CN107904183A - A kind of restructuring yeast strains and construction method and lycopene production process - Google Patents
A kind of restructuring yeast strains and construction method and lycopene production process Download PDFInfo
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- CN107904183A CN107904183A CN201711191989.9A CN201711191989A CN107904183A CN 107904183 A CN107904183 A CN 107904183A CN 201711191989 A CN201711191989 A CN 201711191989A CN 107904183 A CN107904183 A CN 107904183A
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- lycopene
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
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Abstract
A kind of restructuring yeast strains and construction method and lycopene production process, the restructuring yeast strains are included through 2 function element expression cassettes on yeast homologous recombination and integration to genome；The construction method is：Structure includes the function element expression cassette 1 being made of the endogenous gene GGS1 of Yarrowia lipolytica, gene tHMG1 and endogenous strong promoter, terminator and rDNA homology arms first, while builds comprising the function element expression cassette 2 being made of the gene crtB in pantoea agglomerans source, the gene crtI in trispore Bruce mould source and yeast entogenous strong promoter, terminator and rDNA homology arms；Above-mentioned 2 expression cassettes are integrated into the rDNA sites solved on fat formula Yeast genome by Li-acetate method cotransformation, so as to obtain the recombinant bacterial strain of brand-new high yield lycopene；The production method is：Culture in fermentation medium is inoculated in after the activation of YPD culture mediums and reaches 21.0 mg/g DCW with somatic cells extraction lycopene, the unit cell yield of lycopene after shake flask fermentation, is collected.
Description
Technical field
The present invention relates to a kind of restructuring yeast strains and construction method and lycopene production process, belong to gene work
Journey technical field.
Background technology
Lycopene is fat-soluble natural food colour, and carotenoid is belonged in chemical constitution, possesses extremely strong antioxygen
Change power, be the hot spot that functional food composition is studied in the world in recent years.But presently commercially available lycopene about 90% belongs to chemistry
Method synthesizes, and there are food safety hazards, therefore there is an urgent need for biosynthesis replacement.At present in the research of lycopene biosynthesis,
Trispore Bruce mould is natural beta carotene Producing Strain, but it needs the blocking agents such as extra addition imidazoles to accumulate kind
Lycopene, causes the yield especially purity of lycopene not very good, and the addition of blocking agent is also unfavorable for food peace
Entirely, so needing to seek excellent host strain.Realized most in Escherichia coli at present by building Microbial cell factories
High yield has reached 3.52 g/L (50.6 mg/g DCW).And Escherichia coli are compared, saccharomyces cerevisiae and solution fat formula yeast
It is then preferred edible safety bacterial strain, but the yield of lycopene reported at present is undesirable, in saccharomyces cerevisiae most
High yield is 1.65 g/L (55.56 mg/g DCW), and the maximum output only 16 mg/g DCW in fat formula yeast is solved；
Compared to saccharomyces cerevisiae, solution fat formula yeast has liposome and is more advantageous to the efficient of strong-hydrophobicity material as lycopene
Synthesis.To sum up, develop and realize that the high yield of lycopene has very big potentiality and space as host strain to solve fat formula yeast,
Worth researcher goes further to explore.
The content of the invention
It is an object of the invention to overcoming the shortcomings of the prior art, and provide a kind of lycopene that can be applied to
In biosynthesis, and keep the restructuring yeast strains and construction method and lycopene production process of high yield.
The purpose of the present invention is by following technical solution to complete, a kind of restructuring yeast strains, the recombination yeast
Bacterial strain is included through 2 function element expression cassettes on yeast homologous recombination and integration to genome.
A kind of construction method of such as restructuring yeast strains, the construction method are：Structure is included by solution fat first
The Functional Unit that gene GGS1, gene tHMG1 and endogenous strong promoter, terminator and the rDNA homology arms of Ye Shi yeast entogenous form
Part expression cassette 1, at the same build include by the gene crtB in pantoea agglomerans source, the gene crtI in trispore Bruce mould source with
And the function element expression cassette 2 of yeast entogenous strong promoter, terminator and rDNA homology arms composition；Above-mentioned 2 expression cassettes are led to
Peracetic acid lithium method cotransformation is integrated into the rDNA sites on solution fat formula yeast (ATCC MYA2613) genome, so as to obtain complete
The recombinant bacterial strain of new high yield lycopene.
A kind of production method that lycopene is carried out using the restructuring yeast strains, the production method are：In YPD
Culture in fermentation medium is inoculated in after culture medium activation, and, with after shake flask fermentation, collection somatic cells extract lycopene, tomato
The unit cell yield of red pigment reaches 21.0 mg/g DCW.
The present invention utilizes the restructuring yeast strains for when producing lycopene, yield of lycopene to reach 21 mg/g
DCW, is to utilize the highest water for solving fat formula yeast production lycopene higher than existing highest level (16 mg/g DCW)
It is flat.
Embodiment
Below in conjunction with specific embodiment, the present invention will be described in detail：A kind of recombinant yeast of the present invention
Strain, the restructuring yeast strains are included through 2 function element expression cassettes on yeast homologous recombination and integration to genome.
A kind of construction method of such as restructuring yeast strains, the construction method are：Structure is included by solution fat first
The Functional Unit that gene GGS1, gene tHMG1 and endogenous strong promoter, terminator and the rDNA homology arms of Ye Shi yeast entogenous form
Part expression cassette 1, at the same build include by the gene crtB in pantoea agglomerans source, the gene crtI in trispore Bruce mould source with
And the function element expression cassette 2 of yeast entogenous strong promoter, terminator and rDNA homology arms composition；Above-mentioned 2 expression cassettes are led to
Peracetic acid lithium method cotransformation is integrated into the rDNA sites on solution fat formula yeast (ATCC MYA2613) genome, so as to obtain complete
The recombinant bacterial strain of new high yield lycopene.
A kind of production method that lycopene is carried out using the restructuring yeast strains, the production method are：In YPD
Culture in fermentation medium is inoculated in after culture medium activation, and, with after shake flask fermentation, collection somatic cells extract lycopene, tomato
The unit cell yield of red pigment reaches 21.0 mg/g DCW.
Embodiment：Restructuring yeast strains of the present invention are included through 2 work(on yeast homologous recombination and integration to genome
Can element expression cassette；The construction method of the restructuring yeast strains, i.e., by constructing function element expression cassette by function element module
Change is integrated into Yeast genome, is specifically：Structure is included by the endogenous gene GGS1 of Yarrowia lipolytica, gene tHMG1 first
And the function element expression cassette 1 of endogenous strong promoter, terminator and rDNA homology arms composition, while build and include by pantoea agglomerans
The gene crtB in source, the gene crtI and yeast entogenous strong promoter in trispore Bruce mould source, terminator and rDNA
The function element expression cassette 2 of homology arm composition.Above-mentioned 2 expression cassettes are integrated into solution fat formula ferment by Li-acetate method cotransformation
RDNA sites on female (ATCC MYA2613) genome, so as to obtain the recombinant bacterial strain of one plant of brand-new high yield lycopene.
Present invention also offers a kind of method using restructuring yeast strains production lycopene, i.e., by recombinant yeast
Strain is inoculated in fermentation medium after seed culture medium activates cultivates, and somatic cells extraction lycopene is collected after culture, kind
The unit cell yield of Lycopene reaches 21.0 mg/g DCW.
The specific production method of restructuring yeast strains of the present invention has：Restructuring yeast strains are inoculated in the training of 5mL seeds
Support in base, cultivate 14-16h in 30 DEG C, 250rpm, transferred with initial cell concentration OD600=0.2 and trained in fresh 25mL seeds
Support in base, cultivate to mid log phase under the conditions of 30 DEG C, 250rpm, transferred respectively with initial cell concentration OD600=0.5
In 50mL fermentation mediums, cultivated under the conditions of 30 DEG C, 250rpm, fermenting and producing lycopene.
The seed culture medium includes 20g/L or 40g/L glucose, 20g/L peptones and 10g/L yeast extracts；Or
Person adds 10g/L D- galactolipins.
Claims (3)
1. a kind of restructuring yeast strains, it is characterised in that the restructuring yeast strains are included through yeast homologous recombination and integration to gene
2 function element expression cassettes in group.
2. a kind of construction method of restructuring yeast strains as claimed in claim 1, it is characterised in that the construction method is：It is first
First structure includes same by the endogenous gene GGS1 of Yarrowia lipolytica, gene tHMG1 and endogenous strong promoter, terminator and rDNA
The function element expression cassette 1 of source arm composition, while build comprising gene crtB, the trispore Bruce mould by pantoea agglomerans source
The function element expression cassette 2 that gene crtI and yeast entogenous strong promoter, terminator and the rDNA homology arms in source form；Will
Above-mentioned 2 expression cassettes are integrated into the rDNA on solution fat formula yeast (ATCC MYA2613) genome by Li-acetate method cotransformation
Site, so as to obtain the recombinant bacterial strain of brand-new high yield lycopene.
3. a kind of production method that lycopene is carried out using restructuring yeast strains described in claim 1, it is characterised in that described
Production method be：Culture in fermentation medium is inoculated in after the activation of YPD culture mediums and, with after shake flask fermentation, collects somatic cells
Lycopene is extracted, the unit cell yield of lycopene reaches 21.0 mg/g DCW.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711191989.9A CN107904183A (en) | 2017-11-24 | 2017-11-24 | A kind of restructuring yeast strains and construction method and lycopene production process |
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| CN201711191989.9A CN107904183A (en) | 2017-11-24 | 2017-11-24 | A kind of restructuring yeast strains and construction method and lycopene production process |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120142082A1 (en) * | 2006-12-12 | 2012-06-07 | Sharpe Pamela L | Carotenoid production in a recombinant oleaginous yeast |
| CN105087406A (en) * | 2015-07-22 | 2015-11-25 | 天津大学 | Recombinant yeast strain as well as construction method and application thereof |
| CN105779319A (en) * | 2016-03-23 | 2016-07-20 | 天津大学 | Recombinant yeast strain, and building method and application thereof |
-
2017
- 2017-11-24 CN CN201711191989.9A patent/CN107904183A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120142082A1 (en) * | 2006-12-12 | 2012-06-07 | Sharpe Pamela L | Carotenoid production in a recombinant oleaginous yeast |
| CN105087406A (en) * | 2015-07-22 | 2015-11-25 | 天津大学 | Recombinant yeast strain as well as construction method and application thereof |
| CN105779319A (en) * | 2016-03-23 | 2016-07-20 | 天津大学 | Recombinant yeast strain, and building method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 张根林: "酿酒酵母中 β-香树脂醇合成途径的构建与调控", 《中国博士学位论文全文数据库 工程科技I辑》 * |
| 施明雨: "产番茄红素酵母工程菌的构建及发酵工艺优化", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
| 杨振江: "产番茄红素Yarrowia lipolytica基因工程菌的构建", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
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Application publication date: 20180413 |