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5-aminolevulinic acid (ALA) synthetase mutant and host cells and application thereof

A host cell, amino acid technology, applied to mutants of 5-aminolevulinic acid synthase and its host cells and application fields, can solve the problem of not many, no ALA biosynthesis, no research on the thermostability and resistance of mutant enzymes Hemoglobin feedback inhibition ability and other issues

Active Publication Date: 2018-07-06
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are not many studies on the rational design and transformation of ALA synthetase. The only reports mainly involve the ALA synthase derived from eukaryotes. Turbeville et al. expressed two ALA synthetase 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 improved enzyme activity by constructing a mutant library of ALA synthase II in mouse erythrocytes Synthetase mutant (Lendrihas et al. Journal of Biological Chemistry, 2010, 285(18): 13704)
Although the above studies obtained ALA synthase with improved enzyme activity, they did not study the thermostability and anti-heme feedback inhibition ability of the mutant enzyme, and did not use it for ALA biosynthesis

Method used

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  • 5-aminolevulinic acid (ALA) synthetase mutant and host cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Embodiment 1. Construction of ALA synthetase mutation carrier

[0146] Utilize the Stratagene Series Site-directed mutagenesis kit, design 2 pairs of primers (see Table 1), use pET21a-hemA (refer to Zhang et al. Biotechnology Letters, 2013, 35(5): 763-768) wild-type plasmid as a template, use the above primers Perform PCR amplification, respectively mutate the arginine (R) at the 40th and 365th positions of HemA to glycine (G) and lysine (K), the PCR reaction conditions are: 95 ° C for 5 min, 10 cycles (95 ℃30s, 74℃-65℃30s, 68℃7min), 13 cycles (95℃30s, 65℃30s, 68℃7min), 68℃10min. PCR amplification system (50 μL): template 1 μL, upstream and downstream primers 2 μL, dNTP mix 1 μL, 10×Pyrobest Buffer 5 μL, sterilized double distilled water 38.5 μL, Pyrobest DNA polymerase 0.5 μL. PCR products were purified and recovered using a gel recovery kit. The transformants were sequenced by Jinweizhi Company, and the correctly sequenced expression vectors were named pET21a-R40G...

Embodiment 2

[0149] Example 2. Detection of ALA Synthetase Mutant Enzyme Enzymatic Properties

[0150] Transform the pET21a-hemA, pET21a-R40G and pET21a-R365K mutant plasmids into E.coliBL21(DE3), respectively, to obtain recombinant strains BL21(DE3) / pET21a-hemA, BL21(DE3) / pET21a-R40G, BL21(DE3) / pET21a-R365K, used for the expression of different enzymes and the detection of enzymatic properties.

[0151] Inoculate 5 mL of LB liquid medium containing 100 μg / mL ampicillin with a single colony of the above-mentioned recombinant bacteria, and culture at 37° C. and 220 rpm for 12 hours. Transfer to a 500mL Erlenmeyer flask filled with 100mL LB liquid medium containing 100μg / mL ampicillin, culture at OD at 37°C and 220rpm 600 After reaching 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and the cells were collected after 22 hours of induction culture at 20°C and stored at -80°C. The specific purification steps are as follows:

[0152] (1) Resuspend the cells that have been col...

Embodiment 3

[0164] Example 3. Application of ALA Synthetase Mutant Enzymes in ALA Synthesis

[0165] Inoculate 5 mL of LB liquid medium containing 100 μg / mL ampicillin with a single colony of the above-mentioned recombinant bacteria, and culture 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 3h, add IPTG with a final concentration of 0.05mM, collect the fermentation broth after 24h of induction culture, and detect the concentration of ALA. The shake flask fermentation medium formula is: glucose 15g / L, yeast powder 2.0g / L, 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 2.0mM, CaCl 2 0.1mM, glycine 4g / L, adjust the pH to 7.0. The final concentration of ampicillin was 100 μg / mL. ALA detection and glucose analysis methods are described in the "Materials and Methods" section. The fermentation results of the recombinant ba...

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Abstract

The invention provides a 5-aminolevulinic acid (ALA) synthetase. The amino acid sequence of the ALA synthetase has mutation at the amino acid residues of the 40th site, the 365th site, the 75th site,the 29th site and the 44th site corresponding to the amino acid sequence as shown in SEQ ID NO:1. The ALA synthetase provided by the invention obviously improves the activity and also partially relieves the feedback inhibition of hemachrome. Furthermore, the invention also provides methods for preparing and modifying the ALA synthetase, an expression vector containing the ALA synthetase, host cells containing the ALA synthetase, and application of the ALA synthetase in ALA production.

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. At present, ALA is mainly produced by chemical synthesis, and the cost of raw materials and pollutant emissions are high, resulting in high production costs and limiting its large-scale application in agriculture, feed and other fields. In recent years, the production of ALA by microbial fermentation has been applied industrially. There are two main biosynthesis pathways for ALA, one is to sy...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12P13/00
CPCC12N9/1029C12P13/005C12Y203/01037
Inventor 郑平赵晶谭子瑊陈久洲饶德明孙际宾马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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