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Escherichia coli for producing adipic acid precursor namely cis,cis-muconic acid and application of escherichia coli

A technology of Escherichia coli and muconic acid, applied in the biological field, can solve problems affecting the excessive accumulation of target products, and achieve the effect of wide application prospects

Inactive Publication Date: 2014-03-26
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, metabolic pathways in cells are often not independent, but exist in complex metabolic networks, which are subject to various regulations and affect the excessive accumulation of target products

Method used

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  • Escherichia coli for producing adipic acid precursor namely cis,cis-muconic acid and application of escherichia coli
  • Escherichia coli for producing adipic acid precursor namely cis,cis-muconic acid and application of escherichia coli
  • Escherichia coli for producing adipic acid precursor namely cis,cis-muconic acid and application of escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 The optimized design and synthesis of 3-dehydroshikimate dehydratase gene, protocatechuate decarboxylase gene and catechol 1,2-dioxygenase gene

[0018] According to the sequences of the 3-dehydroshikimate dehydratase gene aroZ of Klebsiella pneumoniae A170-40, the protocatechuate decarboxylase gene aroY and the catechol 1,2-dioxygenase gene catA of Acinetobacter calcoaceticus, respectively, through codon optimization Novel genes aroZ-jCat, aroY-jCat and catA-jCat (attached Figure 1-3 ), and then use the method of DNA chemical synthesis to obtain these design-optimized genes, which can be used for the creation of heterologous synthetic pathways.

Embodiment 2

[0019] Example 2 Assembly of heterologous expression modules consisting of 3-dehydroshikimate dehydratase, protocatechuate decarboxylase and catechol 1,2-dioxygenase

[0020] 通过合适的限制性内切酶,把人工设计合成的组成型启动子P3(TCTAGAATATGTTATCTCTGGCGGTGTTGACATTTCCGTTTTTTCATGATATAATCGCTCCTGAGCGGATAACAATTTCAAGGAGGACAGCTCCATCAT)、上述设计合成的新基因aroZ-jCat,aroY-jCat和catA-jCat以及人工设计合成的终止子T1(CCGGCTTATCGGTCAGTTTCACCTGATTTACGTAAAAACCCGCTTCGGCGGGTTTTTGCTTTTGGAGGGGCAGAAAGATGAATGACTGTCCACGACGCTATACCCAAAAGAAA ) to form the heterologous expression module CCMA. The restriction enzymes between P3, aroZ-jCat, aroY-jCat, catA-jCat and T1 are NdeI, SacI, NheI, XhoI, respectively. In addition, an RBS binding region (AAGAAGGAGATATAGTA) was added before the initiation codon ATG of the aroY-jCat and catA-jCat genes, respectively. The schematic diagram and sequence of the CCMA module are shown in the attached Figure 4 and 5 .

Embodiment 3

[0021] Example 3 Construction of Escherichia coli (Escherichia coli) TIB-LEc340 CGMCC No 6435

[0022] ClaI and SalI restriction enzyme cutting sites were added to both ends of the DNA sequence of the above-mentioned construction of 3-dehydroshikimate dehydratase, protocatechuate decarboxylase and catechol 1,2-dioxygenase heterologous expression module CCMA point, and then insert the DNA fragment into the corresponding site of the plasmid pACYC184 to obtain the recombinant plasmid pACYC-CCMA (attached Figure 6 ). Then the recombinant plasmid pACYC-CCMA was transferred into shikimate dehydrogenase (AroE) mutant Escherichia coli AB2834 (Escherichia coli AB2834) to obtain the target strain Escherichia coli (Escherichia coli) TIB-LEc340 CGMCC No 6435.

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Abstract

Cis,cis-muconic acid is an important chemical organic acid, has a wide range of application and can be used as a precursor of adipic acid. The cis,cis-muconic acid can be used for producing polymers including nylon 66 and the like after being converted into adipic acid through hydrogenation. The invention provides a recombinant escherichia coli TIB-LEc340 CGMCC No 6435 for producing cis,cis-muconic acid, wherein the recombinant escherichia coli is established by the steps of combining 3-dehydrogenated shikimic acid dehydrase which comes from different microbes and is optimized by codons, protocatechuic acid decarboxylase, catechol 1,2-dioxidase and artificially designed constitutive promoters and terminators into a heterologous expression module, and then introducing the heterologous expression module into shikimic acid dehydrogenase mutated escherichia coli. The escherichia coli TIB-LEc340 CGMCC No 6435 is fermented for 16-120 hours at 30-40 DEG C to obtain fermentation liquor, and the maximum content of cis,cis-muconic acid in the fermentation liquor can reach 52g / L, so that the escherichia coli has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a metabolic engineering bacterium for producing adipic acid precursor cis, cis-muconic acid, Escherichia coli (Escherichia coli) TIB-LEc340 CGMCC No 6435 and the use of the bacterium to ferment and produce cis, cis-muconic acid method. Background technique [0002] Adipic acid is a six-carbon dibasic acid, which has the general properties of aliphatic dibasic acids, can undergo salt-forming reactions, esterification reactions, and amide reactions, and can condense with diamines to form polymers. Important industrial basic raw materials for the production of hexamethylenediamine, nylon 66, fibers and engineering plastics and their resins (van Duuren JB, Brehmer B, Mars AE, Eggink G, Dos Santos VA, Sanders JP.A limited LCA of bio-adipic acid: Manufacturing the nylon-6, 6 precursor adipic acid using the benzoic acid degradation pathway from different feedstocks. Biotechnol ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/44C12R1/19
Inventor 王钦宏吴元庆陈五九张媛媛彭彦峰元飞马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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