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Engineering bacterium capable of co-producing long-chain dicarboxylic acid and 1,3-propylene glycol and building method thereof

A technology of propanediol dehydrogenase and Clostridium butyricum, applied in the field of bioengineering, can solve the problems of simultaneous production without fermentation

Active Publication Date: 2017-05-10
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although there are now separate fermentation schemes for Candida tropicalis to produce long-chain dibasic acids and Klebsiella to produce 1,3-propanediol, there is no simultaneous production of long-chain dibasic acids and 1,3-propanediol by fermentation of the same strain. Propylene Glycol Related Reports

Method used

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  • Engineering bacterium capable of co-producing long-chain dicarboxylic acid and 1,3-propylene glycol and building method thereof
  • Engineering bacterium capable of co-producing long-chain dicarboxylic acid and 1,3-propylene glycol and building method thereof
  • Engineering bacterium capable of co-producing long-chain dicarboxylic acid and 1,3-propylene glycol and building method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] A method for preparing a recombinant plasmid, comprising the steps of:

[0153] (1) Using the genome of Candida tropicalis as a template, carry out PCR amplification to obtain the promoter pGAPg fragment, and the electrophoresis results are as follows: figure 1 shown;

[0154] The primer nucleotide sequence of described PCR amplification is as follows:

[0155] pGAPg-up:5'- AGATCT AAGAAGAATTTAGTGGTTAGTCG-3',

[0156] pGAPg --down:5'-ATCCTTACTTATCATTGTTAAAATTTAATTTGTAAGTGATTTG-3';

[0157] The single underline is the enzyme cutting site Bgl II;

[0158] The amplification system of the PCR amplification is as follows, the total volume of the system is 50 μl:

[0159] 2×HiFi-PCR Master 25μl, 10μmol / L upstream primer 2μl, 10μmol / L downstream primer 2μl, template 2.5μl, use ddH 2 O supplemented to 50 μl;

[0160] The amplification procedure of described PCR amplification is as follows:

[0161] Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, ann...

Embodiment 2

[0282] A preparation method for transforming oil and co-producing long-chain dibasic acid and 1,3-propanediol Candida tropicalis engineering bacteria, comprising the following steps:

[0283] (i) preparing Candida tropicalis competent cells;

[0284] The preparation of described Candida tropicalis competent cell can adopt the conventional technique in this field, also can follow the steps as follows:

[0285] ① Inoculate Candida tropicalis on a YPD solid medium plate, culture at 30°C overnight, inoculate a single colony on the plate into 25ml of YPD liquid medium, and culture in a shaker at 30°C and 220rpm / min for 20 hours to obtain a seed solution ;

[0286] ② Take 0.5ml of the above seed solution and inoculate it into 25ml of YPD liquid medium, and culture it on a shaker at 220rpm / min at 30°C for 8h, so that the OD 600 Between 1.3 and 1.5, the expanded culture medium was prepared;

[0287] ③ Take 1ml of expanded culture medium into a 1.5ml EP tube, centrifuge at 3000rpm f...

Embodiment 3

[0297] Example 3 Construction and Fermentation Verification of Candida Tropicale Engineering Bacteria

[0298] The Candida tropicalis engineering bacterium constructed and obtained in Example 2 was inoculated in YPD medium, and the culture condition was 30° C. shaker for 12 hours, and then transferred to the Candida tropicalis fermentation medium at a ratio of 10% by volume, at 30 ℃ shaking table fermentation verification.

[0299] The YPD medium components are as follows:

[0300] Peptone 20g, glucose 20g, sodium chloride 10g, water to 1L.

[0301] The fermentation medium components are as follows:

[0302] Glucose 62g, ammonium sulfate 1g, yeast powder 2g, vitamin B1 0.2g, sodium chloride 2g, potassium dihydrogen phosphate 8g, disodium hydrogen phosphate 10g, urea 3g, magnesium sulfate 6g, water to 1L.

[0303] The yield of long-chain dibasic acid after fermentation by Candida tropicalis engineered bacteria was 10g / L, and the yield of 1,3-propanediol produced by convertin...

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Abstract

The invention relates to an engineering bacterium capable of co-producing long-chain dicarboxylic acid and 1,3-propylene glycol and a building method thereof. The engineering bacterium is prepared by transforming a recombinant plasmid. The recombinant plasmid is prepared by the following steps: performing PCR (Polymerase Chain Reaction) amplification to obtain a glycerol dehydratase gene, and connecting the glycerol dehydratase gene with a promoter pGAPg obtained by PCR amplification in an overlapping way to obtain a pGAPg-GD segment; performing PCR amplification to obtain a 1,3-propanediol dehydrogenase gene, connecting the 1,3-propanediol dehydrogenase gene with the promoter pGAPp obtained by the PCR amplification in the overlapping way to obtain a pGAPp-PDE segment; performing PCR amplification to obtain a terminator gene, and preparing a two-way terminator segment; separately connecting the segments to obtain the recombinant plasmid. By adopting the engineering bacterium built by the method, co-expression of a plurality of genes is realized; moreover, the yield of the long-chain dicarboxylic acid is 10g / L, and the yield of the 1,3-propylene glycol generated by transforming glycerine is 13g / L, being remarkably higher than current reported values.

Description

technical field [0001] The invention relates to an engineering bacterium for co-producing long-chain dibasic acid and 1,3-propanediol and a construction method thereof, in particular to a project of Candida tropicalis for converting oil and co-producing long-chain dibasic acid and 1,3-propanediol The invention relates to bacteria and its construction method and application, belonging to the technical field of bioengineering. Background technique [0002] Long-chain dibasic acids refer to straight-chain aliphatic dicarboxylic acids with more than 10 carbon atoms in the carbon chain. An important fine chemical intermediate, which can be used to synthesize a series of high-value-added special chemicals such as spices, special nylon, and polyamide hot-melt adhesives. At present, there are two main methods for producing long-chain dibasic acids at home and abroad: chemical method and fermentation method . Compared with the microbial fermentation method, the production of long-c...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/81C12P7/18C12P7/64C12R1/74
CPCC12N15/66C12N15/81C12N2800/102C12P7/18C12P7/6409
Inventor 汪俊卿郭维伟彭健王瑞明杨晓慧石莹程成修翔
Owner QILU UNIV OF TECH
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