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A recombinant Escherichia coli and its application in the production of 2-butanol

A technology for recombining Escherichia coli and butanol, applied in the directions of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of low product productivity, difficulty in large-scale production, and high cost, and achieve the effects of low cost, low cost and convenient operation.

Active Publication Date: 2019-04-16
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] In the above existing methods, the productivity of the product is low, the cost is high, and it is difficult to produce on a large scale.

Method used

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  • A recombinant Escherichia coli and its application in the production of 2-butanol
  • A recombinant Escherichia coli and its application in the production of 2-butanol
  • A recombinant Escherichia coli and its application in the production of 2-butanol

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Construction of Escherichia coli co-expressing formate dehydrogenase gene fdh and alcohol dehydrogenase gene adh

[0042] (1) Cloning of gene fdh: Genomic DNA of strain Candida boidinii (Candidaboidinii) NCYC 1513 was prepared by conventional methods. Quantitative preparation method, extracting the genomic DNA of Candida boidinii NCYC 1513; using synthetic primers to PCR amplify the formate dehydrogenase gene fdh from the genomic DNA of Candida boidinii NCYC 1513;

[0043] Candida boidinii NCYC 1513 is used as the source bacterium of the fdh gene. According to the genome sequence of the bacterium that has been sequenced, primers are designed to introduce NdeI and XhoI restriction enzyme sites that can be inserted into the plasmid pETDute-1 (Novagen). The primer sequences are as follows:

[0044] Upstream primer 5'- CATATG AAGATCGTTTTAGTCTTATATGATGCTGGTA-3', carrying an NdeI site;

[0045] Downstream primer 5'- CTCGAG TTATTTCTTATCGTGTTTACCGTAAGCTTTG-3', c...

Embodiment 2

[0052] Example 2: Preparation of Whole Cell Catalyst

[0053] (1) Plate culture: Streak Escherichia coli (Escherichia coli) BL21 / pETDuet-fdh-adh on an LB plate containing 1.5-1.8% agar by mass volume ratio and 100 μg / mL ampicillin, culture at 37±1°C 12±1 hours;

[0054] (2) First-class seeds: under sterile conditions, pick a single colony on the flat plate of step (1) with a sterile toothpick, and then inoculate it into 5 mL of LB liquid medium containing 100 μg / mL ampicillin, Shaking culture at 37±1°C for 12±1 hours;

[0055] (3) Secondary seeds: under aseptic conditions, take the bacterial solution cultivated in step (2) and inoculate 100 mL of LB liquid culture medium containing 100 μg / mL of ampicillin with a volume ratio of 1 to 2%. medium, 37±1°C shaker culture for 12±1 hours;

[0056] (4) Shake flask culture: under aseptic conditions, take the bacterial solution obtained in step (3) and inoculate it into 1 L of LB liquid medium with an inoculum volume ratio of 5 to 10...

Embodiment 3

[0059] Embodiment 3: utilize the biocatalyst that embodiment 2 obtains to prepare 2-butanol

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Abstract

The invention discloses recombinant escherichia coli. The recombinant escherichia coli is named Escherichia coli BL21 / pETDuet-fdh-adh and contains formate dehydrogenase fdh and alcohol dehydrogenase adh, wherein the nucleotide sequence of the fdh gene is as shown in SEQ ID No. 1, and the nucleotide sequence of the adh gene is as shown in SEQ ID No. 2. The recombinant escherichia coli is preserved in China Center for Type Culture Collection on September 23rd, 2015, and the preservation number of the recombinant escherichia coli is CCTCC No. M2015572. The invention further discloses application of the recombinant escherichia coli to 2-butanol production which uses the recombinant escherichia coli as a catalyst to catalyze 2-butanone to produce 2-butanol. Experiments prove that the concentration of the 2-butanol produced by using the recombinant escherichia coli can reach above 18.2 g / L (conversion rate reaches 0.68g / g), cofactor regeneration is achieved, and promising industrial application prospect is achieved.

Description

technical field [0001] The present invention relates to a gene recombinant bacteria and its application, specifically, relates to a recombinant Escherichia coli co-expressing formate dehydrogenase (FDH) gene fdh and alcohol dehydrogenase (ADH) gene adh and its ability to catalyze 2- The application of butanone in the production of 2-butanol. Background technique [0002] 2-Butanol is a colorless transparent liquid with wine aroma. Miscible with various organic solvents such as alcohols, esters, ethers, aromatics, etc., it is a valuable biofuel with high octane number, low hygroscopicity, higher energy density than ethanol, and the same as gasoline The function of [1]. When 2-butanol is converted to 2-butene, it can be used in the synthesis of polyesters and other bioplastics [2,3]. [0003] The reported production methods of 2-butanol are divided into traditional chemical method and biological method. The chemical method usually adopts the n-butene hydration method [4], ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P7/16C12R1/19
Inventor 高超褚海培马翠卿许平
Owner SHANDONG UNIV
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