Recombinant escherichia coli of temperature-control coexpression exogenous gene and application thereof

A technology for recombining Escherichia coli and exogenous genes, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of toxic substances, low conversion rate of whole cells, recombinant catalysts that are not suitable for large-scale preparation, etc.

Inactive Publication Date: 2010-10-27
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0025] In view of the above existing catalytic synthesis of N-acetylneuraminic acid, the recombinant catalyst is not suitable for large-scale preparation, and the defects of toxic substances will be introduced during the preparation process, and the conversion rate of whole cells is low, the cost is high, post-processing is troublesome, and it is difficult to scale up. Insufficient scale production, the problem to be solved in the present invention is to provide a strain of recombinant Escherichia coli, that is, a strain of recombinant Escherichia coli that co-expresses foreign genes under temperature control and its application in the preparation of N-acetylneuraminic acid

Method used

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  • Recombinant escherichia coli of temperature-control coexpression exogenous gene and application thereof
  • Recombinant escherichia coli of temperature-control coexpression exogenous gene and application thereof
  • Recombinant escherichia coli of temperature-control coexpression exogenous gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Construction of recombinant genetic engineering strain Escherichia coli (Escherichia coli) DT26 (pBVNsS) CCTCC NO: M 209018

[0076] 1. Cloning of N-acetylneuraminic acid aldolase gene (nanA)

[0077] The genomic DNA of bacterial strain Escherichia coli K12 ATCC 25404 was prepared by a conventional method, and this process can be referred to the method for small-scale preparation of bacterial genome in "Guide to Molecular Biology" published by Science Press; using synthetic primers from Escherichia coli K12 The N-acetylneuraminidase gene nanA was obtained by PCR amplification from the genomic DNA. The fragment was recovered after double digestion with EcoRI and PstI, and connected to the recovered plasmid pBV220 treated with the same enzymes (see Acta Virus Sinica, Vol. 6, No. 2, pp. 111-116).

[0078] Among them, the above-mentioned Escherichia coli K12ATCC 25404 is used as the source bacterium of the nanA gene, and according to the genome sequence of the b...

Embodiment 2

[0116] Example 2: Application of Whole Cell Catalysts in the Synthesis of N-Acetylneuraminic Acid

[0117](1) Plate culture: Streak the above-mentioned Escherichia coli (Escherichia coli) DT26 (pBVNsS) CCTCC NO: M 209018 strain on an ampicillin LB plate containing 1.5% agar with a mass volume ratio of 100 μg / ml, and culture at 37°C 12 hours.

[0118] (2) First-class seeds: under aseptic conditions, pick a single colony on the flat plate of step (1) with a sterile toothpick, then inoculate into 5 ml of liquid medium containing 100 μg / ml ampicillin, 30 Cultivate on a shaker for 12 hours.

[0119] (3) Shake flask culture: under aseptic conditions, take the culture solution cultivated in step (2) as an inoculum size of 1% by volume, and inoculate it into 30 ml of LB liquid medium containing 100 μg / ml of ampicillin, Incubate on a rotary shaker at 30°C at 200 rpm for 2 hours.

[0120] (4) Induction: the shaker flask was moved to a shaker at 45° C., and induced at 180 rpm for 4 ho...

Embodiment 3

[0131] Example 3: Application of Whole-Cell Catalysts in the Synthesis of N-acetylneuraminic Acid

[0132] (1) Plate culture: Streak the Escherichia coli (Escherichia coli) DT26 (pBVNsS) CCTCC NO: M 209018 strain on an ampicillin LB plate containing 1.5% agar with a mass volume ratio of 100 μg / mL, culture at 37°C for 12 Hour.

[0133] (2) First-class seeds: under aseptic conditions, pick a single colony on the plate of step (1) with a sterile toothpick, and then inoculate it into 5 ml of liquid medium containing 100 μg / ml ampicillin, 37 Cultivate on a shaker for 12 hours.

[0134] (3) Secondary seed: under aseptic conditions, get the nutrient solution cultivated in step (2) with a volume ratio of 1% inoculum, inoculate to 500ml LB inorganic salt liquid medium containing ampicillin of 100 μg / ml Incubate at 30°C for 12 hours with shaking.

[0135] (4) Fermentation tank culture: under sterile conditions, take the culture solution obtained in step (3) and inoculate 5 L of the a...

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Abstract

The invention discloses recombinant escherichia coli of a temperature-control coexpression exogenous gene, which is named as Escherichia coli DT26 (pBVNsS) CCTCC NO:M 209018. The genotype of the recombinant escherichia coli is delta nagE::FRT; and the recombinant escherichia coli contain a temperature-control coexpression carrier pBVNsS. Simultaneously, the invention also discloses the application of the recombinant escherichia coli used as a catalyst for the complete cell catalytic preparation of N-acetylneuraminic acid. In the process of the large-scale catalytic production of the N-acetylneuraminic acid, the recombinant escherichia coli of the temperature-control coexpression exogenous gene has the characteristics of safe and convenient preparation of the catalyst, simple operation of the catalytic process, cheap price, higher efficiency, easy extraction and the like.

Description

technical field [0001] The invention relates to a strain of genetically recombined Escherichia coli and its application, in particular to a strain of recombinant Escherichia coli with temperature-controlled co-expression of exogenous genes and its application in the preparation of N-acetylneuraminic acid. Background technique [0002] Sialic acid (Sialic acid) is the general term for neuraminic acid and its derivatives. It is a class of nine-carbon amino sugars containing carboxyl groups. Its biological functions are closely related to important physiological and pathological phenomena, and it has broad application prospects.【Traveling , 1998; Schauer, 2000]. N-acetylneuraminic acid (N-acetyl-D-neuraminicacid, Neu5Ac) is the most widely distributed type of sialic acid, and itself and its analogues have important medicinal prospects. Among them, the structural analogue of N-acetylneuraminic acid, Zanamivir (Zanamivir), was approved for commercialization in 1995 as an anti-in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/26C12R1/19C12N1/21
Inventor 许平张奕南马翠卿
Owner SHANDONG UNIV
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