Construction method of genetic engineering strain for producing shikimic acid

A technology of genetically engineered strains and construction methods, which is applied in the field of construction of genetically engineered strains for shikimic acid production, can solve problems such as cumbersome operations, and achieve the effects of accelerating synthesis reactions, reducing catabolism, and simplifying operations

Inactive Publication Date: 2012-01-04
河南孟成生物药业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010]Most of the existing inventions interrupt the metabolism of shikimic acid by knocking out genes, and introduce shikimic acid synthesis and metabolism genes to achieve effective accumulation of shikimic acid. Knocking out more genes in host cells is relatively cumbersome to operate

Method used

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  • Construction method of genetic engineering strain for producing shikimic acid
  • Construction method of genetic engineering strain for producing shikimic acid
  • Construction method of genetic engineering strain for producing shikimic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A method for constructing a genetically engineered bacterial strain for shikimic acid production, comprising the following steps:

[0052] 1. Construction aroA The knockout Escherichia coli strain W3110 ( ?aroA );

[0053] The principle of Red recombination technology is to introduce two DNA fragments containing the homologous sequence of the target gene into the host bacterium, and under the action of recombinase, the target gene fragment is replaced by other fragments to complete the knockout of the target gene.

[0054] ）a、 Obtained by PCR amplification aroA Homologous replacement sequence DaroA;

[0055] Using plasmid pKD4 (provided by Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) as a template, using primers aroAF (see sequence 1 in the sequence listing) and aroAR (see sequence 2 in the sequence listing), PCR amplifies the can R Gene. The 5' end of the homologous recombination primer is the homologous sequence on both sid...

Embodiment 2

[0162] Example 2 Production of shikimic acid by fermentation

[0163] Will construct successful target engineering strains E. coli W3110 (? aroA ) pAR63 was activated on the slant, inoculated in LB medium, cultured to OD at 37°C, 220r / min 600 At ≈2, inoculate in a 5L fermenter, the fermenter is filled with 2.4L, the fermentation medium is: glucose 15g, yeast extract 1g, K 2 HPO 4 8g, citric acid monohydrate 2.5g, L-Phe 0.7g, L-Tyr 0.7g, L-Trp 0.7g, MgSO 4 0.24g, H 3 BO 3 0.0247g, ZnSO 4 ·7H 2 O 0.0029g, CuSO 4 ·5H 2 O 0.0025g, MnCl 4H 2O 0.0158g, p-hydroxybenzoic acid 0.010g, p-aminobenzoic acid 0.010g, 2,3-dihydroxybenzoic acid 0.010g. The fermentation temperature is 37°C, the initial stirring speed is 50r / min, and the initial ventilation rate is 1:0.1. At first, control the dissolved oxygen at 30% by controlling the speed. When the speed reaches the highest 800r / min, control the dissolved oxygen at 20% by adjusting the ventilation rate. When the ventilatio...

Embodiment 3

[0165] Example 3 Production of shikimic acid by fermentation

[0166] Will construct successful target engineering strains E. coli W3110 (? aroA ) pAR63 was activated on the slant, inoculated in LB medium, cultured to OD at 37°C, 220r / min 600 At ≈2, inoculate in a 5L fermenter, the fermenter is filled with 2.4L, the fermentation medium is glucose 15g, yeast extract 1g, K 2 HPO 4 8g, citric acid monohydrate 2.5g, L-Phe 0.7g, L-Tyr 0.7g, L-Trp 0.7g, MgSO 4 0.24g, H 3 BO 3 0.0247g, ZnSO 4 ·7H 2 O 0.0029g, CuSO 4 ·5H 2 O 0.0025g, MnCl 4H 2 O 0.0158g, p-hydroxybenzoic acid 0.010g, p-aminobenzoic acid 0.010g, 2,3-dihydroxybenzoic acid 0.010g. The fermentation temperature is 37°C, the initial stirring speed is 50r / min, and the initial ventilation rate is 1:0.1. At first, control the dissolved oxygen at 30% by controlling the speed. When the speed reaches the highest 800r / min, control the dissolved oxygen at 20% by adjusting the ventilation rate. When the ventilatio...

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Abstract

The invention discloses a construction method of a genetic engineering strain for producing shikimic acid. The method comprises the following steps of: 1, constructing an escherichia coli strain of which the aroA gene is knocked out; 2, constructing a recombinant expression plasmid pAR63 containing key enzyme genes aroGFBR, aroE, aroB, aroD, tktA and ppsA in the metabolic pathway of the shikimic acid, so that the genes are subjected to transcriptional control of a tryptophan promoter Ptrp; and 3, transferring the recombinant expression plasmid pAR63 into the escherichia coli strain of which the aroA gene is knocked out to obtain a production strain for expressing the shikimic acid. In the method, the aim of interrupting the metabolism of the shikimic acid is fulfilled by the knock-out of the single gene aroA, a small number of genes are knocked out, the operating difficulty is low, and the expression of the genes is in the state of starting and stopping automatically and is started automatically in the middle and late period without the induction of inducers, so the possibility of adding toxic substances into a culture medium is reduced, and the shikimic acid has high yield.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for constructing a genetic engineering bacterial strain for shikimic acid production, and the bacterial strain can be used to ferment and produce shikimic acid. Background technique [0002] Shikimic acid (Shikimate), chemical name [3R-(3α,4α,5β)]-3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid, is the intermediate of metabolic process in organisms The product is also the raw material for the synthesis of many alkaloids, aromatic amino acids and indole derivatives, chiral drugs (such as antiviral drugs), and widely exists in plants and microorganisms. [0003] Shikimic acid affects the metabolism of arachidonic acid, inhibits platelet aggregation, and inhibits the formation of arterial and venous thrombosis and cerebral thrombosis. Shikimic acid not only has anti-inflammatory and analgesic effects, but also can be used as an intermediate of antiviral and a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12R1/19
Inventor 王春阳张蕴才武广君孟军虎任存邦董建辉石玲珑李晓霞乔娟平叶晓冲王娜
Owner 河南孟成生物药业股份有限公司
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