Rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant and application thereof

A technology of aminolevulinic acid and swamp pseudomonas, which is applied in the biological field, can solve the problems of heme feedback inhibition, limited application, and low catalytic efficiency, and achieve the effect of improving release or relief, and improving enzyme activity

Active Publication Date: 2022-03-15
TIANJIN UNIV
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, they all have disadvantages such as low catalytic efficiency and severe heme feedback inhibition, which greatly limit their application in 5-ALA biosynthesis.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Construction of a 5-aminolevulinic acid (5-ALA) synthase mutant from Rhodopseudomonas palustris

[0026] Construction of plasmids: Rhodopseudomonas palustris ( Rhodopseudomonas palustris BisB5)-derived 5-ALA synthase gene hemA , carry out codon optimization according to the codon preference of the host cell Corynebacterium glutamicum, and synthesize the nucleotide sequence shown in SEQ ID NO: 3 in Jinweizhi Company. Using it as a template, using the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 as primers to carry out PCR amplification to obtain fragment 1; using the same nucleotide sequence as a template, using SEQ ID NO: 6 and SEQ ID NO: The sequence shown in ID NO: 7 was used as a primer for PCR amplification to obtain Fragment 2. Gel recovery and purification were performed on the PCR product fragment 1 and fragment 2 obtained above. Subsequently, fragment 1 and fragment 2 were used as templates, and the sequences shown in SEQ ID NO: 4 and SEQ ID...

Embodiment 2

[0038] Example 2. Detection of enzymatic properties of 5-ALA synthetase mutants

[0039] Construction of engineering strains expressing 5-ALA synthetase: the constructed pET28a-hemA and mutant plasmid pET28a-hemA-C132A were respectively introduced into E. coli In BL21 competence, engineered strains expressing 5-ALA synthetase were prepared: BL21-pET28a-hemA and BL21-pET28a-hemA-C132A.

[0040] (1) Cell culture: Inoculate single colonies of the above engineering strains into 5 mL of LB liquid medium containing 40 μg / mL kanamycin, and culture at 37°C and 220 rpm for 12 hours. Transfer to 5 mL of LB liquid medium containing 40 μg / mL kanamycin. After the seeds grow thick, transfer to a 1L Erlenmeyer flask containing 200 mL of LB liquid medium containing 40 μg / mL kanamycin. When the OD600 reaches 0.6-0.8, add IPTG with a final concentration of 0.5mM, induce the temperature at 16°C, induce the time for 12h, centrifuge at 4500rpm, remove the supernatant, and collect the bacteria. ...

Embodiment 3

[0057] Example 3. Application of 5-ALA synthetase mutants in 5-ALA synthesis

[0058] The effects of 5-ALA synthetase mutants on 5-ALA synthesis were verified in Corynebacterium glutamicum.

[0059] Plasmid construction: using the psod* nucleotide sequence (SEQ ID NO: 12) as a template, using the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9 as primers to perform PCR amplification to obtain a psod* fragment; The plasmids pET28a-hemA and pET28a-hemA-C132A constructed in "Example 1" were used as templates, and the sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11 were used as primers for PCR amplification to obtain wild-type hemA fragment 4 and mutant hemA-C132A fragment 5. Then, using the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 11 as primers, fragments 4 and 5 were fused with the psod* fragment respectively, and pEC-ΔlacIq-Δtrc was used as the original plasmid (its nucleoside The acid sequence is shown in SEQ ID NO:13) Select the two restricti...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant and application thereof. The amino acid sequence of the rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant is as shown in SEQ ID NO.1. The invention further discloses a preparation method of the rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant. Compared with non-mutated 5-aminolevulinic acid synthetase, the rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant disclosed by the invention has the advantages that the enzyme activity is improved, and the capability of demodulating feedback inhibition of high-concentration heme is also improved, so that the capability of producing 5-ALA by a host cell disclosed by the invention is remarkably improved and is improved by about 40%.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to Rhodopseudomonas palustris ( Rhodopseudomonas palustris ) source of 5-aminolevulinic acid synthase mutant and application. Background technique [0002] 5-aminolevulinic acid (5-minolevulinic acid, 5-ALA) is a biosynthetic heme, chlorophyll, VB 12 Precursors of tetrapyrrole compounds widely exist in animals, plants and microorganisms. As an endogenous substance, 5-ALA is non-toxic to humans and animals, green and efficient, easy to degrade and has no residue in the environment, so it has been widely used in the fields of medicine, food and agriculture. At present, 5-ALA has been clinically used to improve the diagnosis and treatment of human metabolic diseases, cancer and the treatment of novel coronavirus pneumonia (COVID-19). Studies have shown that 5-ALA can effectively inhibit the infection of cells by the COVID-19 pathogen SARS-CoV-2, and is safe, tolerable and effective. ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/1029C12N15/70C12Y203/01037C12P13/005
Inventor 王智文何桂美姜玫如崔真真孙曦陈涛
Owner TIANJIN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap