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Promoter pYLG and application thereof to construction of Candida tropicalis with high yield of long-chain dicarboxylic acid

A technology of Candida tropicalis and long-chain dibasic acid, applied in the field of bioengineering, can solve the problems of increasing the production cost of long-chain dibasic acid, unfavorable separation of long-chain dibasic acid, energy consumption, etc., and achieves high-efficiency glycerol utilization capacity , the effect of saving fermentation costs and reducing consumption

Active Publication Date: 2017-11-24
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the formation of the above-mentioned by-products is not only unfavorable for the subsequent separation of long-chain dibasic acids, but also greatly increases the production cost of long-chain dibasic acids because the synthesis of by-products consumes a lot of energy.

Method used

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  • Promoter pYLG and application thereof to construction of Candida tropicalis with high yield of long-chain dicarboxylic acid
  • Promoter pYLG and application thereof to construction of Candida tropicalis with high yield of long-chain dicarboxylic acid
  • Promoter pYLG and application thereof to construction of Candida tropicalis with high yield of long-chain dicarboxylic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Connection of resistance marker gene Kanr and expression vector pPICzαA.

[0070] The resistance marker Kanr gene fragment derived from Kanr9k was seamlessly cloned into the pPICzαA expression vector by one-step directional cloning to form the recombinant plasmid pPICzαA-Kanr.

[0071] (i) According to the Kanr sequence in the Kanr9k plasmid annotated in Genbank, two pairs of primers were designed for Kanr: (the single underline is the restriction site BamH I).

[0072] The primer nucleotide sequence of described PCR amplification is as follows:

[0073] Kanr-up: 5'-GACCTTCGTTTGTGC GGATCC TGAGGGAGCCACGGTTGAT-3',

[0074] Kanr-down: 5'-GAAAAGGGGGACGAGGATCGGTTGAGGCCGTTGAGCAC-3'

[0075] Kanr-PCR reaction system 50μl:

[0076] 2×HiFi-PCR Master 25μl, upstream primer (10μmol / L) 2μl, downstream primer (10μmol / L) 2μl, template 2μl, use ddH 2 O to make up 50 μl.

[0077] PCR reaction program:

[0078] (1) 5min at 95°C; (2) 30sec at 95°C; (3) 30sec at 56°C; (4)...

Embodiment 2

[0097] Example 2 Connection of overlapping fragment pYLG-GK and recombinant vector pPICzαA-Kanr

[0098] (i) Overlap junction of promoter gene pYLG and glycerol kinase gene GK

[0099] The promoter gene pYLG derived from Yarrowia lipolytica (Yarrowia lipolytica) and the glycerol kinase gene GK derived from Candida tropicalis were overlapped and joined to form an overlapping gene fragment pYLG-GK.

[0100] Two pairs of primers were designed for pYLG and GK respectively: (the single underline is the restriction site)

[0101] pYLG-up:5'-TTAGACCACTCTTTT GAGCTC GGTTGAAATGAATCGGCCG-3',

[0102] pYLG-down:5'-ACTACGACGTGGCATTGTTGATGTGTGTTTAATTCAAGAATG-3',

[0103] GK-up: 5'-AATTAAATTTTAACAATGCCACGTCGTAGTAGTAA-3',

[0104] GK-down:5'-GAGATGAGTTTTTGT TCTAGA GCTTTATTTTTTTTTGTTCATTAGTTCTAC-3

[0105] 50 μl of pYLG-PCR reaction system:

[0106] 2×HiFi-PCR Master 25μl, upstream primer (10μmol / L) 2μl, downstream primer (10μmol / L) 2μl, template 2μl, use ddH 2 O make up 50 μl;

[0...

Embodiment 3

[0136] Example 3 Transformation of the constructed recombinant vector pPICzαA-Kanr-pYLG-GK into Candida tropicalis primitive bacteria

[0137] The recombinant plasmid pPICzαA-Kanr-pYLG-GK obtained in Example 2 was electrically transformed into Candida tropicalis; the steps for transforming Candida tropicalis were:

[0138] Mix 80 μl of Candida tropicalis competent cells with 10 μl of the linearized plasmid pPICzαA-Kanr-pYLG-GK, and transform it into a 0.2 cm point rotor cup, and put the transformation cup containing the mixture in ice bath for 5 minutes, and adjust The parameters of the gene transfer instrument, 1500v voltage click once, the time is 5ms; immediately add 1ml of pre-cooled 1mol / L sorbic acid solution to the transformation cup and mix well, and transform it into a sterilized centrifuge tube, shake at 30°C Bed for 1 hour, apply the mixed solution on the YPD solid medium plate added with antibiotic G418, apply 200μl mixed solution on each plate, place the plate in ...

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Abstract

The invention relates to a promoter pYLG and an application thereof to construction of Candida tropicalis with high yield of long-chain dicarboxylic acid. The nucleotide sequence of the promoter pYLG is shown as SEQ ID NO.1. The invention further relates to the application of the promoter pYLG to construction of the Candida tropicalis with high yield of the long-chain dicarboxylic acid. The promoter pYLG capable of controlling expression of Candida tropicalis GK (glycerol kinase) gene in the Candida tropicalis is disclosed for the first time, and the foundation is laid for construction of engineering Candida tropicalis with GK gene regulation capacity.

Description

technical field [0001] The invention relates to a promoter pYLG and its application in constructing Candida tropicalis with high yield of long-chain dibasic acids, belonging to the technical field of bioengineering. Background technique [0002] Long chain dicarboxylic acid (Long chain dicarboxylic acid) refers to the aliphatic dicarboxylic acid containing more than 10 carbon atoms in the carbon chain, and its general formula is HOOC-(CH2)n-COOH (n=9~16), which is A class of important fine chemical products with a wide range of uses. Long-chain dibasic acid is an important fine chemical intermediate, which can be used to synthesize artificial musk, copolyamide plastic hot-melt adhesive, nylon engineering plastic, advanced nylon spray, high-temperature electrolyte, high-grade clothing hot-melt adhesive, cold-resistant plasticizer, advanced lubrication Oil, high-grade automotive paint, anti-ultraviolet coating, synthetic optical cable conductor and many other fine chemical pr...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/81C12N15/65C12N1/19C12R1/74
CPCC07K14/39C12N9/1205C12N15/65C12Y207/0103
Inventor 汪俊卿彭健王瑞明修翔薛乐王腾飞苏静杨晓慧
Owner QILU UNIV OF TECH
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