Recombinant escherichia coli and application thereof to 2-butanol production

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

Active Publication Date: 2016-01-27
SHANDONG UNIV
View PDF2 Cites 4 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant escherichia coli and application thereof to 2-butanol production
  • Recombinant escherichia coli and application thereof to 2-butanol production
  • Recombinant escherichia coli and application thereof to 2-butanol production

Examples

Experimental program
Comparison scheme
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 bacterial strain Candida boidinii (Candidaboidinii) NCYC1513 is prepared by a conventional method, and this process can refer to a small amount of genome in "Guide to Molecular Biology" published by Science Press. The preparation method is to extract the genomic DNA of CandidaboidiniiNCYC1513; use synthetic primers to amplify the formate dehydrogenase gene fdh from the genomic DNA of CandidaboidiniiNCYC1513 by PCR;

[0043] CandidaboidiniiNCYC1513 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 AAGATCGTTTTAGTCTTATATGATGCTG...

Embodiment 2

[0052] Example 2: Preparation of Whole Cell Catalyst

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

[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 ...

Embodiment 3

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/16C12R1/19
Inventor 高超褚海培马翠卿许平
Owner SHANDONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap