CN109722459A

CN109722459A - A kind of 5-ALA superior strain and the preparation method and application thereof

Info

Publication number: CN109722459A
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: 2019-05-07
Anticipated expiration: 2038-10-31
Also published as: CN109722459B

Abstract

The invention discloses a kind of oxidation resistances that bacterial strain is produced by enhancing 5-ALA, for example, the method for enhancing the activity of anti-oxidant GAP-associated protein GAP in the bacterial strain to construct 5-ALA superior strain.The 5-ALA yield for the 5-ALA superior strain that the present invention constructs significantly improves, while inversion rate of glucose also correspondinglys increase.5-ALA can be generated efficiently at low cost using bacterial strain of the invention.

Description

A kind of 5-ALA superior strain and the preparation method and application thereof

Technical field

The invention belongs to genetic engineerings and field of microbial fermentation；Specifically, the present invention relates to a kind of 5- glycyls Propionic acid superior strain and its preparation method and application.

Background technique

5-ALA (5-minolevulinic acid, ALA) be organism synthesis ferroheme, chlorophyll, The precursor of the tetrapyrroles such as VB12 is widely present in animal, plant and microorganism.ALA medicine, agricultural, feed and The fields such as health food are widely used, and are the high added value biological-based chemicals of great Development volue.ALA can make in medicine It is used for treatment of cancer and diagnosing tumor for photosensitizer, plant growth regulator had both been can be used as in agriculturally ALA and has promoted crop raw It is long, and can be used as biological pesticide as insecticide and herbicide, in addition, as nutritional ingredient ALA in health food and animal The application of field of nutrition is increasingly developed and is received.

ALA is mainly produced by chemical synthesis at present, and cost of material and pollutant discharge amount are high, leads to its production cost High, product price also remains high, and greatly limits it in the scale application in the fields such as agricultural, feed.In recent years, pass through Industrial application has been obtained in the bacterial fermentation production ALA for producing ALA, and gradually substitutes traditional chemical synthesis, becomes and grinds The emphasis studied carefully and developed.The production bacterial strain of ALA mainly passes through mutation breeding at present or genetic engineering transformation obtains, and with The development of metabolic engineering and synthetic biology technology, engineering strain ALA yield are gradually more than what classic mutagenesis obtained Bacterial strain becomes the following principal concern further researched and developed.The building of the engineered strain of existing literature and patent report is mostly sharp Optimize the metabolic pathway of synthesizing of ALA, with genetic engineering and metabolic engineering technology to realize Product yields and the substrate transformation rate not It is disconnected to be promoted.Such as CN106047916A discloses a kind of corynebacterium glutamicum for producing ALA and its building process, passes through multistep Metabolic engineering shake flask fermentation ALA yield has reached 2.78g/L；CN103710374A discloses a kind of ALA production bacterial strain structure It builds and the method for ALA production, engineering bacteria shake flask fermentation ALA yield in the culture medium of addition pantothenic acid has reached 4.12g/L.Although ALA yield has obtained larger promotion, and production cost has certain decline relative to chemical synthesis, but relative to paddy ammonia The technical level of the biology bases bulk chemical such as acid, lysine, threonine, the ALA of fermentation method synthesis at present is still lower, illustrates existing There are the key factors that other unknown limitation ALA synthesis are still had in bacterial strain system.

Therefore, this field needs to develop the superior strain of 5-ALA, so as to efficiently be given birth to using the bacterial strain Produce 5-ALA.

Summary of the invention

The purpose of the present invention is to provide a kind of superior strains of 5-ALA.

The present invention also provides the preparation method and application of the 5-ALA superior strain.

In a first aspect, the 5-ALA that the present invention provides a kind of raising 5-ALA production bacterial strain produces The method of amount, the method includes enhancing the oxidation resistance of the bacterial strain.

In a particular embodiment, the oxidation resistance for enhancing the bacterial strain refers to anti-oxidant phase in the enhancing bacterial strain Close the activity of albumen.

In a particular embodiment, the anti-oxidant GAP-associated protein GAP is catalase, superoxide dismutase, peroxide Compound enzyme, thiol reduction enzyme, thiol disulfide oxidoreducing enzyme, thioredoxin reductase or methionine sulfoxide reductase.

In a preferred embodiment, the anti-oxidant GAP-associated protein GAP can be the anti-oxidant GAP-associated protein GAP in various sources, Including but not limited to human or animal, plant, microorganism.

In a preferred embodiment, the anti-oxidant GAP-associated protein GAP derives from microorganism.

In a preferred embodiment, the anti-oxidant GAP-associated protein GAP is originated from filamentous fungi, saccharomycete or bacterium.

In preferred embodiment, the filamentous fungi includes but is not limited to aspergillus (Aspergillus), Penicillium (Penicillium), in Humicola (Humicola), trichoderma (Trichoderma) or acremonium category (Acremonium) Filamentous fungi.

In a preferred embodiment, the anti-oxidant GAP-associated protein GAP is originated from aspergillus niger (Aspergillus niger), soil Aspergillus (Aspergillusterreus), Acremonium alabamensis (Acremonium alabamensis), thermophilic ascomycete (Thermoascus aurantiacus), thermophilic leather save spore (Scytalidium thermophilum), Humicola insolens (Humicola insolens) and thermophilic loose mould (Penicillium pinophilum) and grey humicola lanuginosa (Humicola grisea)；

In preferred embodiment, the saccharomycete includes but is not limited to saccharomyces cerevisiae (Saccharomyces Cerevisiae), P. pastoris (Pichia pastoris).

In preferred embodiment, the bacterium includes but is not limited to Escherichia (Escherichia), Corynebacterium (Corynebacterium), Serratia (Serratia), bacillus (Bacillus), Micrococcus (Micrococcus), acinetobacter (Acinetobacter), Arthrobacter (Arthrobacter), Proteus (Proteus), rhizobium (Rhizobium), Stenotrophomonas category (Stenotrophomonas), Lactobacillus brevis category , such as Thermus (Thermus) (Lactobacillus) and thermophilic class bacterium.

In a preferred embodiment, the anti-oxidant GAP-associated protein GAP be originated from Escherichia coli (Escherichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum), serratia marcescens (Serratia marcescens), Bacillus subtilis (Bacillus subtilis), bacillus pumilus (Bacillus pumilus), micrococcus lysodeikticus (Micrococcus lysodeikticus), proteus mirabilis (Proteus mirabilis), germ oligotrophy unit cell (Stenotrophomonas maltophilia), Lactobacillus brevis (Lactobacillus breris), thermus thermophilus (Thermus thermophilus) etc..

In a preferred embodiment, the anti-oxidant GAP-associated protein GAP is originated from Escherichia coli or corynebacterium glutamicum.

In further preferred embodiment, the anti-oxidant GAP-associated protein GAP is derived from the catalase of Escherichia coli I (KatG), catalase I I (KatE), alkyl peroxide enzyme (AhpC, AhpF), glutaredoxin I (GrxA), sulphur oxygen are also Protein I I (TrxC), methionine sulfoxide reductase A (MsrA), superoxide dismutase (SodA, SodB, SodC), or it is originated from paddy Catalase (NCgl0251), mycothiol peroxidase (NCgl2502), mycothiol in propylhomoserin corynebacteria are also Protoenzyme (NCgl1928), mycothiol redox protein (NCgl2445) and thioredoxin (NCgl2985).

In further preferred embodiment, the anti-oxidant GAP-associated protein GAP is such as SEQ ID No.35, SEQ ID Catalase shown in No.36, SEQ ID No.45, or equally from Escherichia coli and amino acid sequence and SEQ ID No.35, SEQ ID No.36, SEQ ID No.45 have 80%, preferably 90%, more preferable 95%, and most preferably 98% or more is same Source property and the polypeptide with catalase function；

The alkyl peroxide enzyme as shown in SEQ ID No.37, SEQ ID No.38, or equally derive from Escherichia coli And amino acid sequence and SEQ ID No.37, SEQ ID No.38 have 80%, preferably 90%, more preferable 95%, most preferably 98% or more homology and the polypeptide with alkyl peroxide enzyme function；

The superoxide dismutase as shown in SEQ ID No.42, SEQ ID No.43, SEQ ID No.44, or it is same next Derived from Escherichia coli and amino acid sequence and SEQ ID No.42, SEQ ID No.43, SEQ ID No.44 are with 80%, preferably 90%, more preferable 95%, most preferably 98% or more homology and with superoxide dismutase function polypeptide；

The glutaredoxin I as shown in SEQ ID No.39, or equally from Escherichia coli and amino acid sequence and SEQ ID No.39 has 80%, preferably 90%, more preferable 95%, and most preferably 98% or more homology simultaneously has the function of glutaredoxin Polypeptide；

The thioredoxin as shown in SEQ ID No.40, or equally from Escherichia coli and amino acid sequence and SEQ ID No.40 has 80%, preferably 90%, more preferable 95%, and most preferably 98% or more homology simultaneously has the function of thioredoxin Polypeptide；Thioredoxin shown in SEQ ID No.49, or equally from Corynebacterium glutamicum and amino acid sequence and SEQ ID No.49 has 80%, preferably 90%, more preferable 95%, and most preferably 98% or more homology simultaneously has the function of thioredoxin Polypeptide；

The mycothiol peroxidase as shown in SEQ ID No.46, or equally derive from Corynebacterium glutamicum and amino Acid sequence and SEQ ID No.46 have 80%, preferably 90%, more preferable 95%, and most preferably 98% or more homology simultaneously has point The polypeptide of branch mercaptan peroxidase function；

The mycothiol reductase as shown in SEQ ID No.47, or equally derive from Corynebacterium glutamicum and amino acid sequence Column have 80%, preferably 90%, more preferable 95% with SEQ ID No.47, and most preferably 98% or more homology simultaneously has branch's sulphur The polypeptide of alcohol reductase function；

The mycothiol redox protein as shown in SEQ ID No.48, or equally derive from Corynebacterium glutamicum and ammonia Base acid sequence and SEQ ID No.48 have 80%, preferably 90%, more preferable 95%, and most preferably 98% or more homology simultaneously has The polypeptide of mycothiol redox protein function；

The methionine sulfoxide reductase A as shown in SEQ ID No.41, or equally derive from Corynebacterium glutamicum and amino Acid sequence and SEQ ID No.41 have 80%, preferably 90%, more preferable 95%, and most preferably 98% or more homology simultaneously has egg The polypeptide of propylhomoserin sulfoxide reductase A function.In further preferred embodiment, the anti-oxidant GAP-associated protein GAP is such as SEQ ID No.35, SEQ ID No.36, catalase shown in SEQ ID No.45, such as SEQ ID No.37, SEQ ID Alkyl peroxide enzyme shown in No.38, the super oxygen as shown in SEQ ID No.42, SEQ ID No.43, SEQ ID No.44 Compound mutase, the glutaredoxin I as shown in SEQ ID No.39, as shown in SEQ ID No.40, SEQ ID No.49 Thioredoxin, the mycothiol peroxidase as shown in SEQ ID No.46, branch's sulphur as shown in SEQ ID No.47 Alcohol reductase, the mycothiol redox protein as shown in SEQ ID No.48, the methionine as shown in SEQ ID No.41 Sulfoxide reductase A.

In a preferred embodiment, the anti-oxidant albumen: (a) ammonia being respectively shown in SEQ ID No.35-49 Base acid sequence；Or

(b) amino acid sequence is respectively shown in by one or several by SEQ ID No.35-49, preferably 1-20, more preferably 1-10, more preferable 1-6, the the replacing, missing or adding for amino acid residue of more preferable 1-3, most preferably 1 and formed and have ammonia The derived protein of the base acid sequence protein function as shown in SEQ ID No.35-49 respectively；Or

(c) both ends of amino acid sequence are respectively shown in by one or several in SEQ ID No.35-49, preferably 1-20, More preferable 1-10, more preferable 1-6, more preferable 1-3, most preferably 1 amino acid residue addition and formed and there is amino acid The derived protein of the sequence protein function as shown in SEQ ID No.35-49 respectively.

In a preferred embodiment, the activity of the anti-oxidant GAP-associated protein GAP of the enhancing can one of by the following method or group It closes to realize: expressing the homologous or heterologous encoding gene of the albumen, and/or increase the copy number of the encoding gene, and/or The promoter of the encoding gene is transformed to enhance transcripting starting speed, and/or modification carries the courier of the encoding gene The translational control area of RNA is to enhance translation intensity.

In a preferred embodiment, the 5-ALA production bacterial strain itself has 5-ALA conjunction At ability or 5-ALA superior strain.

In a preferred embodiment, the strain construction method further include strengthen 5-ALA route of synthesis or Introduce exogenous 5-ALA route of synthesis.

In a preferred embodiment, the 5-ALA route of synthesis refers to that 5-ALA synthesizes phase Close enzyme, including but not limited to 5-aminolevulinate synthetase, Glutamyl-tRNA synthetase, glutamyl-tRNA reductase or paddy Propylhomoserin -1- semialdehyde aminopherase；It is preferred that 5-aminolevulinate synthetase.

In a preferred embodiment, the strain construction method further include enhancing phosphoric acid enol pyruvic acid carboxylase or The activity of pyruvate carboxylase.

In a preferred embodiment, the method also includes measuring the 5-ALA yield of obtained strains.

In a preferred embodiment, the bacterial strain is Escherichia coli (Escherichia coli), glutamic acid rod-like stem Bacterium (Corynebacterium glutamicum), Spherical red antibacterial (Rhodobacter sphaeroides), marsh are red false single Born of the same parents bacterium (Rhodopseudomonas palustris) etc.；It is preferred that Escherichia coli and corynebacterium glutamicum.

In a preferred embodiment, the shaking flask 5-ALA yield of the bacterial strain is higher than 5.6g/L.

In second aspect, the present invention provides a kind of method for constructing 5-ALA superior strain, the method packet It includes:

The step of enhancing the oxidation resistance of the bacterial strain.

In the third aspect, the present invention provides a kind of 5-ALA superior strain, the oxidation resistance of the bacterial strain Enhancing.

In a preferred embodiment, the anti-oxidant GAP-associated protein GAP is originated from aspergillus niger (Aspergillus niger), soil Aspergillus (Aspergillusterreus), Acremonium alabamensis (Acremonium alabamensis), thermophilic ascomycete (Thermoascus aurantiacus), thermophilic leather save spore (Scytalidium thermophilum), Humicola insolens (Humicola insolens) and thermophilic trichosporon spp (Thermomyces), thermophilic loose mould (Penicillium ) and grey humicola lanuginosa (Humicola grisea) pinophilum；

In further preferred embodiment, the anti-oxidant GAP-associated protein GAP is derived from the catalase of Escherichia coli I (KatG), catalase I I (KatE), alkyl peroxide enzyme (AhpC, AhpF), glutaredoxin I (GrxA), sulphur oxygen are also Protein I I (TrxC), methionine sulfoxide reductase A (MsrA), superoxide dismutase (SodA, SodB, SodC), or it is originated from paddy Catalase (NCgl0251), mycothiol peroxidase (NCgl2502), mycothiol in propylhomoserin corynebacteria are also Protoenzyme (NCgl1928), mycothiol redox protein (NCgl2445), thioredoxin (NCgl2985).

The methionine sulfoxide reductase A as shown in SEQ ID No.41, or equally derive from Corynebacterium glutamicum and amino Acid sequence and SEQ ID No.41 have 80%, preferably 90%, more preferable 95%, and most preferably 98% or more homology simultaneously has egg The polypeptide of propylhomoserin sulfoxide reductase A function.

In further preferred embodiment, the anti-oxidant GAP-associated protein GAP is such as SEQ ID No.35, SEQ ID Catalase shown in No.36, SEQ ID No.45；The alkyl peroxy as shown in SEQ ID No.37, SEQ ID No.38 Compound enzyme；The superoxide dismutase as shown in SEQ ID No.42, SEQ ID No.43, SEQ ID No.44；Such as SEQ ID Glutaredoxin I shown in No.39；The thioredoxin as shown in SEQ ID No.40, SEQ ID No.49；Such as SEQ ID Mycothiol peroxidase shown in No.46, the mycothiol reductase as shown in SEQ ID No.47, such as SEQ ID Mycothiol redox protein shown in No.48, the methionine sulfoxide reductase A as shown in SEQ ID No.41.

In a preferred embodiment, the anti-oxidant albumen: (a) ammonia being respectively shown in SEQ ID No.35-49 Base acid sequence；Or amino acid sequence (b) is respectively shown in by one or several by SEQ ID No.35-49, preferably 1-20, more It is preferred that 1-10, more preferable 1-6, the the replacing, missing or adding for amino acid residue of more preferable 1-3, most preferably 1 and formed and have There is the derived protein of the amino acid sequence protein function as shown in SEQ ID No.35-49 respectively；Or

In a preferred embodiment, the 5-ALA in the 5-ALA superior strain synthesizes way Diameter is strengthened or is introduced exogenous 5-ALA route of synthesis.

In a preferred embodiment, the PEP carboxylase in the 5-ALA superior strain The increased activity of enzyme or pyruvate carboxylase.

In fourth aspect, the present invention provides a kind of method for generating 5-ALA, which comprises

1) it cultivates 5-ALA described in the third aspect and produces bacterial strain, to obtain 5-ALA；With

2) 5-ALA optionally is obtained from fermentation culture system 1).

At the 5th aspect, the present invention provides the purposes of 5-ALA superior strain described in the third aspect, described Bacterial strain is for generating 5-ALA and/or generating using 5-ALA as the downstream product of precursor.

In a preferred example, described by the downstream product of precursor of ALA is ferroheme or vitamin B12.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 shows the influence that the expression of anti-oxidant GAP-associated protein GAP in Escherichia coli synthesizes ALA；

Fig. 2 shows the influence that Expression of Superoxide Dismutase synthesizes ALA；

Fig. 3 shows the influence that hydrogen peroxide expression of enzymes synthesizes ALA in corynebacterium glutamicum；

Fig. 4 shows the influence that the anti-oxidant correlative protein expression of other in corynebacterium glutamicum synthesizes ALA.

Specific embodiment

Inventor produces bacterium after extensive and in-depth study, it was unexpectedly found that passing through and enhancing 5-ALA The oxidation resistance of strain, such as enhance the activity of anti-oxidant GAP-associated protein GAP and can effectively improve ALA yield.It completes on this basis The present invention.

Term definition

The term as used herein " exogenous " refers to the substance for containing in certain system and being not present originally.For example, by turning The modes such as change are introduced into the encoding gene being not present originally in the bacterial strain in certain bacterial strain, then the gene is " external source for the bacterial strain Property ".

The term as used herein " endogenous " refers to that in microorganism polypeptide is in the activity of unmodified state, i.e. nature Activity under state.

The term as used herein " activity of enhancing protein " refers to enhancing the albumen in microorganism by modification albumen The intracellular reactive of matter improves intracellular reactive compared with the protein active having with nature.The term as used herein " exogenous " refers to the substance for containing in certain system and being not present originally.E.g., including but be not limited by the modes such as conversion and exist The encoding gene for the enzyme being not present originally in the bacterial strain is introduced into certain bacterial strain, to express the enzyme in the bacterial strain, then the enzyme pair In the bacterial strain be " exogenous ".

The term as used herein " enhancing " not only includes the increase due to protein activity itself and bring is than original function The higher effect of energy, and it can pass through at least one method chosen from the followings and carry out: increase the multicore glycosides of coding protein The copy number of acid is modified the regulating and controlling sequence of the gene of coding protein, with strongly active sequence substitutions chromosome The regulating and controlling sequence of the gene of upper coding protein, the work for increasing protein with the gene of mutated gene permutation encoding protein Property, modification is introduced in the gene of coding protein on chromosome to enhance the activity of protein, can also wrap without limitation Any method is included, as long as the activity of protein or the work of enhancing introducing protein can be enhanced compared with endogeneous activity Property.

The term as used herein " activity for introducing protein " can be carried out by methods known in the art, including but not It is limited to, such as: the polynucleotides of the polynucleotide sequence comprising coding protein being inserted on chromosome, and/or by multicore glycosides Acid, which is cloned on carrier, introduces microorganism, and/or row directly increases the copy number of the polynucleotides on chromosome, and/or changes The polynucleotides promoter of the polynucleotides with coding protein is made to enhance transcripting starting speed, and/or to coding albumen The transcription of the polynucleotides of matter is modified to enhance its activity, and/or modification carries the multicore glycosides of the coding protein The translational control sequence of the mRNA of acid is to enhance translation intensity, and/or the polynucleotides of modification coding protein itself to increase Strong mRNA stability, solves the methods of the feedback inhibition of isolating protein to realize at protein stability, can also wrap without limitation Include any of method that can introduce protein active.

As described above, regulating and controlling sequence includes the promoter that can originate transcription, any manipulation base for transcriptional control Because of sequence, the sequence of suitable mRNA ribosomes binding structural domain, the sequence of regulatory transcription and translation termination are encoded.To regulation sequence The modification of column includes but is not limited to, such as: lacking, is inserted into polynucleotide sequence, conservative variants or non-conservative mutation or its group It closes and introduces modification, it can also be by replacing original polynucleotide sequence with the active polynucleotide sequence of enhancing.Carrier Be include coding target protein polynucleotides polynucleotide sequence DNA construct, it is suitable operationally to be linked to Regulating and controlling sequence is so that target protein can express in host cell.Carrier can be independently of host after being transferred into suitable host cell Cellular genome replicates or works, or can be integrated on the genome of host.These carriers can be not particularly restricted, As long as the carrier is reproducible in host cell, and hot river vector construction known in the art can be used in it.Carrier Example include natural or recombinant plasmid, clay, virus and bacteriophage.For example, pWE15, pET, pUC carrier etc..In addition, passing through Carrier is inserted on the chromosome of host cell, the polynucleotides of encoding endogenous target protein on chromosome can be replaced with The polynucleotides of modification.Any method known in the art can be used in polynucleotides insertion chromosome to carry out, including but not It is limited to, such as: passing through homologous recombination.Polynucleotides include the DNA and RNA of the target protein of coding, can be inserted into any form Onto the chromosome of host cell, as long as it can be expressed in host cell.Including but not limited to, such as: polynucleotides can be with Host cell is introduced in the form of reset condition, and/or expression cassette.Expression cassette be include all required members needed for self-expression The gene construct of part is also possible to be capable of the expression vector of self-replacation, may include being operatively coupled to polynucleotides Promoter, transcription stop signals, ribosomes binding structural domain and translation termination signal.

Oxidation resistance and anti-oxidant GAP-associated protein GAP

The present invention produces the oxidation resistance of bacterial strain by enhancing 5-ALA to improve ALA yield.Based on this The introduction of invention, those skilled in the art could be aware that the various particular technique means for improving the oxidation resistance of bacterial strain.Having In the embodiment of body, enhance the oxidation resistance of high bacterial strain by the activity of anti-oxidant GAP-associated protein GAP in enhancing bacterial strain.

The term as used herein " anti-oxidant GAP-associated protein GAP " refers to egg relevant to the antioxidant activity of the bacterial strain in bacterial strain It is white.Based on the teachings of the present invention, the anti-oxidant GAP-associated protein GAP in bacterial strain as known to those skilled in the art.For example, as described herein Anti-oxidant GAP-associated protein GAP includes but is not limited to catalase, superoxide dismutase, peroxidase, thiol reduction enzyme, oxygen Change reduction albumen, methionine sulfoxide reductase.

More specifically, the catalase, is the enzyme that catalyzing hydrogen peroxide generates oxygen and water, including but not limited to source From the catalase of Escherichia coli catalase I (KatG), catalase I I (KatE) and corynebacterium glutamicum (NCgl0251)；

The superoxide dismutase is superoxide radical (O in energy catalysis biological body^2-) occur disproportionated reaction enzyme, packet It includes but is not limited to superoxide dismutase A (SodA), superoxide dismutase B (SodB), superoxides from Escherichia coli Mutase C (SodC)；

The peroxidase is the enzyme aoxidized using hydrogen peroxide as electron acceptor catalysis substrate, including but not limited to alkyl Peroxidase (AhpC, AhpF), mycothiol peroxidase (NCgl2502), glutathione peroxidase (Gpx)；

The thiol reduction enzyme is the enzyme that catalysis oxidation type small molecule thiol reduction is reduced form mercaptan, including but not limited to Mycothiol reductase (NCgl1928), glutathione reductase (Gor)；

Under the conditions of the thiol disulfide oxidoreducing enzyme is existing for the small molecule mercaptan, protein disulfide is restored As the enzyme of protein dithiol, including but not limited to glutaredoxin (GrxA, GrxB, GrxC), thioredoxin (TrxA, TrxC, NCgl2985), mycothiol redox protein (NCgl2445)；

The methionine sulfoxide reductase is in the form of thioredoxin by methionine-S- sulfoxide (MetSO) It is reduced to the enzyme of methionine, including but not limited to methionine sulfoxide reductase A (MsrA) and methionine sulfoxide reductase B (MsrB)。

" anti-oxidant GAP-associated protein GAP " of the present invention can be the albumen in various sources, including but not limited to human or animal (ox, pig liver), plant, microorganism.

In a preferred embodiment, the anti-oxidant GAP-associated protein GAP is originated from aspergillus niger (Aspergillus niger), soil Aspergillus (Aspergillusterreus), Acremonium alabamensis (Acremonium alabamensis), thermophilic ascomycete (Thermoascus aurantiacus), thermophilic leather save spore (Scytalidium thermophilum), Humicola insolens (Humicola insolens), thermophilic loose mould (Penicillium pinophilum) and grey humicola lanuginosa (Humicola grisea)；

" anti-oxidant GAP-associated protein GAP " of the present invention further includes the regulation egg that can regulate and control anti-oxidant protein transcription translation It is white, such as Escherichia coli oxidative pressure regulatory factor OxyR and SoxR.

In addition, mercaptan peroxidase of the present invention, thiol reduction enzyme and thiol redox protein, wherein mercaptan Include not only glutathione described in the text and mycothiol, further includes being widely present small molecule mercaptan, such as ovum in organism Class mercaptan, ergot mercaptan, gemma mercaptan etc..

Bacterial strain and its construction method of the invention and purposes

The inventors discovered that improving the oxidation resistance of the production bacterial strain of 5-ALA (ALA), such as enhancing life Produce the anti-oxidant GAP-associated protein GAP in bacterial strain, including but not limited to peroxidase, superoxide dismutase, thiol reduction enzyme, paddy The activity of oxygen also albumen, thioredoxin, mycothiol redox protein, methionine sulfoxide reductase, can significantly improve The ALA yield of the production bacterial strain.On this basis, the present invention provides the ALA superior strains and benefit using the method building The method for preparing ALA with the bacterial strain.

Based on the teachings of the present invention and the prior art, it will be understood by those skilled in the art that present invention could apply to this Body has the bacterial strain of certain 5-ALA synthesis capability, also can be applied to itself do not have 5-ALA conjunction At ability, but by, for example, exogenous importing 5-ALA route of synthesis to have 5-ALA synthesis The bacterial strain of ability obtains 5-ALA superior strain whereby；It is raw that the present invention also can be applied to 5-ALA Bacterial strain, even 5-ALA superior strain are produced, so that the 5-ALA for further increasing the bacterial strain is raw Production capacity power.

The term as used herein " 5-ALA route of synthesis " refers to that 5-ALA synthesizes relevant enzyme, That is, the various enzymes for including in the concrete ways of generation 5-ALA in microorganism, including but not limited to 5- glycyl ALA synthase, Glutamyl-tRNA synthetase, glutamyl-tRNA reductase or glutamic acid -1- semialdehyde aminopherase, etc.； It is preferred that 5-aminolevulinate synthetase.

Those skilled in the art will know that many bacterial strains can be used for generating 5-ALA.Although these bacterial strains are not Together, but the synthetic system, approach of their synthesis 5-ALAs are similar.Therefore, those of ordinary skill in the art In view of the teachings of the present invention and the prior art it will be appreciated that bacterial strain of the invention can be and any can be used for generating 5- glycyl The bacterial strain of propionic acid, including but not limited to: Escherichia coli (Escherichia coli), corynebacterium glutamicum (Corynebacterium glutamicum), Spherical red antibacterial (Rhodobacter sphaeroides), the red false unit cell in marsh Bacterium (Rhodopseudomonas palustris) etc..In a particular embodiment, the preferred Escherichia coli of the bacterial strain and paddy Propylhomoserin corynebacteria.

The purposes of 5-ALA superior strain of the invention

It will be understood by those skilled in the art that 5-ALA superior strain of the invention cannot be only used for generating 5- ammonia Base levulic acid, it may also be used for generate using 5-ALA as the derivative of precursor, such as ferroheme or VB12.

Advantages of the present invention:

1. the 5-ALA yield of bacterial strain of the present invention greatly improves；

2. present invention discover that the activity of anti-oxidant GAP-associated protein GAP can be significant in enhancing 5-ALA production bacterial strain The 5-ALA yield of production bacterial strain is improved, to produce bacterial strain or optimization 5- for engineered 5-ALA The production technology of amino-laevulic acid production bacterial strain provides new thinking；

3. the method provided by the invention for improving engineering bacteria ALA yield also can be further improved strain growth performance and substrate Inversion rate of glucose.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Unless otherwise defined, all technical and scientific terms used herein and one skilled in the art of the present invention Normally understood meaning is identical.Implement or examine although can utilize to described herein similar or of equal value any method and material The present invention, but preferably method described herein and material.

Embodiment

Materials and methods

Archaeal dna polymerase used in the embodiment of the present invention is purchased from the Fastpfu of Beijing Quan Shi King Company；Restriction enzyme and DNA ligase etc. is purchased from Fermentas company；

Yeast powder and peptone are purchased from Britain Oxoid Products；Glycine and IPTG are purchased from Promega company；ALA and Paradime thylaminobenzaldehyde etc. is purchased from Sigma company；Agar powder and antibiotic are purchased from Beijing Suo Laibao；Glucose, glacial acetic acid, height Chloric acid, trichloroacetic acid, acetylacetone,2,4-pentanedione, chloroform and other common chemical reagent are purchased from traditional Chinese medicines.

Plasmid extraction kit and agarose gel electrophoresis QIAquick Gel Extraction Kit are purchased from the raw work in Shanghai, and relevant operation is stringent It executes to specifications；

Plasmid construction sequence verification is completed by Jin Weizhi；

DH5 α competent cell is purchased from Beijing Quan Shi King Company.

LB medium component: yeast powder 5g/L, peptone 10g/L, NaCl 10g/L, addition 2% in solid medium Agar powder.

Antibiotic concentration are as follows: 100 μ g/mL of ampicillin.

The detection method of ALA: 100 μ L pH, 4.6 sodium acetate buffer is added in the diluted fermentation liquid of 200 μ L, is then added 5 μ L acetylacetone,2,4-pentanedione, 100 DEG C of water-baths incubate 15min, isometric Ehrlish ' s reagent (42mL ice vinegar are added after being cooled to room temperature Acid, 70% perchloric acid of 8mL, 1g dimethylaminobenzaldehyde) it mixes, the absorbance under 553nm wavelength is surveyed after the 10min that develops the color.

Glucose analysis method is detected using the SBA-40D bio-sensing analyzer of Shandong academy of sciences production.

Embodiment 1.ALA synzyme and the building of phosphoric acid enol pyruvic acid carboxylase coexpression vector

According to NCBI announce Rhodopseudomonas palustris ATCC17001 hemA gene order (GenBank: JQ048720.1) and genome sequence the design primer hemA-F/R and ppc-F/R of Escherichia coli MG1655, and with pZPA6 matter Grain (CN103981203B) is that template PCR amplifications obtain hemA gene and the ppc genetic fragment with own promoter, and PCR expands Increasing parameter is 94 DEG C of 5min；94 DEG C of 20s, 55 DEG C of 20s, 72 DEG C of 1.5min are recycled 30 times；72 DEG C of extension 5min.After segment recycling Digestion processing, connects pTrc99A plasmid, obtains the recombinant vector of hemA and ppc coexpression and is named as pZCA40.

The anti-oxidant correlative protein expression vector construction of 2. Escherichia coli of embodiment

Primer katG-F/R, katE-F/ are separately designed according to the genome sequence of the NCBI Escherichia coli MG1655 announced R, ahpCF-F/R, grxA-F/R, trxC-F/R, msrA-F/R are obtained using MG1655 genome as template by PCR amplification respectively To catalase of Escherichia coli I (amino acid sequence is as shown in SEQ ID No.35), the hydrogen peroxide for having own promoter Enzyme II (amino acid sequence is as shown in SEQ ID No.36), alkyl peroxide enzyme (amino acid sequence such as SEQ ID No.37 and Shown in SEQ ID No.38), glutaredoxin I (amino acid sequence is as shown in SEQ ID No.39), thioredoxin II (amino Acid sequence is as shown in SEQ ID No.40), the volume of methionine sulfoxide reductase A (amino acid sequence is as shown in SEQ ID No.41) Code gene katG, katE, ahpC-ahpF, grxA, trxC and msrA, PCR amplification system are as follows: PCR amplification parameter are as follows: 94 DEG C 5min, 94 DEG C of 20s, 58 DEG C of 20s, 72 DEG C of 2min are recycled 30 times, 72 DEG C of extension 5min.Target fragment is handled with NotI digestion, so Segment will be obtained under DNA ligase effect afterwards to connect with the pZCA40 carrier equally handled, and connection product is transformed into DH5 α competent cell, coating amicillin resistance plate are incubated overnight, and choose positive colony progress bacterium colony PCR verifying and sequencing is tested Card, correct recombinant vector be respectively designated as pZCA40-katG, pZCA40-katE, pZCA40-ahpCF, pZCA40-grxA, PZCA40-trxC and pZCA40-msrA.

Embodiment 3. enhances the influence that Escherichia coli inoxidizability synthesizes ALA

In order to verify the influence strengthening Escherichia coli inoxidizability and synthesizing to ALA, above-mentioned carrier and its control are carried respectively Body pZCA40 is transferred in MG1655 bacterial strain, and competent cell preparation and conversion process are with reference to J. Pehanorm Brooker (Sambrook) etc. " Molecular Cloning:A Laboratory guide " write.Converted product is coated with the LB plate of amicillin resistance, is incubated overnight rear picking sun Property clone extract plasmid verifying, respectively obtain recombinant bacterial strain MG1655/pZCA40, MG1655/pZCA40-katG, MG1655/ PZCA40-katE, MG1655/pZCA40-ahpCF, MG1655/pZCA40-grxA, MG1655/pZCA40-trxC and MG1655/pZCA40-msrA。

Above-mentioned recombinant bacterium single colonie is inoculated with the LB liquid medium that 5mL contains 100 μ g/mL ampicillins respectively, 37 DEG C, 220rpm cultivates 12h.It transfers according to initial OD for 0.05 and the 250mL triangular flask of 50mL fermentation medium is housed, 37 DEG C, Final concentration of 50 μM of IPTG is added after 220rpm culture 2.5h, collects fermentation liquid after Fiber differentiation 28h, detects the concentration of ALA. Wherein fermentation medium is the M9 culture medium for adding yeast powder, main component are as follows: Na₂HPO₄·12H₂O 17.1g/L, KH₂PO₄ 3.0g/L, NaCl 0.5g/L, NH₄Cl 1.0g/L, MgSO₄2mM, CaCl₂0.1mM, glucose 15g/L, yeast powder 2g/L, Glycine 4g/L.The detection method of ALA is as described in " materials and methods " part.

Recombinant bacterium fermentation results are shown in Fig. 1, the yield of ALA after control strain MG1655/pZCA40 (Control) fermentation 28h For 1.46g/L, catalase (KatG and KatE) expression strains A LA yield is 1.81 times and 1.93 of control strain respectively Times, reach 2.64g/L and 2.81g/L, while strain growth also has improvement by a relatively large margin.

Alkyl peroxide expression of enzymes bacterial strain (AhpCF) ALA yield has reached 2.07g/L, is 1.42 times of control strain, But growth is had little effect, therefore the synthesis capability of unit thallus ALA is obviously improved.

Glutaredoxin I (GrxA), thioredoxin II (TrxC) and methionine sulfoxide reductase A (MsrA) express bacterial strain ALA yield is the 2.1 of control strain, 1.48 and 1.54 times respectively, and thioredoxin II, which expresses bacterial strain thalli growth, to be improved obviously, And glutaredoxin and methionine sulfoxide reductase A expression bacterial strain unit thallus ALA yield are 2.26 Hes of control strain respectively 1.39 times, promote significant effect.

The above results show can by the expression promotion thallus oxidation resistance for enhancing the anti-oxidant GAP-associated protein GAP of Escherichia coli To effectively improve engineering bacteria ALA yield.

The building of 4. Expression of Superoxide Dismutase carrier of embodiment

According to NCBI announce Escherichia coli MG1655 genome sequence separately design primer J23101-sodA-F/R, J23101-sodB-F/R and J23101-sodC-F/R is respectively obtained by PCR amplification as template using MG1655 genome and is had The coding base of the Escherichia coli superoxide dismutase (amino acid sequence is as shown in SEQ ID No.42-44) of J23101 promoter Because of sodA, sodB and sodC, PCR amplification system is as follows: PCR amplification parameter are as follows: 94 DEG C of 5min, 94 DEG C of 20s, 58 DEG C of 20s, and 72 DEG C 1min is recycled 30 times, 72 DEG C of extension 5min.Target fragment is handled with XbaI and SacI digestion, then under DNA ligase effect Segment will be obtained to connect with the pZCA9 carrier (CN103710374B) equally handled, and connection product is transformed into DH5 α impression State cell, coating amicillin resistance plate are incubated overnight, and are chosen positive colony and are carried out bacterium colony PCR verifying and sequence verification, just True recombinant vector is respectively designated as pZZCCA3, pZZCCA4 and pZZCCA5.

The influence that 5 Expression of Superoxide Dismutase of embodiment synthesizes ALA

In order to verify the influence that Expression of Superoxide Dismutase synthesizes ALA, will construct correct recombinant vector pZZCA3, PZZCA4, pZZCA5 are converted respectively into E.coli BW25113/pZGA24 (CN103981203B) bacterial strain, obtain recombined engineering Bacterial strain BW25113/pZGA24/pZZCA3, BW25113/pZGA24/pZZCA4 and BW25113/pZGA24/pZZCA5.

Above-mentioned recombinant bacterium single colonie is inoculated with 5mL respectively and contains 100 μ g/mL ampicillins and 34 μ g/mL chloramphenicol In LB liquid medium, 37 DEG C, 220rpm cultivates 12h.It is 0.05 switching equipped with 50mL fermentation medium according to initial OD 250mL triangular flask, 37 DEG C, final concentration of 50 μM of IPTG is added after cultivating 2.5h in 220rpm, collects fermentation after Fiber differentiation 17h Liquid detects the concentration of ALA.Wherein fermentation medium is the LB culture medium for adding succinic acid and glycine, main component are as follows: albumen Peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, succinic acid 10g/L, glucose 10g/L, glycine 4g/L.The detection side of ALA Method is as described in " materials and methods " part.

Recombinant bacterium fermentation results are shown in Fig. 2, and the ALA yield of control strain BW24/pZCA9 is 0.94g/L, superoxide dismutase Enzyme (SodA, SodB and SodC) expression strains A LA yield has reached 1.06g/L, 1.14g/L and 1.13g/L, is control bacterium respectively 1.13,1.21 and 1.20 times of strain show significantly mention by expressing superoxide dismutase enhancement engineering bacterium oxidation resistance High ALA yield.

The building of 6. corynebacterium glutamicum catalase expression vector of embodiment

According to NCBI corynebacterium glutamicum (Corynebacterium glutamicum) ATCC's 13032 announced Catalase (amino acid sequence is as shown in SEQ ID No.45) encoding gene NCgl0251 sequence (GeneID: 1021318), design primer Ncgl0251-F/R obtains purpose base by PCR amplification using 13032 genome of ATCC as template Cause, PCR amplification system are as follows: PCR amplification parameter are as follows: and 94 DEG C of 10min, 94 DEG C of 20s, 58 DEG C of 30s, 72 DEG C of 2min are recycled 30 times, 72 DEG C of extension 5min.Target fragment is handled with XbaI enzyme cutting, and segment and same digestion then will be obtained under DNA ligase effect The pZWA2 carrier (WO2014121724A1) of processing connects, and connection product is transformed into DH5 α competent cell, and coating blocks that Chloramphenicol resistance plate culture 36h chooses positive colony and carries out bacterium colony PCR verifying, and correct transformant carries out sequence verification, will be correct Recombinant vector be named as pZWA2-Ncgl0251.

Meanwhile according to catalase I (the amino acid sequence such as SEQ ID in the source Escherichia coli MG1655 announced NCBI Shown in No.35) encoding gene katG (GeneID:948431) design primer katG-F2/R2, using MG1655 genome as template The genetic fragment that band has additional promoter, PCR amplification parameter are obtained by PCR amplification are as follows: 94 DEG C of 5min, 94 DEG C of 20s, 58 DEG C 20s, 72 DEG C of 2min are recycled 30 times, 72 DEG C of extension 5min.Target fragment is handled with XbaI enzyme cutting, is then acted in DNA ligase The lower segment that will obtain is connect with the pZWA2 carrier that same digestion is handled, and connection product is transformed into DH5 α competent cell, is applied Cloth kalamycin resistance plate culture 36h chooses positive colony and carries out bacterium colony PCR verifying, and correct transformant carries out sequence verification, Correct recombinant vector is named as pZWA2-katG.

The influence that 7. hydrogen peroxide expression of enzymes of embodiment synthesizes corynebacterium glutamicum ALA

In order to verify the influence that hydrogen peroxide expression of enzymes synthesizes ALA, above-mentioned carrier is transferred to glutamic acid rod-like stem respectively In bacterium ATCC13032 bacterial strain.Converted product is coated with the plate of kalamycin resistance, is incubated overnight rear picking positive colony and extracts matter Grain verifying, obtains recombinant bacterial strain 13032/pZWA2-NCgl0251 and 13032/pZWA2-katG respectively.

By above-mentioned recombinant bacterium and its control strain 13032/pZWA2 single colonie be inoculated with respectively 50mL contain 25 μ g/mL cards that The LB liquid medium of mycin, 2/0g/L glucose and 3g/L corn pulp, 30 DEG C, 200rpm cultivates 12h.It is according to initial OD 0.5 switching is equipped with the 500mL triangular flask of 50mL fermentation medium, and 30 DEG C, 200rpm is added final concentration of 100 μM after cultivating 3h Fermentation liquid is collected after IPTG, Fiber differentiation 36h, detects the concentration of ALA.Wherein Medium of shaking flask fermentation formula are as follows: glucose 50g/L, Na₂HPO₄·12H₂O 17.1g/L, KH₂PO₄3.0g/L, NaCl 0.5g/L, NH₄Cl 1.0g/L, MgSO₄2mM, CaCl₂0.1mM, glycine 4g/L, pH 7.0, the final concentration of 25 μ g/mL of kanamycins.PH is adjusted with ammonium hydroxide in fermentation process Stablize in the detection of 7.0 or so, ALA and glucose analysis method as described in " materials and methods " part.

Recombinant bacterium fermentation results are shown in Fig. 3, and the yield of ALA is 3.20g/L, paddy after control strain 13032/pZWA2 fermentation 36h The hydrogen peroxide expression of enzymes strains A LA yield increased bacterial strain of propylhomoserin corynebacteria autogenous improves 15%, and large intestine bar ALA yield increased bacterial strain improves 17%, while grape after the catalase I in bacterium source is expressed in corynebacterium glutamicum Sugared conversion ratio also improves 36% than control strain, and product production cost can be greatly reduced in the raising of yield and the substrate transformation rate.

The building of other anti-oxidant correlative protein expression carriers of 8. corynebacterium glutamicum of embodiment

According to NCBI corynebacterium glutamicum (Corynebacterium glutamicum) ATCC's 13032 announced Genome sequence separately designs primer NCgl2502-F/R, NCgl1928-F/R, NCgl2445-F/R and NCgl2985-F/R, with The genome of ATCC 13032 is that template respectively obtains the corynebacterium glutamicum branch with own promoter by PCR amplification Mercaptan peroxidase (amino acid sequence is as shown in SEQ ID No.46), mycothiol reductase (amino acid sequence such as SEQ Shown in ID No.47), mycothiol redox protein (amino acid sequence is as shown in SEQ ID No.48), thioredoxin Encoding gene NCgl2502 (GeneID:1020537), the NCgl1928 of (amino acid sequence is as shown in SEQ ID No.49) (GeneID:1019960), NCgl2445 (GeneID:1020480) and NCgl2985 (GeneID:1021035), PCR amplification body It is as follows: PCR amplification parameter are as follows: 94 DEG C of 10min, 94 DEG C of 20s, 58 DEG C of 20s, 72 DEG C of 1min are recycled 30 times, 72 DEG C of extensions 5min.Target fragment is handled with XbaI enzyme cutting, and segment and same digestion processing then will be obtained under DNA ligase effect The connection of pZWA2 carrier, and connection product is transformed into DH5 α competent cell, it is coated with kalamycin resistance plate culture 36h, is chosen Positive colony carries out bacterium colony PCR verifying, and correct transformant carries out sequence verification, correct recombinant vector is named as pZWA2- NCgl2502, pZWA2-NCgl1928, pZWA2-NCgl2445 and pZWA2-NCgl2985.

The influence that the anti-oxidant correlative protein expression of 9. corynebacterium glutamicum of embodiment synthesizes ALA

In order to verify the influence that anti-oxidant correlative protein expression synthesizes ALA, above-mentioned recombinant vector is transferred to paddy ammonia respectively In sour corynebacteria A TCC13032 bacterial strain.Converted product is coated with the plate of kalamycin resistance, cultivates after 36h positive gram of picking The verifying of grand extraction plasmid, obtain respectively recombinant bacterial strain 13032/pZWA2-NCgl2502,13032/pZWA2-NCgl1928, 13032/pZWA2-NCgl2445 and 13032/pZWA2-NCgl2985.

Shake flask fermentation verification process is with embodiment 5, and due to the part, albumen relates generally to oxidation reparation, fermentation time 48h is extended to, to give full play to its antioxidant effect.Fermentation results are shown in that Fig. 4, control strain 13032/pZWA2 ferment after 48h The yield of ALA be 3.81g/L, and mycothiol peroxidase, mycothiol reductase, mycothiol redox protein, Thioredoxin expresses strains A LA yield and improves 19%, 40%, 47% and 41% than control strain respectively, while glucose turns Rate and unit thallus ALA yield also have promotion by a relatively large margin, maximum to promote 22% and 31% respectively.Therefore the present invention mentions The method of the raising engineered strain ALA yield of confession is equally applicable to the fermentation industries such as corynebacterium glutamicum and ALA production is common Microorganism.

The sequence of primer used in the embodiment of the present invention and relative antioxidant albumen see the table below:

Discuss: the present invention significantly improves the production of ALA by the activity that enhancing ALA produces anti-oxidant GAP-associated protein GAP in bacterial strain Amount, effect of the technical effect generated even than being directly transformed to the GAP-associated protein GAP in ALA route of synthesis is more significant, More unexpectedly these albumen are not directly related to ALA route of synthesis.

According to CN103981203A and WO2014121724A1, control strain of the invention (ALA+ppc) itself is Art-recognized ALA superior strain, and bacterial strain of the invention no matter ALA absolute yield, or glucose turn It is obviously improved compared with control strain in terms of rate, also has greater advantage compared with state of the art, Actual effect is expected to further be promoted after technique amplification, thus has highly significant economic value and social value.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

SEQUENCE LISTING

<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences

<120>a kind of 5-ALA superior strain and the preparation method and application thereof

<130> P2018-1735

<150> CN2017110476130

<151> 2017-10-31

<160> 49

<170> PatentIn version 3.5

<210> 1

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

gcgcgaattc taactttaag aaggagatat acat 34

<210> 2

<211> 35

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

aatagcggcc gctcaggccg ccttggcgag accga 35

<210> 3

<211> 40

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

agcatctaga tatccgcggt atccgaccta cacctttggt 40

<210> 4

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ctaagcggcc gcggacttct gtggaatgca tagt 34

<210> 5

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

taagcggccg ccctcattac ttgaaggata tg 32

<210> 6

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

attgcggccg catgagagat ccagtgtgta g 31

<210> 7

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

taagcggccg cggacatagc taataatctg g 31

<210> 8

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

taagcggccg ccttgagact gctgacaaac g 31

<210> 9

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

taagcggccg ccagagccag taaaagtatc t 31

<210> 10

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

taagcggccg caggtgaatc ttacttcttc 30

<210> 11

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

atagcggccg cgagtcgctt accgacagca 30

<210> 12

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

atagcggccg ctcattctgg caagagctgg 30

<210> 13

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

atagcggccg cggtacgttt atcagaatca tc 32

<210> 14

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

atagcggccg ccgtaactgg agaacagcgt t 31

<210> 15

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

aatgcggccg cgataacccc tcgacctgta ga 32

<210> 16

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

atagcggccg catgcaaaac tgcctgatac g 31

<210> 17

<211> 78

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

gctctagatt tacagctagc tcagtcctag gtattatgct agcaagaagg agatatacat 60

atgagctata ccctgcca 78

<210> 18

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

cgagctctta ttttttcgcc gcaa 24

<210> 19

<211> 80

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gctctagatt tacagctagc tcagtcctag gtattatgct agcaagaagg agatatacat 60

atgtcattcg aattacctgc 80

<210> 20

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

cgagctctta tgcagcgaga tttttc 26

<210> 21

<211> 80

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

gctctagatt tacagctagc tcagtcctag gtattatgct agcaagaagg agatatacat 60

atgaaacgtt ttagtctggc 80

<210> 22

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

cgagctctta cttaattaca ccacaggc 28

<210> 23

<211> 92

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 23

gctctagaac taatttgtca gcgattggag taatagttaa attaggtaaa cagtgcaagg 60

agatatagat atgtctgaga agtcagcagc ag 92

<210> 24

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 24

gctctagatt aagccttctt ctggaggtaa agc 33

<210> 25

<211> 92

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 25

gctctagaac taatttgtca gcgattggag taatagttaa attaggtaaa cagtgcaagg 60

agatatagat atgagcacgt cagacgatat cc 92

<210> 26

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 26

gctctagatt acagcaggtc gaaacggtcg agg 33

<210> 27

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 27

aactctagac tggatgaggt agtaaccgt 29

<210> 28

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 28

aactctagaa agtgtaactg ttgccactgg 30

<210> 29

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 29

acatctagaa aagagcatcg gatggcttc 29

<210> 30

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 30

aactctagac gcagttaact tgacgtggaa 30

<210> 31

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 31

acctctagag tttggtggag caa 23

<210> 32

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 32

aactctagat gcatggctca cctgttcgat 30

<210> 33

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 33

aactctagat cgcttcgtca ccaacaagac t 31

<210> 34

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 34

aactctagac ctgctccttc catcattcat g 31

<210> 35

<211> 726

<212> PRT

<213>Escherichia coli (Escherichia coli)

<400> 35

Met Ser Thr Ser Asp Asp Ile His Asn Thr Thr Ala Thr Gly Lys Cys

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Glu Phe Ser Lys Leu Asp Tyr Tyr Gly Leu Lys Lys Asp Leu Lys Ala

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Ile Trp Gln Asp Pro Leu Pro Gln Pro Ile Tyr Asn Pro Thr Glu Gln

435 440 445

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

450 455 460

Ser Glu Leu Val Ser Val Ala Trp Ala Ser Ala Ser Thr Phe Arg Gly

465 470 475 480

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

485 490 495

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

500 505 510

Leu Glu Lys Ile Gln Lys Glu Ser Gly Lys Ala Ser Leu Ala Asp Ile

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

<210> 36

<211> 753

<212> PRT

<213>Escherichia coli (Escherichia coli)

<400> 36

Met Ser Gln His Asn Glu Lys Asn Pro His Gln His Gln Ser Pro Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Ser Arg Gly Pro Thr Leu Leu Glu Asp Phe Ile Leu Arg Glu

100 105 110

Lys Ile Thr His Phe Asp His Glu Arg Ile Pro Glu Arg Ile Val His

115 120 125

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

130 135 140

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 kind of method for the 5-ALA yield for improving 5-ALA production bacterial strain, the method includes increasing The step of oxidation resistance of the strong bacterial strain.

2. the method as described in claim 1, which is characterized in that the oxidation resistance for enhancing the bacterial strain refers to the enhancing bacterium The activity of anti-oxidant GAP-associated protein GAP in strain.

3. method according to claim 2, which is characterized in that the anti-oxidant GAP-associated protein GAP is catalase, super oxygen Object mutase, peroxidase, thiol reduction enzyme, thiol disulfide oxidoreducing enzyme, thioredoxin reductase or methionine Sulfoxide reductase.

4. a kind of method for constructing 5-ALA superior strain, which comprises

The step of enhancing the oxidation resistance of the bacterial strain.

5. method as claimed in claim 4, which is characterized in that the oxidation resistance for enhancing the bacterial strain refers to the enhancing bacterium The activity of anti-oxidant GAP-associated protein GAP in strain.

6. a kind of 5-ALA superior strain, the oxidation resistance enhancing of the bacterial strain.

7. bacterial strain as claimed in claim 6, which is characterized in that the oxidation resistance for enhancing the bacterial strain refers to the enhancing bacterium The activity of anti-oxidant GAP-associated protein GAP in strain.

8. bacterial strain as claimed in claim 7, which is characterized in that the anti-oxidant GAP-associated protein GAP is catalase, peroxidating Object enzyme, thiol reduction enzyme, thiol disulfide oxidoreducing enzyme, thioredoxin reductase or methionine sulfoxide reductase.

9. a kind of method for generating 5-ALA, which comprises

1) it cultivates 5-ALA described in any one of claim 6-8 and produces bacterial strain, to obtain 5- glycyl Propionic acid；With

2) 5-ALA optionally is obtained from fermentation culture system 1).

10. the purposes of 5-ALA superior strain described in any one of claim 6-8, the bacterial strain is for generating 5-ALA and/or generation are using 5-ALA as the downstream product of precursor.