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5-aminolevulinic acid high-yielding strain and its preparation method and application

A kind of aminolevulinic acid, technology for producing strains, applied in the fields of genetic engineering and microbial fermentation

Active Publication Date: 2020-10-30
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

However, it is questionable whether this conclusion is generally applicable given the complexity of microbial metabolism
[0005] At present, the output of ALA in the prior art still has a lot of room for improvement, so this field still needs to further increase the output of ALA, so as to prepare ALA with high efficiency, low cost and low pollution.

Method used

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  • 5-aminolevulinic acid high-yielding strain and its preparation method and application
  • 5-aminolevulinic acid high-yielding strain and its preparation method and application
  • 5-aminolevulinic acid high-yielding strain and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. pZGA24 and pZPA6 transformed iclR gene deletion strain

[0070]In order to verify the effect of strengthening the glyoxylate cycle on ALA synthesis, pZGA24 and pZPA6 (refer to CN103981203B for the construction process) vectors were transformed into the aceBAK operon repressor protein coding iclR deletion strain JW3978 (from the KeioCollection Escherichia coli single gene deletion strain library), and the competent For the cell preparation and transformation process, refer to the "Molecular Cloning Experiment Guide" written by J. Sambrook et al. The transformation product was coated on an ampicillin-resistant LB plate, and after overnight culture, positive clones were picked to extract plasmids for verification, and recombinant strains ΔiclR / pZGA24 and ΔiclR / pZPA6 were obtained respectively.

Embodiment 2

[0071] Example 2. The Effect of Strengthening the Glyoxylate Cycle on ALA Synthesis

[0072] A single colony of the above-mentioned recombinant bacteria and its control strains BW25113 / pZGA24 and BW25113 / pZPA6 were inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin, respectively, and cultured at 37°C and 220 rpm for 12 hours. According to the initial OD of 0.05, transfer to a 250mL Erlenmeyer flask containing 50mL fermentation medium, culture at 37°C, 220rpm for 2.5h, add IPTG with a final concentration of 50μM, and collect the fermentation broth after 24h of induction culture to detect the concentration of ALA. Wherein the fermentation medium is the M9 medium added with yeast powder, the main components are: Na 2 HPO 4 12H 2 O 17.1g / L, KH 2 PO 4 3.0g / L, NaCl 0.5g / L, NH 4 Cl 1.0g / L, MgSO 4 2mM, CaCl 2 0.1mM, glucose 15g / L, yeast powder 2g / L, glycine 4g / L. The detection method of ALA is as follows: add 100 μL pH 4.6 sodium acetate buffer solution...

Embodiment 3

[0074] Example 3. pZGA24 and pZPA6 were respectively transformed into glyoxylate cycle weakened strains

[0075] In order to verify the effect of glyoxylate cycle weakening on ALA synthesis, the vectors pZGA24 and pZPA6 (refer to CN103981203B for construction process) were transferred into JW3975 (isocitrate lyase encoding gene aceA deletion strain) or JW3974 (malate synthase encoding gene aceB In the deletion strain), the competent cell preparation and transformation process refer to the "Molecular Cloning Experiment Guide" written by J. Sambrook (Sambrook) et al. The transformation product was coated on an ampicillin-resistant LB plate, and after overnight culture, positive clones were picked to extract plasmids for verification, and recombinant strains ΔaceA / pZGA24, ΔaceB / pZGA24, ΔaceA / pZPA6 and ΔaceB / pZPA6 were obtained respectively.

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Abstract

In the invention, a method of constructing a 5-aminolevulinic acid producing bacterial strain is disclosed, namely, on the basis that the 5-aminolevulinic acid producing bacterial strain expresses ALAsynthetase (ALAS) and phosphoenolpyruvate carboxylase (PPC), a glyoxylate cycle route of the 5-aminolevulinic acid producing bacterial strain is weakened. The invention also discloses the ALA highly-producing bacterial strain constructed by the method, and a method of preparing the ALA through the bacterial strain. By means of the bacterial strain, yield of the ALA is significantly increased.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and microbial fermentation. Specifically, the present invention relates to a high-yield strain of 5-aminolevulinic acid and its preparation method and application. Background technique [0002] 5-Aminolevulinic acid (ALA) is an important high value-added bio-based chemical product, widely used in agriculture, medicine and other fields. At present, ALA is mainly synthesized by chemical synthesis, which has high cost and heavy pollution, which limits its popularization and application in various fields. Due to the advantages of low cost and no pollution, the synthesis of ALA by microbial fermentation has gradually become a research hotspot. [0003] There are mainly two synthetic pathways (C4 pathway and C5 pathway) for ALA in organisms, and the expression of key enzymes in the enhanced pathway has been widely used in the construction of ALA high-yielding strains as a routine pathway ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/77C12N15/74C12N1/21C12P13/00
CPCC12N15/70C12N15/74C12N15/77C12P13/005
Inventor 郑平陈久洲孙际宾周文娟孙村民郭轩马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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