5-aminolevulinic acid synthetase mutant as well as host cell and application thereof

一种氨基乙酰丙酸、宿主细胞的技术，应用在5-氨基乙酰丙酸合成酶的突变体及其宿主细胞和应用领域，能够解决不多、没有用ALA生物合成、未研究突变酶热稳定性等问题

Active Publication Date: 2022-02-11
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, 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 synthetase with improved enzyme activity, the thermostability of the mutant enzyme was not studied, and it was not used for the biosynthesis of ALA.

Method used

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  • 5-aminolevulinic acid synthetase mutant as well as host cell and application thereof
  • 5-aminolevulinic acid synthetase mutant as well as host cell and application thereof
  • 5-aminolevulinic acid synthetase mutant as well as host cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Embodiment 1. Construction of ALA synthetase mutation carrier

[0129] Utilize the Stratagene Series XL-II site-directed mutagenesis kit, designed 19 pairs of primers (see Table 1), using the pET21a-hemA wild-type plasmid as a template (refer to Zhang et al.Biotechnology Letters, 2013,35(5):763-768 for the construction process), using The above primers were amplified by PCR, and the 15th amino acid residue of HemA was mutated into lysine (K) and arginine (R), and the 29th amino acid residue was mutated into glutamine (Q) and arginine respectively. acid (R), tyrosine (Y), lysine (K), the 39th amino acid residue is mutated to tyrosine (Y), phenylalanine (F), arginine (R), the Amino acid residue 176 is mutated to asparagine (N), lysine (K), aspartic acid (D), and amino acid residue 206 is mutated to tyrosine (Y) and phenylalanine (F ), the amino acid residue at position 236 was mutated to aspartic acid (D), asparagine (N), and arginine (R), the amino acid residue at pos...

Embodiment 2

[0133] Example 2. Expression of ALA synthase mutant enzyme and detection of enzymatic properties

[0134] The expression vectors constructed in Example 1 were transformed into E.coli BL21(DE3) to obtain recombinant strains BL21(DE3) / pET21a-hemA, BL21(DE3) / pET21a-15K, BL21(DE3) / pET21a-15R, BL21(DE3) / pET21a-29Q, BL21(DE3) / pET21a-29R, BL21(DE3) / pET21a-29Y, BL21(DE3) / pET21a-29K, BL21(DE3) / pET21a-39Y, BL21(DE3) / pET21a-39F, BL21(DE3) / pET21a-39R, BL21(DE3) / pET21a-176N, BL21(DE3) / pET21a-176K, BL21(DE3) / pET21a-176D, BL21(DE3) / pET21a-206Y, BL21 (DE3) / pET21a-206F, BL21(DE3) / pET21a-236D, BL21(DE3) / pET21a-236N, BL21(DE3) / pET21a-236R, BL21(DE3) / pET21a-293Y, BL21(DE3) / pET21a -304M and BL21(DE3) / pET21a-304Q are used for the expression of different enzymes and the detection of enzymatic properties.

[0135]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. Trans...

Embodiment 3

[0146] Example 3. Application of ALA Synthetase Mutant Enzymes in ALA Synthesis

[0147] Inoculate 5 mL of LB liquid culture medium containing 100 μg / mL ampicillin with the preserved bacteria in glycerol tubes of the recombinant bacteria in Example 2, and culture them at 37° C. and 220 rpm for 12 hours. According to the initial OD of 0.05, transfer to a 250mL Erlenmeyer flask containing 30mL fermentation medium, culture at 37°C, 220rpm for 2.5h, then add IPTG with a final concentration of 0.05mM, induce culture for 9.5h, collect the fermentation broth, 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. After det...

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Abstract

The invention provides 5-aminolevulinic acid synthetase (ALA synthetase). The amino acid sequence of the ALA synthetase is mutated at a part of amino acid residues corresponding to the amino acid sequence shown in SEQ ID NO: 1. According to the ALA synthetase disclosed by the invention, the activity and the yield of ALA are remarkably improved. Meanwhile, the invention also provides a method for preparing and modifying the enzyme, an expression vector containing the enzyme, a host cell and application of the enzyme in ALA production.

Description

[0001] This application was submitted on July 13, 2018, the application number is 201810772331.5, and the invention title is a divisional application of the invention patent application named "5-aminolevulinic acid synthase mutant and its host cell and application". technical field [0002] 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 [0003] 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, result...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N15/77C12N1/21C12P13/02C12R1/19C12R1/15
CPCC12N9/1029C12P13/02C12Y203/01037C12N15/70C12N15/77
Inventor 郑平赵晶谭子瑊陈久洲孙际宾马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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