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Yarrowia lipolytica genetic engineering bacterium for producing beta-carotene and application of yarrowia lipolytica genetic engineering bacterium

A technology of Yarrowia lipolytica and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of low yield, low consumption, hindered extraction efficiency and the like

Inactive Publication Date: 2020-06-23
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the route of chemical synthesis of β-carotene is very complicated, and the yield is not high; extraction of β-carotene from natural sources such as potato, carrot, seabuckthorn and corn is affected by many conditions, such as seasons, climate, etc., and these The content of β-carotene in plant sources is relatively low, resulting in high cost of naturally extracted β-carotene products, high energy consumption and low extraction efficiency hinder the practical use of these methods

Method used

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  • Yarrowia lipolytica genetic engineering bacterium for producing beta-carotene and application of yarrowia lipolytica genetic engineering bacterium
  • Yarrowia lipolytica genetic engineering bacterium for producing beta-carotene and application of yarrowia lipolytica genetic engineering bacterium
  • Yarrowia lipolytica genetic engineering bacterium for producing beta-carotene and application of yarrowia lipolytica genetic engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0155] Example 1 Construction of CRISPR / Cas9 gene knock-in plasmid pair

[0156] The plasmid constructions for different sgRNAs are all based on the purchased plasmid pCRISPRyl, which has a restriction site AvrII, and the upstream of the restriction site contains the promoter SCR1'-tRNAGly to initiate the expression of the sgRNA. The 20bp sequence was inserted into this site to obtain the sgRNA plasmid. Plasmid pCRISPRyl was purified after single digestion with AvrII restriction endonuclease to obtain linearized plasmid pCRISPRyl. The linearized plasmid was respectively combined with sgRNA fragments designed for POX2, POX3, POX4, LIP1, E1, A1, B1, A2, POX6, E2 sites (see SEQ ID NO: 1-SEQ ID NO: 10 in Table 1 ) for seamless cloning to obtain vectors pCRISPRyl_POX2, pCRISPRyl_POX3, pCRISPRyl_POX4, pCRISPRyl_LIP1, pCRISPRyl_E1, pCRISPRyl_A1, pCRISPRyl_B1, pCRISPRyl_A2, pCRISPRyl_POX6, pCRISPRyl_E2.

[0157] (1) Construction of plasmid pHR_POX2_hrGFP: Using the Yarrowia lipolyti...

Embodiment 2

[0167] Example 2 Construction of Yarrowia lipolytica genetically engineered bacteria producing β-carotene

[0168] The schematic diagram of the construction of Yarrowia lipolytica genetic engineering bacteria producing β-carotene of the present invention is as follows Figure 4 shown.

[0169] (1) Based on the existing CRISPR / Cas9 operating system, two pairs of knock-in plasmid pairs were constructed, respectively constructing a knock-in plasmid pair containing an optimized carRP gene and constructing a knock-in plasmid pair containing an optimized carB gene.

[0170] The donor plasmids in the two pairs of knock-in plasmids both contain restriction sites for NheI and BssHII.

[0171] The donor plasmid also contains promoter and / or terminator sequences. More preferably, the donor plasmid also contains a promoter UAS1B8-TEF (136).

[0172] The knock-in plasmid pair in this example is a conventional recombinant vector in the field, wherein the sgRNA plasmid contains a leucine ...

Embodiment 3

[0182] Embodiment 3 Determination of strain produces the output of β-carotene

[0183] The strain Yarrowia lipolytica Po1f and the strains XK2, XK3, XK4, XK5, XK6, XK7, XK8, XK10, XK11, XK12, XK13, XK14, XK15, XK16, XK17 and XK19 prepared in Example 2 were inoculated in 2mL respectively YPD medium (this YPD medium is made up of 2% glucose, 2% peptone and 1% yeast extract, balance is water, and described percentage is mass percentage), cultivated for 24 hours, then with initial OD 600 The inoculum of 0.01 was inoculated into new 50mL YPD medium for culture. After 4 days of fermentation, β-carotene was extracted using DMSO and acetone.

[0184] The results of the test are shown in Table 3 and figure 2 .

[0185] The output of the β-carotene of different bacterial strains of table 3

[0186]

[0187] Table 3 and figure 2 The results show that the ability of strain XK19 to produce β-carotene has been greatly improved. By overexpressing each gene in the MVA pathway, the o...

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Abstract

The invention provides a yarrowia lipolytica genetic engineering bacterium for producing beta-carotene. The yarrowia lipolytica genetic engineering bacterium for producing beta-carotene is prepared bythe following steps: knocking a carRP gene and a carB gene into chromosomes of uracil and leucine auxotrophic yarrowia lipolytica, then knocking a GGGS1 gene, a HMG gene, two copied carRP gene, one copied of carB gene, and an ERG13 gene into the chromosomes, and finally performing backfilling by two auxotrophic screening markers of uracil and leucine. Continuous feeding fermentation is performed,and the yield of beta-carotene can reach 4.5g / L. The yarrowia lipolytica genetic engineering bacteria constructed according to the invention has the advantages of simple and convenient operation andstable and reliable performance, and can be applied to large-scale commercial production, and the obtained beta-carotene can be safely used, so that a good prospect is achieved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a Yarrowia lipolytica genetically engineered bacterium producing β-carotene and its application. Background technique [0002] β-carotene is a natural orange pigment synthesized by plants and microorganisms. In addition to being a precursor of vitamin A and playing an important role in photosynthesis, this compound has many physiological effects, such as scavenging free radicals, enhancing immune response, enhancing Gap junctions. At present, β-carotene has a wide range of applications in the fields of food, aquaculture, cosmetics and pharmaceuticals: colorants, food additives, antioxidants, anti-cancer agents, and prevention of heart disease. [0003] The production methods of β-carotene mainly include chemical synthesis, natural extraction and microbial fermentation. However, the route of chemical synthesis of β-carotene is very complicated, and the yield is not...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/54C12N15/53C12N15/81C12N15/90C12P23/00C12P19/44C12R1/645
CPCC12N15/52C12N9/1085C12N9/0006C12N9/1029C12N15/815C12N15/905C12P23/00C12P19/44C12Y205/01029C12Y101/01088C12Y203/01009
Inventor 花强张鑫锎韦柳静汪丹妮陈骏
Owner EAST CHINA UNIV OF SCI & TECH
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