5-aminolevulinic acid synthetase mutant and its host cells and use

Abstract

To provide: a 5-aminolevulinic acid synthetase (ALA synthetase) improved in activity for producing 5-aminolevulinic acid (ALA) present widely in animals, plants and microbes as a precursor of a tetrapyrrole compound such as heme, chlorophyll and VB12 synthesized by organisms; a method of enhancing ALA synthetase activity; and a method of producing ALA using the obtained ALA synthetase.SOLUTION: The invention provides an ALA synthetase having a specific amino acid sequence or an active fragment thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to mutants of 5-aminolevulinic acid synthase and host cells thereof use and

Background Art

[0002] 5-aminolevulinic acid (ALA) widely exists in animals, plants, and microorganisms as a precursor of tetrapyrrole compounds such as heme, chlorophyll, and VB12 synthesized by organisms. ALA is a high-value-added bio-based chemical substance widely used in fields such as pharmaceuticals, agriculture, feed, and health foods, and has high development value. In recent years, the fermentative production of ALA via the C4 pathway has been industrially use performed using microorganisms.

[0003] 5-aminolevulinic acid synthetase (ALA synthetase) is a crucial enzyme and rate-limiting enzyme for the synthesis of ALA and tetrapyrrole compounds via the C4 pathway, and the superiority or inferiority of its enzymatic properties directly affects the efficiency of ALA synthesis. In recent years, a large number of ALA synthetases from various sources, including Agrobacterium radiobacter (CN1322132C), Rhodoblastus acidophilus (CN1974758B), Rhodobacter sphaeroides (CN103146694B), and Rhodopseudomonas palustris (CN103981203B), have been cloned and identified, and high-yielding ALA strains have been constructed. However, all of the aforementioned ALA synthetases are sources of enzymes that naturally exist in different organisms, and the activity of these enzymes themselves is not high (Meng et al. Biotechnology Letters, 2015, 37(11): 2247-2253; Meng et al. Biotechnology Letters, 2015, 37(11): 2247-2253), so in engineering bacteria... use It's difficult to do.

[0004] In recent years, with the rapid development of enzyme engineering and protein engineering technologies, improving the properties of enzymes, such as catalytic activity, through rational design has become widespread. useHowever, there is currently little research on the rational design and improvement of ALA synthetases. Previous reports have mainly focused on eukaryotic ALA synthetases. Turbeville et al. were able to increase the catalytic efficiency after fusion expression of two mouse-derived ALA synthetases (Turbeville et al. Archives of Biochemistry & Biophysics, 2011, 511(1): 107-117), and Lendrihas et al. constructed a library of mutants of ALA synthetase II in mouse erythrocytes and screened for ALA synthetase mutants with improved enzyme activity (Lendrihas et al. Journal of Biological Chemistry, 2010, 285(18): 13704). While the above studies obtained ALA synthetases with improved enzyme activity, eukaryotic ALA synthetases, although having low expression activity in bacteria, were not used in ALA biosynthesis.

[0005] Furthermore, as an important enzyme in the ALA synthesis process, improving ALA synthetase activity accelerates the catalytic efficiency of engineered bacteria, reduces energy loss during the synthesis and degradation of their unique proteins, improves the stability and productivity of engineered bacteria, reduces the production cost of ALA, and increases the use of ALA in fields such as agriculture and livestock farming. use It has great value in promoting this.

[0006] Therefore, in order to produce ALA using a low-cost method, there is an urgent need in this field to develop a highly active ALA synthetase. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide an ALA synthetase with improved activity, a method for improving ALA synthetase activity, and a method for producing ALA using the obtained ALA synthetase. [Means for solving the problem]

[0008] In a first embodiment, the present invention provides ALA synthetase or an active fragment thereof. a. The C-terminal amino acid residue of the amino acid sequence of the ALA synthetase is deleted, non-alanine, and / or b. The amino acid sequence of the ALA synthetase is missing amino acid residue 403, which corresponds to the amino acid sequence shown in SEQ ID NO:1, or is non-alanine, and / or c. The amino acid sequence of the ALA synthetase is missing amino acid residue 401, which corresponds to the amino acid sequence shown in SEQ ID NO:43, or is non-alanine, and / or d. The ALA synthetase-like amino acid sequence is missing amino acid residue 403 or 407 corresponding to the amino acid sequence shown in SEQ ID NO:42, or is non-alanine, and / or e. The present invention is characterized by adding one or more arbitrary amino acid residues to the C-terminus of the ALA synthetase, preferably 1 to 10 arbitrary amino acid residues, more preferably 1 to 6, more preferably 1 to 3, most preferably 1 arbitrary amino acid residue and a polypeptide having ALA synthetase function.

[0009] In a preferred embodiment, the ALA synthetase is obtained by mutation. In a preferred embodiment, prior to the mutation, the ALA synthetase is a polypeptide having ALA synthetase activity, wherein the amino acid residues at positions 401, 403, 407, or the C-terminal amino acid residue are alanine.

[0010] In a preferred embodiment, the ALA synthetase can be derived from a variety of species, including but not limited to Agrobacterium radiobacter, Rhodoblastus acidophilus, Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas palustris, with Rhodopseudomonas palustris being preferred.

[0011] In a more preferred embodiment, the amino acid sequence of the ALA synthetase has 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96%, 97%, 98%, and 99% homology with the amino acid sequence shown in SEQ ID NO:1, or the amino acid sequence of the ALA synthetase and the amino acid sequence shown in SEQ ID NO:42 have 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96%, 97%, 98%, and 99% homology with the amino acid sequence of the ALA synthetase and the amino acid sequence shown in SEQ ID NO:43.

[0012] In a preferred embodiment, the ALA synthetase is a. The 403rd or C-terminal amino acid residue is deleted, or the 403rd or C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:1 is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and / or b. The 401st or C-terminal amino acid residue or the 401st or C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:43 is deleted, or is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and / or c. The 403rd, 407th, or C-terminal amino acid residue is missing, or the 403rd, 407th, or C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:42 is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and / or One to ten arbitrary amino acid residues are added to the dC terminus, more preferably one to six, more preferably one to three, and most preferably one arbitrary amino acid residue and a polypeptide having ALA synthetase function, wherein the arbitrary amino acid residue is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and Ala.

[0013] In a preferred embodiment, the ALA synthetase is a. The 403rd or C-terminal amino acid residue is deleted, or the 403rd or C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:1 is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and / or b. The 401st or C-terminal amino acid residue or the 401st or C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:43 is deleted, or is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and / or c. The 403rd, 407th, or C-terminal amino acid residue is missing, or the 403rd, 407th, or C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:42 is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and / or d. Add 1-6, more preferably 1-3, and most preferably 1 arbitrary amino acid residue and a polypeptide having ALA synthetase function to the C-terminus of the ALA synthetase, wherein the arbitrary amino acid residue is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and Ala.

[0014] In a preferred embodiment, the amino acid residue added to the C-terminus of the ALA synthetase is Glu or Gln. In a preferred embodiment, the amino acid sequence of the ALA synthetase is shown in SEQ ID NO:1, a. The 403rd or C-terminal amino acid residue is deleted, or is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, and / or One to six, more preferably one to three, and most preferably one arbitrary amino acid residue and a polypeptide having ALA synthetase function are added to the bC-terminus, wherein the arbitrary amino acid residue is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and Ala.

[0015] In a preferred embodiment, the amino acid sequence of the ALA synthetase is shown in SEQ ID NO:43, a. The 401st or C-terminal amino acid residue is deleted, or is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, and / or One to six, more preferably one to three, and most preferably one arbitrary amino acid residue and a polypeptide having ALA synthetase function are added to the bC-terminus, wherein the arbitrary amino acid residue is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and Ala.

[0016] In a preferred embodiment, the amino acid residue added to the C-terminus of the ALA synthetase is Glu or Gln. In a preferred embodiment, the amino acid sequence of the ALA synthetase is shown in SEQ ID NO:42, a. The amino acid residue at position 403 or 407 or the C-terminal amino acid residue is deleted, or is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, and / or b. Add a polypeptide having 1 to 6, more preferably 1 to 3, and most preferably 1 arbitrary amino acid residue and ALA synthase function to the C-terminus, and the arbitrary amino acid residue is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, Ala.

[0017] In a preferred embodiment, the amino acid residue added to the C-terminus of the ALA synthase is Glu or Gln. In a second aspect, the present invention provides a coding nucleic acid sequence of the ALA synthase according to the first aspect or an active fragment thereof.

[0018] In a third aspect, the present invention provides the expression of a coding nucleic acid sequence containing the ALA synthase according to the first aspect or an active fragment thereof vector and provides. In a fourth aspect, the present invention provides a host cell containing the ALA synthase according to the first aspect or an active fragment thereof.

[0019] In a preferred embodiment, the host cell contains the expression according to the third aspect vector or its genome integrated with the coding nucleic acid sequence according to the second aspect. In a preferred embodiment, the host cell is Escherichia coli, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopseudomonas palustris, and more preferably Escherichia coli or Corynebacterium glutamicum.

[0020] In a fifth embodiment, the present invention relates to an ALA synthetase or an active fragment thereof according to the first embodiment, or a coding nucleic acid sequence according to the second embodiment, or an expression according to the third embodiment. vector , or a fourth aspect of host cell generation in ALA production use To provide.

[0021] In a sixth embodiment, the present invention provides a method for manufacturing ALA, the method being Step a, which involves culturing host cells according to the fourth embodiment or producing resting cells from host cells according to the fourth embodiment to generate ALA, and The process includes step b, which involves selectively separating the ALA produced in step a from the culture medium.

[0022] In a seventh embodiment, the present invention provides a method for manufacturing ALA, the method being Step a, which involves catalyzing the synthesis of ALA from succinyl-CoA and glycine using an ALA synthetase or an active fragment thereof according to the first embodiment, and The process includes step b, which involves selectively separating ALA from the reaction system.

[0023] In the eighth embodiment, the present invention provides a method for producing ALA synthetase according to the first embodiment, the method being Step a to obtain the coding sequence of ALA synthetase according to the first embodiment, The coding sequence obtained from step a is directly transfected into appropriate host cells, vector Step b involves introducing the cell into a suitable host cell, Step c involves culturing the host cells obtained from step b, and The process includes step c, and step d, which involves separating the ALA synthetase produced in the host cells from the culture system obtained from step c.

[0024] In a preferred embodiment, the method further includes the step of measuring the activity of the obtained ALA synthetase and the ALA production of the strain. In a ninth embodiment, the present invention provides an improved ALA synthetase and a method for improving its activity, the method being Compared to the amino acid sequence of the original ALA synthetase, the amino acid residues at positions 401, 403, or 407 or the C-terminus of the improved ALA synthetase are deleted or replaced with non-alanine, and / or 1-N arbitrary amino acid residues are added to the C-terminus of the ALA synthetase, preferably 1-10 arbitrary amino acid residues, more preferably 1-6, more preferably 1-3, and most preferably 1 arbitrary amino acid residue, and / or The amino acid sequence of the improved ALA synthetase is compared with the amino acid sequence shown in SEQ ID NO:1, and the coding sequence of the improved ALA synthetase is modified so that the amino acid residue at position 403 or the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:1 is deleted or replaced with a non-alanine residue. The coding sequence of the improved ALA synthetase is modified by comparing the amino acid sequence of the improved ALA synthetase with the amino acid sequence shown in SEQ ID NO:42, and by deleting or replacing with a non-alanine amino acid residue at position 403, 407, or the C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:42 in the coded amino acid sequence, and / or Step a: Compare the amino acid sequence of the improved ALA synthetase with the amino acid sequence shown in SEQ ID NO:43 and improve the coding sequence of the improved ALA synthetase by deleting or replacing the amino acid residue at position 401 or the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:43 with a non-alanine residue. The coding sequence obtained from step a is directly transfected into appropriate host cells, vector Step b involves introducing the cell into a suitable host cell, Step c involves culturing the host cells obtained from step b, and The process includes step c, and step d, which involves separating the ALA synthetase produced in the host cells from the culture system obtained from step c.

[0025] In preferred embodiments, the 403rd or C-terminal amino acid residue of the amino acid sequence of the ALA synthetase is deleted, or the 403rd or C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:1 is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and / or The amino acid residue at position 403, 407, or the C-terminal amino acid residue of the amino acid sequence of the ALA synthetase is either deleted, or it is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and / or The amino acid residue at position 401 or the C-terminal amino acid residue of the amino acid sequence of the ALA synthetase is either deleted or is one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and / or 1-N arbitrary amino acid residues are added to the C-terminus of the ALA synthetase, preferably 1-10 arbitrary amino acid residues, more preferably 1-6, more preferably 1-3, and more preferably 1 arbitrary amino acid residue and a polypeptide having ALA synthetase function, wherein the arbitrary amino acid residues are one of the amino acids selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, Met, and Ala.

[0026] In a preferred embodiment, the amino acid residue added to the C-terminus of the ALA synthetase is Glu or Gln. In a preferred embodiment, the method further includes the step of measuring the activity of the obtained ALA synthetase and the ALA production of the engineered strain containing the ALA synthetase. [Effects of the Invention]

[0027] It should be understood that, within the scope of the present invention, the above technical features of the present invention and each of the technical features specifically described below (e.g., in the examples) can be combined to form novel or preferred technical solutions. Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0028] [Figure 1] This shows the cocrystal structure of ALA synthetase from Rhodobacter capsulatus and succinyl-CoA. The purple mark indicates the region related to substrate binding. [Figure 2] The amino acid sequence alignment results for different subspecies of Rhodopseudomonas palustris, HemA, are shown. [Figure 3] The results of amino acid sequence alignment of different species of ALA synthetase are shown. [Modes for carrying out the invention]

[0029] Through extensive and thorough research, the inventors discovered that known ALA synthases have relatively low homology to their amino acid sequences, but that the C-terminus or position 403 is entirely alanine. Further research revealed that by deleting or mutating at position 403 or the C-terminus of ALA synthetase, or by adding multiple arbitrary amino acid residues to the C-terminus of ALA synthetase, the resulting ALA synthetase not only exhibits improved enzyme activity but also enhances the ALA production of the engineered strain, demonstrating superior performance. use It was unexpectedly discovered that it had value.

[0030] In relation to the present invention, ALA synthetase is SEQ ID It can be a protein corresponding to the amino acid sequence shown in NO:1 (MNYEAYFRRQLDGLHREGRYRVFADLERHAGSFPRATHHRPEGAGDVTVWCSNDYLGMGQHPAVLTAMHEALDSCGAGAGGTRNIAGTNHYHVLLEQELAALHGKESALLFTSGYVSNWASLSTLASRMPGCVILSDELNHASMIEGIRHSRSETRIFAHNDPRDLERKLADLDPHAPKLVAFESVYSMDGDIAPIAEICDVADAHNAMTYLDEVHGVGLYGPNGGGIADREGISHRLTIIEGTLAKAFGVVGGYIAGSSAVCDFVRSFASGFIFSTSPPPAVAAGALASIRHLRASSAERERHQDRVARLRARLDQAGVAHMPNPSHIVPVMVGDAALCKQISDELISRYGIYVQPINYPTVPRGTERLRITPSPQHTDADIEHLVQALSEIWTRVGLAKAA), SEQ ID The ALA synthetase of the present invention may not be a protein corresponding to the amino acid sequence shown in NO:1; in other words, the ALA synthetase of the present invention may have relatively high homology to the amino acid sequence shown in SEQ ID NO:1, or it may have relatively low homology. The ALA synthetase of the present invention may be derived from various species, including, but not limited to, Agrobacterium radiobacter, Rhodoblastus acidophilus, Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas palustris, and any protein having ALA synthetase activity, with the corresponding 403rd or C-terminal amino acid residue deleted or mutated, and / or with 1-N arbitrary amino acid residues added to its C-terminus, thereby improving its activity compared to its natural state, falls within the scope of the present invention.In particular, the inventors discovered a new mutation site from the amino acid sequence of wild-type ALA synthetase, pioneering a novel idea to obtain an ALA synthetase with superior activity, and thus laid the foundation for the material. Based on this, the present invention was completed.

[0031] As used herein, the term "natural state" refers to the activity of a polypeptide in its unmodified state, i.e., its activity in its natural state. "ALA synthetase" As used herein, the terms “5-aminolevulinic acid synthetase” and the “ALA synthetase” and “polypeptide of the present invention” are interchangeable and have meanings generally understood by those skilled in the art. The ALA synthetase of the present invention has the activity to catalyze the synthesis of ALA from succinyl-CoA and glycine.

[0032] Specifically, the amino acid residue at position 403 or the C-terminal amino acid residue of the ALA synthetase of the present invention is deleted or non-alanine, and the amino acid sequence of the ALA synthetase corresponds to the amino acid sequence shown in SEQ ID NO:1, where the amino acid residue at position 403 or the C-terminal amino acid residue is deleted or non-alanine, and / or 1-N arbitrary amino acid residues are added to the C-terminus of the ALA synthetase, preferably 1-10, preferably 1-6, more preferably 1-3, and most preferably 1 arbitrary amino acid residue, and a polypeptide having ALA synthetase function is added.

[0033] Those skilled in the art know that, in order to improve activity, it is more important to mutate the wild-type polypeptide than to find the site of interest that is necessary. Accordingly, based on the teachings of the present invention, those skilled in the art have measured the relevant activity of mutants by deleting or mutating the corresponding 403rd or C-terminal amino acid residue of the improved ALA synthetase, or by adding 1-N arbitrary amino acid residues to the C-terminus.

[0034] In specific embodiments, the 403rd or C-terminal amino acid residue of the ALA synthetase of the present invention may be deleted from or mutated to (but not limited to) Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ile, Leu, Lys, or Met.

[0035] Furthermore, it is not difficult for those skilled in the art to know that, in certain regions of a polypeptide, altering a small number of amino acid residues does not fundamentally change its biological activity; for example, a sequence obtained by appropriately substituting specific amino acids does not affect its activity. (See Watson et al., Molecular Biology of The Gene, 4th edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Therefore, those skilled in the art can make these substitutions and ensure that the resulting molecule still possesses the desired biological activity.

[0036] Therefore, the present invention includes conserved variants of the ALA synthetase of the present invention. These conserved variants can be generated by amino acid substitutions as shown in the table below.

[0037] [Table 1] TIFF0007845639000002.tif85170

[0038] The present invention further provides polynucleotides encoding the polypeptide of the present invention. The term “polynucleotide encoding polypeptide” may include the polynucleotide encoding this polypeptide and may further include additional coding and / or non-coding sequences.

[0039] The ALA synthetase gene can be a probe produced under strict conditions for the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:42, and SEQ ID NO:43, for example, DNA hybridized with a sequence complementary to part or all of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:42, and SEQ ID NO:43, as long as initial function is maintained. The aforementioned "strict conditions" refer to conditions under which so-called specific hybridization can be formed and non-specific hybridization cannot be formed. For example, this means conditions in which DNA with high homology, such as DNA with 80% or more homology, is hybridized with each other, while DNA with less than 80% homology is not hybridized with each other, or the usual Southern hybridization washing conditions, i.e., washing once, preferably two to three times, at a salt concentration and temperature of 60°C, 1 * SSC, 0.1% SDS, preferably 60°C, 0.1 * SSC, 0.1% SDS, more preferably 68°C, 0.1 * SSC, 0.1% SDS.

[0040] Furthermore, since codon denaturation varies depending on the host, any codon in the ALA synthetase gene can be replaced with a corresponding equivalent codon; that is, the ALA synthetase gene can be a mutation of any of the ALA synthetase genes in the above example due to denaturation of the gene code. For example, the ALA synthetase gene can be a gene that has been modified to have the optimal codon depending on the frequency of the codon in the host used.

[0041] Therefore, in specific embodiments, the ALA synthetase gene can be DNA having a homology or sequence identity of 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more, and more preferably 96%, 97%, 98%, or 99% with the DNA encoding the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:42, and SEQ ID NO:43.

[0042] Therefore, as used herein, “contains,” “has,” or “includes” includes “contains,” “mainly consists of,” “basically consists of,” and “consist of,” and “mainly consists of,” “basically consists of,” and “consist of” are sub-concepts of “contains,” “has,” or “includes.”

[0043] As used herein, the term “active fragment” has the same or similar meaning as commonly understood by those skilled in the art, and all such fragments have an amino acid sequence that is part of the amino acid sequence of a complete protein or polypeptide, but the fragment has the same or similar function or activity as the complete protein or polypeptide. Specifically, in the present invention, “active fragment” refers to any amino acid fragment having ALA synthetase activity obtained from the ALA synthetase of the present invention.

[0044] Based on the teachings of the present invention and the ALA synthetase specifically obtained by the present invention, it is not difficult for those skilled in the art to obtain active fragments having the same or similar activity or function, and these active fragments must, of course, fall within the scope of protection of the present invention.

[0045] "handle" As used herein, the term "corresponding" has the meaning generally understood by those skilled in the art. Specifically, "corresponding" means that, after comparing the homology or sequence identity of two stranded sequences, the single-stranded sequence indicates a position that corresponds to a position specified in the other single-stranded sequence. Therefore, for example, in the case of "the 11th amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:1," when a 6×His tag is added to one end of the amino acid sequence shown in SEQ ID NO:1, the resulting mutant can have the 11th and 17th amino acids corresponding to the amino acid sequence shown in SEQ ID NO:1.

[0046] As described above, the amino acid sequence homology of known ALA synthetases is not high, but the C-terminus or site 403 is basically alanine. Therefore, the ALA synthetase of the present invention does not necessarily need to have high sequence homology or identity with the amino acid sequence shown in SEQ ID NO:1. For example, the homology or sequence identity can be 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more. In specific embodiments, the homology or sequence identity can be 80% or more, 90% or more, preferably 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more.

[0047] Methods for measuring sequence homology or identity known to those skilled in the art include: Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, edited by Gribskov, M. and Develeux, J., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073. This includes, but is not limited to, (1988). The preferred method for measuring homology is to obtain the greatest match between the sequences being tested. Methods for measuring identity have been compiled into publicly available computer programs. Preferred computer program methods for measuring identity between double-stranded sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., 1990). The public can obtain the BLASTX program from NCBI and other sources (BLAST Handbook, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). Identity can also be measured using the well-known Smith-Waterman algorithm.

[0048] host cell As used herein, the term “host cell” has the meaning commonly understood by those skilled in the art, namely, a host cell capable of producing the ALA synthetase of the present invention. In other words, the present invention can utilize any host cell, as long as the ALA synthetase of the present invention can be expressed in the host cell.

[0049] For example, the host cells to which the present invention is applied may be derived from (but are not limited to) Escherichia coli, Corynebacterium glutamicum, Rhodobacter sphaeroides, or Rhodopseudomonas palustris, and more preferably Escherichia coli or Corynebacterium glutamicum.

[0050] Immobilized enzymes As used herein, the term “immobilized enzyme” has the meaning generally understood by those skilled in the art. Specifically, the term refers to a water-soluble enzyme that has been treated by physical or chemical means to form a water-insoluble macromolecule. vector This indicates that the enzyme is bound to or embedded within a membrane, causing it to form a water-soluble gel or semipermeable microcapsule, thus reducing its fluidity.

[0051] Immobilized enzymes still possess enzymatic activity and act on substrates in a solid state in catalytic reactions. Generally, after immobilization, enzymes become more stable, easier to separate from reaction systems, easier to control, can be reused multiple times, are convenient for transport and storage, and are advantageous for automated production. Immobilized enzymes are enzymes developed over the past decade. use In terms of technology, it is attractive in fields such as industrial production, chemical analysis, and pharmaceuticals. use We have this outlook.

[0052] In light of the teachings herein, it will not be difficult for those skilled in the art to process the ALA synthetase of the present invention into the form of an immobilized enzyme or a whole-cell catalyst for catalyzing the production of ALA from glycine and succinyl-CoA.

[0053] This invention use and advantages 1. The ALA synthetase of the present invention can be applied industrially and enables low-cost production of ALA. 2. The ALA synthetase of the present invention is highly active and has a wide range of applications in industry. use With the outlook, 3. The present invention provides mutation sites for improved ALA synthetase, and mutations at these sites can effectively enhance the enzymatic activity of the improved ALA synthetase.

[0054] The present invention will be further described below in relation to specific examples. Please understand that these examples are merely for illustrative purposes and do not limit the scope of the present invention. Experimental methods in the following examples that do not explicitly state specific experimental conditions should follow normal conditions, for example, conditions based on Sambrook et al., Molecular Clones: Laboratory Handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions proposed by the manufacturer.

[0055] Materials and methods The DNA polymerase used in the examples of this invention was purchased from Pyrobest of Dalian Bao Biological Company, and restriction enzymes and DNA ligases were all purchased from Fermentas. Yeast powder and peptone were purchased from Oxoid Company in the UK, glycine and IPTG from Promega, ALA and p-dimethylaminobenzaldehyde, etc. from Sigma, perchlorheme, agar powder and antibiotics from Beijing Solarbio, and glucose, glacial acetic acid, perchloric acid, trichloroacetic acid, acetylacetone chloroform and other commonly used chemical reagents were all purchased from TCM.

[0056] Plasmid extraction kits and agarose gel electrophoresis recovery kits were purchased from Shanghai Sangon, and the relevant procedures were strictly carried out according to the manual. Plasmid construction sequence validation was completed by GENEWIZ. E. coil DH5α-eligible cells were purchased from Beijing TransGen.

[0057] LB medium components: 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, and 2% agar powder were added to the solid medium. M9 medium components: 17.1 g / L Na2HPO4·12H2O, 3.0 g / L KH2PO4, 0.5 g / L NaCl, 1.0 g / L NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, 20 g / L glucose, and 2 g / L yeast powder.

[0058] The antibiotic concentrations are 100 μg / mL ampicillin and 50 μg / mL kanamycin. ALA detection method: 200 μL of diluted fermentation broth or the sample after the enzyme activity measurement reaction was completed was added to 100 μL of pH 4.6 sodium acetate buffer, then 5 μL of acetylacetone was added, and the mixture was incubated in a 100 °C wet bath for 15 minutes. After cooling to room temperature, the same volume of Ehrlish's reagent (42 mL of glacial acetic acid, 8 mL of 70% perchloric acid, and 1 g of dimethylaminobenzaldehyde) was added and mixed, and after color development for 10 minutes, the absorbance at a wavelength of 553 nm was measured.

[0059] The procedure for measuring crude enzyme activity is as follows: (1) Bacterial cells collected by centrifugation and kept at a temperature of -80°C (OD 600 = 25) is resuspended in 1 mL of Tris-HCl buffer (pH 7.5, containing 100 mM NaCl), (2) Cell destruction with MP: Destroy cells 7 times under the conditions of 6 M / S, 30 seconds. (3) The cell disruption solution obtained in the above step is centrifuged and the supernatant is collected. (4) Protein concentration was measured using the Thermo Scientific BCA Protein Quantification Kit, following the instructions in the manual.

[0060] Measurement of enzyme activity: The measurement reaction was carried out in a 50 mM Tris-HCl buffer system (pH 7.5, containing 100 mM NaCl) with a total volume of 200 μL. The components of the enzyme reaction system were as shown in Table 1. 10 μL of cell disruption supernatant was added, and the reaction was shaken in a metal bath at 37 °C for 6 minutes. Then, 100 μL of 10% trichloroacetic acid solution was added to the system to terminate the reaction.

[0061] [Table 2]

[0062] Example 1. Structural analysis of ALA synthetase The literature reports the protein structure of ALA synthetase (RcA) derived from Rhodobacter capsulatus (PDB database number: 2BWN), as well as its co-crystallized protein structures with its substrates, glycine (PDB database number: 2BWP) and succinyl-CoA (PDB database number: 2BWO). Here, the Arg374 site directly interacts with glycine, and the Thr365 site directly interacts with succinyl-CoA (Astner et al. EMBO Journal 2005; 24: 3166-3177). After analyzing the structure of the enzyme, it was found that two regions of its amino acid sequence, 332-348 and 358-374 (shown in purple), are related to substrate binding (see Figure 1). These regions include the Arg374 site, which directly interacts with glycine, and the Thr365 site, which directly interacts with succinyl-CoA. The flexible C-terminus is located close to the spatial position of these two regions. The inventors hypothesized that mutations in the flexible C-terminal residue could cause different interactions between the two regions of the substrate structure, thereby affecting substrate binding and ALA synthetase activity.

[0063] Furthermore, the inventors also simulated the structure of HemA (RpA) derived from Rhodopseudomonas palustris ATCC17001. Although the amino acid sequence of the enzyme showed only 56% sequence identity with that of Rhodobacter capsulata ALA synthetase (RcA), whose protein structure had been analyzed, the protein structure predicted by the simulation was similar to the aforementioned structure, and it had a similar flexible region at the C-terminus (position 403). The inventors speculated that mutations at C-terminus 403 could affect substrate binding and ALA synthetase activity.

[0064] Example 2. Sequence analysis of ALA synthetase derived from different species First, the inventors compared and analyzed the amino acid sequences of HemA, a different reported subspecies of Rhodopseudomonas palustris, and found that the sequence identity between the different strains was very high, reaching 94%, and that the C-terminal amino acid residue 403 was highly conserved, all of which was alanine (see Figure 2).

[0065] The inventors further expanded the scope of sequence alignment and performed Blast alignment of the rhodopseudomonas pulsetris ATCC17001 HemA(RpA) amino acid sequence in NCBI to analyze sequences annotated as ALA synthetase. Although the reported ALA synthetase species have different origins, the C-terminus or 403rd amino acid of ALA synthetase sequences is almost always alanine. The specific alignment results are shown in Figure 3.

[0066] For example, the ALA synthetase (RcA) of Rhodobacter capsulata, whose protein structure has been analyzed, is 59% identical to the RpA amino acid sequence of Rhodopseudomonas palustris, but its C-terminal position 401 is also alanine. As another example, the ALA synthetase (RsA) of Rhodobacter sphaeroides, which is most commonly used in ALA biosynthesis, is 57% identical to the RpA amino acid sequence of Rhodopseudomonas palustris, but its positions 403 and C-terminal 407 are still alanine. Combining the protein structure analysis in Example 1, the inventors further hypothesized that mutations in the C-terminus or position 403 of ALA synthetase may enhance the activity of ALA synthetases from different species.

[0067] Example 3. Rhodopseudomonas palustris ATCC17001 HemA(RpA) mutant vector Construction Using the Stratagene series QuikChange® XL-II site-directed mutagenesis kit, 39 pairs of primers (see Table 2) were designed, and the pEC-XK99E-hemA wild-type plasmid was used as a template (the construction process was described in *Construction and Optimization of the 5-aminolevulinic acid synthesis pathway by Corynebacterium glutamicum [J]. Biotechnology Bulletin, 2017, 33(01):148-156.). The above primers were used for PCR amplification to synthesize Rhodopseudomonas palustris ATCC17001. The 403rd amino acid residue of HemA(RpA) is mutated to 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), and valine (V). The PCR reaction conditions are as follows: 10 cycles of 5 minutes at 95°C (30 seconds at 95°C, 30 seconds at 74°C-65°C, 4.5 minutes at 72°C), 13 cycles of 30 seconds at 95°C, 30 seconds at 65°C, 4.5 minutes at 72°C, and 10 minutes at 72°C. The PCR amplification system (50 μL) consists of 1 μL of template, 1 μL each of upstream and downstream primers, 4 μL of dNTP mix, 10 μL of 5×TransStart® Fast Pfu Fly Buffer, 32 μL of sterile double distilled water, and 1 μL of TransStart® Fast Pfu Fly DNA Polymerase. The above PCR product is purified and recovered using a gel recovery kit, transformed into E. coli DH5α-compatible cells, coated onto LB plates containing kanamycin, cultured overnight at 37°C, and verified by sequencing of the transformants for accurate expression. vector These were named p403R, p403N, p403D, p403C, p403Q, p403E, p403G, p403H, p403I, p403L, p403K, p403M, p403F, p403P, p403S, p403T, p403W, p403Y, and p403V, ​​respectively.

[0068] [Table 3] TIFF0007845639000005.tif177170

[0069] Example 4. Construction of recombinant strain and verification of fermentation. Precise expression of RpA mutations vector The C. glutamicum strain ATCC13032 was transformed to obtain an engineered strain. The RpA mutant engineered strain was subjected to ALA fermentation in a 24-well plate, and the mutant engineered strain was cultured for 16 hours in a 24-well plate of M9 medium containing yeast powder to obtain seed solutions, with an initial OD of 0.5. 600 The samples were transferred to a 24-well plate containing M9 medium, fermentation was verified, and the culture was incubated for 3 hours. Inducer IPTG was added at a final concentration of 100 μM. After 24 hours, fermentation was complete, and the crude ALA synthetase enzyme activity and ALA production were measured. The methods for detecting crude enzyme activity, ALA detection, and glucose analysis are as described in "Materials and Methods." After detection, the crude enzyme activity of each mutant was improved compared to the wild-type strain. There was a certain difference in ALA production among the recombinant strains, and as shown in Table 3, the ALA production of the wild-type strain was set to 1.

[0070] The results showed that, compared to the wild type, a mutation in position 403 of RpA to any amino acid other than non-alanine could improve ALA production, with the improvement ranging from 3% to 40%.

[0071] [Table 4]

[0072] Example 5: Verification of a 5 L fermentation tank We selected a strain expressing the effective mutant described above (where cell 403 is mutated to Asp, Gln) and tested it in a 5 L fermentation tank. The fermentation medium consisted of 5 g / L (NH4)2SO4, 5 g / L KH2PO4, 2 g / L corn steep liquor dry powder, 2 g / L MgSO47H2O, and 1 mg / L thiamine hydrochloride. The control temperature for the fermentation process was 30 °C, pH 6.5, and dissolved oxygen was above 30%. As a result, the ALA production of the above strains reached 20.6 g / L and 22 g / L, respectively, which is 20% and 28.4% higher than the control strain (17.2 g / L), indicating that the engineered strain expressing the above mutant can effectively increase the production of the product ALA at the fermentation tank level.

[0073] Example 6. Verification of the C-terminal alanine mutation in Rhodobacter capsulata ALA synthetase (RcA) and its effects. To further confirm the crucial role of non-alanine at position 401 of the C-terminus of ALA synthetase in ALA synthetase activity, we conducted a structural study that was relatively clear and successful in ALA biosynthesis. use Using ALA synthetase (RcA) derived from Rhodobacter capsulata (Yang et al. Applied and Environmental Microbiology 2016; 82: 2709-2717), three amino acids with different characteristics (Asp, His, Tyr) were selected for C-terminal mutation testing.

[0074] First, the corresponding DNA coding sequence (SEQ ID NO: 44) was synthesized according to the RcA amino acid sequence (SEQ ID NO: 43; GenBank: AML83926.1) published by NCBI. Using the synthesized gene fragment as a template, primers were designed according to the DNA sequence (see Table 4). PCR amplification was performed to replace the alanine at C-terminal position 401 with aspartic acid (Asp), histidine (His), tyrosine (Tyr) gene fragments and the wild-type gene fragment, respectively. Simultaneously, pEC-XK99E-hemA was reverse-amplified using pEC-Rc-F / R primers to obtain a plasmid fragment from which the original heMA gene had been removed. PCR reaction conditions: 5 minutes at 95°C, 10 cycles (30 seconds at 95°C, 30 seconds at 65°C-55°C, 4 minutes at 72°C), 20 cycles (30 seconds at 95°C, 30 seconds at 55°C, 4 minutes at 72°C), 5 minutes at 72°C. PCR amplification system (50 μL): 1 μL template, 1 μL upstream and downstream primers each, 4 μL dNTP mix, 10 μL 5×TransStart® Fast Pfu Fly Buffer, 32 μL sterile redistilled water, 1 μL TransStart® FastPfu Fly DNA polymerase. The PCR products were purified and recovered using a gel recovery kit, the target gene fragments and plasmid fragments were recombined using a recombination kit, transformed into E. coli T1 competent cells, coated with an LB plate containing kanamycin, incubated overnight at 37 °C, and the transformants were sequenced. The correct recombinant vectors were named pRc401WT, pRc401D, pRc401H, and pRc401Y.

[0075] Next, the vector was transformed into C. glutamicum ATCC13032 strain to obtain the corresponding recombinant strain. The strain was fermented using a 24-well plate, and the culture process and parameters were the same as in Example 4. The fermentation results showed that mutations of alanine at C-terminal position 401 of Rhodobacter capsulata ALA synthetase (RcA) to aspartic acid, histidine, and tyrosine significantly increased enzyme activity, and the ALA yield of the recombinant strain increased by 19%, 15%, and 11%, respectively, compared to the control strain expressing the wild-type enzyme.

[0076] [Table 5]

[0077] SEQ ID NO: 43, Rhodobacter capsulatus ALA synthetase (RcA) amino acid sequence MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQWNRPDGGKQDITVWCGNDYLGMGQHPVVLAAMHEALEAVGAGSGGTRNISGTTAYHRRLEAEIADLHGKEAALVFSSAYIANDATLSTLRVLFPGLIIYSDSLNHASMIEGIKRNAGPKRIFRHNDVAHLRELIAADDPAAPKLIAFESVYSMDGDFGPIKEI CDIADEFGALTYIDEVHAVGMYGPRGAGVAERDGLMHRIDIFNGTLAKAYGVFGGYIAASAKMVDAVRSYAPGFIFSTSLPPAIAAGAQASIAFLKTAEGQKLRDAQQMHAKVLKMRLKALGMPIIDHGSHIVPVVIGDPVHTKAVSDMLLSDYGVYVQPINFPTVPRGTERLRFTPSPVHDLKQIDGLVHAMDLLWARCA SEQ ID NO:44: Rhodobacter capsulatus ALA synthetase (RcA) DNA sequence Example 7. Mutations in the C-terminus and position 403 of Rhodobacter ferroides ALA synthetase (RsA) and verification of their effects. To reaffirm the crucial role of non-alanine at the C-terminus or position 403 in ALA synthetase activity, we further selected ALA synthetase (RsA) from Rhodobacter sphaeroides, the most commonly used ALA biosynthesis (see amino acid sequence SEQ ID NO: 42), and replaced its alanine at positions 403 and C-terminus 407, respectively, with Asp to test the mutagenesis effect.

[0078] First, primers (as shown in Table 5) were designed based on the RshemA gene sequence (SEQ ID NO: 45) from Rhodobacter spheroides disclosed in the literature (Meng et al. Biotechnology Letters, 2015, 37(11): 2247-2253). PCR amplification was performed using pRsA (Chen et al. Biotechnology for Biofuels, 2020, 13: 41) as a template to obtain plasmid fragments in which alanine at position 403 and C-terminal position 407 were replaced with aspartic acid (Asp). PCR reaction conditions: 5 minutes at 95 °C, 10 cycles (30 seconds at 95 °C, 30 seconds at 65 °C-55 °C, 4 minutes at 72 °C), 20 cycles (30 seconds at 95 °C, 30 seconds at 55 °C, 4 minutes at 72 °C), 5 minutes at 72 °C. PCR amplification system (50 μL): 1 μL template, 1 μL upstream and downstream primers each, 4 μL dNTP mix, 10 μL 5×TransStart® Fast Pfu Fly Buffer, 32 μL sterile redistilled water, 1 μL TransStart® Fast Pfu Fly DNA polymerase. The PCR product was purified and recovered using a gel recovery kit, phosphorylated with T4 PNK, and ligated with T4 ligase to transform E. coli T1 competent cells. The cells were coated with LB plates containing kanamycin, incubated overnight at 37°C, and the transformants were sequenced to identify the correct recombinant vectors as pRs403D and pRs407D.

[0079] Next, the aforementioned strain and a control strain possessing pRsA were examined by fermentation using a 24-well plate. Recombinant strains were cultured in LB medium as a seed solution for 12 hours, and the initial OD was measured. 600The cultures were transferred to 24-well plates containing 0.1 ml of M9 medium to confirm fermentation. After 3 hours of incubation, a final concentration of 50 μM IPTG was added to induce fermentation. After 20 hours, fermentation was complete, and ALA production was detected. The fermentation results showed that mutations of alanine at positions 403 and 407 to aspartic acid significantly increased the activity of Rhodobacter ferroides ALA synthetase (RsA), and recombinant ALA yields increased by 11% and 22%, respectively, compared to the control strain expressing the wild-type enzyme.

[0080] [Table 6]

[0081] SEQ ID NO:42, Rhodobacter sphaeroides ALA synthetase (RsA) amino acid sequence MDYNLALDTALNRLHTEGRYRTFIDIERRKGAFPKAMWRKPDGSEKEITVWCGNDYLGMGQHPVVLGAMHEALDSTGAGSGGTRNISGTTLYHKRLEAELADLHGKEAALVFSSAYIANDATLSTLPQLIPGLVIVSDKLNHASMIEGIRRSGTEKHIFKHNDLDDLRRILTSIGKDRPILVAFESVYSMDGDFGRIEEICDI ADEFGALKYIDEVHAVGMYGPRGGGVAERDGLMDRIDIINGTLGKAYGVFGGYIAASSKMCDAVRSYAPGFIFSTSLPPVVAAGAAASVRHLKGDVELREKHQTQARILKMRLKGLGLPIIDHGSHIVPVHVGDPVHCKMISDMLLEHFGIYVQPINFPTVPRGTERLRFTPSPVHDSGMIDHLVKAMDVLWQHCALNRAEVVA SEQ ID NO: 45, Rhodobacter sphaeroides ALA synthetase (RsA) DNA sequence Example 8. Rhodopseudomonas palustris ATCC17001 with deletion of HemA(RpA) 403 and verification of fermentation. To investigate the importance of ALA synthetase 403 or the C-terminus in ALA synthetase activity, the inventors took RpA as an example, deleted 403, and examined the effects after deletion. Using the pEC-XK99E-hemA wild-type plasmid as a template (see Construction and Optimization of the 5-aminolevulinic acid synthesis pathway in Corynebacterium glutamicum [J]. Biotechnology Bulletin, 2017, 33(01): 148-156. for the construction process), primers were designed (as shown in Table 6), and PCR amplification was performed using these primers to mutate the amino acid residue 403 of RpA into a stop codon. The PCR reaction conditions, amplification system, and transformation validation were the same as in Example 3. The correct expression vector p403Stop was obtained, and the expression vector was transformed into the C. glutamicum ATCC13032 strain to obtain the engineered strain C. glutamicum ATCC13032 / p403Stop. 24-well plate ALA fermentation was performed, and crude enzyme activity and ALA production were detected. The well-plate fermentation process is as shown in Example 4. Methods for detecting crude enzyme activity, ALA detection, and glucose analysis are described in the "Materials and Methods" section. After detection, crude enzyme activity increased compared to the wild-type strain with the stop codon mutation (i.e., deletion at position 403), and ALA production after deletion at position 403 increased by nearly 10% compared to the wild-type strain.

[0082] [Table 7]

[0083] Example 9. Testing the C-end extension effect of ALA synthetase from different sources. Based on the above data and results, the inventors hypothesized that C-terminal elongation may be beneficial in increasing the activity of ALA synthetase without altering the amino acid residue at position 403 or the C-terminus. To test this hypothesis, the inventors used the above three different ALA synthetases (RpA, RcA, and RsA) and randomly added glutamic acid (Glu) or glutamine (Gln) to the C-terminus, and observed the effects of C-terminal elongation. use Test the effect did .

[0084] Primers were designed (as shown in Table 7) according to the sequences of the genes encoding ALA synthetase from the different origins mentioned above. Using the aforementioned pEC-XK99E-hemA, pRcWT, and pRsA as templates, PCR amplification was performed to obtain the corresponding plasmid fragments. PCR reaction conditions: 5 minutes at 95 °C, 10 cycles (30 seconds at 95 °C, 30 seconds at 65 °C-55 °C, 4 minutes at 72 °C), 20 cycles (30 seconds at 95 °C, 30 seconds at 55 °C, 4 minutes at 72 °C), 5 minutes at 72 °C. PCR amplification system (50 μL): 1 μL template, 1 μL upstream and downstream primers each, 4 μL dNTP mix, 10 μL 5×TransStart® Fast Pfu Fly Buffer, 32 μL sterile redistilled water, 1 μL TransStart® FastPfu Fly DNA polymerase. The PCR products were purified and recovered using a gel recovery kit, phosphorylated with T4 PNK, and then ligated with T4 ligase to transform E. coli T1 competent cells. These cells were coated with LB plates containing kanamycin, incubated overnight at 37 °C, and the transformants were sequenced. The correct recombinant vectors were named pRpE, pRpQ, pRcE, pRcQ, pRsE, and pRsQ.

[0085] The above strains and their corresponding control strains were fermented using 24-well plates, with the same culture process and parameters as in Example 4. The fermentation results showed that the ALA yield increased by 10%-25% after adding the above amino acid residues to the C-terminus of ALA synthetase derived from different species.

[0086] [Table 8]

[0087] All documents referenced in this invention are incorporated by reference to this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of this invention, those skilled in the art should understand that various changes or modifications can be made to the invention, and these equivalent forms are also within the scope defined by the claims attached to this application.

Claims

1. 5-aminolevulinic acid synthetase (ALA synthetase), The amino acid sequence of the ALA synthetase has 95% or more identity with the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence of the ALA synthetase has 95% or more identity with the amino acid sequence shown in SEQ ID NO: 42, or the amino acid sequence of the ALA synthetase has 95% or more identity with the amino acid sequence shown in SEQ ID NO: 43, Furthermore, the ALA synthetase is derived from Agrobacterium radiobacter, Rhodoblastus acidophilus, Rhodobacter spheroides, Rhodobacter capsulata, or Rhodopseudomonas palustris. a. The C-terminal amino acid residue of the amino acid sequence of the ALA synthetase is deleted, non-alanine, and / or b. The amino acid sequence of the ALA synthetase is missing or non-alanine residue 403, which corresponds to the amino acid sequence shown in SEQ ID NO: 1, and / or c. The amino acid sequence of the ALA synthetase is missing or non-alanine residue 401, which corresponds to the amino acid sequence shown in SEQ ID NO: 43, and / or d. The 5-aminolevulinic acid synthetase (ALA synthetase) wherein the amino acid sequence of the ALA synthetase is missing or non-alanine amino acid residue 403 or 407, which corresponds to the amino acid sequence shown in SEQ ID NO:

42.

2. A nucleic acid molecule coding for ALA synthetase or its active fragment, as described in claim 1.

3. An expression vector comprising a coding nucleic acid sequence of the ALA synthetase or its active fragment according to claim 1.

4. A host cell comprising the ALA synthetase or an active fragment thereof as described in claim 1.

5. The use of the ALA synthetase described in claim 1, or the coding nucleic acid molecule described in claim 2, or the expression vector described in claim 3, or the host cell described in claim 4, in ALA generation.

6. A method for producing ALA, Step a, which involves culturing the host cells described in claim 4 or producing the host cells described in claim 4 into resting cells to produce ALA, A method for producing ALA, comprising step b, which is to selectively separate the ALA produced in step a from the culture medium.

7. A method for producing ALA, Step a, which involves catalyzing the synthesis of ALA from succinyl CoA and glycine using the ALA synthetase described in claim 1, A method for producing ALA, comprising step b, selectively separating ALA from the reaction system.

8. A method for producing ALA synthetase according to claim 1, Step a to obtain the coding sequence of the ALA synthetase described in claim 1, Step b involves either directly infecting a suitable host cell with the coding sequence obtained from step a, or introducing it into a suitable host cell via a vector. Step c involves culturing the host cells obtained from step b, A method for producing ALA synthetase according to claim 1, comprising step d of separating the ALA synthetase produced in the host cells from the culture system obtained from step c.

9. A method for providing an improved ALA synthetase and enhancing its activity, Compared to the amino acid sequence of the original ALA synthetase, the amino acid residues at positions 401, 403, or 407, or the C-terminal amino acid residue, of the improved ALA synthetase sequence are deleted or replaced with non-alanine, and / or The coding sequence of the improved ALA synthetase is modified by comparing the amino acid sequence of the improved ALA synthetase with the amino acid sequence shown in SEQ ID NO:1, and by deleting or replacing the amino acid residue at position 403 or the C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:1 with a non-alanine residue, and / or The coding sequence of the improved ALA synthetase is modified by comparing the amino acid sequence of the improved ALA synthetase with the amino acid sequence shown in SEQ ID NO: 42, and by deleting or replacing with a non-alanine amino acid residue at position 403, 407, or the C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO: 42 in the coded amino acid sequence, and / or Step a: Compare the amino acid sequence of the improved ALA synthetase with the amino acid sequence shown in SEQ ID NO: 43, and improve the coding sequence of the improved ALA synthetase by deleting or replacing the 401st or C-terminal amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO: 43 with a non-alanine residue. Step b involves either directly infecting a suitable host cell with the coding sequence obtained from step a, or introducing it into a suitable host cell via a vector. Step c involves culturing the host cells obtained from step b, Step d comprises separating the ALA synthetase produced in the host cells from the culture system obtained from step c, The amino acid sequence of the improved ALA synthetase has 95% or more identity with the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence of the improved ALA synthetase has 95% or more identity with the amino acid sequence shown in SEQ ID NO: 42, or the amino acid sequence of the improved ALA synthetase has 95% or more identity with the amino acid sequence shown in SEQ ID NO: 43, A method for providing and improving the activity of the improved ALA synthetase, wherein the improved ALA synthetase is derived from Agrobacterium radiobacter, Rhodoblastus acidophilus, Rhodobacter spheroides, Rhodobacter capsulata, or Rhodopseudomonas palustris.

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