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Recombinant escherichia coli and application to synthesis of tetrahydropyrimidine

A technology for recombining Escherichia coli and tetrahydropyrimidine is applied in the fields of genetic engineering and biology, and can solve the problems of difficulty in advancing the research progress of tetrahydropyrimidine, low yield of tetrahydropyrimidine and tetrahydropyrimidine, and high cost of chemical synthesis

Inactive Publication Date: 2019-01-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the chiral structure of ectoine and the extremely low yield of ectoine that can be isolated initially, and the high cost of chemical synthesis, it is difficult to be directly applied to commercial applications, making it difficult to advance the research progress of ectoine

Method used

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  • Recombinant escherichia coli and application to synthesis of tetrahydropyrimidine
  • Recombinant escherichia coli and application to synthesis of tetrahydropyrimidine
  • Recombinant escherichia coli and application to synthesis of tetrahydropyrimidine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of Escherichia coli ectoine metabolic pathway

[0026] 1. Knockout of lysA gene

[0027] Gene manipulation method using Red homologous recombination

[0028] According to the lysA gene sequence and pKD3 plasmid sequence of E. coli MG1655, primers lysA-1 and lysA-2 were designed to amplify the resistant fragment using pKD3 as a template. The program of PCR amplification: 30 cycles, denaturation at 98°C for 30 sec, annealing at 55°C for 15 sec, extension at 72°C for 30 sec.

[0029] Table 1 PCR amplification system

[0030]

[0031] Table 2 Primer Nucleic Acid Sequence

[0032]

[0033] Electrotransform the amplified resistant fragment (see SEQ ID NO.2 for the nucleotide sequence) into E.coli MG1655 containing the PkD46 plasmid, and spread it on a plate containing chloramphenicol resistance (30 μg / mL) , Colony PCR identification and screening of positive transformants, that is, the lysA knockout strain E.coli MG1655-1 was obtained.

[00...

Embodiment 2

[0042] Embodiment 2: Engineering bacterium E.coli Hect fermentation preparation ectoine

[0043] 1. Fermentation of engineering bacteria E.coli Hect

[0044] The engineered bacteria E.coli Hect, E.coliMG1655-1-pTrc99a, and E.coli MG1655 were inoculated in the seed medium for shaker culture respectively. The culture conditions: temperature 37°C, rotation speed 200rpm, culture time 12h to logarithmic growth period to obtain seed solution. Seed medium composition: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, solvent is deionized water, pH7.0.

[0045] The seed solution was added to the fermentation medium with an inoculation amount of 10% volume concentration, and the culture temperature was 37 ° C, 200 rpm shaking culture until the OD600 was 0.6, 50 mg / mL inducer IPTG was added, and the culture temperature was 30 ° C, 200 rpm shaking culture for 12 hours, Obtain a fermented broth. After the fermentation, the fermentation broth was centrifuged at 5000 rpm at 4° C...

Embodiment 3

[0050] Embodiment 3: Optimization of the whole cell biocatalysis system

[0051] Centrifuge to collect the bacteria after fermentation 12 in Step 1 of Example 1, and resuspend the bacteria to OD with PBS buffer 600 =5, on the basis of Example 2, investigate the influence of different conversion liquid compositions and reaction temperature and time on the preparation of ectoine respectively, according to Example 2 conversion liquid (L-sodium aspartate 10g / L, glycerol 10g / L, Potassium Chloride 10g / L, solvent is PBS buffer solution, pH6.5), investigates the influence of one element at a time, all the other element concentrations are constant.

[0052] Sodium L-aspartate (5, 10, 20, 40, 80g / L), potassium chloride (0, 5, 10, 20, 40g / L), glycerin (5, 10, 20, 40, 80g / L) L), pH value (6.0, 6.5, 7.0, 7.5, 8.0), reaction temperature (25, 30, 35, 40, 45°C), reaction time (6, 12, 18, 24, 36, 48h).

[0053] The result is as image 3 As shown, the best whole-cell biocatalytic system is ...

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Abstract

The invention provides recombinant escherichia coli and application to synthesis of tetrahydropyrimidine. The recombinant escherichia coli is obtained by knocking out a diaminapimelate decarboxylase lysA gene of escherichia coli E.coli MG1655 and introducing a tetrahydropyrimidine synthetic gene luster ectABC with a nucleotide sequence shown as SEQ ID NO. 1. The nucleotide sequence of the diaminapimelate decarboxylase lysA gene is shown as SEQ ID NO. 2. A thallus subjected to induced expression takes L-sodium aspartate as a substrate and the substrate is biologically converted to prepare the tetrahydropyrimidine. After conversion is carried out for 20 to 30h, the yield of the tetrahydropyrimidine reaches 2.5 to 3.5g / L, and therefore, the recombinant escherichia coli has relatively good industrial application value.

Description

(1) Technical field [0001] The invention relates to a method for transforming whole cells of recombinant Escherichia coli to synthesize ectoine, belonging to the fields of genetic engineering and biotechnology. (2) Background technology [0002] In order to adapt to the high-salt conditions in the deep sea, marine microorganisms need to rely on a strategy called Compatible-Solutes (Strategy) to counteract the negative effects of the external salt environment and maintain osmotic pressure balance. According to the chemical structure, compatible solutes mainly include: ① sugars and derivatives: sucrose, trehalose, glycerol glucoside, etc.; ② polyols and derivatives: glycerol sorbitol, mannitol, etc.; ③ amino acids and derivatives: Proline, glutamic acid, alanine, ectoine, 5-hydroxy ectoine, etc.; ④ betaines: betaine, glycine betaine, proline betaine. Compatible solutes, as osmotic pressure protectors for cells, have a good function of stabilizing biological macromolecules, an...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P17/12C12R1/19
CPCC12N9/88C12N15/70C12P17/12C12Y401/0102C12N1/205C12R2001/19
Inventor 王鸿陈俊储消和柳鹏福陈建伟章华伟
Owner ZHEJIANG UNIV OF TECH
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