A kind of biotransformation method for synthesizing hexahydropyridazine-3-carboxylic acid

A technology of hexahydropyridazine and biotransformation, applied in the biological field, can solve the problems of high cost, complex process, low stereoselectivity, etc., achieve good technical application and industrialization prospects, mild and easy to control reaction conditions, and specific stereoselectivity sex high effect

Active Publication Date: 2021-10-08
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The object of the present invention is to provide a method for biotransformation and synthesis of hexahydropyridazine-3-carboxylic acid, utilizing Escherichia coli genetic engineering bacteria containing intracellular expression of L-ornithine hydroxylase and L-piperazine Whole cells or crude enzyme solution after broken cells catalyze L-ornithine to prepare hexahydropyridazine-3-carboxylic acid. However, problems such as low stereoselectivity, serious pollution, complicated process, high cost and low yield

Method used

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  • A kind of biotransformation method for synthesizing hexahydropyridazine-3-carboxylic acid
  • A kind of biotransformation method for synthesizing hexahydropyridazine-3-carboxylic acid
  • A kind of biotransformation method for synthesizing hexahydropyridazine-3-carboxylic acid

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Experimental program
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Embodiment 1

[0045] Construction of a recombinant plasmid containing the gene ktzI encoding L-ornithine hydroxylase and the gene ktzT encoding L-piperazine acid synthase

[0046] According to the amino acid sequences of L-ornithine hydroxylase and L-piperazine acid synthetase, as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, and codon optimization is carried out according to the codon preferred by Escherichia coli, according to The characteristics of the expression vector pETDuet-1, design PCR primers, design enzyme cutting sites NcoI and HindIII at both ends of the L-ornithine hydroxylase coding gene ktzI, and design enzymes at both ends of the L-piperazine synthase coding gene ktzT The cleavage sites NdeI and XhoI are used to synthesize KtzI and KtzT gene DNA fragments in a fully synthetic manner through conventional genetic engineering operations.

[0047] The DNA fragment of the ktzI gene and the expression plasmid pETDuet-1 were digested with NcoI and HindIII restriction endonucl...

Embodiment 2

[0049] Construction of host Escherichia coli E.coli BL21-ΔargI that does not consume the substrate L-ornithine itself

[0050] According to the position of ornithine carbamoyltransferase coding gene argI (NCBI database number: AJH11494) on the genome of Escherichia coli E.coli BL21 (DE3), the fragments of homology arms at both ends of argI were designed, and the fragments containing Apramyces The pIJ773 plasmid with the antibiotic resistance marker was used as a template, and the homologous recombination fragment containing the apramycin resistance marker was obtained by PCR, and the gene argI encoding the ornithine carbamoyltransferase was replaced by the conventional E. coli red recombination method Apramycin resistance marker, spread on the LB solid medium plate containing apramycin (final concentration: 50 μg / ml), cultivate overnight at 37°C, and in the colony grown on the plate the next day Randomly pick clones and extract plasmids, sequence them with identification prime...

Embodiment 3

[0055] Construction of genetically engineered bacteria containing genes encoding L-ornithine hydroxylase and L-piperazine acid synthase

[0056] The recombinant plasmid pETDuet-ktzI-ktzT constructed in Example 1 was transformed into the E.coliBL21-ΔargI host bacterium constructed in Example 2, and spread on LB solid medium containing ampicillin (final concentration: 50 μg / ml) On the plate, cultivate overnight at 37°C to construct the genetically engineered bacteria E.coli BL21-ΔargI / pETDuet-ktzI-ktzT.

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Abstract

The invention discloses a method for biotransforming and synthesizing hexahydropyridazine-3-carboxylic acid, which belongs to the field of biotechnology. The method comprises: (1) constructing a recombinant plasmid containing a gene encoding L-ornithine hydroxylase and a gene encoding L-piperazine acid synthase; (2) constructing ornithine transcarbamylase by gene knockout Escherichia coli genetically engineered bacteria whose coding gene argI is deleted; (3) transforming the recombinant plasmid constructed in step (1) into the Escherichia coli genetically engineered bacteria transformed in step (2) to obtain recombinant genetically engineered bacteria; (4) induction culture Recombinant genetically engineered bacteria, collect the bacteria, use the whole cell of the bacteria or the crude enzyme solution after the cell is broken as the catalyst, and use L-ornithine as the substrate to prepare hexahydropyridazine-3-carboxylate through biotransformation reaction acid. The biocatalytic method has high stereoselective specificity, high substrate conversion rate, mild and easy-to-control reaction conditions, and meets the requirements of environmental protection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for biotransforming and synthesizing hexahydropyridazine-3-carboxylic acid. Background technique [0002] Hexahydropyridazine-3-carboxylic acid (L-piperazicacid) and its structural derivatives are a unique class of non-protein amino acids with nitrogen-nitrogen (N-N) linkages. Hexahydropyridazine-3-carboxylic acid exists in many natural products with significant biological activity, such as natural immunosuppressant sanglifehrin A and antitumor active peptide luzopeptins this structural unit. In addition, the rigid conformation of hexahydropyridazine-3-carboxylic acid allows it to be used as a structural unit to introduce β-turns in peptide synthesis. These unique properties have inspired synthetic chemists to develop chemical synthetic pathways for hexahydropyridazine-3-carboxylic acid as an important synthetic intermediate of drugs and chemicals. Therefore, it is of gre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/12C12N15/70C12N1/21C12R1/19
CPCC12N9/00C12N9/0071C12N15/70C12P17/12
Inventor 杜艺岭潘海峰
Owner ZHEJIANG UNIV
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