5-aminolevulinic acid synthetase mutant and its host cell and application

A technology of aminolevulinic acid and host cells, which is applied to mutants of 5-aminolevulinic acid synthase, host cells and application fields thereof, and can solve the problems of few and low expression activity.

Active Publication Date: 2020-06-26
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
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Problems solved by technology

However, there are not many studies on the rational design and transformation of ALA synthase. The existing reports mainly involve the ALA synthase derived from eukaryotes. Turbeville et al. expressed two ALA synthase derived from mouse fusion, Improve its catalytic efficiency (Turbeville et al.Archives of Biochemistry&Biophysics, 2011,511(1):107-117), Lendrihas et al. obtained ALA with impro

Method used

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  • 5-aminolevulinic acid synthetase mutant and its host cell and application

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Experimental program
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Effect test

Embodiment 1

[0094] Embodiment 1. Construction of ALA synthetase mutation carrier

[0095] Utilize the Stratagene Series XL-II site-directed mutagenesis kit, designed 19 pairs of primers (see Table 2), using the pEC-XK99E-hemA wild-type plasmid as a template (the construction process refers to the construction and fermentation of the pathway for the synthesis of 5-aminolevulinic acid by Corynebacterium glutamicum Optimization [J]. Biotechnology Bulletin, 2017,33(01):148-156.), using the above primers for PCR amplification, the 11th amino acid residue of HemA was mutated into alanine (A), sperm amino acid (R), cysteine ​​(C), isoleucine (I), methionine (M), serine (S), threonine (T), valine (V). The PCR reaction conditions are: 95°C for 5min, 10 cycles (95°C for 30s, 74°C-65°C for 30s, 72°C for 4.5min), 13 cycles (95°C for 30s, 65°C for 30s, 72°C for 4.5min), 72°C 10min. PCR amplification system (50 μL): template 1 μL, upstream and downstream primers 1 μL, dNTP mix 4 μL, 5× Fast Pfu F...

Embodiment 2

[0098] Example 2. Recombinant strain construction and fermentation verification

[0099] Transform the expression vector of the correct ALA synthetase mutation into C. glutamicum ATCC13032 strain to obtain the engineering strain, carry out 24-well plate ALA fermentation on the ALA synthase mutant engineering strain, and the mutant engineering strain is in the M9 medium containing yeast powder. Cultured in 24-well plate for 16h as seed solution, according to the initial OD 600Transfer 0.5 to a 24-well plate containing M9 medium for fermentation verification, culture for 3 hours, add the inducer IPTG with a final concentration of 100uM, and finish the fermentation after 24 hours, and measure the crude enzyme activity of ALA synthase and ALA production. The detection methods of crude enzyme activity, ALA detection and glucose analysis are as described in the "Materials and Methods" section. After testing, compared with the wild-type strain, the crude enzyme activity of each muta...

Embodiment 35

[0103] Example 3.5L fermenter verification

[0104] Select the mutant (p11I) expressing bacterial strain with the best above-mentioned effect to carry out 5L fermenter verification, and fermentation medium composition is: (NH 4 ) 2 SO 4 5g / L, KH 2 PO 4 5g / L, corn steep liquor dry powder 2g / L, MgSO 4 7H 2 O 2g / L, thiamine hydrochloride 1mg / L, fermentation process control temperature 30°C, pH 6.5, dissolved oxygen not less than 30%. The results showed that the ALA production of the above strains reached 20g / L respectively, which was 16.2% higher than that of the control strain (17.2g / L), indicating that the engineering strains expressing mutants could also effectively improve the production of ALA at the fermenter level.

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Abstract

The invention provides a 5-aminolevulinic acid synthetase (ALA synthetase). Amino acid residues of the ALA synthetase at the 11 position of the amino acid sequence shown in SEQ ID NO:1 are Ile, Ser, Met, Cys and Val. The invention also provides a method for preparing the ALA synthetase, an expression vector and host cell which contain the ALA synthetase, the application of the ALA synthetase in ALA production and a method for modifying the ALA synthetase to improve the activity of the ALA synthetase.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to mutants of 5-aminolevulinic acid synthetase, host cells and applications thereof. Background technique [0002] 5-aminolevulinic acid (5-minolevulinic acid, ALA) is the precursor of tetrapyrrole compounds such as heme, chlorophyll, and VB12, which are widely found in animals, plants and microorganisms. ALA is widely used in the fields of medicine, agriculture, feed and health food, and is a high value-added bio-based chemical with great development value. In recent years, the use of microorganisms to ferment and produce ALA through the C4 pathway has been industrialized. [0003] 5-Aminolevulinic acid synthase (ALA synthase) is the key enzyme and rate-limiting enzyme for the synthesis of ALA and tetrapyrrole compounds in the C4 pathway, and its enzymatic properties directly affect the efficiency of ALA synthesis. In recent years, including Agr...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/77C12N1/21C12R1/15
CPCC12N9/1029C12N15/77C12Y203/01037
Inventor 孙际宾陈久洲郑平朱成超郭轩周文娟马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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