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Yarrowia lipolytica with high yield of [beta]-carotene and application thereof

A technology of Yarrowia lipolytica and carotene, applied in the field of engineering bacteria, can solve the problem of low yield of production strains, achieve wide industrial application prospects, and reduce the consumption of manpower and material resources

Active Publication Date: 2021-05-25
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the above-mentioned research background, the object of the present invention is to overcome the defect of low yield of existing beta-carotene production strains, aiming to provide a high-yielding strain of beta-carotene

Method used

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  • Yarrowia lipolytica with high yield of [beta]-carotene and application thereof
  • Yarrowia lipolytica with high yield of [beta]-carotene and application thereof
  • Yarrowia lipolytica with high yield of [beta]-carotene and application thereof

Examples

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Embodiment 1

[0087] 1. Materials and methods

[0088] 1. The gene synthesis in the present invention was completed by Nanjing GenScript Biotechnology Co., Ltd.; the primer synthesis and sequencing in the present invention were completed by Beijing Qingke Xinye Biotechnology Co., Ltd.

[0089] 2. The experimental methods used in the following examples include plasmid construction, enzyme digestion, preparation of competent cells, transformation, etc., unless otherwise specified, are conventional methods. The specific experimental conditions can be determined through simple experiments if necessary.

[0090] 3. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0091] 4. The original strain of Yarrowia lipolytica involved in the present invention (ATCC number MYA-2613; genotype MATAura3-3021leu2-270xpr2-322axp2-deltaNU49XPR2::SUC2) was purchased from ATCC.

[0092] 5. The genes involved in the present invention, w...

Embodiment 2

[0117] Example 2 Construction of Yarrowia lipolytica engineering strain GVD-A producing β-carotene

[0118] 1. Based on the existing CRISPR / Cas9 operating system, the plasmids p-car-M3 and pCRISPRyl-car containing the gene expression cassettes of CarB and CarRP were introduced into Yarrowia lipolytica and integrated into the zeta site of the genome to obtain the β- Carotene recombinant strain T1.

[0119] The specific method is as follows: (1) After Yarrowia lipolytica po1fΔTRP was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose), the competent cells were prepared by conventional yeast lithium acetate competent preparation method. (2) Add 2 μL of pCRISPRyl-car and p-car-M3 plasmids to 40 μL of competent cells, then add 2 μL of salmon sperm DNA, and incubate in a water bath at 30°C for 15 minutes. (3) Add 350 μL PEG 4000-LithiumAcetate (0.1M pH 6.0) and 16 μL 1MDTT (40 mM) to the above system, and incubate statically in a water b...

Embodiment 3

[0122] Embodiment 3 GVD-A bacterial strain continuous feeding fermentation culture

[0123] The Yarrowia lipolytica GVD-A with high β-carotene production of GVD-A constructed by genetic engineering described in Example 2 was subjected to a continuous fed-feed fermentation experiment in a 1L fermenter, and the culture medium used in this example was 2XYPD culture base (4% tryptone, 2% yeast extract, 6% glucose) with an initial volume of 0.6 L. Take out the bacterial solution from the glycerin seed preservation tube, streak on the YPD solid plate, and cultivate for 36 h. Inoculate the grown monoclonal into a shaker flask containing 50mL of YPD medium, cultivate at 30°C and 220rpm for 24h, and inoculate the seeds into the fermenter. The set temperature of the fermenter is 30°C, the ventilation rate is 2.0VVM, the dissolved oxygen is set to 20%, the stirring speed (400-1000) is coupled with the dissolved oxygen, and no pH adjustment is performed. Sampling and detection of the gl...

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Abstract

The invention particularly relates to yarrowia lipolytica with high yield of [beta]-carotene and application thereof. The strain can utilize common carbon sources and nitrogen sources, after high-density culture, the yield of [beta]-carotene reaches 7.8 g / L, the content reaches 161 mg / g DCW, and the strain is an excellent strain for high yield of [beta]-carotene. According to the engineering strain, a low-yield [beta]-carotene yarrowia lipolytica strain T1 which integrates CarRP and CarB genes to a zeta site at a fixed point is taken as a starting strain, endogenous key enzymes HMGs, HMGCR, ERG20, GGS1, ERG8, ERG12, ERG19 and IDI are subjected to multiple rounds of random integration, and exogenous GGPP synthetase GPS (which can directly catalyze IPP and DMAPP into GGPP) and hemoglobin VHb are introduced; and finally, the key gene AAL8 for reducing lipidosome degradation is knocked out to obtain the high-yield strain. As a [beta]-carotene production engineering bacterium, the strain has a good application prospect in the fields of food, feed, medicine and cosmetics.

Description

technical field [0001] The invention belongs to the technical field of engineering bacteria, and in particular relates to a strain of Yarrowia lipolytica (Yarrowialipolytica) GVD-A, its construction method, and the application of the strain as an engineering bacteria for producing β-carotene; the invention also includes the above-mentioned strains Bacteria agent, and its application in food industry, feed industry, medicine and cosmetics industry. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily to be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] So far, more than 700 kinds of carotenoids have been reported, and plants, bacteria, algae and yeasts can all be their sources, and β-carotene is one of them. β-carotene ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P23/00C12R1/645
CPCC12N15/52C07K14/805C12N9/1085C12N9/90C12N9/1025C12N9/0006C12N9/1205C12N9/88C12N9/1229C12N9/93C12N15/815C12P23/00C12Y205/01032C12Y505/01019C12Y205/01029C12Y203/0301C12Y101/01034C12Y207/01036C12Y401/01033C12Y207/04002C12Y503/03002C12Y205/0101C12Y602/01001Y02E50/10
Inventor 祁庆生侯进刘萌萌张瑾
Owner SHANDONG UNIV
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