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Method for producing L-piperidinecarboxylic acid by mixed bacteria fermentation

A technology of pipecolinic acid and mixed bacteria, applied in the field of fermentation, can solve the problems of high construction difficulty and poor stability

Active Publication Date: 2020-10-23
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this transformation method requires the co-expression of multiple proteins, which has disadvantages such as high construction difficulty and poor stability.

Method used

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  • Method for producing L-piperidinecarboxylic acid by mixed bacteria fermentation
  • Method for producing L-piperidinecarboxylic acid by mixed bacteria fermentation
  • Method for producing L-piperidinecarboxylic acid by mixed bacteria fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of a recombinant plasmid expressing lysine cyclizing deaminase.

[0041] Cloning is from S. pristinaespiralis (ATCC25486) lysine cyclizing deaminase gene (SpLCD) (gene is synthesized by Supkin Company, its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO .4.) Add XhoI and NcoI restriction sites at both ends, and insert the gene fragment into the corresponding restriction sites of the expression vector pET28a by double restriction digestion and ligation, and place it under the control of the T7 promoter to obtain The recombinant plasmid pET28a-SpLCD was obtained.

Embodiment 2

[0042] Example 2: Modification of lysine cyclizing deaminase Ile61.

[0043] By analyzing the structure of SpLCD, the bottleneck amino acid site Ile61 in the process of substrate binding and product release was found. From figure 1 It can be seen in b that the distance between Ile61 and Asp236 is the narrowest in the entire channel, which becomes the bottleneck that restricts the substrate small molecule lysine to enter the active center. From figure 2 a It can be seen that Ile61 and NAD + The distance between them causes a large steric hindrance at the product release site 1, which becomes a bottleneck that restricts the product small molecule L-piperidinecarboxylic acid from leaving the active center. Through whole plasmid PCR and saturation mutation screening, the optimal mutant Val61-SpLCD and recombinant plasmid pET28a-Val61-SpLCD were obtained.

Embodiment 3

[0044] Example 3: Modification of lysine cyclizing deaminase Ile94.

[0045] By analyzing the structure of SpLCD, the bottleneck amino acid site Ile94 in the process of substrate binding and product release was found. From figure 2 As can be seen in b, Ile94 and NAD + The distance between them causes a large steric hindrance at the product release site 2, which becomes a bottleneck that restricts the product small molecule L-piperidinecarboxylic acid from leaving the active center. The optimal mutant strain Val94-SpLCD and recombinant plasmid pET28a-Val94-SpLCD were obtained by whole plasmid PCR and saturation mutation screening.

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Abstract

The invention discloses a method for producing L-piperidine formic acid by fermenting mixed bacteria, a strain capable of producing lysine and a strain excessively expressing lysine cyclodeaminase aretaken as fermentation strains to produce the L-piperidine formic acid, wherein the nucleotide sequence of the lysine cyclodeaminase is shown as in SEQ ID NO.1. The method takes glucose as a substrate, and the L-piperidine formic acid is produced by fermenting two strains together.

Description

technical field [0001] The invention relates to a method for producing L-piperidinecarboxylic acid by fermentation of mixed bacteria, which belongs to the technical field of fermentation. Background technique [0002] Pipecolinic acid is a chiral molecule, and it is an important precursor for the production and synthesis of various heterocyclic polyketide chiral drugs. Such as rapamycin, FK506 and FK520 and so on. They have potential immunosuppressant activity, neurotrophic activity and antifungal activity. Currently, rapamycin and its analogues can be clinically used to prevent immune rejection after organ transplantation, treat advanced renal cancer, and be used as drugs in the fields of dermatology and cardiology. Piperidine carboxylic acid derivatives are responsible for constituting a common core structural feature of this class of compounds: the FKBP12-binding motif, which is involved in their binding to FKBP12. Pipecolinic acid is essential for the biosynthesis of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/12C12N15/70C12N15/60
CPCC12N9/88C12N15/70C12P17/12C12Y403/01028
Inventor 陈可泉刘易辰应晗笑王昕欧阳平凯
Owner NANJING TECH UNIV