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Recombinant escherichia coli as well as preparation method and application thereof

A technology of recombinant Escherichia coli and Escherichia coli, which is applied in the field of bioengineering, can solve the problems of low production efficiency, low product yield, high product purity, etc., and achieve the effects of low cost, simple operation, and reduced production cost

Pending Publication Date: 2022-07-12
WANHUA CHEM GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two main production routes. One is hydrolysis of malonate with sulfuric acid at 80-90°C. This method is characterized by short process route and production cycle and less waste, but the hydrolysis process is a reversible reaction, and malonate is subjected to high temperature. Decarboxylation reaction is easy to decompose to generate acetic acid, water and carbon dioxide, so the product yield is low, but this method is easy to control the generation of impurities, so the purity of the product is high; the second is the transesterification process, as long as the purity of the raw materials is controlled, the content of the product can be prepared High-purity malonic acid with high purity, less impurities, and low moisture, and the obtained by-products can be used to prepare ethyl acetate with higher purity. The disadvantages of this method are: acetic acid is needed, it has a pungent sour smell, and there is another by-product of acetic acid A mixed solution of ethyl vinegar and ethanol is produced, which is not easy to handle
However, the malonyl-CoA pathway is less efficient at producing metabolites than the aspartate pathway, so using the aspartate pathway to produce malonate is a more efficient method

Method used

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  • Recombinant escherichia coli as well as preparation method and application thereof
  • Recombinant escherichia coli as well as preparation method and application thereof
  • Recombinant escherichia coli as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Construction of recombinant plasmid and acquisition of BL21 (TPP) recombinant bacteria

[0075] 1. Construction of recombinant plasmid pRSF-yneI-pa0132

[0076] Succinate semialdehyde dehydrogenase gene (yneI) (sequence shown in SEQ ID NO: 1) and β-alanine pyruvate aminotransferase gene (pa0132) (sequence shown in SEQ ID NO: 2) were provided by Suzhou Jinweizhi Synthesized by Biotechnology Co., Ltd.;

[0077] Using the succinate semialdehyde dehydrogenase gene (yneI) as a template, primers yneI-F (5'-CACACAGGAAACAGACCATGACCATTACTCCGGCAACTC-3', SEQ ID NO: 3) and yneI-R (5'-TTCTTTACCAGACTCGAGTCAGATCCGGTCTTTCCACAC-3', SEQ ID NO:4) carry out PCR amplification, obtain yneI fragment;

[0078] Using β-alanine pyruvate aminotransferase gene (pa0132) as a template, primers pa0132-F (5'-CACACAGGAAACAGACCATGAATCAGCCCCTGAATGTC-3', SEQ ID NO: 5) and pa0132-R (5'-GCCGGATGATTAATTGTCAAAAGCTTTCAGGCAATTCCGTTCAGAG-3', SEQ ID NO: 5) were used as the template. ID NO: 6) carry ...

Embodiment 2

[0094] Example 2: Construction of recombinant plasmid and acquisition of BL21 (SCR) recombinant bacteria

[0095] 1. The linker gene (sequence shown in SEQ ID NO: 25) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the encoded amino acid sequence is (SSSSG) 4 (SEQ ID NO: 26), and codon-optimized according to E. coli preference.

[0096] 2. Construction of recombinant plasmid pCDF-ppc-linker-aspC

[0097] Using the linker gene as a template, use primers linker-pCDF-F (5'-TAATACCGGCGAATTCTCTTCAAGCTCTGGTAGCTCGTC-3', SEQ ID NO:27), linker-pCDF-R (5'-CGGCGGTAATGTTCTCAAACATTCCGGAGCTCGAACTGCC-3', SEQ ID NO:28) to carry out PCR amplification to obtain linker fragments;

[0098] Using the pCDF-ppc-aspC plasmid as a template, use primers pCDF-linker-F (5'-ATGTTTGAGAACATTACCGCCG-3', SEQ ID NO: 29), pCDF-linker-R (5'-GAATTCGCCGGTATTACGCA-3', SEQ ID NO: 29) : 30) carry out PCR amplification, obtain linearized pCDF-ppc-aspC plasmid fragment;

[0099] The linker fragment...

Embodiment 3

[0107] Example 3: Shake flask fermentation of recombinant E. coli

[0108] 1. Preparation of seed solution: The recombinant strains BL21 (TPP) and BL21 (SCR) obtained by LB solid plate activation were respectively inoculated into 25 mL of liquid LB medium, cultured at 37°C and 230 r / min for 12 h, and then inoculated with secondary seed solution. 1 mL was inoculated into 25 mL of liquid LB medium, and cultured at 37 °C and 230 r / min for 12 h.

[0109] 2. Fermentation: 2% of the inoculum was inoculated into SOB medium with a liquid volume of 50 mL / 250 mL, and cultured at a constant temperature of 37°C and 230 r / min. SOB medium: peptone 20g / L, yeast extract 5g / L, MgSO 4 ·7H 2 O 2.47g / L, NaCl 0.5g / L, KCl 0.186g / L, and antibiotics ampicillin (Amp), kanamycin (Kan) and streptomycin (Str) were added to the medium with a final concentration of 1mM.

[0110] Analysis of the results: During the fermentation process, samples were taken every 12 hours, 1 mL each time, centrifuged at 13...

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Abstract

The invention discloses recombinant escherichia coli as well as a preparation method and application thereof. The recombinant escherichia coli disclosed by the invention can be used for synthesizing malonic acid by taking glucose as a raw material through an oxaloacetic acid-aspartic acid way; according to the invention, phosphoenolpyruvate carboxylase, aspartic acid transaminase and aspartic acid-alpha-decarboxylase which are derived from escherichia coli and beta-alanine pyruvate transaminase which is derived from pseudomonas aeruginosa are subjected to constitutive overexpression according to modules; the succinic semialdehyde dehydrogenase is derived from escherichia coli; and the pyruvate carboxylase is derived from corynebacterium glutamicum. According to the method, the malonic acid can be synthesized by using the escherichia coli through a whole biological method, and the method is simple to operate, low in cost and capable of being used for industrial production.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a recombinant Escherichia coli and a preparation method and application thereof, in particular to a method for synthesizing malonate by using Escherichia coli total biosynthesis. Background technique [0002] Malonic acid is mainly used in the production of spices, adhesives and resin additives, etc. It can also be used as a surface treatment agent for leather products or aluminum products, foam foaming agents and chemical cleaning agents for nuclear reactors. For example, malonic acid is used in the production of pesticides and fungicides. Raw materials for Fuji No. 1, herbicide Kuzada, and plant growth regulator indoxyl ester; the pharmaceutical industry uses malonic acid to prepare diuretic sulfinpyrazone, anti-inflammatory drug oxybutazone, sedative brommethoxine etc.; Malonic acid generates carbon dioxide and water after heating, and there is no pollution problem, so it can be di...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/60C12N15/54C12N15/53C12N15/52C12P7/46C12R1/19
CPCC12N9/88C12N9/1096C12N9/0008C12N9/93C12N15/70C12P7/46C12Y401/01031C12Y206/01001C12Y401/01011C12Y206/01018C12Y102/01016C12Y102/01024C12Y102/01076C12Y102/01079C12Y604/01001Y02A50/30
Inventor 张雅萍邓禹赵运英张光祥
Owner WANHUA CHEM GRP CO LTD
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