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Recombinant Escherichia coli construction and (S)-2-hydroxy-3-phenylpropionic acid synthesis method

A technology for recombining Escherichia coli and Bacillus megaterium, applied in the biological field, can solve the problems of inability to produce L-PLA and low production efficiency of synthetic PLA, and achieve the effects of increasing yield and yield, wide application, and simple culture conditions

Inactive Publication Date: 2015-09-02
CHANGSHU INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first object of the present invention is to provide a kind of L-lactate dehydrogenase gene, solve the problem that the production efficiency of PLA synthesis of Bacillus bacterium is low at present, can only catalyze and produce D-PLA, but can not produce the problem of L-PLA

Method used

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  • Recombinant Escherichia coli construction and (S)-2-hydroxy-3-phenylpropionic acid synthesis method
  • Recombinant Escherichia coli construction and (S)-2-hydroxy-3-phenylpropionic acid synthesis method
  • Recombinant Escherichia coli construction and (S)-2-hydroxy-3-phenylpropionic acid synthesis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This example illustrates recombinant E. coli E. coli Construction of BL21(DE3) / pET-28a-ldhL.

[0036] 1. L-lactate dehydrogenase derived from Bacillus megaterium Bacillus megaterium Z2013513 (CCTCC NO: M2013244), optimizes the codons of the L-LDH gene according to the codon preference of Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO:1.

[0037] 2. The codon-optimized L-lactate dehydrogenase gene was obtained by artificial synthesis, and the upstream and downstream primers were designed. The primer sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3. After PCR amplification, the nucleic acid agarose gel electrophoresis gel was recovered, connected to the pMD19-T vector, and the cloning vector was transformed into E. coli JM109 gets E. coli JM109-pMD-ldhL, the recombinant plasmid was extracted for double enzyme digestion verification, and the verified positive clones were E. coli The nucleotide sequence of JM109-pMD-ldhL was sequenced. ...

Embodiment 2

[0039] This example illustrates the preparation of recombinant E. coli whole cells.

[0040] (1) Strain activation: pick E. coli A single colony of BL21(DE3) / pET-28a-ldhL was inoculated into 10 mL LB liquid medium (containing 50 mg / L kanamycin), and cultured overnight at 37 °C and 200 r / min.

[0041] (2) Seed culture: Add the culture solution obtained in the above step 1) into 100 mL LB medium (containing 50 mg / L kanamycin) according to the inoculum amount (volume fraction) of 1%, at 37 °C, 200 r / min cultured cell OD 600 It is 0.4~1.0.

[0042](3) Induced expression: IPTG was added to the bacterial liquid obtained in the above 4), and cultured at 25 °C, 200 r / min for 4-6 h to the mid-log phase of cell growth, and the obtained culture liquid was incubated at 4 °C, 6000 r / min Centrifuge for 5 min, wash twice with sterilized sodium phosphate buffer (0.1 mol / L, pH=7.0) to obtain resting cells.

Embodiment 3

[0044] This example illustrates whole cell batch catalytic synthesis of L-PLA.

[0045] (1) Transformation of whole cells: suspend the cells obtained in Example 2 above with 5 ml of transformation solution, and pour into a 100 ml Erlenmeyer flask. The transformation solution contained 0.1 mol / L sodium phosphate buffer and 100 mmol / L glucose. After the bacteria slime is fully and evenly suspended in the transformation solution, add 5 ml of 70 mmol / L PPA solution to obtain a final concentration of 35 mmol / L, mix well and carry out the transformation reaction on a shaker at a reaction temperature of 37 °C and 200 r / min Shake the reaction for 60 min.

[0046] (2) Centrifuge the reaction conversion liquid obtained in the above step 1) at 4 °C and 10000 r / min for 2 min, then take the supernatant, and use high performance liquid chromatography to measure the content of L-PLA. The detection conditions are: chiral column OJ -RH, mobile phase (volume ratio): acetonitrile: methanol: tr...

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Abstract

The invention relates to an L- lactate dehydrogenase gene, and the nucleotide sequence represents as SEQ ID NO. 1. The invention further provides an expression vector with the gene, a recombinant Escherichia coli, a recombinant Escherichia coli construction method and the application thereof during phenylpyruvic acid conversion and (S)-2-hydroxy-3-phenylpropionic acid synthesis. The method includes after performing codon optimization on (Bacillus megaterium Z2013513) CCTCC NO. M2013244 L- lactate dehydrogenase gene, constructing the expression vector, inducing expression of the Escherichia coli, and obtaining the L-PLA production strain with simple culture condition, convenience for usage and wide application. The yield for the recombinant Escherichia coli converting the L-PLA in whole-cell and single-batch manners is 27.13mmol / L, the converting rate is 77.5%, and the basis is provided for increasing the L-PLA yield and producing rate for subsequent fermentor continuous feeding.

Description

Technical field [0001] The present invention is a biotechnology field, which specializes in the construction of a reorganization of E. coli and its synthesis (S) -2-hydroxyl-3-benzal propyate. Background technique [0002] Phenyl lactic acid (PLA), that is, 2-hydroxyl-3-benzaline, 3-benzene lactate or β-phenyl acid.PLA is a pale yellow powder with a slightly special smell, molecular formula C 9 H 9 O 3 The relative molecular weight is 166, and the melting point is 121 ~ 125 ° C.PLA is a new type of natural preservatives discovered in recent years. It exists in lactic acid bacteria fermented products and honey. It is a metabolic product of phenylalanine in a variety of microorganisms (mainly lactic acid bacteria).The study of aromatic organic acids generated by the metabolism of lactic acid bacteria recognized as safe has shown non -toxic to humans and animal cells.PLA is the first generation of bacteriostatic substances such as lactic acid bacteria, such as lactic acid, acetic ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/70C12N1/21C12P7/42C12R1/19
Inventor 朱益波王立梅齐斌王颖梁剑光
Owner CHANGSHU INSTITUTE OF TECHNOLOGY
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