CN106520558B - A kind of chlorella mutation algae strain and its cultural method of production luteole and beta carotene - Google Patents
A kind of chlorella mutation algae strain and its cultural method of production luteole and beta carotene Download PDFInfo
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Abstract
The chlorella mutation algae strain of a kind of high-yield corn flavine and the required carotenoid of other people bodies and its cultural method, chlorella Chlorella zofingiensisATCC 30412 is subjected to NTG mutant treatment, treated, and frustule is coated on the Kuhl solid medium containing DPA cultivates, it selects single algae on culture medium and falls in Kuhl fluid nutrient medium and cultivate, glucose is added when to the logarithm middle and later periods and carries out carotenogenesis induction, pass through the variation of carotenoid type and content in detection frond, it obtains a plant height and produces luteole, lutein and the strain of beta carotene algae are simultaneously named as CZ-ZEA1.CZ-ZEA1 biomass under the conditions of the glucose induction of 30g/L reaches 12.95g/L, and the content of luteole, lutein and beta carotene respectively reaches 2.176mg/g, 1.10mg/g and 1.211mg/g.The mutation algae strain belongs to the edible algae of green alga, containing a large amount and optimal proportion carotenoid needed by human, the especially very rare luteole of natural resources is the new resources functional food of ideal prevention middle-aged and the old's eye degeneration and lesion, has good development and application prospect.
Description
Technical field
The invention belongs to field of microorganism engineering, and in particular to produce maize using the plant height that nitrosoguanidine mutagenesis generates
The mutation chlorella strain and its cultural method of element and beta carotene.
Technical background
Luteole is a kind of hydroxylating carotenoid of yellow, scientific name 3,3 '-dihydroxy-beta carotene,
As beta carotene, luteole has very strong coloring and anti-oxidation function.Luteole and its isomer leaf
Flavine is the primary pigments ingredient of eye retina macula lutea, their content ratios in macula lutea are 2:1.Recent studies indicate that
Luteole facilitates the oxidation of prevention, Age related macular regression, cancer and low-density lipoprotein, furthermore maize
Element is also widely used in poultry and fish culture as feed addictive.The main source of luteole currently on the market
It is biological extraction, natural luteole is mainly extracted from the plants such as hot red pepper, yellow maize.But corn in these plants
The content of flavine is only up to 400 μ g/g, and plant growing cycle is long, it is more also to be occupied using plant production luteole
Soil and water resource.Microbial resources especially plant progenitor monoplast green alga, growth cycle is short, and biomass is high, is ideal
Carotenoid resource.If Dunaliella salina is for commercially producing natural beta-carotin, haematococcus pluvialis is for producing shrimp
Green element.Plant and green alga usually contain the lutein of higher amount but do not accumulate the luteole of a large amount.Dunaliella salina mutant
Zea1 can accumulate luteole because being catalyzed luteole and losing function at the gene of downstream carotenoid and be up to 6mg/g cell
Dry weight is the green alga that there are commercial applications to be worth.However photoautotrophic Dunaliella salina biomass is low (about 1g/L), limits
The application potential of the algae.The green alga new resources of high-yield corn flavine and other carotenoid needed by human are researched and developed with great
Business application meaning.
So far, a kind of production luteole and beta carotene be there are no in the prior art, be chlorella
The report of the mutagenesis algae strain ZEA1 of (Chlorella zofingiensis, ATCC 30412).
Summary of the invention
The purpose of the present invention is to provide the algae strains of a kind of production luteole and beta carotene, are chlorella
The mutagenesis algae strain ZEA1 of (Chlorella zofingiensis, ATCC 30412) and the luteole and β-carrot
The cultural method of plain high yield algae strain ZEA1.
In order to realize above-mentioned purpose of the invention, the present invention provides the following technical solutions:
A kind of mutation chlorella strain producing luteole and beta carotene is chlorella Chlorella
The mutagenesis algae strain ZEA1 of zofingiensis, ATCC 30412, preservation mechanism and deposit number are CGMCC No.12948.
A kind of mutation chlorella strain CGMCC No.12948 producing luteole and beta carotene as mentioned, the mutation
For chlorella strain under the conditions of the glucose induction of 30g/L, growth rate is higher than wild type, and biomass is 1.2 times of wild type,
Up to 12.95g/L, the content of luteole, lutein and beta carotene is respectively 2.176mg/g, 1.10mg/g and
1.211mg/g。
A kind of mutation chlorella strain CGMCC No.12948 producing luteole and beta carotene as mentioned, the mutation
The edible algae of chlorella strain category green alga, containing a large amount and optimal proportion luteole: the class needed by human of lutein 2: 1
Carrotene and the rare luteole of natural resources.
A kind of mutation chlorella strain CGMCC No.12948 producing luteole and beta carotene as mentioned, the mutation
Beta carotene angulation Huang matter and luteole are catalyzed in chlorella strain into assimilation enzyme gene BKT1 gene 74 of astaxanthin
Base has been replaced as guanine G by thymidine T, and cause brings it about nonsense mutation.
A kind of mutation chlorella strain CGMCC No.12948 producing luteole and beta carotene as mentioned, the mutation
Chlorella strain is measured through HPLC, and accumulation is up to 2.176mg/g and 1.17mg/g under the condition of culture of Kuhl+30g/L glucose
Luteole and lutein, the ratio 2: 1 of their ratio 1.86: 1 very close to both pigments in eyes macula lutea.
A kind of mutation chlorella strain CGMCC No.12948 producing luteole and beta carotene as mentioned, the mutation
Chlorella strain contains 1.211mg/g beta carotene needed by human, and the content of various carotenoid and ratio are in difference
Show highly stable in the frond of algebra, the yield of luteole, lutein and beta carotene respectively reaches
36.79mg/L, 28.65mg/L and 26.18mg/L.
The present invention also provides the mutation chlorella strains of any production luteole and beta carotene
CGMCCNo.12948 is obtained by following methods: first by chlorella Chlorella zofingiensisATCC
30412 carry out NTG mutant treatments, and treated, and frustule is coated on the Kuhl solid medium containing DPA cultivates, and choose
It selects single algae on culture medium to fall in Kuhl fluid nutrient medium, glucose is added when culture is to the logarithm middle and later periods and carries out carotenoids
Element synthesis induction detects the variation of carotenoid type and content in frond, and the ingredient of the Kuhl culture medium is (L):
1.01g KNO3；0.62g NaH2PO4·H2O；0.089g Na2HPO4·2H2O；0.247g MgSO4·7H2O；14.7mg
CaCl2·2H2O；6.95mg FeSO4·7H2O；0.061mg H3BO3；0.169mg MnSO4·H2O；0.287mg ZnSO4·
7H2O；0.0025mg CuSO4·5H2O；and 0.01235mg(NH4)6MO7O24·4H2O；PH is 6.8.
As mentioned it is any produce luteole and beta carotene mutation chlorella strain CGMCC No.12948, be
It is obtained by following methods:
1) 30412 algae solution of chlorella Chlorella zofingiensisATCC of logarithmic growth phase is diluted to 105It is a
Cell/ml takes algae solution 5ml, 2000rpm to be centrifuged 10min, abandons supernatant, is resuspended with 7.0 buffer of 20mL 50mM PBS, pH,
2000rpm is centrifuged 10min；
2) abandon supernatant, add 10mL 50mM PBS, pH is that cell is resuspended in 7.0 and 100 μ l 20mg/mL MNNG, after mixing in
25 DEG C of constant-temperature table shading treatment 1h；
3) by treated in step 2), cell 2000rpm is centrifuged 10min, abandons supernatant, and frustule precipitating cleans 2 with PBS
Secondary, frustule adds fresh Kuhl fluid nutrient medium, is incubated for for 24 hours in constant-temperature table shading；The ingredient of the Kuhl culture medium is
(L): 1.01g KNO3；0.62g NaH2PO4·H2O；0.089g Na2HPO4·2H2O；0.247g MgSO4·7H2O；
14.7mg CaCl2·2H2O；6.95mg FeSO4·7H2O；0.061mg H3BO3；0.169mg MnSO4·H2O；0.287mg
ZnSO4·7H2O；0.0025mg CuSO4·5H2O；and 0.01235mg(NH4)6MO7O24·4H2O；PH is 6.8；
4) the frustule 2000rpm of step 3) is centrifuged 10min, frustule fresh culture dilutes and is coated on mutagenesis
The Kuhl plate containing 30 μM of diphenylamines of screening, in 25 DEG C of constant temperature illumination box cultures；
5) it from picking chlorella monoclonal on step 4) mutagenesis screening plate and numbers, is inoculated in equipped with 3mL containing 30 μM two
It in the 12mL culture tube of the Kuhl fluid nutrient medium of aniline, is placed in constant-temperature table culture 10 days, later connects algae solution with 10%
Kind amount is inoculated in the 100mL conical flask of the Kuhl culture medium containing 30 μM of diphenylamines containing 10mL, Yu Hengwen illumination shaking table culture 4
It；
6) frustule of step 5) is taken to be inoculated in the 250mL conical flask of the culture medium of Kuhl containing 50mL with 10% inoculum concentration
In, Yu Hengwen illumination shaking table culture 4 days to mid-log phase, then add various concentration 0g/L, 10g/L, 15g/L, 30g/L,
The glucose of 50g/L, 60g/L are induced, and the variation of its pigment composition and content is detected by HPLC, and HPLC detects luring
The deposit number that the luteole of high-content, lutein and beta carotene can be accumulated under the conditions of leading is the algae of CGMCC No.12948
Strain.
Any mutation chlorella strain CGMCC No.12948 for producing luteole and beta carotene as mentioned, it is described
Mutation chlorella strain CGMCC No.12948 be with chlorella be set out algae strain, through nitrosoguanidine mutagenesis, diphenylamines screening obtain
, it is obtained by following methods culture:
1) algae prepares: being algae strain of setting out with chlorella Chlorella zofingiensisATCC 30412, by bead
Algae is inoculated on Kuhl agar slant culture-medium and cultivates, and a ring frond is chosen from inclined-plane and is seeded to shaking flask culture；The Kuhl
The ingredient of culture medium is (L): 1.01g KNO3；0.62g NaH2PO4·H2O；0.089g Na2HPO4·2H2O；0.247g
MgSO4·7H2O；14.7mg CaCl2·2H2O；6.95mg FeSO4·7H2O；0.061mg H3BO3；0.169mg MnSO4·
H2O；0.287mg ZnSO4·7H2O；0.0025mg CuSO4·5H2O；and 0.01235mg(NH4)6MO7O24·4H2O；PH is
6.8；
2) behind frustule culture 4 days in shaking flask, new Kuhl the preparation of mutagenized cell: is inoculated into 1: 10 volume ratio
In the shaking flask of fluid nutrient medium, culture to logarithm middle and later periods；
3) nitrosoguanidine mutagenesis: the chlorella algae solution of logarithmic growth phase is diluted to 105A cell/ml, centrifugation are used in combination
PBS buffer solution is washed 2 times, and the PBS buffer solution is K2HPO4·3H20 6.82g/L, KH2PO4 2.6g/L, PH 7.0, later
It is resuspended in PBS, MNNG is added into the PBS buffer solution, in constant-temperature table shading treatment after mixing；
4) screening of diphenylamines: mutagens treated algae solution is centrifuged, and is washed 3 times with PBS buffer solution, and will be final
Frond is resuspended in Kuhl fluid nutrient medium, is taken to be coated on the Kuhl solid medium containing diphenylamines after gradient dilution and be cultivated
Screening, selects the bigger algae of color change and falls；
5) pigment detection: the monoclonal algae colony selected being inoculated into Kuhl fluid nutrient medium and is cultivated, culture to logarithm
The glucose induction of various concentration is added after mid-term, and measures the content of related pigment, takes the highest algae strain of luteole content
CGMCC No.12948 after continuously culture is more instead of, measures pigment content, verifies the stability of its subculture.
A kind of luteole and beta carotene high yield algae strain of the present invention is chlorella (Chlorella
Zofingiensis, ATCC30412) mutagenesis algae strain ZEA1, algae strain has been stored in Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, number is CGMCC No.12948.
Compared with prior art, excellent benefit of the invention is:
The present invention is by carrying out nitrosoguanidine mutagenesis screening to chlorella Chlorella zofingiensisATCC30412
To mutant strain CZ-ZEA1 (the biomaterial name: ZEAl, systematic name: Chlorella that can accumulate luteole
Zofingienesis) (it is common that the mutant strain has been stored in China Committee for Culture Collection of Microorganisms on September 7th, 2016
Microorganism center, Institute of Microorganism, Academia Sinica, number are CGMCC No.12948, address: the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, phone: 010-64807355, postcode: 100101).Culture of the mutant strain in Kuhl+30g/L glucose
Growth rate in base is higher than wild type, and biomass is 1.2 times of wild type, reaches 12.95g/L (Fig. 1,2).It was found that CZ-
Beta carotene angulation Huang matter and luteole are catalyzed in ZEA1 into assimilation enzyme gene BKT1 gene 74 bases of astaxanthin
It has been replaced as guanine (G) by thymidine (T), cause brings it about nonsense mutation (Fig. 3).HPLC measures CZ-ZEA1 in Kuhl+
Can be accumulated under the condition of culture of 30g/L glucose up to 2.176mg/g and 1.17mg/g luteole and lutein (Fig. 4,
5), ratio (2: 1) of their ratio (1.86: 1) very close to both pigments in eyes macula lutea.Furthermore the algae also contains
Beta carotene (Fig. 4,5) 1.211mg/g needed by human, and the content of various carotenoid and ratio are in different algebra
It is shown in frond highly stable.
Detailed description of the invention
Fig. 1 wild type WT and growth curve of the mutagenic fungi CZ-ZEA1 under Kuhl culture medium condition, abscissa indicate training
Number of days is supported, ordinate indicates cell concentration (cells/ml).
The dry cell weight of Fig. 2 wild type WT and mutagenic fungi CZ-ZEA1.Abscissa indicates that cultivated days, ordinate indicate thin
Born of the same parents' dry weight (g/L).
The BKT1 gene order of Fig. 3 chlorella WT and mutant strain ZEA1 compare, and show the base of mutation in black.
Fig. 4 chlorella WT and mutant strain ZEA1HPLC carotenoid analyze map.1- astaxanthin (free body), 2- leaf
Flavine, 3- luteole, 4- beta carotene, 5- astaxanthin (esterification body).
Fig. 5 wild type WT and mutagenic fungi CZ-ZEA1 cylinder accumulation under conditions of the Kuhl culture medium of 30g/L glucose
Luteole (Zeaxanthin), lutein (Lutein) and beta carotene (β-carotene) content (mg/g)；
Upper figure is wild type algae strain, and the following figure is mutagenic fungi CZ-ZEA1；Abscissa is cultivated days, and ordinate is pigment content (mg/g).
Fig. 6 mutagenic fungi CZ-ZEA1 luteole under conditions of the Kuhl culture medium of 30g/L glucose
(Zeaxanthin), the yield (mg/L) of lutein (Lutein) and beta carotene (β-carotene)；Abscissa is training
Number of days is supported, ordinate is pigment production (mg/L).
Specific embodiment
With reference to the accompanying drawing, essentiality content of the invention is further illustrated with the embodiment of the present invention, but not with
This limits the present invention.
Embodiment 1
1) will set out algae chlorella (Chlorella zofingiensis, ATCC 30412) (American Type
Culture Collection, ATCC, Rockville, MD, USA) from the Kuhl agar slant culture-medium (glucose containing 3g/L
With 1.5% agar) on be inoculated into the Kuhl fluid nutrient medium containing 10ml and cultivate 4 days.Condition of culture are as follows: 25 degrees Celsius of temperature,
Shaking speed 150rpm, intensity of illumination 30umol m-2s-1.The ingredient of Kuhl culture medium is (L): 1.01g KNO3；0.62g
NaH2PO4·H2O；0.089g Na2HPO4·2H2O；0.247g MgSO4·7H2O；14.7mg CaCl2·2H2O；6.95mg
FeSO4·7H2O；0.061mg H3BO3；0.169mg MnSO4·H2O；0.287mg ZnSO4·7H2O；0.0025mg
CuSO4·5H2O；and 0.01235mg(NH4)6MO7O24·4H2O；PH is 6.8.
2) 4 days frustules will have been cultivated in above-mentioned bottle, and fresh 50ml Kuhl liquid training is inoculated into 1: 10 ratio
It supports and is cultivated 4 days in base.
3) 2) frustule after step culture 4 day is collected by centrifugation, and washes 2 times with PBS buffer solution and is collected by centrifugation, be resuspended in
In PBS buffer solution, so that cell concentration is 105A cell/ml, preliminary experiment show 100 μ l 20mg/mL MNNG Mutagenic Effects most
It is good, therefore the MNNG for 100 μ l 20mg/mL being added into the PBS re-suspension liquid, in constant-temperature table shading treatment 1h after mixing.
4) frustule is collected by centrifugation, frustule is resuspended in the Kuhl liquid of 1ml after washing frustule 3 times with PBS buffer solution
In culture medium, the Kuhl+3g/L grape of various concentration (0,10,20,30,60,100 μM) diphenylamines is coated on after gradient dilution
It is screened on sugar culture-medium.The algae grown from screening and culturing medium falls number and determines that the concentration of most suitable diphenylamines is 30 μM.
5) picking 4) color changes (it is orange or green to become yellow from red) on step screening and culturing medium algae falls,
It is inoculated into 10ml Kuhl fluid nutrient medium and cultivates, be inoculated into 50ml Kuhl Liquid Culture in 1: 10 ratio after culture 4 days
It is cultivated in base, the glucose processing of various concentration (0,10,15,30,50,60g/L) is added after 4 days, every 48h measurement pigment
Content and component.
6) measurement of pigment content: taking 2ml frustule, and 2000rpm is centrifuged 5min, uses ddH2O is centrifuged after washing 2 times, is resuspended
Pigment is extracted in 1ml acetone.Extracting solution crosses 0.22 μm of Millipore organic film, filtered extracting solution Agilent
1290 Ultra Performance Liquid Chromatography instrument analysis detections, chromatographic column be Waters YMC Carotenoid column (4.6 ×
250mm).The mobile phase of chromatography are as follows: A, methanol；B, methyl tertiary butyl ether(MTBE).Pillar elution condition are as follows: 70%A phase and
30%B phase, sample volume are 10 μ l, flow velocity 1ml/min.
7) the algae strain of the wild type and mutagenesis that grow to the logarithm middle and later periods, dilution the measurement of biomass and growth curve: are taken
To 1.5x106Cell/ml is cultivated in 50ml Kuhl fluid nutrient medium, every 24 hours measurement cell numbers, is surveyed every 48h
Determine biomass.The measurement of cell number is using counting method of blood cell；The measurement of biomass is to take the algae solution of 4ml, and algae is collected by centrifugation
Body is resuspended and uses ddH2O is washed 2 times, claims dry weight after filtering drying.
8) mutant strain (biomaterial name: ZEAl, systematic name: Chlorella is measured with HPLC and the method for surveying dry weight
Zofingienesis) (it is common that the mutant strain has been stored in China Committee for Culture Collection of Microorganisms on September 7th, 2016
Microorganism center, Institute of Microorganism, Academia Sinica, number are CGMCC No.12948, address: the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, phone: 010-64807355, postcode: the 100101) pigment of every generation and life after continuous subculture 20 times
Object amount, mutant strain reach the condition of maximum luteole yield to be added in Kuhl fluid nutrient medium after culture to stationary phase
The glucose of 30g/L.After culture 8 days, the content and yield of the luteole of mutant strain are respectively 2.176mg/g and 36.79mg/
L, content beta-carotene and yield are respectively 1.211mg/g and 26.18mg/L.
<110>Kunming Inst. of Botany, Chinese Academy of Sciences
<120>a kind of chlorella mutation algae strain for producing luteole and beta carotene and its cultural method
<160> 2
<210> 1
<211> 936
<212> DNA
<213>chlorella mutagenic fungi ZEA1(Chlorella zofingiensis ZEA1)
<400> 1
ATGGCGCCAG ATGTGACACA TGTGCAGCCA CGTGTACAGT CCCCGGCTGG CCCCGATGAT 60
GAGGATGACG CGTGAAGCTT GTGGAAAGCC CAATATCCTA TGCCGGAGGA GAAGGGTACA 120
GTATCCAAAC CTCAAGCCGC ACTCAAATAC AGGCCACCAC GCAGTGACTG GAAGGGTGTA 180
TCAATTGCAT GCACTGTCAT CACCCTATGG ACAGCTGTCT TTTACCATGG CTGCTGGCAA 240
ATCAAACTCA CAGGCCCTGA TAAGTCAGCC TGGTGGGACG TTGTTGCAAC GTTTCTGGCA 300
CTGGAGTTCC TCAACACTGG GCTTTTCATC ACCACGCATG ATGCCATGCA TGGGACTATT 360
GCCATCAGGA ACCGTCGTTT GAATGACCTA CTTGGCAATA TAGCCATCAG CCTATATGCC 420
TGGTTTGACT ATGACATGCT GCACAAGAAG CACTGGGAGC ATCACAACTT CACTGGGTTA 480
CCACATAAAG ACCCAGACTT CCATCGAGGC GATCCTGCGC TACATAAGTG GTTTGGCAGG 540
TTTATGTGGG AGTATGCAAC ACCACTCCAG TTTGCCAAGA TCTTCGCATA CACCTTCTTC 600
CTACAATCCT TACGGGTGCA ATACCCCAAT TTATGCGTCT TTCTGGCGGC TGCACCCCTG 660
GTCAGTGCGT TCCGATTGTT CTATTTTGGC ACCTATTTAC CCCACCTCCC CTCCAATGCT 720
CAGGAGACAA TGCCCTGGGA GAAATCTCAC AGTGCTGATG ACCCTCGGCC GCTGTCATTC 780
TTGAAATGTT ATCACTTTGA TTATCACTGG GAGCATCACA GGTGGCCTTA TGCCCCTTGG 840
TGGGAGTTAC CCGTGTGTAA GCGCATCACA AAGACACTGG ATGCTGCAGT TCCAGGAGTA 900
CAGTCAGACG GCACGAAGAA GAGTCAGTTG GTGAAC 936
<210> 1
<211> 312
<212> PRT
<213>chlorella mutagenic fungi ZEA1(Chlorella zofingiensis ZEA1)
<400> 2
Met Ala Pro Asp Val Thr His Val Gln Pro Arg Val Gln Ser Pro
5 10 15
Ala Gly Pro Asp Asp Glu Asp Asp Ala * Ser Leu Trp Lys Ala
20 25 30
Gln Tyr Pro Met Pro Glu Glu Lys Gly Thr Val Ser Lys Pro Gln
35 40 45
Ala Ala Leu Lys Tyr Arg Pro Pro Arg Ser Asp Trp Lys Gly Val
50 55 60
Ser Ile Ala Cys Thr Val Ile Thr Leu Trp Thr Ala Val Phe Tyr
65 70 75
His Gly Cys Trp Gln Ile Lys Leu Thr Gly Pro Asp Lys Ser Ala
80 85 90
Trp Trp Asp Val Val Ala Thr Phe Leu Ala Leu Glu Phe Leu Asn
95 100 105
Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly Thr Ile
110 115 120
Ala Ile Arg Asn Arg Arg Leu Asn Asp Leu Leu Gly Asn Ile Ala
125 130 135
Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Asp Met Leu His Lys Lys
140 145 150
His Trp Glu His His Asn Phe Thr Gly Leu Pro His Lys Asp Pro
155 160 165
Asp Phe His Arg Gly Asp Pro Ala Leu His Lys Trp Phe Gly Arg
170 175 180
Phe Met Trp Glu Tyr Ala Thr Pro Leu Gln Phe Ala Lys Ile Phe
185 190 195
Ala Tyr Thr Phe Phe Leu Gln Ser Leu Arg Val Gln Tyr Pro Asn
200 205 210
Leu Cys Val Phe Leu Ala Ala Ala Pro Leu Val Ser Ala Phe Arg
215 220 225
Leu Phe Tyr Phe Gly Thr Tyr Leu Pro His Leu Pro Ser Asn Ala
230 235 240
Gln Glu Thr Met Pro Trp Glu Lys Ser His Ser Ala Asp Asp Pro
245 250 255
Arg Pro Leu Ser Phe Leu Lys Cys Tyr His Phe Asp Tyr His Trp
260 265 270
Glu His His Arg Trp Pro Tyr Ala Pro Trp Trp Glu Leu Pro Val
275 280 285
Cys Lys Arg Ile Thr Lys Thr Leu Asp Ala Ala Val Pro Gly Val
290 295 300
Gln Ser Asp Gly Thr Lys Lys Ser Gln Leu Val Asn
305 310 312
Claims (6)
1. a kind of mutation chlorella strain for producing luteole and beta carotene is chlorella Chlorella
The mutagenesis algae strain ZEA1 of zofingiensis, ATCC 30412, preservation mechanism and deposit number are CGMCC No. 12948.
2. a kind of mutation chlorella strain for producing luteole and beta carotene as described in claim 1, it is characterised in that should
Chlorella strain is mutated under the conditions of the glucose induction of 30g/L, growth rate is higher than wild type, and biomass is wild type
1.2 times, 12.95g/L is reached, the content of luteole, lutein and beta carotene is respectively 2.176 mg/g, 1.10 mg/g
With 1.211 mg/g.
3. a kind of mutation chlorella strain for producing luteole and beta carotene as described in claim 1, it is characterised in that should
It is mutated the edible algae of chlorella strain category green alga, the luteole of content ratio about 2: 1 and the carotenoid of lutein, and
Beta carotene.
4. a kind of mutation chlorella strain for producing luteole and beta carotene as described in claim 1, it is characterised in that should
It is mutated in chlorella strain and is catalyzed beta carotene angulation Huang matter and luteole into the assimilation enzyme gene BKT1 gene 74 of astaxanthin
The base of position has been replaced as guanine G by thymidine T, and cause brings it about nonsense mutation.
5. a kind of mutation chlorella strain for producing luteole and beta carotene as described in claim 1, it is characterised in that should
Mutation chlorella strain measured through HPLC, under the condition of culture of Kuhl+30g/L glucose accumulation be up to 2.176mg/g and
The luteole and lutein of 1.17mg/g, their ratio are 1.86: 1.
6. a kind of mutation chlorella strain for producing luteole and beta carotene as described in claim 1, it is characterised in that should
Mutation chlorella strain contains 1.211 mg/g beta carotene needed by human, and the content of various carotenoid and ratio exist
Show highly stable in the frond of different algebra, the yield of luteole, lutein and beta carotene respectively reaches
36.79 mg/L, 28.65 mg/L and 26.18 mg/L.
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