Engineering bacteria and application thereof

A technology for engineering strains and genetically engineered bacteria, applied in the field of bioengineering, can solve the problems of high cost of racemic α-hydroxycarboxylic acid and expensive substrates, etc.

Active Publication Date: 2017-10-27
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Chinese patent 201610080101.3 discloses a method for producing optically pure 2-hydroxyacid by treating racemic α-hydroxycarboxylic acid with multiple enzymes, but the cost of chemically synthesized racemic α-hydroxycarboxylic acid is relatively high
Rantwijka reviewed the Enzymatic cascadesynthesis of (S)-2-hydroxycarboxylic amides and acids: Cascade reactions employing a hydroxynitrole lyase, nitrogen-converting enzymes and an amidase, Journal of Molecular Catalysis B: Enzymatic, 2015, 114, 25-30), but the substrates of this method are relatively expensive

Method used

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  • Engineering bacteria and application thereof
  • Engineering bacteria and application thereof
  • Engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Construction of three-gene co-expression system.

[0043] (1) Primer design

[0044] Design primers, the primer sequences are shown in Table 1

[0045] Table 1 Primers used for gene amplification

[0046]

[0047]

[0048] (2) PCR amplification

[0049]According to the instructions provided by the manufacturer, Genomic DNA Purification Kit (Takara) was used to extract the genomic DNA of the strains in logarithmic growth phase, and the primers in Table 1 were used for PCR amplification from the corresponding strains. Amplification system: PrimeSTAR HS DNA Polymerase (2.55U / μL) 0.5μL, 10×PrimeSTAR Buffer 10μL, dNTP Mixture (2.5mM each) 4μL, template DNA 1μL, Up primer (20μM) 1μL, Down primer (20μM) 1μL ,ddH 2 O to make up to 50 μL. The amplification program is: 94°C, 10min; 94°C, 30sec; 55°C, 30sec; 72°C, 2min, a total of 30 cycles; 72°C, 10min. PCR products were sent to Huada Genomics for confirmation.

[0050] From Lactobacillus plantarum ATCC 8041, Enteroc...

Embodiment 2

[0067] Induced expression of the genetically engineered bacteria obtained in Example 1.

[0068] Pick the single bacterium colony of genetically engineered bacteria that has been constructed and inoculate in 10mL LB medium (containing 0.1g / L ampicillin), cultivate at 37 ℃ for 12 hours, inoculate in LB medium (200mL of 1000mL shake flask liquid) at a volume ratio of 2%. , containing 0.1g / L ampicillin), continue to cultivate at 37°C for 2.5hr, until the bacterial logarithmic growth phase (OD 600 0.6-0.8), add IPTG to a concentration of 0.4mM, and culture at 20°C and 200rmp for 8h. After induction of expression, the cells were collected by centrifugation at 20° C., 8000 rpm, and 20 minutes. According to the amount of cells required for transformation, the number of shake flasks can be increased to obtain sufficient cells.

Embodiment 3

[0070] According to the induced expression method described in Example 2, the recombinant E. coli M15 / pQLinkN-cmaao-lpldhd-bsgdh was induced and expressed, and the bacterial cells were collected. In a reaction volume of 100ml, the transformation of different substrates mixed with whole cells was investigated. , the final concentration of the substrate is 0.5g / L, the glucose concentration is 10g / L, the pH is adjusted to 7.0, the weight of fresh whole cells is added to 20g (wet weight), the temperature is 30°C, and the results are measured after 24 hours of transformation. Various substrates The responses are shown in the table below.

[0071] Table 2 Transformation of different substrates by E. coli M15 / pQLinkN-cmaao-lpldhd-bsgdhcf

[0072]

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Abstract

The invention discloses escherichia coli genetically engineered bacteria co-expressed by three enzymes. The escherichia coli genetically engineered bacteria are characterized in that an L-amino-acid oxidase gene, a (D/L)-alpha-hydroxyl carboxylic acid dehydrogenase gene and a gene capable of reducing NAD(P) are imported. The invention also discloses a construction method and an application of the recombinant escherichia coli. The method is used to biologically synthesize optically pure alpha-hydroxyl carboxylic acid, has the characteristics of simple operation, low cost, high product synthesizing efficiency and high optical purity, and has a good industrial prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the construction of an Escherichia coli genetically engineered bacterium co-expressing three enzymes, and the application of the engineered bacterium in the production of α-hydroxycarboxylic acid. Background technique [0002] α-hydroxycarboxylic acid (α-hydroxy acid, 2-hydroxy acid, α-hydroxy-carboxylic acid, 2-hydroxy-carboxylic acid) widely exists in nature, and its structure is characterized by a hydroxyl group at the C position on the carboxyl side. Optically pure α-hydroxycarboxylic acids are important fine chemical intermediates and chiral drug precursors. [0003] Usually, α-keto carboxylic acids (α-keto carboxylic acids) can be used as substrates for chemical chiral hydrogenation to synthesize α-hydroxy carboxylic acids, or enzymatic synthesis (Chinese patent 201410818165.X) α-hydroxy carboxylic acids , but α-keto carboxylic acid and chiral catalyst a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/42C12R1/19
CPCC12N9/0004C12N9/0006C12N9/0008C12N9/0022C12P7/42C12Y101/9901C12Y102/01002C12Y104/03002C12Y120/01001
Inventor 蔡宇杰熊天真刘金彬丁彦蕊白亚军郑晓晖
Owner JIANGNAN UNIV
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