

The molecular formula of β-carotene is C ₄₀h ₅₆, molecular weight is 536.88, and it has alltrans, 9-cis and 15-cis 3 kinds of structures.The method of current production β-carotene mainly contains Nature inorganic bone method, chemical synthesis and microbe fermentation method.Roche Holding Ag uses chemical synthesis to produce β-carotene for 1954 at first, within 1972, BASF AG also brings into use chemical synthesis to produce, natural beta-carotin is trans and mix-configuration that is cis, chemosynthesis be then alltrans configuration, and As time goes on go deep into research, it is found that and adopt the β-carotene of chemical method synthesis owing to not being absorbed by the body completely, and to human body generation toxic side effect to a certain degree, long-term taking also can produce irreversible pathology to human body, therefore the β-carotene of chemosynthesis has been prohibited to be used as foodstuff additive in western countries.In addition, β-carotene can extract from the natural phant such as Radix Dauci Sativae, sea-buckthorn, tomato, potato, matrimony vine and corn, but content beta-carotene is very low in natural goods, extraction process is complicated, cost is high, product purity is low, and a large amount of plantation needs to expend a lot of land resources, and crop cycle is longer.Comparatively speaking, biological fermentation process produces the shortcoming that β-carotene then overcomes above two kinds of methods, has the advantages such as production technique is simple, the cycle is short, cost is low, good product quality.

Research finds, as photosynthetic bacterium (Rhodobactersphaeroides) etc. in bacterium, yeast is as phaffiafhodozyma (Xanthophyllomycesdendrorhous), rhodotorula mucilaginosa (Rhodotorulamucilaginosa), rhodotorula (Rhodotorulaglutinis), rhodothece rubra (Rhodotorularubra) etc., mould is as three spore cloth Laplaces mould (Blakesleatrispro), phycomyces blakesleeanus (Phycomycesblakesleeanus) and volume branch Mucor (Mucorcircinelloides) etc. and algae as Dunaliella salina (Dunaliella) etc. can synthesize and intracellular accumulation carotenoid (Zhang Chuan etc. different microorganisms produces the present Research of carotenoid, 2011, 32 (2): 179-183), β-carotene all containing different ratios in total carotinoid.

The fermentation production technology comparative maturity of Dunaliella salina (Dunaliella) and three spore cloth Laplaces mould (Blakesleatrispro), all enter suitability for industrialized production, wherein three spore cloth Laplaces mould (Blakesleatrispro) cultivate 5 days β-carotene output can up to 800 ~ 900mg/L, Dunaliella salina (Dunaliella) ferments the content of β-carotene in dry mycelium can up to 10%.But the reasons such as the mould mode of reproduction due to mould of three spore cloth Laplaces, its proterties easily fails, and algal culture conditional request is harsh, and these deficiencies limit its development, so seek strain excellent to become particularly important.

Summary of the invention

In order to overcome deficiency of the prior art, the present invention aims to provide a kind of high-yield strains of β-carotene.First, by genome packing technique, 2 genes carB, carRP of the β-carotene route of synthesis of MucorcircinelloidesATCC90680 will be come from, and two gene GGS1 and tHmg1 coming from Yarrowia lipolytica NRRLY-1095 introduce Yarrowia lipolytica ATCCMYA2613, are configured to the engineering bacteria producing β-carotene.Then further mutagenic and breeding is carried out to the engineering bacteria Yarrowia lipolytica CIBTS-1177 bacterial strain built, finally obtain high yield and the β-carotene producing strains Yarrowia lipolytica of stable yield (different name: separate the sub-sieve yeast of fat) (Yarrowialipolytica) HCCB08563, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on March 20th, 2014, culture presevation number is CGMCCNo.8940.

Therefore, first object of the present invention is to provide a kind of Yarrowia lipolytica (Yarrowialipolytica), and the preserving number of this bacterial classification is CGMCCNo.8940.

Second object of the present invention is to provide described Yarrowia lipolytica CGMCCNo.8940 for the production of the application of β-carotene.

3rd object of the present invention is the production method providing a kind of β-carotene, and obtain β-carotene by the Yarrowia lipolytica CGMCCNo.8940 fermenting described, the culture condition of described fermentation is as follows:

Slant medium is: glucose 1.5 ~ 4%, peptone 0.5 ~ 3%, yeast extract powder 0.5 ~ 2%, agar 2%;

Seed culture medium is: glucose 0.5 ~ 4%, peptone 0.2 ~ 3%, yeast extract powder 0.5 ~ 3.5%;

Fermention medium is: glucose 1 ~ 7%, peptone 0.5 ~ 3%, yeast extract powder 1 ~ 3.5%, potassium primary phosphate 0.01 ~ 0.5%, dipotassium hydrogen phosphate 0.01 ~ 0.5%.

According to a preferred embodiment of the invention, the culture condition of described fermentation is as follows:

Slant medium: glucose 2%; Tryptones 2%; Yeast extract powder 1%; Agar 2%;

Seed culture medium: glucose 2%; Peptone 2%; Yeast extract powder 1%;

Fermention medium: glucose 3%; Peptone 2%; Yeast extract powder 1%; Potassium primary phosphate 0.08%; Dipotassium hydrogen phosphate 0.08%.

Beneficial effect of the present invention: the present invention with Yarrowia lipolytica ATCCMYA2613 for starting strain, build and mutagenesis screening through genetically engineered, obtain a strain Yarrowia lipolytica CGMCCNo.8940, it can utilize common Carbon and nitrogen sources, after fermentation culture, β-carotene output can reach 4.5g/L, is the strain excellent of high yield β-carotene.

The invention provides a kind of new Yarrowia lipolytica, can be used for high yield β-carotene, there is prospects for commercial application widely.

Accompanying drawing explanation

Fig. 1 is each netic module integration assembling sequence schematic diagram on chromosome.

Fig. 2 is the PCR qualification result that β-carotene produces bacterium.

Embodiment

Below in conjunction with specific embodiment, the present invention will be further described.Should be understood that following examples only for illustration of the present invention but not for limiting the scope of the invention.

Yarrowia lipolytica of the present invention (different name: separate the sub-sieve yeast of fat) (Yarrowialipolytica) HCCB08563 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on March 20th, 2014, preservation address is No. 1, Chaoyang District Beijing North Star West Road No. 3 Institute of Microorganism, Academia Sinica of institute, preserving number CGMCCNo.8940.

embodiment 1, engineering bacteria structure

1.1, experimental program and design of primers

At Yarrowia lipolytica ATCCMYA2613 (purchased from ATCC, USA, bacterial strain information: MATAura3-302leu2-270xpr2-322axp2-deltaNU49XPR2::SUC2) in express and come from two gene carB (GenBank:AJ238028.1) of the β-carotene route of synthesis of MucorcircinelloidesATCC90680, carRP (GenBank:AJ250827.1), and come from two gene GGS1 and tHmg1 of Yarrowia lipolytica NRRLY-1095, and express URA3 gene as selection markers simultaneously, select yeast chromosomal rDNA site as integration site, each netic module integration assembling sequence on chromosome as shown in Figure 1.Each promotor, terminator and gene are numbered respectively, as shown in table 1.

Table 1, each promotor, terminator and gene are numbered

Promotor is numbered	Promotor title	Gene is numbered	Gene Name	Terminator is numbered	Terminator title
						A	TEF1p	1	GGS1	a	xpr2t
B	EXP1p	2	carB	b	mig1t
						C	GPDp	3	carRP	c	lip2t
		4	tHmg1

Build following genetic expression module: rDNAu-TEF1p-GGS1-xpr2t, EXP1p-carB-mig1t, GPDp-carRP-lip2t, EXP1p-tHmg1-xpr2t-URA3-rDNAd, i.e. A1aB2bC3cB4a combination.

Design primer sequence is for the above each small segment that increases, and primer sequence is as shown in table 2.

Table 2, primer sequence

1.2, the structure of plasmid

CarB, carRP gene order deriving from Mucorcircinelloides is cloned into carrier pUC57 (purchased from Nanjing Genscript Biotechnology Co., Ltd.), obtain plasmid pUC57-carB, pUC57-carRP is transformed into E.coli bacterial strain DH5 α, preserves plasmid.

1.3, the extraction of template DNA

The extracting method of Yarrowia lipolytica chromosomal DNA is as follows: the full single bacterium colony of picking is connected to 3mlYPD liquid nutrient medium (glucose 2%, Tryptones 2%, yeast extract powder 1%) test tube, 30 DEG C, incubated overnight is about about 18h, collect bacterium liquid, extract chromosomal DNA (with reference to the operation of Lai Feng bio tech ltd, Shanghai Yeast genome extraction agent box specification sheets).

The extracting method of plasmid is as follows: the DH5 α bacterium of inoculation containing corresponding plasmid is in 3mlLB liquid nutrient medium, wherein containing penbritin final concentration is 100 μ g/ml, 37 DEG C, incubated overnight is about about 15h, extracting plasmid pUC57-carB, pUC57-carRP (with reference to the operation of Axygen plasmid extraction test kit specification sheets).

1.4, the pcr amplification of little DNA fragmentation and the structure of genetic expression module

Utilize the primer described in table 2 to carry out the pcr amplification of each small segment, PCR primer used the agarose gel electrophoresis of 1% concentration to be separated, rubber tapping, purifying reclaims fragment, and makes the heavy molten concentration of each fragment keep quite, saving backup.

The each little DNA fragmentation obtained with PCR, for template, adopts OverlapPCR to build each genetic expression module rDNAu-TEF1p-GGS1-xpr2t, EXP1p-carB-mig1t, GPDp-carRP-lip2t, EXP1p-tHmg1-xpr2t-URA3-rDNAd.OverlapPCR product is used the agarose gel electrophoresis of 1% concentration, rubber tapping, purifying reclaims fragment, saves backup and conversion.

2.4, transform

Adopt lithium acetate transformation method by genetic expression module rDNAu-TEF1p-GGS1-xpr2t, EXP1p-carB-mig1t, GPDp-carRP-lip2t, EXP1p-tHmg1-xpr2t-URA3-rDNAd cotransformation Yarrowia lipolytica ATCCMYA2613.

Working method is as follows: picking list bacterium colony connects YPD liquid nutrient medium, and in 30 DEG C, 200rpm/min overnight incubation, fresh 25mlYPD liquid nutrient medium of transferring, 30 DEG C, 200rpm/min shaking culture, treats bacterium liquid OD ₆₀₀when reaching 0.8 ~ 1.0,4 DEG C, 4000rpm, 5min collected by centrifugation thalline, washs thalline respectively with ice bath sterilized water and 100mMLiAc, then with the resuspended thalline of 100mMLiAc to cell concn about 2 × 10 ⁸individual/mL, obtained competent cell.Get 50 μ l competent cells, 4 DEG C, 13000rpm, 30s collected by centrifugation thalline, add the PEG6000 of 240 μ l50%, the LiAc of 36 μ l1M, 50 μ l strand milt DNA, the aseptic ddH of 24 μ l successively ₂the DNA of O and 10 μ about l, vortex mixes, then 30 DEG C of water-bath 30min, 42 DEG C of water-bath 25min, 13000rpm, 30s collected by centrifugation thalline, with the resuspended thalline of 1mlYPD liquid nutrient medium, in 30 DEG C of shaking culture recovery 6h, 13000rpm after recovery, 30s is centrifugal, the resuspended thalline of 100 μ l sterilized water, coating SC-ura (the basic nitrogenous source 1.7g/l of glucose 20g/l, YNB, the each 50mg/l of Lys and Leu) solid medium, cultivate 3 ~ 4 days in 30 DEG C.

2.5, identify

Select the bacterium colony that color in transformant is darker, be inoculated in 3mlYPD liquid nutrient medium test tube, 30 DEG C, 200rpm/min shaking culture is about 18h, get bacterium liquid, extract genome, C1-F/R, C2-F/R, C3-F/R, C4-F/R primer pair PCR of mark in table 2 is used to verify (C1:2457bp, C2:3649bp, C3:3477bp, C4:5487bp), by PCR primer electrophoretic analysis, result as shown in Figure 2, obtains a strain PCR and verifies the genotype positive colony that four bands all become clear, by engineering bacteria called after CIBTS-1177.

embodiment 2, induction mutation of bacterium screening and optimizing

2.1, strain culturing

Zhi Shi yeast (different name: separate the sub-sieve yeast of fat) (Yarrowialipolytica) CIBTS-1177 freeze pipe bacterial classification will be separated through YPD solid medium (glucose 2%, Tryptones 2%, yeast extract powder 1%, agar powder 2%) be inoculated in YPD culture medium slant after plate isolation purifying, cultivate 3 ~ 4 days for 28 DEG C.

2.2, natural separation

The fresh inclined-plane getting bacterial strain adds appropriate amounts of sterilized water, is scraped gently by slant pore, pour into and be equipped with in the triangular flask of sterile glass beads, shake well 30min with inoculation shovel, filters, obtain monospore suspension with the aseptic funnel being plugged with absorbent cotton.To coat on YPD solid medium flat board after monospore suspension gradient dilution, cultivate and obtain single bacterium colony.

2.3, mutagenic treatment

(1) ultraviolet mutagenesis process

The continuous ultraviolet mutagenesis treatment process of bacteria suspension: by monospore bacterial suspension inoculation in the 50ml triangular flask containing 10mlYPD liquid nutrient medium and magnetic agitation rotor, and to be placed in power be mutagenic treatment 40s under the ultraviolet lamp of 30W, wavelength 253.7nm, irradiation distance 33cm, mutagenesis liquid is applied on YPD solid medium flat board.

(2) EMS (ethylmethane sulfonate) mutagenic treatment

By centrifugal for bacteria suspension 10000r/min 10min, abandon supernatant, after the phosphoric acid salt of thalline 0.05MpH7.0 rushes liquid washing, be suspended in the iodine flask containing 10ml phosphate buffered saline buffer, add 200 μ lEMS, oscillation treatment 20min on shaking table, adds the hypo solution termination reaction that 10ml concentration is 5% after mutagenic treatment.Finally draw 5ml treatment solution in centrifuge tube, under 10000r/min, use brine twice after centrifugal 10min, be coated on after gradient dilution on YPD solid medium flat board.

2.4, shaking flask screening

By the single colony inoculation YPD culture medium slant obtained through seed selection process, carry out shaking flask second order fermentation after cultivating 3 ~ 4 days in 28 DEG C, and detect the output of β-carotene with HPLC.

(method is with reference to WO2009/126890A2) result is detected for foundation with HPLC, strain Yarrowia lipolytica (Yarrowialipolytica) superior strain HCCB08563 is finally obtained through repeated screening, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on March 20th, 2014, culture presevation number is CGMCCNo.8940.

2.5, fermention medium is investigated

According to the characteristic of β-carotene superior strain Yarrowia lipolytica (Yarrowialipolytica) CGMCCNo.8940 adopted, β-carotene fermention medium, fermentation condition are investigated.Confirm that the available seed of this superior strain and fermention medium kind comprise carbon source: glucose, sucrose, Zulkovsky starch, starch, glycerine, soya-bean oil, vegetable seed wet goods; Nitrogenous source: peptone, yeast powder, analysis for soybean powder, ammonium sulfate etc.; And various phosphoric acid salt, various metallic salt comprise magnesium salts, sodium salt, calcium salt, molysite, zinc salt, cobalt salt etc.

Better suited slant medium is: glucose 1.5 ~ 4%, peptone 0.5 ~ 3%, yeast extract powder 0.5 ~ 2%, agar 2%.

More suitable seed culture medium is: glucose 0.5 ~ 4%, peptone 0.2 ~ 3%, yeast extract powder 0.5 ~ 3.5%.

More suitable fermention medium is: glucose 1 ~ 7%, peptone 0.5 ~ 3%, yeast extract powder 1 ~ 3.5%, potassium primary phosphate 0.01 ~ 0.5%, dipotassium hydrogen phosphate 0.01 ~ 0.5%.

embodiment 3, fermentation

3.1, shake flask fermentation

Yarrowia lipolytica (Yarrowialipolytica) CGMCCNo.8940 is inoculated on the inclined-plane containing following slant medium: glucose 2%; Tryptones 2%; Yeast extract powder 1%; Agar 2%, after inclined plane inoculating, cultivates 3 days in 28 DEG C.

Adopt dig block method by inclined plane inoculating in containing 30ml seed culture fluid 250ml triangle shaking flask in.Seed culture medium: glucose 2%; Peptone 2%; Yeast extract powder 1%; 24h, shaking speed 220r/m is cultivated in 28 DEG C.

Cultured seed liquor is pressed 5 ~ 8% inoculum size transferred speciess in the 250ml triangle shaking flask containing 30ml fermentation culture, in 28 DEG C, shaking speed 220r/m, cultivates 5 days.Fermention medium: glucose 4%; Peptone 2%; Yeast extract powder 1%.Terminal fermentation yield (often liter of fermented liquid contains the grams of β-carotene) about 2.1g/L.4 batches of experimental results as indicated at 3.

Table 3, shake flask fermentation result

3.2,7.5L glass pot fermentation

Bacterial classification is Yarrowia lipolytica (Yarrowialipolytica) CGMCCNo.8940, and inclined-plane and seed culture mode are with embodiment 3.1.Culture temperature: 28 DEG C; Shaking speed 220r/m; Incubation time: 24h.

Seed is inoculated in the 7.5L glass fermentation tank containing 3.5L fermention medium by transferred species amount 5 ~ 8%.Fermention medium: glucose 3%; Peptone 2%; Yeast extract powder 1%; Potassium primary phosphate 0.08%; Dipotassium hydrogen phosphate 0.08%.

7.5L glass pot ferments: leavening temperature 28 DEG C, air flow 1:1 (vol:vol), mixing speed 550r/m, add the glucose of 50% in fermenting process, speed is 3ml/h.l, and fermentation period is 5 days, mean yield 4.5g/L.4 batch fermentation results are as shown in table 4.

Table 4, glass pot fermentation results

Claims (5)

1. a Yarrowia lipolytica (Yarrowialipolytica), is characterized in that, the preserving number of this bacterial classification is CGMCCNo.8940.

2. Yarrowia lipolytica CGMCCNo.8940 as claimed in claim 1 is for the production of the application of β-carotene.

3. a production method for β-carotene, is characterized in that, by fermenting, Yarrowia lipolytica CGMCCNo.8940 as claimed in claim 1 obtains β-carotene.

4. production method as claimed in claim 3, it is characterized in that, the culture condition of described fermentation is as follows:

Slant medium is: glucose 1.5 ~ 4%, peptone 0.5 ~ 3%, yeast extract powder 0.5 ~ 2%, agar 2%;

Seed culture medium is: glucose 0.5 ~ 4%, peptone 0.2 ~ 3%, yeast extract powder 0.5 ~ 3.5%;

Fermention medium is: glucose 1 ~ 7%, peptone 0.5 ~ 3%, yeast extract powder 1 ~ 3.5%, potassium primary phosphate 0.01 ~ 0.5%, dipotassium hydrogen phosphate 0.01 ~ 0.5%.

5. production method as claimed in claim 4, it is characterized in that, the culture condition of described fermentation is as follows:

Slant medium: glucose 2%; Tryptones 2%; Yeast extract powder 1%; Agar 2%;

Seed culture medium: glucose 2%; Peptone 2%; Yeast extract powder 1%;

Fermention medium: glucose 3%; Peptone 2%; Yeast extract powder 1%; Potassium primary phosphate 0.08%; Dipotassium hydrogen phosphate 0.08%.