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Microbial strain for efficiently synthesizing L-piperazinic acid as well as construction method and application of microbial strain

A piperazinic acid and microbial technology, applied in the field of genetic engineering, can solve the problems of difficult reaction, low total yield, high cost, etc., achieve the effects of environmental friendliness, mild and easy-to-control reaction conditions, and increase production

Active Publication Date: 2021-04-16
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Piperazic acid can be synthesized by two methods, one of which uses chiral compounds as starting materials, such as L-(+) glutamic acid, and obtains this compound through nine-step synthesis. This method has long lines, high cost and complicated process , the reaction difficulty is large, and the total yield is low (less than 30%); the second method is to use achiral compound as raw material, through Diels-Alder reaction, although this process is relatively simple, the yield is also high, but The obtained product is racemized and requires chemical resolution, and general chemical resolution agents are relatively expensive

Method used

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  • Microbial strain for efficiently synthesizing L-piperazinic acid as well as construction method and application of microbial strain
  • Microbial strain for efficiently synthesizing L-piperazinic acid as well as construction method and application of microbial strain
  • Microbial strain for efficiently synthesizing L-piperazinic acid as well as construction method and application of microbial strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Transformation of piperazine acid synthase genes from different sources

[0045] Construction of yeast strains expressing piperazine acid synthase gene includes:

[0046] The homologous genes of L-piperazine acid synthase from six different sources obtained by NCBI were codon-optimized and sent to the company for synthesis. A series of recombinant plasmids were obtained after vector construction; each recombinant plasmid was transformed into the DF1 strain respectively, thereby obtaining a series of bacterial strains expressing L-piperazonic acid synthase genes from different sources, as shown in Table 1 below:

[0047] Table 1

[0048]

[0049] 1. Construction of recombinant plasmids:

[0050] (1) Plasmids were synthesized by SalI and SpeI linearization companies;

[0051] (2) Link the linearized plasmids to the expression vector pAMEXLoxp-1 by recombinase, the specific steps are as follows:

[0052] a. Ligation system: pAMEXLoxp-1 vector 2uL, gene fr...

Embodiment 2

[0100] Embodiment 2: Construction of SidA-KtzT co-expression strain

[0101] Construct the recombinant plasmid pAMEXLoxp-1-SidA-KtzT containing L-ornithine hydroxylase coding gene SidA and L-piperazine acid synthase coding gene ktzT according to the aforementioned method;

[0102] The constructed recombinant plasmid pAMEXLoxp-1-SidA-KtzT was transformed into the DF1 host bacteria with the original SidA knocked out by electroporation, spread on the YPD solid medium plate containing the corresponding antibiotics, and cultured overnight at 28°C to construct the gene Engineering bacteria DFAK1.

[0103] DFAK1 was fermented in CM medium for 5 days, and the output of piperazine acid was 694.87±41.36 mg / L as detected by HPLC. The results are shown in Table 3.

Embodiment 3

[0104] Embodiment 3: the influence of iron ion on piperazine acid output

[0105] In order to verify whether the DFAK1 strain is released from the inhibition of iron ions, in this experiment, DFK1 and DFAK1 were fermented in MSP medium and high-iron medium (adding ferric chloride with a final concentration of 10 μM on the basis of MSP) for 5 days respectively, and piperazine was detected after 120 hours. acid production, see Figure 8 , it can be seen from the results that the DFAK1 strain has completely released the inhibition of iron ion.

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Abstract

The invention relates to the technical field of gene engineering, and discloses a microbial strain for efficiently synthesizing L-piperazinic acid as well as a construction method and application of the microbial strain. The microbial strain provided by the invention further transforms a carrier containing an Lpiperazine acid synthetase encoding gene ktzT or hmtC in an aureobasidium melanogenum DOLCF strain, or integrates the Lpiperazine acid synthetase encoding gene ktzT or hmtC into an aureobasidium melanogenum DOLCF strain genome. According to the invention, the L-piperazine acid synthase gene with a proper source is selected to construct the microbial strain with high Lpiperazine acid yield, and the inhibition of iron ions can be further relieved by introducing the L-ornithine hydroxylase encoding gene SidA, so that the yield of L-piperazine acid is further improved. Therefore, the microbial strain provided by the invention directly converts fermentation raw materials to produce the Lpiperazinic acid, the synthesis process does not need to depend on chemically synthesized platform compounds, the reaction conditions are mild and easy to control, and the method is environment-friendly.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, more specifically relate to a kind of microbial strain for efficiently synthesizing L-piperazine acid and its construction method and application. Background technique [0002] Piperazinic acid is a unique non-protein amino acid containing a nitrogen-nitrogen (N-N) linkage. Piperazinic acid exists in many natural products with significant biological activity, such as natural immunosuppressant sanglifehrin A and antitumor active peptide luzopeptins. Piperazine is the first-line antihypertensive drug Cilazapril Synthetic precursors. In addition, the rigid conformation of piperazine acid allows it to be used as a structural unit to introduce a β-turn structure in peptide synthesis. These unique properties have inspired synthetic chemists to develop chemical synthetic pathways for piperazinic acid as an important intermediate in the synthesis of drugs and chemicals. Therefore...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/60C12N15/53C12N15/80C12P17/12C12R1/645
Inventor 池哲池振明孔存翠
Owner OCEAN UNIV OF CHINA