[Beta]-carotenoid ketolase mutant, recombinant expression vector, genetic engineering bacterium and application thereof

A technology of carotene ketolase mutant and carotene ketolase, which is applied in the field of genetic engineering and enzyme engineering, can solve the problems of low enzyme activity and non-specific effect, and achieve the effect of improving catalytic activity

Active Publication Date: 2017-03-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the substrates of these enzymes derived from plan

Method used

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  • [Beta]-carotenoid ketolase mutant, recombinant expression vector, genetic engineering bacterium and application thereof
  • [Beta]-carotenoid ketolase mutant, recombinant expression vector, genetic engineering bacterium and application thereof
  • [Beta]-carotenoid ketolase mutant, recombinant expression vector, genetic engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Molecular cloning and expression of β-carotene ketolase mutant gene

[0036] 1.1 Materials and methods

[0037] 1.1.1 Reagent and medium formula

[0038] High Fidelity Enzyme DNA Polymerase Prime STAR TM HS DNA polymeras, T4 DNA ligase, and DNA restriction endonuclease were purchased from Takara Company, Easy Taq DNA polymerase was purchased from Beijing Quanshijin Biotechnology Co., Ltd., DNAmarker (1kb DNAL ladder) (Thermo Scientific), PCR product purification reagents Kit, DNA gel purification kit (Axygen, Hangzhou) and other molecular biology kits are used for gene cloning and other operations; PCR primer synthesis and sequencing services are provided by Shanghai Bioengineering Co., Ltd. or Boshang Biotechnology Co., Ltd.

[0039] Amino yeast nitrogen base (Yeast nitrogen base without amino acids, YNB) (Bidey Medical Devices, Shanghai) was used for the preparation of synthetic medium; astaxanthin standard was provided by Zhejiang Xinhecheng Co., Ltd., c...

Embodiment 2

[0075] Example 2 Construction and linearization of Saccharomyces cerevisiae expression vector comprising β-carotene ketolase mutant

[0076] 2.1 Expression vector construction

[0077] The plasmid containing the positive mutant obtained in Example 1.2 was digested with NotI and SacI to obtain the PUMRI-11 plasmid (NCBI number: KM216413 ) connection for conversion.

[0078] Double enzyme digestion system: total system 20μl

[0079] Gene / plasmid: 17μl

[0080] 10×buffer: 2μl

[0081] NotI enzyme: 0.5 μl

[0082] SacI enzyme: 0.5 μl

[0083] Digestion conditions: place at 37°C, digest for 1.5-2 hours

[0084] Connection system:

[0085] Gene: 5.5 μl, plasmid 3 μl, 10×T4 DNA ligase buffer 1 μl, T4 DNA ligase; 0.5 μl.

[0086]Transformation of competent cells: Add 10 μl of the ligation product to 100 μl of competent cells melted on ice, mix gently with a pipette gun, and place in an ice bath for 20-40 minutes; heat shock at 42°C for 90 seconds, and quickly place in ice. Ic...

Embodiment 3

[0091] Example 3 Integration of β-carotene ketolase mutants into the staining of Saccharomyces cerevisiae producing β-carotene for further verification

[0092] The recombinant linearized plasmid obtained in Example 2 was integrated into the chromosome of Saccharomyces cerevisiae producing β-carotene according to the method in Example 1.1.3, and spread on the YPD plate containing G418 resistance. The mutants OBKTM1, OBKTM2, and OBKTM3 showed increased activity and promoted the conversion of β-carotene to astaxanthin. Compared with wild-type results such as image 3 shown.

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Abstract

The invention provides a [beta]-carotenoid ketolase mutant, which is obtained through a directed evolution means, nucleotide sequences for encoding the mutant, a recombinant expression vector containing a mutant gene, expression in saccharomyces cerevisiae and application of a genetic engineering bacterium in the synthesis of keto-carotenoids. According to the invention, a mutant strain with catalytic activity on [beta]-carotenoid enhanced is screened by virtue of a high-throughput screening method, and through gene sequencing analysis, mutant sites are subjected to combined mutation, so that the catalytic activity is further enhanced; and the screened mutant strain is applied to the production of saccharomyces cerevisiae keto-carotenoids.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a β-carotene ketolase mutant derived from a strain of Haematococcus pluvialis obtained by directed evolution, a nucleic acid sequence encoding it, and a gene containing the mutant Recombinant expression vector, expression in Saccharomyces cerevisiae and its application in ketocarotenoid synthesis. Background technique [0002] Keto carotenoids are an important class of natural pigments that exist in plants, algae, marine bacteria and fungi. They belong to C40 isoprene compounds, which contain many conjugated double bonds and unsaturated rings on the terminal six-membered ring. The ketone group can effectively quench the singlet oxygen and has a strong antioxidant capacity. Therefore, it has a variety of physiological and nutritional functions, and can be widely used in various industries such as food, medicine, health care products and cosmeti...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P23/00C12R1/865
CPCC12N9/0069C12N15/81C12P23/00
Inventor 于洪巍周萍萍叶丽丹
Owner ZHEJIANG UNIV
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