CN109722459B

CN109722459B - 5-aminolevulinic acid high-yield strain and preparation method and application thereof

Info

Publication number: CN109722459B
Application number: CN201811287908.XA
Authority: CN
Inventors: 郑平; 陈久洲; 孙际宾; 饶德明; 朱成超; 潘丹丹; 周文娟; 马延和
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2017-10-31
Filing date: 2018-10-31
Publication date: 2021-12-24
Anticipated expiration: 2038-10-31
Also published as: CN109722459A

Abstract

Disclosed is a method for constructing a 5-aminolevulinic acid-producing strain by enhancing the antioxidant ability of a 5-aminolevulinic acid-producing strain, for example, enhancing the activity of an antioxidant-related protein in the strain. The 5-aminolevulinic acid yield of the 5-aminolevulinic acid high-yield strain constructed by the invention is obviously improved, and the glucose conversion rate is correspondingly improved. The strain can be used for producing the 5-aminolevulinic acid with high efficiency and low cost.

Description

5-aminolevulinic acid high-yield strain and preparation method and application thereof

Technical Field

The invention belongs to the field of genetic engineering and microbial fermentation; specifically, the invention relates to a 5-aminolevulinic acid high-yield strain, and a preparation method and application thereof.

Background

5-aminolevulinic acid (ALA) is a precursor for synthesizing tetrapyrrole compounds such as heme, chlorophyll, VB12 and the like by organisms and is widely present in animals, plants and microorganisms. ALA is widely applied in the fields of medicine, agriculture, feed, health food and the like, and is a high-added-value bio-based chemical with great development value. ALA can be used as photosensitizer in cancer treatment and tumor diagnosis in medicine, ALA can be used as plant growth regulator to promote crop growth in agriculture, and can be used as pesticide and herbicide as biopesticide, and in addition, ALA as nutrient component is developed and accepted in the fields of health food and animal nutrition.

At present, ALA is mainly produced by a chemical synthesis method, the cost of raw materials and the emission of pollutants are high, so that the production cost is high, the price of the product is high, and the large-scale application of ALA in the fields of agriculture, feed and the like is greatly limited. In recent years, ALA production through ALA-producing bacterial fermentation has been industrially applied and gradually replaces the traditional chemical synthesis method, and is the key point of research and development. ALA producing strains are mainly obtained by mutation breeding or genetic engineering at present, and along with the development of metabolic engineering and synthetic biological technology, the ALA yield of the genetic engineering strains is gradually superior to that of strains obtained by traditional mutation, so that the ALA producing strains become a main focus of further development in the future. The construction of engineering strains reported in the prior literature and patents mostly utilizes genetic engineering and metabolic engineering techniques to optimize the anabolic pathway of ALA so as to realize the continuous improvement of product yield and substrate conversion rate. For example, CN106047916A discloses a Corynebacterium glutamicum strain for producing ALA and a construction process thereof, wherein ALA yield is up to 2.78g/L by using a multi-step metabolic engineering modification shake flask to ferment ALA; CN103710374A discloses a method for ALA production strain construction and ALA production, wherein ALA yield is up to 4.12g/L by shake flask fermentation of engineering bacteria in a culture medium added with pantothenic acid. Although ALA yield is greatly improved and production cost is reduced to a certain extent relative to a chemical synthesis method, compared with the bio-based bulk chemicals such as glutamic acid, lysine, threonine and the like, the technical level of ALA synthesis by the fermentation method is still lower at present, which indicates that other unknown key factors for limiting ALA synthesis still exist in the existing strain system.

Therefore, there is a need in the art to develop a highly productive strain of 5-aminolevulinic acid so that 5-aminolevulinic acid can be efficiently produced using the strain.

Disclosure of Invention

The invention aims to provide a high-yield strain of 5-aminolevulinic acid.

The invention also provides a preparation method and application of the 5-aminolevulinic acid high-producing strain.

In a first aspect, the present invention provides a method for increasing the production of 5-aminolevulinic acid by a 5-aminolevulinic acid-producing strain, the method comprising the step of enhancing the antioxidant capacity of the strain.

In a specific embodiment, enhancing the antioxidant capacity of said strain refers to enhancing the activity of an antioxidant-related protein in said strain.

In a specific embodiment, the antioxidant-related protein is catalase, superoxide dismutase, peroxidase, thiol reductase, sulfhydryl-disulfide oxidoreductase, thioredoxin reductase or methionine sulfoxide reductase.

In a preferred embodiment, the antioxidant related protein may be antioxidant related protein of various sources, including but not limited to human or animal, plant, microorganism.

In a preferred embodiment, the antioxidant-related protein is derived from a microorganism.

In a preferred embodiment, the antioxidant-related protein is derived from a filamentous fungus, a yeast or a bacterium.

In a preferred embodiment, the filamentous fungus includes, but is not limited to, a filamentous fungus from the genus Aspergillus (Aspergillus), Penicillium (Penicillium), Humicola (Humicola), Trichoderma (Trichoderma) or Acremonium (Acremonium).

In a preferred embodiment, the antioxidant-related protein is derived from Aspergillus niger (Aspergillus niger), Aspergillus terreus (Aspergillus terreus), Acremonium (Acremonium arabinans), ascosphaera thermophilus (Thermoascus aurantiacus), colletotrichum thermophilum (Scytalidium thermophilum), Humicola insolens (Humicola insolens) and Penicillium pinophilum (Humicola) and Humicola grisea (Humicola grisea);

in preferred embodiments, the yeast includes, but is not limited to, Saccharomyces cerevisiae, Pichia pastoris.

In preferred embodiments, the bacteria include, but are not limited to, Escherichia (Escherichia), Corynebacterium (Corynebacterium), Serratia (Serratia), Bacillus (Bacillus), Micrococcus (Micrococcus), Acinetobacter (Acinetobacter), Arthrobacter (Arthrobacter), Proteus (Proteus), Rhizobium (Rhizobium), Stenotrophomonas (Stenotrophoromonas), Lactobacillus brevis (Lactobacillus), and thermophilic bacteria such as Thermus (Thermus), among others.

In a preferred embodiment, the antioxidant-related protein is derived from Escherichia coli (Escherichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Serratia marcescens (Serratia marcescens), Bacillus subtilis (Bacillus subtilis), Bacillus pumilus (Bacillus pumilus), Micrococcus lyticus (Micrococcus lysodeikticus), Proteus mirabilis (Proteus mirabilis), Stenotrophomonas maltophilia (Stenotrophorus malphilus), Lactobacillus brevis (Lactobacillus brevis), Thermus thermophilus (Thermus thermophilus), and the like.

In a preferred embodiment, the antioxidant-related protein is derived from Escherichia coli or Corynebacterium glutamicum.

In a further preferred embodiment, the antioxidant-related proteins are catalase i (katg), catalase ii (kate), alkylperoxidase (AhpC, AhpF), glutaredoxin i (grxa), thioredoxin ii (trxc), methionine sulfoxide reductase a (msra), superoxide dismutase (SodA, SodB, SodC), or catalase (NCgl0251), branched thiol peroxidase (NCgl2502), branched thiol reductase (NCgl1928), branched thiol redox protein (NCgl2445) and thioredoxin (NCgl2985) derived from e.

In a further preferred embodiment, the anti-oxidation related protein is catalase as shown in SEQ ID No.35, SEQ ID No.36 and SEQ ID No.45, or polypeptide which is also derived from Escherichia coli and has more than 80%, preferably 90%, more preferably 95% and most preferably 98% homology of amino acid sequence with SEQ ID No.35, SEQ ID No.36 and SEQ ID No.45 and catalase function;

alkyl peroxidase shown as SEQ ID No.37 and SEQ ID No.38, or polypeptide which is also derived from Escherichia coli, has 80%, preferably 90%, more preferably 95%, most preferably 98% homology of amino acid sequence with SEQ ID No.37 and SEQ ID No.38, and has alkyl peroxidase function;

superoxide dismutase as shown in SEQ ID No.42, SEQ ID No.43 and SEQ ID No.44, or polypeptide which is also derived from Escherichia coli, has 80%, preferably 90%, more preferably 95% and most preferably 98% homology of amino acid sequence with SEQ ID No.42, SEQ ID No.43 and SEQ ID No.44, and has superoxide dismutase function;

glutaredoxin I as shown in SEQ ID No.39, or polypeptide which is also derived from Escherichia coli, has more than 80%, preferably 90%, more preferably 95% and most preferably 98% of homology with the amino acid sequence of SEQ ID No.39 and has glutaredoxin function;

thioredoxin shown as SEQ ID No.40, or polypeptide which is also derived from Escherichia coli, has more than 80%, preferably 90%, more preferably 95% and most preferably 98% homology of amino acid sequence and SEQ ID No.40 and has thioredoxin function; thioredoxin represented by SEQ ID No.49, or a polypeptide which is also derived from Corynebacterium glutamicum and has an amino acid sequence which has homology of 80%, preferably 90%, more preferably 95%, most preferably 98% or more with SEQ ID No.49 and has a thioredoxin function;

a branched thiol peroxidase as shown in SEQ ID No.46, or a polypeptide which is also derived from Corynebacterium glutamicum and has an amino acid sequence which has 80%, preferably 90%, more preferably 95%, most preferably 98% or more homology with SEQ ID No.46 and has a branched thiol peroxidase function;

a branched thiol reductase represented by SEQ ID No.47, or a polypeptide which is also derived from Corynebacterium glutamicum and has an amino acid sequence which has 80%, preferably 90%, more preferably 95%, most preferably 98% or more homology with SEQ ID No.47 and has a branched thiol reductase function;

a branched thiol redox protein as shown in SEQ ID No.48, or a polypeptide which is also derived from Corynebacterium glutamicum and has an amino acid sequence which has more than 80%, preferably 90%, more preferably 95%, most preferably 98% homology with SEQ ID No.48 and has the function of a branched thiol redox protein;

methionine sulfoxide reductase A shown as SEQ ID No.41, or polypeptide which is also derived from Corynebacterium glutamicum and has more than 80%, preferably 90%, more preferably 95%, and most preferably 98% homology with SEQ ID No.41 in amino acid sequence and has methionine sulfoxide reductase A function. In a further preferred embodiment, the antioxidant related protein is catalase as shown in SEQ ID No.35, SEQ ID No.36 and SEQ ID No.45, alkylperoxidase as shown in SEQ ID No.37 and SEQ ID No.38, superoxide dismutase as shown in SEQ ID No.42, SEQ ID No.43 and SEQ ID No.44, glutaredoxin I as shown in SEQ ID No.39, thioredoxin as shown in SEQ ID No.40 and SEQ ID No.49, thiol peroxidase as shown in SEQ ID No.46, thiol reductase as shown in SEQ ID No.47, thiol redox protein as shown in SEQ ID No.48, methionine sulfoxide reductase A as shown in SEQ ID No. 41.

In a preferred embodiment, the antioxidant protein: (a) has amino acid sequences shown in SEQ ID No.35-49 respectively; or

(b) A derivative protein which is formed by substituting, deleting or adding one or more, preferably 1-20, more preferably 1-10, more preferably 1-6, more preferably 1-3 and most preferably 1 amino acid residue in the amino acid sequences shown in SEQ ID No.35-49 respectively and has the functions of the proteins shown in SEQ ID No.35-49 respectively; or

(c) Derived proteins which are formed by adding one or more, preferably 1-20, more preferably 1-10, more preferably 1-6, more preferably 1-3, most preferably 1 amino acid residue at both ends of the amino acid sequences shown in SEQ ID Nos. 35-49 respectively and have the functions of the proteins shown in SEQ ID Nos. 35-49 respectively.

In a preferred embodiment, the enhancing of the activity of the antioxidant-related protein can be achieved by one or a combination of the following methods: expressing the homologous or heterologous coding gene of the protein, and/or increasing the copy number of the coding gene, and/or modifying the promoter of the coding gene to enhance the transcription initiation rate, and/or modifying the translation regulatory region of the messenger RNA carrying the coding gene to enhance the translation strength.

In a preferred embodiment, the 5-aminolevulinic acid-producing strain has 5-aminolevulinic acid-synthesizing ability as such or is a 5-aminolevulinic acid-producing strain.

In preferred embodiments, the strain construction method further comprises enhancing the 5-aminolevulinic acid synthesis pathway or introducing an exogenous 5-aminolevulinic acid synthesis pathway.

In a preferred embodiment, the 5-aminolevulinic acid synthesis pathway refers to a 5-aminolevulinic acid synthesis-related enzyme, including, but not limited to, 5-aminolevulinic acid synthase, glutamyl-tRNA synthetase, glutamyl-tRNA reductase or glutamate-1-semialdehyde aminotransferase; 5-Aminolevulinic acid synthase is preferred.

In a preferred embodiment, the strain construction method further comprises enhancing the activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase.

In a preferred embodiment, the method further comprises determining the 5-aminolevulinic acid production of the resulting strain.

In a preferred embodiment, the strain is Escherichia coli (Escherichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Rhodobacter sphaeroides (Rhodobacter sphaeroides), Rhodopseudomonas palustris (Rhodopseudomonas palustris), or the like; escherichia coli and Corynebacterium glutamicum are preferred.

In a preferred embodiment, the shake flask 5-aminolevulinic acid yield of the strain is higher than 5.6 g/L.

In a second aspect, the present invention provides a method for constructing a 5-aminolevulinic acid highly productive strain, the method comprising:

a step of enhancing the antioxidant capacity of said strain.

In a third aspect, the present invention provides a 5-aminolevulinic acid-producing strain having an enhanced antioxidant ability.

In a preferred embodiment, the antioxidant-related protein is derived from Aspergillus niger (Aspergillus niger), Aspergillus terreus (Aspergillus terreus), Acremonium (Acremonium arabinans), ascothermophilus (Thermoascus aurantiacus), colletotrichum thermophilum (Scytalidium thermophilum), Humicola insolens (Humicola insolens) and myceliophthora thermophila (Thermomyces), Penicillium pinophilum (Penicillium pinophilum) and Humicola grisea (Humicola grisea);

In a further preferred embodiment, the antioxidant-related protein is catalase i (katg), catalase ii (kate), alkylperoxidase (AhpC, AhpF), glutaredoxin i (grxa), thioredoxin ii (trxc), methionine sulfoxide reductase a (msra), superoxide dismutase (SodA, SodB, SodC), or catalase (NCgl0251), branched thiol peroxidase (NCgl2502), branched thiol reductase (NCgl1928), branched thiol redox protein (NCgl2445), thioredoxin (NCgl2985) derived from e.

methionine sulfoxide reductase A shown as SEQ ID No.41, or polypeptide which is also derived from Corynebacterium glutamicum and has more than 80%, preferably 90%, more preferably 95%, and most preferably 98% homology with SEQ ID No.41 in amino acid sequence and has methionine sulfoxide reductase A function.

In a further preferred embodiment, the antioxidant related protein is catalase as shown in SEQ ID No.35, SEQ ID No.36 and SEQ ID No. 45; alkyl peroxidase shown as SEQ ID No.37 and SEQ ID No. 38; superoxide dismutase as shown in SEQ ID No.42, SEQ ID No.43 and SEQ ID No. 44; glutaredoxin I as shown in SEQ ID No. 39; thioredoxin shown as SEQ ID No.40 and SEQ ID No. 49; a branching thiol peroxidase shown as SEQ ID No.46, a branching thiol reductase shown as SEQ ID No.47, a branching thiol redox protein shown as SEQ ID No.48, and a methionine sulfoxide reductase A shown as SEQ ID No. 41.

In a preferred embodiment, the antioxidant protein: (a) has amino acid sequences shown in SEQ ID No.35-49 respectively; or (b) a derivative protein which is formed by substituting, deleting or adding one or more, preferably 1-20, more preferably 1-10, more preferably 1-6, more preferably 1-3 and most preferably 1 amino acid residue in the amino acid sequences shown in SEQ ID No.35-49 respectively and has the functions of the proteins shown in SEQ ID No.35-49 respectively; or

In a preferred embodiment, the 5-aminolevulinic acid synthesis pathway in the 5-aminolevulinic acid producing strain is enhanced or an exogenous 5-aminolevulinic acid synthesis pathway is introduced.

In a preferred embodiment, the activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase is enhanced in said 5-aminolevulinic acid-producing strain.

In a fourth aspect, the present invention provides a method of producing 5-aminolevulinic acid, the method comprising:

1) culturing the 5-aminolevulinic acid-producing strain of the third aspect, thereby obtaining 5-aminolevulinic acid; and

2) optionally obtaining 5-aminolevulinic acid from the fermentation culture system of 1).

In a fifth aspect, the present invention provides the use of a 5-aminolevulinic acid-producing strain according to the third aspect for producing 5-aminolevulinic acid and/or producing a downstream product from 5-aminolevulinic acid as a precursor.

In a preferred embodiment, the downstream product from ALA is heme or vitamin B12.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the effect of expression of antioxidant-related proteins on ALA synthesis in E.coli;

FIG. 2 shows the effect of superoxide dismutase expression on ALA synthesis;

FIG. 3 shows the effect of catalase expression in C.glutamicum on ALA synthesis;

FIG. 4 shows the effect of expression of other antioxidant-related proteins on ALA synthesis in C.glutamicum.

Detailed Description

The inventors have made extensive and intensive studies and have unexpectedly found that ALA production can be effectively increased by enhancing the antioxidant ability of a 5-aminolevulinic acid-producing strain, for example, enhancing the activity of an antioxidant-related protein. The present invention has been completed based on this finding.

Definition of terms

The term "exogenous" as used herein means that a system contains material that was not originally present. For example, a coding gene that is not originally present in a strain is introduced into the strain by transformation or the like, and the gene is "exogenous" to the strain.

The term "endogenous" as used herein refers to an activity of a polypeptide in a microorganism in an unmodified state, i.e., an activity in the natural state.

The term "enhancing the activity of a protein" as used herein refers to enhancing the intracellular activity of a protein in a microorganism by modifying the protein to increase the intracellular activity as compared with the activity of the protein possessed in a natural state. The term "exogenous" as used herein means that a system contains material that was not originally present. For example, including but not limited to, introducing a gene encoding an enzyme that is not originally present in a strain into the strain by transformation or the like, thereby expressing the enzyme in the strain, the enzyme is "exogenous" to the strain.

The term "enhancing" as used herein includes not only higher effects than the original functions due to the increase in the activity of the protein itself, but also it can be performed by at least one method selected from the group consisting of: increasing the copy number of a polynucleotide encoding a protein, modifying a regulatory sequence of a gene encoding a protein, replacing a regulatory sequence of a gene encoding a protein on a chromosome with a sequence having strong activity, replacing a gene encoding a protein with a mutant gene to increase the activity of a protein, introducing a modification in a gene encoding a protein on a chromosome to enhance the activity of a protein, and may also include, without limitation, any of the existing methods as long as the activity of a protein can be enhanced or the activity of an introduced protein can be enhanced as compared with the endogenous activity.

The term "activity of a protein introduced" as used herein can be carried out by methods known in the art, including, but not limited to, such as: inserting a polynucleotide comprising a polynucleotide sequence encoding a protein into a chromosome, and/or cloning a polynucleotide into a vector, and/or directly increasing the copy number of the polynucleotide on the chromosome, and/or engineering a polynucleotide promoter having a polynucleotide encoding a protein to enhance the transcription initiation rate, and/or the transcription of a polynucleotide encoding a protein is modified to enhance its activity, and/or the translation regulatory sequence of a messenger RNA carrying the polynucleotide encoding the protein is modified to enhance the translation strength, and/or modifying the polynucleotide encoding the protein itself to enhance mRNA stability, protein stability, release of feedback inhibition of the protein, and the like, and may include, without limitation, any known method by which protein activity may be introduced.

As described above, the control sequences include a promoter capable of initiating transcription, any operator sequence for transcriptional control, sequences encoding suitable mRNA ribosome binding domains, sequences which control termination of transcription and translation. Modifications to regulatory sequences include, but are not limited to, such as: modifications introduced by deletions, insertions, conservative or non-conservative mutations, or combinations thereof in a polynucleotide sequence may also be made by replacing the original polynucleotide sequence with a polynucleotide sequence having enhanced activity. A vector is a DNA construct comprising a polynucleotide sequence encoding a polynucleotide of a target protein operably linked to suitable control sequences to allow expression of the target protein in a host cell. The vector may replicate or function independently of the host cell genome, or may be integrated into the genome of the host cell, after being transferred into a suitable host cell. These vectors may not be particularly limited as long as the vector is replicable in host cells, and it may be constructed using a hot river vector known in the art. Examples of vectors include natural or recombinant plasmids, cosmids, viruses, and phages. For example, pWE15, pET, pUC vectors and the like. In addition, by inserting the vector into the chromosome of the host cell, a polynucleotide encoding the endogenous target protein on the chromosome can be replaced with a modified polynucleotide. Insertion of the polynucleotide into the chromosome can be performed using any method known in the art, including, but not limited to, such as: by homologous recombination. Polynucleotides include DNA and RNA encoding target proteins, which may be inserted into the chromosome of a host cell in any form so long as they are capable of expression in the host cell. Including, but not limited to, such as: the polynucleotide may be introduced into the host cell in its native state, and/or in the form of an expression cassette. An expression cassette is a genetic construct that includes all the necessary elements for self-expression, and may also be an expression vector capable of self-replication, and may include a promoter operably linked to a polynucleotide, a transcription termination signal, a ribosome binding domain, and a translation termination signal.

Antioxidant capacity and antioxidant related protein

The invention improves ALA yield by enhancing the antioxidant capacity of the 5-aminolevulinic acid producing strain. Based on the teaching of the present invention, those skilled in the art can know various specific technical means for improving the antioxidant ability of the strain. In particular embodiments, the antioxidant capacity of the high strain is enhanced by enhancing the activity of an antioxidant-related protein in the strain.

The term "antioxidant-related protein" as used herein refers to a protein in a strain that is associated with the antioxidant activity of the strain. Based on the teachings of the present invention, one skilled in the art is aware of antioxidant-related proteins in strains. For example, antioxidant related proteins described herein include, but are not limited to, catalase, superoxide dismutase, peroxidase, thiol reductase, redox protein, methionine sulfoxide reductase.

More specifically, the catalase, which is an enzyme catalyzing hydrogen peroxide to generate oxygen and water, includes, but is not limited to, catalase derived from Escherichia coli catalase I (KatG), catalase II (KatE), and Corynebacterium glutamicum catalase (NCgl 0251);

the superoxide dismutase can catalyze superoxide radical (O) in organism^2-) Enzymes that undergo disproportionation reactions, including but not limited to superoxide dismutase a (soda), superoxide dismutase b (sodb), superoxide dismutase c (sodc) from e.coli;

the peroxidase is an enzyme which catalyzes substrate oxidation by taking hydrogen peroxide as an electron acceptor, and includes but is not limited to alkyl peroxidase (AhpC, AhpF), branched thiol peroxidase (NCgl2502) and glutathione peroxidase (Gpx);

the thiol reductase is an enzyme which catalyzes the reduction of oxidized small molecule thiol into reduced thiol, and includes but is not limited to branched thiol reductase (NCgl1928), glutathione reductase (Gor);

the thiol-disulfide oxidoreductase is an enzyme that reduces protein disulfide bonds to protein dithiols in the presence of small molecule thiols, including but not limited to glutaredoxin (GrxA, GrxB, GrxC,), thioredoxin (TrxA, TrxC, NCgl2985), branched thiol redox protein (NCgl 2445);

the methionine sulfoxide reductase is an enzyme that reduces methionine-S-sulfoxide (MetSO) to methionine in the form of a thioredoxin, including but not limited to methionine sulfoxide reductase a (msra) and methionine sulfoxide reductase b (msrb).

The antioxidant related protein can be protein of various sources, including but not limited to human or animals (cattle and pig livers), plants and microorganisms.

In a preferred embodiment, the antioxidant-related protein is derived from Aspergillus niger (Aspergillus niger), Aspergillus terreus (Aspergillus terreus), Acremonium (Acremonium arabinans), ascosphaera thermophila (Thermoascus aurantiacus), colletotrichum thermophilum (Scytalidium thermophilum), Humicola insolens (Humicola insolens), Penicillium pinophilum (Penicillium pinophilum), and Humicola grisea (Humicola grisea);

The antioxidant-related protein of the invention also comprises a regulatory protein capable of regulating the transcription and translation of the antioxidant protein, such as Escherichia coli oxidative stress regulatory factor OxyR and SoxR.

In addition, the thiol peroxidases, thiol reductases, and thiol redox proteins of the present invention include not only glutathione and branched thiols as described herein, but also small molecule thiols such as ovine thiol, ergot thiol, spore thiol, and the like, which are widely present in organisms.

The strain of the invention, and the construction method and the use thereof

The present inventors have found that increasing the antioxidant capacity of a 5-aminolevulinic acid (ALA) producing strain, e.g. enhancing the activity of antioxidant related proteins in the producing strain, including but not limited to peroxidase, superoxide dismutase, thiol reductase, glutaredoxin, thioredoxin, branched thiol redox protein, methionine sulfoxide reductase, significantly increases the ALA yield of the producing strain. On the basis, the invention provides an ALA high-producing strain constructed by the method and a method for preparing ALA by using the strain.

Based on the teaching of the present invention and the prior art, those skilled in the art will understand that the present invention can be applied to a strain which itself has a certain 5-aminolevulinic acid synthesizing ability, and can also be applied to a strain which itself does not have a 5-aminolevulinic acid synthesizing ability, but has a 5-aminolevulinic acid synthesizing ability by, for example, exogenously introducing a 5-aminolevulinic acid synthesizing pathway, thereby obtaining a 5-aminolevulinic acid high-producing strain; the present invention can also be applied to 5-aminolevulinic acid-producing strains, even 5-aminolevulinic acid-producing strains, thereby further improving the 5-aminolevulinic acid-producing ability of the strains.

The term "5-aminolevulinic acid synthesis pathway" as used herein refers to 5-aminolevulinic acid synthesis-related enzymes, i.e., various enzymes included in a particular pathway for producing 5-aminolevulinic acid in a microorganism, including, but not limited to, 5-aminolevulinic acid synthase, glutamyl-tRNA synthetase, glutamyl-tRNA reductase or glutamate-1-semialdehyde aminotransferase, and the like; 5-Aminolevulinic acid synthase is preferred.

Those skilled in the art know that many strains can be used to produce 5-aminolevulinic acid. Although different, the strains have similar synthesis system and pathway for synthesizing 5-aminolevulinic acid. Thus, as will be apparent to those of ordinary skill in the art in view of the teachings of the present invention and the prior art, the strain of the present invention can be any strain useful for producing 5-aminolevulinic acid, including, but not limited to: escherichia coli (Escherichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Rhodobacter sphaeroides (Rhodobacter sphaeroides), Rhodopseudomonas palustris (Rhodopseudomonas palustris), and the like. In a particular embodiment, the strain is preferably Escherichia coli and Corynebacterium glutamicum.

The application of the 5-aminolevulinic acid high-producing strain

It will be appreciated by those skilled in the art that the 5-aminolevulinic acid-producing strain of the invention can be used not only for producing 5-aminolevulinic acid, but also for producing derivatives which are precursors for 5-aminolevulinic acid, such as heme or VB 12.

The invention has the advantages that:

1. the yield of the 5-aminolevulinic acid of the strain is greatly improved;

2. the invention discovers that the enhancement of the activity of the antioxidant-associated protein in the 5-aminolevulinic acid production strain can obviously improve the yield of the 5-aminolevulinic acid of the production strain, thereby providing a new idea for engineering the 5-aminolevulinic acid production strain or optimizing the production process of the 5-aminolevulinic acid production strain;

3. the method for improving the ALA yield of the engineering bacteria can further improve the growth performance of the bacterial strain and the conversion rate of the substrate glucose.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials described herein are preferred.

Examples

Materials and methods

The DNA polymerase used in the examples of the present invention was purchased from Fastpfu, a Kyoto Total gold company; restriction enzymes, DNA ligases and the like were purchased from Fermentas;

yeast powder and peptone were purchased from Oxoid, England; glycine and IPTG were purchased from Promega; ALA and p-dimethylaminobenzaldehyde and the like were purchased from Sigma; agar powder and antibiotics were purchased from Beijing Solibao; glucose, glacial acetic acid, perchloric acid, trichloroacetic acid, acetylacetone, chloroform and other common chemical reagents are all purchased from Chinese herbs.

The plasmid extraction kit and the agarose gel electrophoresis recovery kit are purchased from Shanghai workers, and related operations are executed strictly according to the instructions;

plasmid construction sequencing verification is completed by Jinzhi;

DH 5. alpha. competent cells were purchased from Tokyo Total gold, Inc.

LB medium composition: 5g/L of yeast powder, 10g/L of peptone and 10g/L of NaCl, and 2% of agar powder is added into a solid culture medium.

The antibiotic concentrations were: ampicillin 100. mu.g/mL.

The detection method of ALA comprises the following steps: mu.L of the diluted fermentation broth was added to 100. mu.L of pH 4.6 sodium acetate buffer, then 5. mu.L of acetylacetone was added, incubated in a water bath at 100 ℃ for 15min, cooled to room temperature, mixed with an equal volume of Ehrlish's reagent (42mL glacial acetic acid, 8mL 70% perchloric acid, 1g dimethylaminobenzaldehyde) and developed for 10min before measuring the absorbance at 553 nm.

The glucose analysis method adopts SBA-40D biosensing analyzer produced by Shandong academy of sciences to detect.

Example 1 construction of co-expression vector for ALA synthetase and phosphoenolpyruvate carboxylase

Primers hemA-F/R and ppc-F/R are designed according to a hemA gene sequence (GenBank: JQ048720.1) of rhodopseudomonas palustris ATCC17001 and a genome sequence of escherichia coli MG1655 which are published by NCBI, and a hemA gene and a ppc gene fragment with a self promoter are obtained by PCR amplification by taking a pZPA6 plasmid (CN103981203B) as a template, wherein the PCR amplification parameter is 94 ℃ for 5 min; circulating for 30 times at 94 deg.C for 20s, 55 deg.C for 20s, and 72 deg.C for 1.5 min; extension at 72 ℃ for 5 min. After the fragment is recovered, the fragment is cut by enzyme and is connected with pTrc99A plasmid to obtain a hemA and ppc co-expression recombinant vector which is named as pZCA 40.

Example 2 construction of expression vector for antioxidation-related protein of Escherichia coli

Primers katG-F/R, katE-F/R, ahpCF-F/R, grxA-F/R, trxC-F/R, msrA-F/R are respectively designed according to a genome sequence of Escherichia coli MG1655 published by NCBI, Escherichia coli catalase I (an amino acid sequence is shown as SEQ ID No. 35), catalase II (an amino acid sequence is shown as SEQ ID No. 36), alkyl peroxidase (an amino acid sequence is shown as SEQ ID No.37 and SEQ ID No. 38), glutaredoxin I (an amino acid sequence is shown as SEQ ID No. 39), thioredoxin II (an amino acid sequence is shown as SEQ ID No. 40), and coding genes katG of methionine sulfoxide reductase A (an amino acid sequence is shown as SEQ ID No. 41) are respectively obtained by PCR amplification by taking an MG1655 genome as a template, katE, ahpC-ahpF, grxA, trxC and msrA, the PCR amplification system is as follows: the PCR amplification parameters were: at 94 deg.C for 5min, at 94 deg.C for 20s, at 58 deg.C for 20s, at 72 deg.C for 2min, and circulating for 30 times, and extending at 72 deg.C for 5 min. The target fragment was digested with NotI, the fragment was ligated with pZCA40 vector treated in the same way under the action of DNA ligase, the ligation product was transformed into DH 5. alpha. competent cells, plated ampicillin resistant plates were grown overnight, positive clones were selected for colony PCR and sequencing verification, and the correct recombinant vectors were named pZCA40-katG, pZCA40-katE, pZCA40-ahpCF, pZCA40-grxA, pZCA40-trxC and pZCA40-msrA, respectively.

Example 3 enhancing the Effect of Escherichia coli antioxidant Activity on ALA Synthesis

In order to verify the influence of the enhanced Escherichia coli oxidation resistance on ALA synthesis, the vector and a control vector pZCA40 thereof are respectively transferred into an MG1655 strain, and the preparation and transformation processes of competent cells refer to molecular cloning experimental guidance written by J. Sambruke (Sambrook) and the like. The transformants were plated on ampicillin-resistant LB plates, cultured overnight, and plasmids were extracted from positive clones for validation to obtain recombinant strains MG1655/pZCA40, MG1655/pZCA40-katG, MG1655/pZCA40-katE, MG1655/pZCA40-ahpCF, MG1655/pZCA40-grxA, MG1655/pZCA40-trxC, and MG1655/pZCA40-msrA, respectively.

The single colonies of the recombinant bacteria were inoculated into 5mL of LB liquid medium containing 100. mu.g/mL of ampicillin, respectively, and cultured at 37 ℃ and 220rpmAnd (5) cultivating for 12 h. Transferring into 250mL triangular flask containing 50mL fermentation medium according to initial OD of 0.05, culturing at 37 deg.C and 220rpm for 2.5h, adding IPTG with final concentration of 50 μ M, inducing and culturing for 28h, collecting fermentation liquid, and detecting ALA concentration. Wherein the fermentation medium is an M9 culture medium added with yeast powder, and comprises the following main components: na (Na)₂HPO₄·12H₂O 17.1g/L，KH₂PO₄3.0g/L，NaCl 0.5g/L，NH₄Cl 1.0g/L，MgSO₄ 2mM，CaCl₂0.1mM, 15g/L glucose, 2g/L yeast powder and 4g/L glycine. The detection of ALA is described in the materials and methods section.

The fermentation result of the recombinant bacteria is shown in figure 1, the ALA yield of a Control strain MG1655/pZCA40(Control) after 28 hours of fermentation is 1.46g/L, the ALA yield of catalase (KatG and KatE) expression strains is 1.81 times and 1.93 times of that of the Control strain respectively, 2.64g/L and 2.81g/L are achieved, and meanwhile, the growth of the strains is greatly improved.

The ALA yield of the alkyl peroxidase expression strain (AhpCF) reaches 2.07g/L which is 1.42 times of that of a control strain, but the growth is hardly influenced, so that the unit cell ALA synthesis capacity is obviously improved.

The ALA yields of the glutaredoxin I (GrxA), the thioredoxin II (TrxC) and the methionine sulfoxide reductase A (MsrA) expression strains are respectively 2.1 times, 1.48 times and 1.54 times of those of the control strains, the thallus growth of the thioredoxin II expression strain is obviously improved, the ALA yields of the glutaredoxin and the methionine sulfoxide reductase A expression strains are respectively 2.26 times and 1.39 times of those of the control strains, and the improvement effect is obvious.

The results show that the yield of the engineering bacterium ALA can be effectively improved by enhancing the expression of the escherichia coli antioxidant related protein and improving the oxidation resistance of the thalli.

Example 4 construction of superoxide dismutase expression vectors

Primers J23101-sodA-F/R, J23101-sodB-F/R and J23101-sodC-F/R are respectively designed according to a genome sequence of Escherichia coli MG1655 published by NCBI, genes encoding Escherichia coli superoxide dismutase (amino acid sequence is shown in SEQ ID No. 42-44) with J23101 promoter, sodA, sodB and sodC are respectively obtained by PCR amplification by taking MG1655 genome as a template, and a PCR amplification system is as follows: the PCR amplification parameters were: at 94 deg.C for 5min, at 94 deg.C for 20s, at 58 deg.C for 20s, at 72 deg.C for 1min, and circulating for 30 times, and extending at 72 deg.C for 5 min. The target fragment is cut by XbaI and SacI, then the obtained fragment is connected with pZCA9 vector (CN103710374B) which is processed in the same way under the action of DNA ligase, the connection product is transformed into DH5 alpha competent cells, ampicillin resistant plates are coated for overnight culture, colonies are selected for PCR verification and sequencing verification, and correct recombinant vectors are named as pZZCCA3, pZZCCA4 and pZZCCA5 respectively.

Example 5 Effect of superoxide dismutase expression on ALA Synthesis

In order to verify the influence of superoxide dismutase expression on ALA synthesis, correct recombinant vectors pZZCA3, pZZCA4 and pZZCA5 are constructed and transformed into E.coli BW25113/pZGA24(CN103981203B) strains respectively to obtain recombinant engineering strains BW25113/pZGA24/pZZCA3, BW25113/pZGA24/pZZCA4 and BW25113/pZGA24/pZZCA 5.

The single colonies of the recombinant bacteria were inoculated into 5mL of LB liquid medium containing 100. mu.g/mL ampicillin and 34. mu.g/mL chloramphenicol, respectively, and cultured at 37 ℃ and 220rpm for 12 hours. Transferring into 250mL triangular flask containing 50mL fermentation medium according to initial OD of 0.05, culturing at 37 deg.C and 220rpm for 2.5h, adding IPTG with final concentration of 50 μ M, inducing and culturing for 17h, collecting fermentation liquid, and detecting ALA concentration. Wherein the fermentation medium is LB medium added with succinic acid and glycine, and the main components are as follows: 10g/L of peptone, 5g/L, NaCl 10g/L of yeast powder, 10g/L of succinic acid, 10g/L of glucose and 4g/L of glycine. The detection of ALA is described in the materials and methods section.

The fermentation result of the recombinant bacteria is shown in figure 2, the ALA yield of a control strain BW24/pZCA9 is 0.94g/L, and the ALA yields of superoxide dismutase (SodA, SodB and SodC) expression strains reach 1.06g/L, 1.14g/L and 1.13g/L which are respectively 1.13, 1.21 and 1.20 times of those of the control strain, which shows that the ALA yield can be obviously improved by enhancing the antioxidant capacity of the engineering bacteria by expressing the superoxide dismutase.

Example 6 construction of Corynebacterium glutamicum Catalase expression vector

According to the sequence (GeneID:1021318) of the NCgl0251 coded by catalase (amino acid sequence is shown in SEQ ID No. 45) of Corynebacterium glutamicum ATCC13032 published by NCBI, primers Ncgl0251-F/R are designed, and a target gene is obtained by PCR amplification by using ATCC13032 genome as a template, wherein the PCR amplification system is as follows: the PCR amplification parameters were: 10min at 94 ℃, 20s at 94 ℃, 30s at 58 ℃, 2min at 72 ℃, 30 times of circulation and 5min of extension at 72 ℃. The target fragment is cut by XbaI, then the obtained fragment is connected with pZWA2 vector (WO2014121724A1) which is cut by the same enzyme under the action of DNA ligase, the connection product is transformed into DH5 alpha competent cells, kanamycin-resistant plates are coated for 36h, positive clones are selected for colony PCR verification, the correct transformant is sequenced and verified, and the correct recombinant vector is named as pZWA2-Ncgl 0251.

Meanwhile, a primer katG-F2/R2 is designed according to katG (GeneID:948431) encoding gene katG (GeneID:948431) of Escherichia coli MG1655 derived from NCBI, and a gene fragment with an additional promoter is obtained by PCR amplification by taking MG1655 genome as a template, wherein the PCR amplification parameters are as follows: at 94 deg.C for 5min, at 94 deg.C for 20s, at 58 deg.C for 20s, at 72 deg.C for 2min, and circulating for 30 times, and extending at 72 deg.C for 5 min. The target fragment is cut by XbaI, then the obtained fragment is connected with pZWA2 vector which is cut by the same enzyme under the action of DNA ligase, the connection product is transformed into DH5 alpha competent cells, kanamycin resistant plate culture is carried out for 36h, positive clone is selected for colony PCR verification, the correct transformant is carried out for sequencing verification, and the correct recombinant vector is named as pZWA 2-katG.

Example 7 Effect of catalase expression on C.glutamicum ALA Synthesis

To verify the effect of catalase expression on ALA synthesis, the above vectors were each transferred into Corynebacterium glutamicum ATCC13032 strain. The transformants were plated on kanamycin-resistant plates, cultured overnight, and then plasmid-verified by picking out positive clones to obtain recombinant strains 13032/pZWA2-NCgl0251 and 13032/pZWA2-katG, respectively.

The recombinant strain and the control strain 13032/pZWA2 were inoculated with 50mL of single colony containing 25. mu.g/mL kanamycin and 2/0 g-L glucose and 3g/L corn steep liquor LB liquid medium, 30 degrees, 200rpm culture for 12 h. Transferring into 500mL triangular flask containing 50mL fermentation medium according to initial OD of 0.5, culturing at 30 deg.C and 200rpm for 3h, adding IPTG with final concentration of 100 μ M, inducing and culturing for 36h, collecting fermentation liquid, and detecting ALA concentration. Wherein the formula of the shake flask fermentation medium is as follows: glucose 50g/L, Na₂HPO₄·12H₂O 17.1g/L，KH₂PO₄ 3.0g/L，NaCl 0.5g/L，NH₄Cl 1.0g/L，MgSO₄ 2mM，CaCl₂0.1mM, glycine 4g/L, pH 7.0, kanamycin to a final concentration of 25. mu.g/mL. The pH is adjusted by ammonia water during fermentation and stabilized at about 7.0, and the detection of ALA and the glucose analysis method are as described in the section of materials and methods.

The fermentation result of the recombinant bacteria is shown in figure 3, the ALA yield is 3.20g/L after the control strain 13032/pZWA2 is fermented for 36h, the ALA yield of the catalase expression strain derived from the corynebacterium glutamicum is improved by 15 percent compared with that of the control strain, the ALA yield of the catalase I derived from escherichia coli is improved by 17 percent compared with that of the control strain after the catalase I is expressed in the corynebacterium glutamicum, meanwhile, the glucose conversion rate is also improved by 36 percent compared with that of the control strain, and the product production cost can be greatly reduced by improving the yield and the substrate conversion rate.

Example 8 construction of expression vectors for other antioxidant-related proteins of Corynebacterium glutamicum

Primers NCgl2502-F/R, NCgl1928-F/R, NCgl2445-F/R and NCgl2985-F/R are designed according to the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI (Corynebacterium glutamicum) respectively, and the genes NCgl2502 (amino acid sequence is shown in SEQ ID No. 46), branched thiol reductase (amino acid sequence is shown in SEQ ID No. 47), branched thiol redox protein (amino acid sequence is shown in SEQ ID No. 48), thioredoxin (amino acid sequence is shown in SEQ ID No. 49) coding genes NCgl2502(GeneID:1020537), NCgl1928(GeneID:1019960), NCgl2445(GeneID:1020480) and NCgl2985(GeneID:1021035) of the Corynebacterium glutamicum with a self-promoter are obtained by PCR amplification with the genome of ATCC13032 as a template, wherein the PCR amplification system comprises the following steps: the PCR amplification parameters were: at 94 deg.C for 10min, at 94 deg.C for 20s, at 58 deg.C for 20s, at 72 deg.C for 1min, and circulating for 30 times, and extending at 72 deg.C for 5 min. The target fragment was digested with XbaI, the resulting fragment was ligated with pZWA2 vector digested with DNA ligase, the ligation product was transformed into DH 5. alpha. competent cells, plated on kanamycin-resistant plates for 36h, positive clones were selected for colony PCR verification, correct transformants were sequenced, and the correct recombinant vectors were named pZWA2-NCgl2502, pZWA2-NCgl1928, pZWA2-NCgl2445, and pZWA2-NCgl 2985.

Example 9 Effect of Corynebacterium glutamicum expression of Oxidation resistance-related proteins on ALA Synthesis

In order to verify the effect of expression of antioxidant-related proteins on ALA synthesis, the above recombinant vectors were transferred into Corynebacterium glutamicum ATCC13032 strain, respectively. The transformed products were plated on kanamycin-resistant plates, and after culturing for 36 hours, positive clones were picked up and plasmid-verified to obtain recombinant strains 13032/pZWA2-NCgl2502, 13032/pZWA2-NCgl1928, 13032/pZWA2-NCgl2445 and 13032/pZWA2-NCgl2985, respectively.

The shake flask fermentation verification process is the same as that in example 5, because the part of protein is mainly involved in oxidation repair, the fermentation time is prolonged to 48 hours, so as to fully exert the antioxidant effect. The fermentation result is shown in figure 4, the yield of ALA after 48 hours of fermentation of the reference strain 13032/pZWA2 is 3.81g/L, the yields of ALA of the branched thiol peroxidase, branched thiol reductase, branched thiol redox protein and thioredoxin expression strain are respectively increased by 19%, 40%, 47% and 41% compared with the reference strain, and simultaneously, the glucose conversion rate and the yield of ALA of unit thallus are greatly increased by 22% and 31% respectively. Therefore, the method for improving the ALA yield of the engineering strain provided by the invention is also suitable for the fermentation industry of corynebacterium glutamicum and the like and common microorganisms for ALA production.

The sequences of the primers and related antioxidant proteins used in the examples of the invention are shown in the following table:

discussion: the invention obviously improves the yield of ALA by enhancing the activity of antioxidant related proteins in ALA producing strains, has more obvious technical effect than the effect of directly modifying related proteins in an ALA synthesis path, and is more unexpected that the proteins are not directly related to the ALA synthesis path.

According to the descriptions of CN103981203A and WO2014121724A1, the control strain (ALA + ppc) per se is an ALA high-producing strain recognized in the field, and the strain of the invention is remarkably improved in terms of both the absolute yield of ALA and the glucose conversion rate compared with the control strain, and has great advantages compared with the prior art in the field, and the actual effect is expected to be further improved after the process is amplified, so that the strain has extremely remarkable economic value and social value.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

SEQUENCE LISTING

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> 5-aminolevulinic acid high-producing strain, preparation method and application thereof

<130> P2018-1735

<150> CN2017110476130

<151> 2017-10-31

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<170> PatentIn version 3.5

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Thr Arg Asp Trp Trp Pro Asn Gln Leu Arg Val Asp Leu Leu Asn Gln

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His Ser Asn Arg Ser Asn Pro Leu Gly Glu Asp Phe Asp Tyr Arg Lys

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Leu Leu Thr Glu Ser Gln Pro Trp Trp Pro Ala Asp Trp Gly Ser Tyr

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Ala Gly Leu Phe Ile Arg Met Ala Trp His Gly Ala Gly Thr Tyr Arg

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Ser Ile Asp Gly Arg Gly Gly Ala Gly Arg Gly Gln Gln Arg Phe Ala

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Pro Leu Asn Ser Trp Pro Asp Asn Val Ser Leu Asp Lys Ala Arg Arg

130 135 140

Leu Leu Trp Pro Ile Lys Gln Lys Tyr Gly Gln Lys Ile Ser Trp Ala

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Asp Leu Phe Ile Leu Ala Gly Asn Val Ala Leu Glu Asn Ser Gly Phe

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Arg Thr Phe Gly Phe Gly Ala Gly Arg Glu Asp Val Trp Glu Pro Asp

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Leu Asp Val Asn Trp Gly Asp Glu Lys Ala Trp Leu Thr His Arg His

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Pro Glu Ala Leu Ala Lys Ala Pro Leu Gly Ala Thr Glu Met Gly Leu

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Ile Tyr Val Asn Pro Glu Gly Pro Asp His Ser Gly Glu Pro Leu Ser

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Ala Ala Ala Ala Ile Arg Ala Thr Phe Gly Asn Met Gly Met Asn Asp

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Glu Glu Thr Val Ala Leu Ile Ala Gly Gly His Thr Leu Gly Lys Thr

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His Gly Ala Gly Pro Thr Ser Asn Val Gly Pro Asp Pro Glu Ala Ala

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Pro Ile Glu Glu Gln Gly Leu Gly Trp Ala Ser Thr Tyr Gly Ser Gly

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Val Gly Ala Asp Ala Ile Thr Ser Gly Leu Glu Val Val Trp Thr Gln

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Thr Pro Thr Gln Trp Ser Asn Tyr Phe Phe Glu Asn Leu Phe Lys Tyr

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Glu Trp Val Gln Thr Arg Ser Pro Ala Gly Ala Ile Gln Phe Glu Ala

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Val Asp Ala Pro Glu Ile Ile Pro Asp Pro Phe Asp Pro Ser Lys Lys

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Arg Lys Pro Thr Met Leu Val Thr Asp Leu Thr Leu Arg Phe Asp Pro

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Glu Phe Glu Lys Ile Ser Arg Arg Phe Leu Asn Asp Pro Gln Ala Phe

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Asn Glu Ala Phe Ala Arg Ala Trp Phe Lys Leu Thr His Arg Asp Met

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Gly Pro Lys Ser Arg Tyr Ile Gly Pro Glu Val Pro Lys Glu Asp Leu

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Asp Ile Ile Asp Leu Lys Phe Ala Ile Ala Asp Ser Gly Leu Ser Val

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Gln Arg Asp Trp Asp Val Asn Ala Ala Ala Val Arg Ala Leu Pro Val

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Gly Leu Ser Ile His Val Pro Phe Ala Pro Gly Arg Val Asp Ala Arg

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Gln Asp Gln Thr Asp Ile Glu Met Phe Glu Leu Leu Glu Pro Ile Ala

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Asp Gly Phe Arg Asn Tyr Arg Ala Arg Leu Asp Val Ser Thr Thr Glu

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Ser Leu Leu Ile Asp Lys Ala Gln Gln Leu Thr Leu Thr Ala Pro Glu

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Met Thr Ala Leu Val Gly Gly Met Arg Val Leu Gly Ala Asn Phe Asp

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Gly Ser Lys Asn Gly Val Phe Thr Asp Arg Val Gly Val Leu Ser Asn

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Asp Phe Phe Val Asn Leu Leu Asp Met Arg Tyr Glu Trp Lys Ala Thr

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Asp Glu Ser Lys Glu Leu Phe Glu Gly Arg Asp Arg Glu Thr Gly Glu

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Val Lys Phe Thr Ala Ser Arg Ala Asp Leu Val Phe Gly Ser Asn Ser

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Val Leu Arg Ala Val Ala Glu Val Tyr Ala Ser Ser Asp Ala His Glu

690 695 700

Lys Phe Val Lys Asp Phe Val Ala Ala Trp Val Lys Val Met Asn Leu

705 710 715 720

Asp Arg Phe Asp Leu Leu

725

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His Asp Ser Ser Glu Ala Lys Pro Gly Met Asp Ser Leu Ala Pro Glu

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Asp Gly Ser His Arg Pro Ala Ala Glu Pro Thr Pro Pro Gly Ala Gln

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Pro Thr Ala Pro Gly Ser Leu Lys Ala Pro Asp Thr Arg Asn Glu Lys

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Leu Asn Ser Leu Glu Asp Val Arg Lys Gly Ser Glu Asn Tyr Ala Leu

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Ala Arg Gly Ser Ala Ala His Gly Tyr Phe Gln Pro Tyr Lys Ser Leu

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Ser Asp Ile Thr Lys Ala Asp Phe Leu Ser Asp Pro Asn Lys Ile Thr

145 150 155 160

Pro Val Phe Val Arg Phe Ser Thr Val Gln Gly Gly Ala Gly Ser Ala

165 170 175

Asp Thr Val Arg Asp Ile Arg Gly Phe Ala Thr Lys Phe Tyr Thr Glu

180 185 190

Glu Gly Ile Phe Asp Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile

195 200 205

Gln Asp Ala His Lys Phe Pro Asp Phe Val His Ala Val Lys Pro Glu

210 215 220

Pro His Trp Ala Ile Pro Gln Gly Gln Ser Ala His Asp Thr Phe Trp

225 230 235 240

Asp Tyr Val Ser Leu Gln Pro Glu Thr Leu His Asn Val Met Trp Ala

245 250 255

Met Ser Asp Arg Gly Ile Pro Arg Ser Tyr Arg Thr Met Glu Gly Phe

260 265 270

Gly Ile His Thr Phe Arg Leu Ile Asn Ala Glu Gly Lys Ala Thr Phe

275 280 285

Val Arg Phe His Trp Lys Pro Leu Ala Gly Lys Ala Ser Leu Val Trp

290 295 300

Asp Glu Ala Gln Lys Leu Thr Gly Arg Asp Pro Asp Phe His Arg Arg

305 310 315 320

Glu Leu Trp Glu Ala Ile Glu Ala Gly Asp Phe Pro Glu Tyr Glu Leu

325 330 335

Gly Phe Gln Leu Ile Pro Glu Glu Asp Glu Phe Lys Phe Asp Phe Asp

340 345 350

Leu Leu Asp Pro Thr Lys Leu Ile Pro Glu Glu Leu Val Pro Val Gln

355 360 365

Arg Val Gly Lys Met Val Leu Asn Arg Asn Pro Asp Asn Phe Phe Ala

370 375 380

Glu Asn Glu Gln Ala Ala Phe His Pro Gly His Ile Val Pro Gly Leu

385 390 395 400

Asp Phe Thr Asn Asp Pro Leu Leu Gln Gly Arg Leu Phe Ser Tyr Thr

405 410 415

Asp Thr Gln Ile Ser Arg Leu Gly Gly Pro Asn Phe His Glu Ile Pro

420 425 430

Ile Asn Arg Pro Thr Cys Pro Tyr His Asn Phe Gln Arg Asp Gly Met

435 440 445

His Arg Met Gly Ile Asp Thr Asn Pro Ala Asn Tyr Glu Pro Asn Ser

450 455 460

Ile Asn Asp Asn Trp Pro Arg Glu Thr Pro Pro Gly Pro Lys Arg Gly

465 470 475 480

Gly Phe Glu Ser Tyr Gln Glu Arg Val Glu Gly Asn Lys Val Arg Glu

485 490 495

Arg Ser Pro Ser Phe Gly Glu Tyr Tyr Ser His Pro Arg Leu Phe Trp

500 505 510

Leu Ser Gln Thr Pro Phe Glu Gln Arg His Ile Val Asp Gly Phe Ser

515 520 525

Phe Glu Leu Ser Lys Val Val Arg Pro Tyr Ile Arg Glu Arg Val Val

530 535 540

Asp Gln Leu Ala His Ile Asp Leu Thr Leu Ala Gln Ala Val Ala Lys

545 550 555 560

Asn Leu Gly Ile Glu Leu Thr Asp Asp Gln Leu Asn Ile Thr Pro Pro

565 570 575

Pro Asp Val Asn Gly Leu Lys Lys Asp Pro Ser Leu Ser Leu Tyr Ala

580 585 590

Ile Pro Asp Gly Asp Val Lys Gly Arg Val Val Ala Ile Leu Leu Asn

595 600 605

Asp Glu Val Arg Ser Ala Asp Leu Leu Ala Ile Leu Lys Ala Leu Lys

610 615 620

Ala Lys Gly Val His Ala Lys Leu Leu Tyr Ser Arg Met Gly Glu Val

625 630 635 640

Thr Ala Asp Asp Gly Thr Val Leu Pro Ile Ala Ala Thr Phe Ala Gly

645 650 655

Ala Pro Ser Leu Thr Val Asp Ala Val Ile Val Pro Cys Gly Asn Ile

660 665 670

Ala Asp Ile Ala Asp Asn Gly Asp Ala Asn Tyr Tyr Leu Met Glu Ala

675 680 685

Tyr Lys His Leu Lys Pro Ile Ala Leu Ala Gly Asp Ala Arg Lys Phe

690 695 700

Lys Ala Thr Ile Lys Ile Ala Asp Gln Gly Glu Glu Gly Ile Val Glu

705 710 715 720

Ala Asp Ser Ala Asp Gly Ser Phe Met Asp Glu Leu Leu Thr Leu Met

725 730 735

Ala Ala His Arg Val Trp Ser Arg Ile Pro Lys Ile Asp Lys Ile Pro

740 745 750

Ala

<210> 37

<211> 187

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 37

Met Ser Leu Ile Asn Thr Lys Ile Lys Pro Phe Lys Asn Gln Ala Phe

1 5 10 15

Lys Asn Gly Glu Phe Ile Glu Ile Thr Glu Lys Asp Thr Glu Gly Arg

20 25 30

Trp Ser Val Phe Phe Phe Tyr Pro Ala Asp Phe Thr Phe Val Cys Pro

35 40 45

Thr Glu Leu Gly Asp Val Ala Asp His Tyr Glu Glu Leu Gln Lys Leu

50 55 60

Gly Val Asp Val Tyr Ala Val Ser Thr Asp Thr His Phe Thr His Lys

65 70 75 80

Ala Trp His Ser Ser Ser Glu Thr Ile Ala Lys Ile Lys Tyr Ala Met

85 90 95

Ile Gly Asp Pro Thr Gly Ala Leu Thr Arg Asn Phe Asp Asn Met Arg

100 105 110

Glu Asp Glu Gly Leu Ala Asp Arg Ala Thr Phe Val Val Asp Pro Gln

115 120 125

Gly Ile Ile Gln Ala Ile Glu Val Thr Ala Glu Gly Ile Gly Arg Asp

130 135 140

Ala Ser Asp Leu Leu Arg Lys Ile Lys Ala Ala Gln Tyr Val Ala Ser

145 150 155 160

His Pro Gly Glu Val Cys Pro Ala Lys Trp Lys Glu Gly Glu Ala Thr

165 170 175

Leu Ala Pro Ser Leu Asp Leu Val Gly Lys Ile

180 185

<210> 38

<211> 521

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 38

Met Leu Asp Thr Asn Met Lys Thr Gln Leu Lys Ala Tyr Leu Glu Lys

1 5 10 15

Leu Thr Lys Pro Val Glu Leu Ile Ala Thr Leu Asp Asp Ser Ala Lys

20 25 30

Ser Ala Glu Ile Lys Glu Leu Leu Ala Glu Ile Ala Glu Leu Ser Asp

35 40 45

Lys Val Thr Phe Lys Glu Asp Asn Ser Leu Pro Val Arg Lys Pro Ser

50 55 60

Phe Leu Ile Thr Asn Pro Gly Ser Asn Gln Gly Pro Arg Phe Ala Gly

65 70 75 80

Ser Pro Leu Gly His Glu Phe Thr Ser Leu Val Leu Ala Leu Leu Trp

85 90 95

Thr Gly Gly His Pro Ser Lys Glu Ala Gln Ser Leu Leu Glu Gln Ile

100 105 110

Arg His Ile Asp Gly Asp Phe Glu Phe Glu Thr Tyr Tyr Ser Leu Ser

115 120 125

Cys His Asn Cys Pro Asp Val Val Gln Ala Leu Asn Leu Met Ser Val

130 135 140

Leu Asn Pro Arg Ile Lys His Thr Ala Ile Asp Gly Gly Thr Phe Gln

145 150 155 160

Asn Glu Ile Thr Asp Arg Asn Val Met Gly Val Pro Ala Val Phe Val

165 170 175

Asn Gly Lys Glu Phe Gly Gln Gly Arg Met Thr Leu Thr Glu Ile Val

180 185 190

Ala Lys Ile Asp Thr Gly Ala Glu Lys Arg Ala Ala Glu Glu Leu Asn

195 200 205

Lys Arg Asp Ala Tyr Asp Val Leu Ile Val Gly Ser Gly Pro Ala Gly

210 215 220

Ala Ala Ala Ala Ile Tyr Ser Ala Arg Lys Gly Ile Arg Thr Gly Leu

225 230 235 240

Met Gly Glu Arg Phe Gly Gly Gln Ile Leu Asp Thr Val Asp Ile Glu

245 250 255

Asn Tyr Ile Ser Val Pro Lys Thr Glu Gly Gln Lys Leu Ala Gly Ala

260 265 270

Leu Lys Val His Val Asp Glu Tyr Asp Val Asp Val Ile Asp Ser Gln

275 280 285

Ser Ala Ser Lys Leu Ile Pro Ala Ala Val Glu Gly Gly Leu His Gln

290 295 300

Ile Glu Thr Ala Ser Gly Ala Val Leu Lys Ala Arg Ser Ile Ile Val

305 310 315 320

Ala Thr Gly Ala Lys Trp Arg Asn Met Asn Val Pro Gly Glu Asp Gln

325 330 335

Tyr Arg Thr Lys Gly Val Thr Tyr Cys Pro His Cys Asp Gly Pro Leu

340 345 350

Phe Lys Gly Lys Arg Val Ala Val Ile Gly Gly Gly Asn Ser Gly Val

355 360 365

Glu Ala Ala Ile Asp Leu Ala Gly Ile Val Glu His Val Thr Leu Leu

370 375 380

Glu Phe Ala Pro Glu Met Lys Ala Asp Gln Val Leu Gln Asp Lys Leu

385 390 395 400

Arg Ser Leu Lys Asn Val Asp Ile Ile Leu Asn Ala Gln Thr Thr Glu

405 410 415

Val Lys Gly Asp Gly Ser Lys Val Val Gly Leu Glu Tyr Arg Asp Arg

420 425 430

Val Ser Gly Asp Ile His Asn Ile Glu Leu Ala Gly Ile Phe Val Gln

435 440 445

Ile Gly Leu Leu Pro Asn Thr Asn Trp Leu Glu Gly Ala Val Glu Arg

450 455 460

Asn Arg Met Gly Glu Ile Ile Ile Asp Ala Lys Cys Glu Thr Asn Val

465 470 475 480

Lys Gly Val Phe Ala Ala Gly Asp Cys Thr Thr Val Pro Tyr Lys Gln

485 490 495

Ile Ile Ile Ala Thr Gly Glu Gly Ala Lys Ala Ser Leu Ser Ala Phe

500 505 510

Asp Tyr Leu Ile Arg Thr Lys Thr Ala

515 520

<210> 39

<211> 85

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 39

Met Gln Thr Val Ile Phe Gly Arg Ser Gly Cys Pro Tyr Cys Val Arg

1 5 10 15

Ala Lys Asp Leu Ala Glu Lys Leu Ser Asn Glu Arg Asp Asp Phe Gln

20 25 30

Tyr Gln Tyr Val Asp Ile Arg Ala Glu Gly Ile Thr Lys Glu Asp Leu

35 40 45

Gln Gln Lys Ala Gly Lys Pro Val Glu Thr Val Pro Gln Ile Phe Val

50 55 60

Asp Gln Gln His Ile Gly Gly Tyr Thr Asp Phe Ala Ala Trp Val Lys

65 70 75 80

Glu Asn Leu Asp Ala

85

<210> 40

<211> 139

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 40

Met Asn Thr Val Cys Thr His Cys Gln Ala Ile Asn Arg Ile Pro Asp

1 5 10 15

Asp Arg Ile Glu Asp Ala Ala Lys Cys Gly Arg Cys Gly His Asp Leu

20 25 30

Phe Asp Gly Glu Val Ile Asn Ala Thr Gly Glu Thr Leu Asp Lys Leu

35 40 45

Leu Lys Asp Asp Leu Pro Val Val Ile Asp Phe Trp Ala Pro Trp Cys

50 55 60

Gly Pro Cys Arg Asn Phe Ala Pro Ile Phe Glu Asp Val Ala Gln Glu

65 70 75 80

Arg Ser Gly Lys Val Arg Phe Val Lys Val Asn Thr Glu Ala Glu Arg

85 90 95

Glu Leu Ser Ser Arg Phe Gly Ile Arg Ser Ile Pro Thr Ile Met Ile

100 105 110

Phe Lys Asn Gly Gln Val Val Asp Met Leu Asn Gly Ala Val Pro Lys

115 120 125

Ala Pro Phe Asp Ser Trp Leu Asn Glu Ser Leu

130 135

<210> 41

<211> 212

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 41

Met Ser Leu Phe Asp Lys Lys His Leu Val Ser Pro Ala Asp Ala Leu

1 5 10 15

Pro Gly Arg Asn Thr Pro Met Pro Val Ala Thr Leu His Ala Val Asn

20 25 30

Gly His Ser Met Thr Asn Val Pro Asp Gly Met Glu Ile Ala Ile Phe

35 40 45

Ala Met Gly Cys Phe Trp Gly Val Glu Arg Leu Phe Trp Gln Leu Pro

50 55 60

Gly Val Tyr Ser Thr Ala Ala Gly Tyr Thr Gly Gly Tyr Thr Pro Asn

65 70 75 80

Pro Thr Tyr Arg Glu Val Cys Ser Gly Asp Thr Gly His Ala Glu Ala

85 90 95

Val Arg Ile Val Tyr Asp Pro Ser Val Ile Ser Tyr Glu Gln Leu Leu

100 105 110

Gln Val Phe Trp Glu Asn His Asp Pro Ala Gln Gly Met Arg Gln Gly

115 120 125

Asn Asp His Gly Thr Gln Tyr Arg Ser Ala Ile Tyr Pro Leu Thr Pro

130 135 140

Glu Gln Asp Ala Ala Ala Arg Ala Ser Leu Glu Arg Phe Gln Ala Ala

145 150 155 160

Met Leu Ala Ala Asp Asp Asp Arg His Ile Thr Thr Glu Ile Ala Asn

165 170 175

Ala Thr Pro Phe Tyr Tyr Ala Glu Asp Asp His Gln Gln Tyr Leu His

180 185 190

Lys Asn Pro Tyr Gly Tyr Cys Gly Ile Gly Gly Ile Gly Val Cys Leu

195 200 205

Pro Pro Glu Ala

210

<210> 42

<211> 206

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 42

Met Ser Tyr Thr Leu Pro Ser Leu Pro Tyr Ala Tyr Asp Ala Leu Glu

1 5 10 15

Pro His Phe Asp Lys Gln Thr Met Glu Ile His His Thr Lys His His

20 25 30

Gln Thr Tyr Val Asn Asn Ala Asn Ala Ala Leu Glu Ser Leu Pro Glu

35 40 45

Phe Ala Asn Leu Pro Val Glu Glu Leu Ile Thr Lys Leu Asp Gln Leu

50 55 60

Pro Ala Asp Lys Lys Thr Val Leu Arg Asn Asn Ala Gly Gly His Ala

65 70 75 80

Asn His Ser Leu Phe Trp Lys Gly Leu Lys Lys Gly Thr Thr Leu Gln

85 90 95

Gly Asp Leu Lys Ala Ala Ile Glu Arg Asp Phe Gly Ser Val Asp Asn

100 105 110

Phe Lys Ala Glu Phe Glu Lys Ala Ala Ala Ser Arg Phe Gly Ser Gly

115 120 125

Trp Ala Trp Leu Val Leu Lys Gly Asp Lys Leu Ala Val Val Ser Thr

130 135 140

Ala Asn Gln Asp Ser Pro Leu Met Gly Glu Ala Ile Ser Gly Ala Ser

145 150 155 160

Gly Phe Pro Ile Met Gly Leu Asp Val Trp Glu His Ala Tyr Tyr Leu

165 170 175

Lys Phe Gln Asn Arg Arg Pro Asp Tyr Ile Lys Glu Phe Trp Asn Val

180 185 190

Val Asn Trp Asp Glu Ala Ala Ala Arg Phe Ala Ala Lys Lys

195 200 205

<210> 43

<211> 193

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 43

Met Ser Phe Glu Leu Pro Ala Leu Pro Tyr Ala Lys Asp Ala Leu Ala

1 5 10 15

Pro His Ile Ser Ala Glu Thr Ile Glu Tyr His Tyr Gly Lys His His

20 25 30

Gln Thr Tyr Val Thr Asn Leu Asn Asn Leu Ile Lys Gly Thr Ala Phe

35 40 45

Glu Gly Lys Ser Leu Glu Glu Ile Ile Arg Ser Ser Glu Gly Gly Val

50 55 60

Phe Asn Asn Ala Ala Gln Val Trp Asn His Thr Phe Tyr Trp Asn Cys

65 70 75 80

Leu Ala Pro Asn Ala Gly Gly Glu Pro Thr Gly Lys Val Ala Glu Ala

85 90 95

Ile Ala Ala Ser Phe Gly Ser Phe Ala Asp Phe Lys Ala Gln Phe Thr

100 105 110

Asp Ala Ala Ile Lys Asn Phe Gly Ser Gly Trp Thr Trp Leu Val Lys

115 120 125

Asn Ser Asp Gly Lys Leu Ala Ile Val Ser Thr Ser Asn Ala Gly Thr

130 135 140

Pro Leu Thr Thr Asp Ala Thr Pro Leu Leu Thr Val Asp Val Trp Glu

145 150 155 160

His Ala Tyr Tyr Ile Asp Tyr Arg Asn Ala Arg Pro Gly Tyr Leu Glu

165 170 175

His Phe Trp Ala Leu Val Asn Trp Glu Phe Val Ala Lys Asn Leu Ala

180 185 190

Ala

<210> 44

<211> 173

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 44

Met Lys Arg Phe Ser Leu Ala Ile Leu Ala Leu Val Val Ala Thr Gly

1 5 10 15

Ala Gln Ala Ala Ser Glu Lys Val Glu Met Asn Leu Val Thr Ser Gln

20 25 30

Gly Val Gly Gln Ser Ile Gly Ser Val Thr Ile Thr Glu Thr Asp Lys

35 40 45

Gly Leu Glu Phe Ser Pro Asp Leu Lys Ala Leu Pro Pro Gly Glu His

50 55 60

Gly Phe His Ile His Ala Lys Gly Ser Cys Gln Pro Ala Thr Lys Asp

65 70 75 80

Gly Lys Ala Ser Ala Ala Glu Ser Ala Gly Gly His Leu Asp Pro Gln

85 90 95

Asn Thr Gly Lys His Glu Gly Pro Glu Gly Ala Gly His Leu Gly Asp

100 105 110

Leu Pro Ala Leu Val Val Asn Asn Asp Gly Lys Ala Thr Asp Ala Val

115 120 125

Ile Ala Pro Arg Leu Lys Ser Leu Asp Glu Ile Lys Asp Lys Ala Leu

130 135 140

Met Val His Val Gly Gly Asp Asn Met Ser Asp Gln Pro Lys Pro Leu

145 150 155 160

Gly Gly Gly Gly Glu Arg Tyr Ala Cys Gly Val Ile Lys

165 170

<210> 45

<211> 502

<212> PRT

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 45

Met Arg Pro Lys Leu Ser Gly Asn Thr Thr Arg His Asn Gly Ala Pro

1 5 10 15

Val Pro Ser Glu Asn Ile Ser Ala Thr Ala Gly Pro Gln Gly Pro Asn

20 25 30

Val Leu Asn Asp Ile His Leu Ile Glu Lys Leu Ala His Phe Asn Arg

35 40 45

Glu Asn Val Pro Glu Arg Ile Pro His Ala Lys Gly His Gly Ala Phe

50 55 60

Gly Glu Leu His Ile Thr Glu Asp Val Ser Glu Tyr Thr Lys Ala Asp

65 70 75 80

Leu Phe Gln Pro Gly Lys Val Thr Pro Leu Ala Val Arg Phe Ser Thr

85 90 95

Val Ala Gly Glu Gln Gly Ser Pro Asp Thr Trp Arg Asp Val His Gly

100 105 110

Phe Ala Leu Arg Phe Tyr Thr Glu Glu Gly Asn Tyr Asp Ile Val Gly

115 120 125

Asn Asn Thr Pro Thr Phe Phe Leu Arg Asp Gly Met Lys Phe Pro Asp

130 135 140

Phe Ile His Ser Gln Lys Arg Leu Asn Lys Asn Gly Leu Arg Asp Ala

145 150 155 160

Asp Met Gln Trp Asp Phe Trp Thr Arg Ala Pro Glu Ser Ala His Gln

165 170 175

Val Thr Tyr Leu Met Gly Asp Arg Gly Thr Pro Lys Thr Ser Arg His

180 185 190

Gln Asp Gly Phe Gly Ser His Thr Phe Gln Trp Ile Asn Ala Glu Gly

195 200 205

Lys Pro Val Trp Val Lys Tyr His Phe Lys Thr Arg Gln Gly Trp Asp

210 215 220

Cys Phe Thr Asp Ala Glu Ala Ala Lys Val Ala Gly Glu Asn Ala Asp

225 230 235 240

Tyr Gln Arg Glu Asp Leu Tyr Asn Ala Ile Glu Asn Gly Asp Phe Pro

245 250 255

Ile Trp Asp Val Lys Val Gln Ile Met Pro Phe Glu Asp Ala Glu Asn

260 265 270

Tyr Arg Trp Asn Pro Phe Asp Leu Thr Lys Thr Trp Ser Gln Lys Asp

275 280 285

Tyr Pro Leu Ile Pro Val Gly Tyr Phe Ile Leu Asn Arg Asn Pro Arg

290 295 300

Asn Phe Phe Ala Gln Ile Glu Gln Leu Ala Leu Asp Pro Gly Asn Ile

305 310 315 320

Val Pro Gly Val Gly Leu Ser Pro Asp Arg Met Leu Gln Ala Arg Ile

325 330 335

Phe Ala Tyr Ala Asp Gln Gln Arg Tyr Arg Ile Gly Ala Asn Tyr Arg

340 345 350

Asp Leu Pro Val Asn Arg Pro Ile Asn Glu Val Asn Thr Tyr Ser Arg

355 360 365

Glu Gly Ser Met Gln Tyr Ile Phe Asp Ala Glu Gly Glu Pro Ser Tyr

370 375 380

Ser Pro Asn Arg Tyr Asp Lys Gly Ala Gly Tyr Leu Asp Asn Gly Thr

385 390 395 400

Asp Ser Ser Ser Asn His Thr Ser Tyr Gly Gln Ala Asp Asp Ile Tyr

405 410 415

Val Asn Pro Asp Pro His Gly Thr Asp Leu Val Arg Ala Ala Tyr Val

420 425 430

Lys His Gln Asp Asp Asp Asp Phe Ile Gln Pro Gly Ile Leu Tyr Arg

435 440 445

Glu Val Leu Asp Glu Gly Glu Lys Glu Arg Leu Ala Asp Asn Ile Ser

450 455 460

Asn Ala Met Gln Gly Ile Ser Glu Ala Thr Glu Pro Arg Val Tyr Asp

465 470 475 480

Tyr Trp Asn Asn Val Asp Glu Asn Leu Gly Ala Arg Val Lys Glu Leu

485 490 495

Tyr Leu Gln Lys Lys Ala

500

<210> 46

<211> 159

<212> PRT

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 46

Met Thr Ser Ile His Asp Ile Ser Val Thr Leu Asn Asp Gly Thr Glu

1 5 10 15

Thr Thr Met Ala Asp Trp Ala Gly His Leu Leu Leu Ile Val Asn Val

20 25 30

Ala Ser Lys Cys Gly Leu Thr Pro Gln Tyr Glu Gly Leu Gln Lys Leu

35 40 45

Tyr Glu Glu Tyr Gln Asp Arg Gly Phe Phe Val Ile Gly Val Pro Cys

50 55 60

Asn Gln Phe Asn Gly Gln Glu Pro Gly Thr Asp Ala Glu Val Cys Ala

65 70 75 80

Phe Ala Gln Asn Gln Tyr Asp Val Thr Phe Pro Leu Leu Ser Lys Thr

85 90 95

Glu Val Asn Gly Glu Gly Ala His Pro Leu Tyr Lys Val Leu Lys Glu

100 105 110

Ala Thr Asp Gly Ser Glu Ile Glu Trp Asn Phe Glu Lys Phe Leu Val

115 120 125

Asp Ala Glu Gly Asn Thr Ile Lys Arg Phe Ala Pro Arg Thr Glu Pro

130 135 140

Ser Ala Ala Glu Val Val Glu Ala Ile Glu Glu Asn Leu Pro Ile

145 150 155

<210> 47

<211> 465

<212> PRT

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 47

Met Ser Glu Gln Pro Ala Ser Ile Lys His Tyr Asp Leu Ile Ile Ile

1 5 10 15

Gly Thr Gly Ser Gly Asn Ser Ile Pro Gly Pro Glu Phe Asp Asp Lys

20 25 30

Ser Ile Ala Ile Val Glu Lys Gly Ala Phe Gly Gly Thr Cys Leu Asn

35 40 45

Val Gly Cys Ile Pro Thr Lys Met Tyr Val Tyr Ala Ala Asp Ile Ala

50 55 60

Gln Glu Ile Gln Glu Ser Ala Arg Leu Gly Ile Asp Ala Thr Val Asn

65 70 75 80

Ser Val Asp Trp Pro Ser Ile Val Ser Arg Val Phe Asp Lys Arg Ile

85 90 95

Asp Leu Ile Ala Gln Gly Gly Glu Ala Tyr Arg Arg Gly Pro Glu Thr

100 105 110

Pro Asn Ile Asp Val Tyr Asp Met His Ala Ser Phe Val Asp Ser Lys

115 120 125

Thr Ile Ser Thr Gly Ile Ala Gly Gln Glu Gln Leu Ile Ser Gly Thr

130 135 140

Asp Ile Val Ile Ala Thr Gly Ser Arg Pro Tyr Ile Pro Glu Ala Ile

145 150 155 160

Ala Glu Ser Gly Ala Arg Tyr Tyr Thr Asn Glu Asp Ile Met Arg Leu

165 170 175

Ala Gln Gln Pro Glu Ser Leu Val Ile Val Gly Gly Gly Phe Ile Ala

180 185 190

Leu Glu Phe Ala His Val Phe Glu Ala Leu Gly Thr Lys Val Thr Ile

195 200 205

Leu Asn Arg Ser Asp Val Leu Leu Arg Glu Ala Asp Ala Asp Ile Ser

210 215 220

Ala Lys Ile Leu Glu Leu Ser Lys Lys Arg Phe Asp Val Arg Leu Ser

225 230 235 240

Thr Ala Val Thr Ala Val His Asn Lys Ala Asp Gly Gly Val Lys Ile

245 250 255

Ser Thr Asp Thr Gly Asp Asp Ile Glu Ala Asp Ile Leu Leu Val Ala

260 265 270

Thr Gly Arg Thr Pro Asn Gly Asn Gln Met Asn Leu Asp Ala Ala Gly

275 280 285

Ile Glu Met Asn Gly Arg Ser Ile Lys Val Asp Glu Phe Gly Arg Thr

290 295 300

Ser Val Glu Gly Val Trp Ala Leu Gly Asp Val Ser Ser Pro Tyr Lys

305 310 315 320

Leu Lys His Val Ala Asn Ala Glu Met Arg Ala Ile Lys His Asn Leu

325 330 335

Ala Asn Pro Asn Asp Leu Gln Lys Met Pro His Asp Phe Val Pro Ser

340 345 350

Ala Val Phe Thr Asn Pro Gln Ile Ser Gln Val Gly Met Thr Glu Gln

355 360 365

Glu Ala Arg Glu Ala Gly Leu Asp Ile Thr Val Lys Ile Gln Asn Tyr

370 375 380

Ser Asp Val Ala Tyr Gly Trp Ala Met Glu Asp Lys Asp Gly Phe Val

385 390 395 400

Lys Leu Ile Ala Asp Lys Asp Thr Gly Lys Leu Val Gly Ala His Ile

405 410 415

Ile Gly Ala Gln Ala Ser Thr Leu Ile Gln Gln Leu Ile Thr Val Met

420 425 430

Ala Phe Gly Ile Asp Ala Arg Glu Ala Ala Thr Lys Gln Tyr Trp Ile

435 440 445

His Pro Ala Leu Pro Glu Val Ile Glu Asn Ala Leu Leu Gly Leu Glu

450 455 460

Phe

465

<210> 48

<211> 77

<212> PRT

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 48

Met Ala Ile Thr Val Tyr Thr Lys Pro Ala Cys Val Gln Cys Asn Ala

1 5 10 15

Thr Lys Lys Ala Leu Asp Arg Ala Gly Leu Glu Tyr Asp Leu Val Asp

20 25 30

Ile Ser Leu Asp Glu Glu Ala Arg Glu Tyr Val Leu Ala Leu Gly Tyr

35 40 45

Leu Gln Ala Pro Val Val Val Ala Asp Gly Ser His Trp Ser Gly Phe

50 55 60

Arg Pro Glu Arg Ile Arg Glu Met Ala Thr Ala Ala Ala

65 70 75

<210> 49

<211> 124

<212> PRT

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 49

Met Asn Val Gly Phe Pro Arg Ser Pro Val Ile Val Asn Leu Gly Glu

1 5 10 15

Thr Met Ser Asn Val Val Ala Val Thr Glu Gln Thr Phe Lys Ser Thr

20 25 30

Val Ile Asp Ser Asp Lys Pro Val Ile Val Asp Phe Trp Ala Glu Trp

35 40 45

Cys Gly Pro Cys Lys Lys Leu Ser Pro Ile Ile Glu Glu Ile Ala Gly

50 55 60

Glu Tyr Gly Asp Lys Ala Val Val Ala Ser Val Asp Val Asp Ala Glu

65 70 75 80

Arg Thr Leu Gly Ala Met Phe Gln Ile Met Ser Ile Pro Ser Val Leu

85 90 95

Ile Phe Lys Asn Gly Ala Lys Val Glu Glu Phe Val Gly Leu Arg Pro

100 105 110

Lys Asn Glu Ile Val Glu Lys Leu Glu Lys His Leu

115 120

Claims

1.A method for increasing the production of 5-aminolevulinic acid by a 5-aminolevulinic acid-producing strain, the method comprising the steps of enhancing the antioxidant capacity of the strain and co-expressing 5-aminolevulinic acid synthase and phosphoenolpyruvate carboxylase;

enhancing the antioxidant capacity of the strain refers to enhancing the activity of antioxidant-related proteins in the strain;

the antioxidant-related protein is catalase I, catalase II, alkyl peroxidase, glutaredoxin I, thioredoxin II, methionine sulfoxide reductase A, superoxide dismutase derived from Escherichia coli, or catalase, thiol peroxidase, thiol reductase, thiol redox protein, and thioredoxin derived from Corynebacterium glutamicum.

2. The method according to claim 1, wherein the antioxidant related protein is catalase as shown in SEQ ID number 35, SEQ ID number 36 and SEQ ID number 45, alkylperoxidase as shown in SEQ ID number 37 and SEQ ID number 38, superoxide dismutase as shown in SEQ ID number 42, SEQ ID number 43 and SEQ ID number 44, glutaredoxin I as shown in SEQ ID number 39, thioredoxin as shown in SEQ ID number 40 and SEQ ID number 49, thiol peroxidase as shown in SEQ ID number 46, thiol reductase as shown in SEQ ID number 47, thiol redox protein as shown in SEQ ID number 48, methionine sulfoxide reductase A as shown in SEQ ID number 41.

3. The method according to claim 1, wherein the 5-aminolevulinic acid-producing strain has 5-aminolevulinic acid-synthesizing ability as such or is a 5-aminolevulinic acid-producing strain.

4. The method of claim 1, wherein the strain construction method further comprises enhancing the 5-aminolevulinic acid synthesis pathway or introducing an exogenous 5-aminolevulinic acid synthesis pathway; the 5-aminolevulinic acid synthetic pathway is a glutamyl-tRNA synthetase, a glutamyl-tRNA reductase, or a glutamate-1-semialdehyde aminotransferase.

5. The method of claim 1, wherein the strain is constructed by further comprising enhancing pyruvate carboxylase activity.

6. The method of claim 1, wherein the strain is E.coli (E.coli)Escherichia coli) Corynebacterium glutamicum (C.glutamicum)Corynebacterium glutamicum) Rhodobacter sphaeroides (A), (B), (C)Rhodobacter sphaeroides) Or Rhodopseudomonas palustris (Rhodopseudomonas palustris)。

7. The method of claim 6, wherein the strain is Escherichia coli or Corynebacterium glutamicum.

8. A method according to any one of claims 1 to 7, wherein the strain has a shake flask 5-aminolevulinic acid yield of more than 5.6 g/L.

9. A method of constructing a 5-aminolevulinic acid high-producing strain, the method comprising:

a step of enhancing the antioxidant ability of the strain and co-expressing 5-aminoacetylpropionate synthetase and phosphoenolpyruvate carboxylase;

10. The method of claim 9, wherein the strain construction method further comprises enhancing the 5-aminolevulinic acid synthesis pathway or introducing an exogenous 5-aminolevulinic acid synthesis pathway; the 5-aminolevulinic acid synthetic pathway is a glutamyl-tRNA synthetase, a glutamyl-tRNA reductase, or a glutamate-1-semialdehyde aminotransferase.

11. The method of claim 9, wherein the strain construction method further comprises enhancing pyruvate carboxylase activity.

12. A 5-aminolevulinic acid high-producing strain which co-expresses 5-aminolevulinic acid synthase and phosphoenolpyruvate carboxylase and has enhanced antioxidant ability;

the antioxidant related protein is catalase I, catalase II, alkyl peroxidase, glutaredoxin I, thioredoxin II, methionine sulfoxide reductase A and superoxide dismutase from Escherichia coli, or catalase, thiol peroxidase, thiol reductase, thiol redox protein and thioredoxin from Corynebacterium glutamicum.

13. The strain of claim 12, wherein the antioxidant-related protein is catalase as shown in SEQ ID number 35, SEQ ID number 36, and SEQ ID number 45; alkyl peroxidase as shown in SEQ ID number 37 and SEQ ID number 38; superoxide dismutase as shown in SEQ ID number 42, SEQ ID number 43 and SEQ ID number 44; glutaredoxin I as shown in SEQ ID number 39; thioredoxin as shown in SEQ ID number 40 and SEQ ID number 49; a branching thiol peroxidase shown as SEQ ID number 46, a branching thiol reductase shown as SEQ ID number 47, a branching thiol redox protein shown as SEQ ID number 48, and a methionine sulfoxide reductase A shown as SEQ ID number 41.

14. The strain of claim 12, wherein the 5-aminolevulinic acid synthesis pathway in the 5-aminolevulinic acid producing strain is enhanced or an exogenous 5-aminolevulinic acid synthesis pathway is introduced; the 5-aminolevulinic acid synthetic pathway is a glutamyl-tRNA synthetase, a glutamyl-tRNA reductase, or a glutamate-1-semialdehyde aminotransferase.

15. The strain of claim 12, wherein pyruvate carboxylase activity is enhanced in the 5-aminolevulinic acid-producing strain.

16. The strain of claim 12, wherein the strain is escherichia coli (e.coli: (a), (b), or (c)Escherichia coli) Corynebacterium glutamicum (C.glutamicum)Corynebacterium glutamicum) Rhodobacter sphaeroides (A), (B), (C)Rhodobacter sphaeroides) Or Rhodopseudomonas palustris (Rhodopseudomonas palustris)。

17. The strain of claim 16, wherein the strain is escherichia coli or corynebacterium glutamicum.

18. A strain according to any of claims 12 to 17, wherein the strain has a shake flask 5-aminolevulinic acid yield of more than 5.6 g/L.

19. A method of producing 5-aminolevulinic acid, the method comprising:

1) cultivating the 5-aminolevulinic acid-producing strain of any one of claims 12-18, thereby obtaining 5-aminolevulinic acid; and

20. Use of a 5-aminolevulinic acid-producing strain according to any one of claims 12 to 18 for producing 5-aminolevulinic acid and/or producing a downstream product with 5-aminolevulinic acid as a precursor.

21. The use of claim 20, wherein the downstream product derived from ALA is heme or vitamin B12.