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Mutant of 5-aminolevulinic acid synthase from Rhodobacter capsulata and its application

A technology of aminolevulinic acid and capsulated rhodobacter, applied in the direction of bacteria, application, acyltransferase, etc., can solve the problems affecting the synthesis of 5-ALA, and achieve the effect of high relative enzyme activity

Active Publication Date: 2022-04-26
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when 5-ALA accumulates to a certain extent, the heme produced by the downstream pathway will have a feedback inhibitory effect on ALAS, thereby affecting the synthesis of 5-ALA

Method used

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  • Mutant of 5-aminolevulinic acid synthase from Rhodobacter capsulata and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1. Construction of Rhodobacter capsularis 5-ALA synthetase mutant

[0037] Select Rhodobacter capsulata in the KEGG database ( R. capsulatusla SB1003) sourced from hemA The gene is used as a template, and the codon is optimized according to the codon preference of the host cell Corynebacterium glutamicum, and the nucleotide sequence shown in SEQ ID NO.9 is synthesized in Jinweizhi Company. Using this sequence as a template, using SEQ ID NO.3 / SEQ ID NO.5, SEQ ID NO.6 / SEQ ID NO.4 as upstream and downstream primers to amplify fragment 1 and fragment 2 respectively, and then using SEQ ID NO. 3 / SEQ ID NO.4 is the primer for fusion PCR amplification of fragment 1 and fragment 2 to obtain the gene hemA The PCR fragment whose cysteine ​​(C) at position 201 is mutated into alanine (A) is denoted as fragment 3, and its nucleotide sequence is as shown in SEQ ID NO.2 (the gene encoding is as shown in SEQ ID NO.1 5-ALA synthetase mutants shown). Using SEQ ID NO.3 / ...

Embodiment 2

[0043] Example 2. Detection of Enzymatic Properties of Rhodobacter capsularis 5-ALA Synthetase Mutant

[0044] The wild-type plasmid pET28a-RC and the mutant plasmid pET28a-RC-C201A were transformed into E. coli BL21, the strains BL21-pET28a-RC and BL21-pET28a-RC-C201A were obtained.

[0045] (1) Bacteria culture: Inoculate the above-mentioned single recombinant colonies 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. Waiting for OD 600 At 0.6-0.8, add IPTG with a final concentration of 0.5mM, induce the temperature at 16°C, and induce the time for about 14 hours, centrifuge at 4500rpm, remove the supernatant, and collect the bacteria.

[0046] (2) Protein purification:

[0047] Binding buffer A: 25mM Tris, 150mM NaC...

Embodiment 3

[0061] Embodiment 3. Capsulated Rhodobacter 5-ALA synthetase mutant is on the influence of 5-ALA synthesis in Corynebacterium glutamicum

[0062] Using plasmids pET28a-RC and pET28a-RC-C201A as templates, and using SEQ ID NO.7 / SEQ ID NO.8 as upstream and downstream primers, PCR amplification was performed to obtain fragment 5 and fragment 6, respectively. Fragment 5, fragment 6 and plasmid vector pXMJ19 (commercial product) were double digested with restriction endonuclease HindIII / XbaI respectively, and the digested fragments and plasmid were respectively connected, and the plasmid pX-RC was finally obtained after the sequencing was correct. , pX-RC-C201A. The constructed plasmids were respectively electroporated into the Corynebacterium glutamicum engineering strain CB8 constructed in the early stage of the laboratory (the source of Corynebacterium glutamicum CB8 is detailed in Chinese patent CN106636171 A, the invention name is: production of 5-aminolevulinic acid glutamic...

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Abstract

The present invention provides a rhodobacter capsulated 5-aminolevulinic acid synthase mutant and its application. The amino acid sequence of the capsulated rhodobacter 5-aminolevulinic acid synthase mutant is shown in SEQ ID NO.1. Compared with the wild-type Rhodobacter capsula 5-aminolevulinic acid synthase mutant of the present invention, the production of 5-aminolevulinic acid in host cells is increased by about 22%; in the presence of 20 μM heme, the mutant 5‑aminolevulinic acid synthase C201A can maintain a relatively high relative enzyme activity.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, in particular to a kind of capsulated rhodobacter ( Rhodopseudomonas palustris ) 5-aminolevulinic acid synthase mutant and its application. Background technique [0002] 5-aminolevulinic acid (5-aminolevulinic acid, 5-ALA), is the biosynthetic vitamin B 12 , heme, chlorophyll and other important precursors of tetrapyrrole compounds, which widely exist in microorganisms, animal and plant cells. Due to its green, non-toxic, easy to degrade and no residue characteristics, it has been widely used in the fields of medicine and agriculture as photodynamic drugs, biodegradable herbicides, plant growth regulators and feed additives. In addition, in the face of the major threat of the 2019 novel coronavirus (COVID-19) to global health and life safety, 5-ALA has shown great application potential in the prevention and treatment of COVID-19 infection ([1] Bando H. Some measures for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/77C12N1/21C12P13/00C12R1/15
CPCC12N9/1029C12N15/77C12P13/005C12Y203/01037
Inventor 王智文姜玫如何桂美孙曦崔真真陈涛
Owner TIANJIN UNIV
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