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Recombinant halomonas, construction method of recombinant halomonas and application of recombinant halomonas in preparation of itaconic acid by catalyzing citric acid

A technology of halophilic bacterium and construction method, which is applied in the field of recombinant halophilic bacterium and its construction method and the application field of cell catalyzed citric acid to prepare itaconic acid, which can solve the problems such as no modified halophilic bacterium , to achieve the effect of mild conditions, high efficiency and short reaction cycle

Active Publication Date: 2021-10-08
TIANJIN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] After searching, there is no relevant report on the transformation of Halomonas bacterium and its use in catalyzing the production of itaconic acid from citric acid

Method used

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  • Recombinant halomonas, construction method of recombinant halomonas and application of recombinant halomonas in preparation of itaconic acid by catalyzing citric acid
  • Recombinant halomonas, construction method of recombinant halomonas and application of recombinant halomonas in preparation of itaconic acid by catalyzing citric acid

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1 Construction of Halomonas Chassis Cells

[0050] In the wild-type Halomonas sp.TD01 (preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee on November 19, 2010, the preservation registration number is CGMCC No.4353, and the preservation address: Chaoyang District, Beijing No. 3, No. 1 Yard, Beichen West Road) integrated the Mmp1 RNA polymerase expression unit into the genome to obtain Halomonassp.TD1.0;

[0051] Then replace the start codon ATG of the isocitrate dehydrogenase coding gene icd on the Halomonas sp.TD1.0 genome with TTG to obtain Halomonas sp.TD2.0;

[0052] The nucleotide sequence of the isocitrate dehydrogenase encoding gene icd is shown in SEQ ID NO.1;

[0053] Specifically include the following steps:

[0054] (1) Integrate the Mmp1 RNA polymerase expression unit on the wild-type Halomonas sp.TD01 genome to obtain Halomonas sp.TD1.0, so as to realize the activated transcription of t...

Embodiment 2

[0056] Embodiment 2: Construction of expression vector

[0057] (1) Construction of high-copy expression vector pN59-P Mmp1 -RBS-cadA-acn

[0058] P shown in SEQ ID No.9 Mmp1 -F1 and RBS-R1 shown in SEQ ID No.10 as a primer, P shown in SEQ ID No.3 Mmp1 The fragment was used as a template, and PCR amplification obtained P Mmp1 -RBS fragment (wherein said RBS is Escherichia coli Escherichia coli MG1655 strong ribosome binding site, its nucleotide sequence is shown in SEQ ID NO.4); Shown in SEQ ID No.11 cadA-F and in SEQ ID The cadA-R shown in No.12 is a primer, and the artificially synthesized cadA gene shown in SEQ ID No.5 is used as a template, and PCR amplification obtains the cadA fragment; acn-R1 shown in .14 is a primer, and the genome of Corynebacterium glutamicum ATCC13032 is used as a template, and PCR amplification obtains the acn fragment (the acn nucleotide sequence is shown in SEQ ID NO.6); Mmp1 -RBS, cadA and acn fusion to get P Mmp1 -RBS-cadA-acn fusion frag...

Embodiment 3

[0070] Embodiment 3: Construction of recombinant halophilic monas

[0071] Containing pN59-P in embodiment 2 Mmp1 - E. coli S17-1λpri of the RBS-cadA-acn plasmid was conjugated with Halomonas sp. TD2.0 as described in Example 1.

[0072] Specifically for the pN59-P containing Mmp1 -Escherichia coli S17-1λpri of the RBS-cadA-acn plasmid was inoculated in 5 mL of LB liquid medium containing 25 mg / L chloramphenicol resistance, and Halomonas sp.TD2.0 was inoculated in 5 mL of 60LB liquid medium, respectively. Cultivate at 37°C, 200rpm for 12h. Collect 1mL containing pN59-P Mmp1 -Escherichia coli S17-1λpri bacteria liquid of RBS-cadA-acn plasmid and 1mL Halomonas sp.TD2.0 bacterial liquid were washed twice with LB liquid medium and 60LB liquid medium respectively, mixed at a ratio of 1:1, and dropped Sow on 20LB solid medium, conjugate at 37°C for 12h. Contain 25mg / L chloramphenicol resistance on the 60LB solid flat plate at last, pick single bacterium colony PCR verification ...

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Abstract

The invention discloses recombinant halomonas, a construction method of the recombinant halomonas and application of the recombinant halomonas to preparation of itaconic acid by catalyzing citric acid. The construction method of the recombinant halomonas is characterized by comprising the following steps of: integrating an Mmp1 RNA polymerase expression unit on a wild type halomonas sp. TD01 genome, and replacing an initiation codon ATG of an icd gene of the genome with TTG to obtain Halomonas sp. TD2.0; sequentially introducing a high-copy expression vector pN59-PMmp1-RBS-cadA-acn and a low-copy expression vector pN85-PMmp1-RBS-GroESL-PMmp1-RBS-acn into the Halomonas sp. TD2.0; and obtaining the recombinant halomonas sp. IA02. The itaconic acid can be efficiently prepared through cell catalysis of the bacterium. The recombinant halomonas is short in period, simple, convenient and high in yield.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant halophilic bacterium, a construction method and the application of cells to catalyze citric acid to prepare itaconic acid. Background technique [0002] Itaconic acid (IA), a C5 unsaturated dibasic acid, is one of the twelve most valuable bio-based platform compounds announced by the U.S. Department of Energy, and is widely used in synthetic resins, plastics, synthetic fibers and pharmaceutical field, etc. At present, it is mainly produced through the fermentation method of Aspergillus terreus, and the highest itaconic acid production can reach 160g / L. There are two key enzymes involved in the synthesis of itaconic acid from citric acid in microorganisms, as summarized in figure 1 , that is, citric acid, an intermediate product of the TCA cycle, is catalyzed by aconitase (ACN) to generate cis-aconitic acid, which is further decarboxylated by cis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/53C12N15/60C12N15/74C12P7/46C12R1/01
CPCC12N9/0006C12N9/1247C12N9/88C12N15/74C12P7/46C12Y207/07006C12Y401/01006C12Y402/01003
Inventor 陈涛张静晋彪王智文陈国强
Owner TIANJIN UNIV
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