5-aminolevulinic acid synthetase mutant, and host cell and application thereof

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

Active Publication Date: 2020-12-01
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 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 have obtained ALA synthase with improved enzyme activity, the ALA synthase derived from eukaryotes has low expression activity in bacteria, but it has not been used for ALA biosynthesis

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0149] Example 1. Structural Analysis of ALA Synthetase

[0150] The literature reported the protein structure of ALA synthase (RcA) derived from Rhodobacter capsulata (PDB database number: 2BWN) and its substrates glycine (PDB database number: 2BWP) and succinyl-CoA (PDB Database number: 2BWO) co-crystallized protein structure, in which Arg374 site directly interacts with glycine, and Thr365 site directly interacts with succinyl-CoA (Astner et al. EMBO Journal 2005; 24:3166-3177). After structural analysis of the enzyme, it was found that the two regions of 332-348 and 358-374 in its amino acid sequence (shown in purple) are related to substrate binding (such as figure 1 shown), the above region includes the Arg374 site that directly interacts with glycine and the Thr365 site that directly interacts with succinyl-CoA, and its flexible C-terminus is relatively close to the spatial position of the above two regions. The inventors speculate that the mutation of the flexible C-t...

Embodiment 2

[0152] Example 2: Sequence Analysis of ALA Synthetase from Different Species

[0153] First, the inventors compared and analyzed the amino acid sequences of HemA of different Rhodopseudomonas palustris subspecies that have been reported, and found that the sequence identity between different strains was very high, reaching 94%, and the 403rd position of the C-terminal Amino acid residues are highly conserved, all of which are alanine (such as figure 2 shown).

[0154] The present inventors further expanded the scope of sequence alignment, and performed Blast alignment at NCBI with the amino acid sequence of Rhodopseudomonas palustris ATCC17001 HemA (RpA), and analyzed the sequence annotated as ALA synthetase. Although the sources of reported ALA synthetases are different, most of the amino acid sequences of ALA synthetase have alanine at the C-terminal end or the 403rd position. The specific comparison results are as image 3 shown.

[0155] For example, the ALA synthetas...

Embodiment 3

[0156] Embodiment 3. Construction of Rhodopseudomonas palustris ATCC17001HemA (RpA) mutant vector

[0157] Utilize the Stratagene Series XL-II site-directed mutagenesis kit, designed 39 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, respectively mutating the 403rd amino acid residue of Rhodopseudomonas palustris ATCC17001HemA(RpA) Arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), Histidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F), Proline (P ), Serine (S), Threonine (T), Tryptophan (W), Tyrosine (Y), Valine (V). The PCR reaction conditions are: 95°C for 5min, 10 cycle...

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Abstract

The invention provides a 5-aminolevulinic acid synthetase (ALA synthetase). The C-terminal tail amino acid residue of the ALA synthetase is deleted or is non-alanine; and/or, the amino acid residue atthe 403rd position corresponding to SEQ ID NO:1 of the amino acid sequence of the ALA synthetase is deleted or is non-alanine; and/or, 1-N arbitrary amino acid residues are added to the tail positionof the C terminal of the ALA synthetase, and have the ALA synthetase activity. The invention also provides a method for preparing the enzyme, an expression vector containing the enzyme, a host cell,application of the enzyme 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N15/74C12N15/77C12N15/78C12N1/21C12R1/19C12R1/15C12R1/38C12R1/01
CPCC12N9/1029C12Y203/01037C12N15/70C12N15/74C12N15/77C12N15/78C12N15/52C12N15/67C12P13/00
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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