CN1322132C - Engineering bacteria for producing 5-amino acetyl propionic acid and its constructing method - Google Patents
Engineering bacteria for producing 5-amino acetyl propionic acid and its constructing method Download PDFInfo
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Abstract
The present invention relates to an engineering bacterium for producing 5-amino levulinic acid, and a construction method thereof. The engineering bacterium contains a 5-amino levulinic acid synthetase gene of agrobacterium radiobacter. The construction method comprises the following steps: (1) a total genome DNA is extracted from agrobacterium radiobacter solution; (2) a 5-amino levulinic acid synthetase gene is enlarged through the chain reaction of polymeric enzyme; (3) the enlarged 5-amino levulinic acid synthetase gene is connected with a cloning vector pGEM-T to carry out DNA sequence; (4) the target gene segment after being sequenced is connected with an expression vector pET28a to construct a recombinant pET28a-A. R-hemA; (5) the recombinant pET28a-A. R-hemA is converted to a parasitifer bacterium. The present invention has high expression of soluble 5-amino levulinic acid synthetase genes, and the output of the extracellular ALA can achieve 4.8 g/l after being optimized to express.
Description
Technical field
The present invention relates to produce the engineering bacteria and the construction process thereof of 5-amino-laevulic acid.
Background technology
(5-aminolev μ linic acid ALA) extensively is present in the biology 5-amino-laevulic acid, is first mixture in the PBP, also is to form protoheme, cytopigment, vitamins B
12Common precursor Deng tetrapyrrole.In addition, (photodynamic agent PDT), has purposes widely in agrochemicals and medical field to ALA as a kind of photodynamics agent.At agriculture field, ALA has weeding, desinsection, increase stress resistance of plant and promotes multiple function such as plant-growth, and the noresidue and to people and animals' nontoxicity of easily degrading becomes the nuisanceless green agrochemicals that have development prospect.At medical field, ALA has the effect of selectivity kill cancer cell, be called as s-generation photodynamics medicine (photodynamic medicine), have normal cytotoxic little, patient's lucifuge time is short, good effect, distinguishing features such as noresidue are used to treat multiple cancers such as skin carcinoma, bladder cancer, colorectal carcinoma and carcinoma of the pancreas.Therefore, the study on the synthesis of ALA causes extensive attention.
Because synthetic ALA complex steps of chemical method and productive rate are low, so adopt biosynthetic means.First biological induced-mutation method is optimized mutant strain CR 720 (the Rhodobacter sphaeroides CR720) culture condition of the red bacterium of class ball, and the output of ALA can reach 27mmol/L, but its culture condition complexity, the cycle is long and cost is higher.It two is gene engineering method, (1999) such as Mariet J.Vander Werf etc. (1996) and Choi have made up the engineering bacteria of the 5-aminolevulinate synthetase gene (hemA) that contains class ball red bacterium Rhodobacter sphaeroides and the living slowly root nodule bacterium Bradyrhizobi of soybean μ m japonic μ m. respectively, are used to produce ALA.But yet there are no the report of the 5-aminolevulinate synthetase genes produce ALA that utilizes agrobacterium radiobacter so far.
Summary of the invention
The engineering bacteria and the construction process thereof that the purpose of this invention is to provide a kind of 5-of production amino-laevulic acid.
The engineering bacteria of production 5-amino-laevulic acid of the present invention is the engineering bacteria that contains the 5-aminolevulinate synthetase gene of agrobacterium radiobacter, in the common micro-organisms center preservation of China Committee for Culture Collection of Microorganisms of specified depositary institution of Patent Office of the People's Republic of China, deposit number is: CGMCC No.1332, preservation date: 2005.3.18, classification name: colon bacillus, depositary institution address: Institute of Microorganism, Academia Sinica.
Above-mentioned 5-aminolevulinate synthetase gene has the sequence shown in the SEQ ID NO.1.
What above-mentioned 5-aminolevulinate synthetase had the sequence shown in the SEQ ID NO.2 or reduced, substitutes, increases one or more amino-acid residues on its basis has an active aminoacid sequence of 5-aminolevulinate synthetase.
Produce the construction process of the engineering bacteria of 5-amino-laevulic acid, it is characterized in that may further comprise the steps:
1) from the bacterium liquid of agrobacterium radiobacter, extracts total genomic dna;
2) go out the 5-aminolevulinate synthetase gene with PCR amplification;
3) the 5-aminolevulinate synthetase gene that amplifies is connected with cloning vector pGEM-T, carries out dna sequencing, obtain the nucleotide sequence shown in the SEQ ID NO.1;
4) the goal gene segment after will checking order is connected with expression vector pET28a or pTRC99a, constructs recon pET28a-A.R-hemA or pTRC99a-A.R-hemA;
5) recon pET28a-A.R-hemA or pTRC99a-A.R-hemA are converted in the host bacterium, get final product.
For obtaining the output of higher 5-amino-laevulic acid, the host bacterium among the present invention is advisable to adopt e. coli bl21 (DE3).
The present invention inserts expression vector pET28a transformed into escherichia coli with a kind of 5-aminolevulinate synthetase gene of agrobacterium radiobacter, under inductor isopropyl-(IPTG) effect, have higher solvable 5-aminolevulinate synthetase to express, and after optimization expression outside the born of the same parents output of ALA up to 4.8g/L.
Description of drawings
Fig. 1 is the design of graphics of recombinant plasmid pET28-A.R-hemA, and among the figure, A.R-hemA is the 5-aminolevulinate synthetase gene, and Kana+ is a kalamycin resistance gene, and EcoRI and HindIII are restriction endonuclease sites;
Fig. 2 is engineering bacteria BL21 (DE3) pET28-A.R-hemA5 abduction delivering synoptic diagram.
Embodiment
Further specify the present invention below in conjunction with embodiment
Embodiment 1
The present invention produces the construction process of the engineering bacteria of 5-amino-laevulic acid, may further comprise the steps:
1. the extraction of agrobacterium radiobacter genomic dna
1) the bacterium liquid with incubated overnight is placed in the 1.5ml centrifuge tube, removes supernatant behind the centrifugal 1min of 13,000 * g;
2) with after the dissolving of 475 μ l TE damping fluids, add N,O-Diacetylmuramidase, 37 ℃ of insulation 20min to final concentration 100 μ g/ml;
3) add 10% (w/v) dodecyl semi-annular jade pendant acid sodium (SDS) solution to final concentration 2% (w/v), add the 20mg/ml Proteinase K simultaneously to final concentration 100 μ g/ml, behind the mixing, 37 ℃ of insulation 1h;
4) add 1/5 volume 5mol/L NaCl mixing, add 1/5 volume, 2 * bromination n-Hexadecane trimethyl ammonium (CTAB) again, 65 ℃ of insulation 30min;
5) add the mixed solution of isopyknic phenol, chloroform and primary isoamyl alcohol, phenol: chloroform: the volume ratio of primary isoamyl alcohol is 25: 24: 1, the centrifugal 5min of 13,000 * g;
6) get the mixed solution that supernatant liquor adds isopyknic chloroform and primary isoamyl alcohol, chloroform: the volume ratio of primary isoamyl alcohol is 24: 1, the centrifugal 5min of 13,000 * g;
7) get supernatant liquor and add the dehydrated alcohol of 2 times of volumes and the 3mol/L sodium acetate of 1/10 volume (pH 4.6), after leaving standstill 10min under-20 ℃, the centrifugal 5min of 13,000 * g;
8) go the DNA precipitation of supernatant liquor gained to wash 2 times with 70% ethanol after, behind the natural air drying, be dissolved in the TE damping fluid and (contain 20 μ g/ml RNase), 55 ℃ of water bath processing 15min promptly obtain genomic dna.
2. amplify the 5-aminolevulinate synthetase gene with polymerase chain reaction (PCR)
1) get the genomic dna 20 μ g of said extracted respectively, concentration is two primers of 10 μ mol/L
Each 1 μ l, (5 ' end primer is GGAATTCGGATCCATGGACTTCGAGGCATTT, 3 ' end primer is TTAAGCTTCCTCACGCCACCGCACGCGC), 10 * reaction buffer, 2 μ l, (5unit/ μ l) PfuDNA polysaccharase 0.5 μ l, 4 kinds of deoxynucleotides (dNTP) concentration is the mixed solution 0.5 μ l of 10mmol/L, and in the 0.5mlPCR pipe, all the other supply 20 μ l with distilled water;
2) with above-mentioned 20 μ l reaction solutions, put into the PCR instrument, reaction conditions is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s then, 58 ℃ of renaturation 30s, 72 ℃ are extended 2min, totally 30 circulations, last 72 ℃ are extended 10min;
3) the product behind the pcr amplification, add 5 μ l sample-loading buffers, identify the band of a treaty 1.2kb with 1% agarose gel electrophoresis;
3. the 5-aminolevulinate synthetase gene that amplifies is connected with cloning vector pGEM-T, carries out dna sequencing, obtain the nucleotide sequence shown in the SEQ ID NO.1
1) (QIAquick Gel ExtractionKit (50) cuts glue and reclaims the hemA gene of pcr amplification to be reclaimed test kit with glue;
2) in the reaction system of 10 μ l, get the gene segment that 7 μ l reclaim and add 0.5 μ l, 200 μ mol/L deoxyadenylic acids (dATP), 0.3 μ l25mmol/LMgCl
2, 0.2 μ l (5unit/ μ l) TaqDNA polysaccharase, 1 μ l10 * reaction buffer, all the other use the distilled water polishing, and this reactive system is at 70 ℃ of following reaction 30min, adds deoxyadenylic acid (A ') end;
3) will have the terminal goal gene of A ' and under 16 ℃, be connected 2h, then transformed competence colibacillus cell TG1 (see figure 1) with cloning vector pGEM-T;
4) on the LB culture medium flat plate of the penbritin that contains 100 μ g/ μ l and 5-bromo-4-hydrogen-3-indoles-β-D-galactoside (X-gal) and each 800 μ g of IPTG, go out positive colony, promptly choose 8 white clones and identify (1 liter of LB substratum contains 10 grams and contains peptone, 5 gram yeast powders, 10 gram sodium-chlor pH 7.0) from transforming ware through blue hickie screening system;
5) to these 8 clones with EcoRHI and HindIII double digestion, wherein 5 have band at about 3kb and 1.2kb place respectively, the gene segment with 1.2kb is thought successful connection, all the other 3 only have band at the 3kb place, the illustration purpose gene is less than in the connection;
6) identify clone's (being recon) called after pGEM-A.R-hemA of correct insertion with EcoRI and HindIII double digestion, carry out dna sequencing, obtain the nucleotide sequence shown in the SEQ ID NO.1.
4. the goal gene segment after will checking order is connected with expression vector pET28a, constructs recon pET28a-A.R-hemA, is converted into intestinal bacteria with this recon
1) cuts recombinant plasmid pGEM-A.R-hemA with restriction enzyme EcoRI and HindIII enzyme; Downcut the 1.2kb segment and carry out the glue recovery;
2) handle expression vector pET28a with restriction endonuclease EcoRI and HindIII, identify, cut the big segment that glue reclaims 5.0kb with 1% agarose gel electrophoresis;
3) two kinds are reclaimed product connection 6h under 16 ℃, transformed competence colibacillus cell BL21 (DE3) then, the hemA gene is with regard to directed EcoRI and the HindIII site that is connected to expression vector pET28a like this, with this expression vector called after pET28a-A.R-hemA, and contain engineering bacteria called after BL21 (DE3) pET28a-A.R-hemA of this expression vector, be the engineering bacteria (see figure 1) of production 5-amino-laevulic acid of the present invention.
Embodiment 2
The expression of 5-aminolevulinate synthetase under inductor isopropyl-(IPTG) effect
1) shaking in the bottle of the 250ml that engineering bacteria BL21 of the present invention (DE3) pET28a-A.R-hemA is cultivated at the LB substratum that contains 50ml, earlier at 37 ℃, 200rpm cultivates 1.5h down; Be cooled to 28 ℃ then, induce, after continuing to cultivate 4h, get 30ml bacterium liquid and remove supernatant liquor in the centrifugal 10min of 8000 * g with 4 μ l1mol/L IPTG;
2) resuspended with 3ml50mmol/L TrisCl (pH 7.5), ultrasonic broken born of the same parents, its condition is 400w work 5min, intermittently 5min repeats the centrifugal 10min of 13,000 * g 30 times;
3) get supernatant liquor and carry out the 10%SDS-polyacrylamide gel electrophoresis, deposition condition is 200v, 500mA, and the time is 1h10min;
4) have the soluble protein that accounts for total protein concentration 23.7% to express (see figure 2) at about 44.0kDa place according to electrophoresis result, this aminoacid sequence size basically identical big or small and according to SEQ ID NO.2 thinks that promptly this albumen is 5-aminolevulinate synthetase.
Embodiment 3
The ratio vitality test of 5-aminolevulinate synthetase
1) 500 μ l is contained 50mmol/L Tris-HCl (pH7.5), 20mmol/L MgCl
2, the cell extract behind 0.1mol/L glycine, 0.1mmol/L pyridoxal phosphate, 0.2mmol/L succinate coenzyme A and the broken born of the same parents mixed solution, in 37 ℃ of reaction 10min;
2) getting the above-mentioned reaction solution of 300 μ l adds 150 μ l10% trichoroacetic acid(TCA)s mix the centrifugal 5min of 13,000 * g in the 1.5mL centrifuge tube;
3) get 300 μ l supernatant liquors and mix boiling water bath 15min with 400 μ l1mol/L acetate buffers (pH 4.6) and 35 μ l methyl ethyl diketones;
4) treat its cooling after, the Ehrlich reagent that adds the new preparation of 700 μ l again is (in the 50ml graduated cylinder, the glacial acetic acid that adds 30ml successively, 1g is right-dimethylaminobenzaldehyde, the perchloric acid of 5ml70%, 5ml water, the dissolving back is diluted to 50ml with glacial acetic acid), behind the reaction 5min in its light absorption value of 554nm place survey, the concentration of ALA (mg/L)=26.35 * OD
554-0.09 (R
2=0.9993);
5) protein concentration is measured by determination of protein concentration test kit (BCA Protein Assay Kit), and the ratio vigor of measuring the 5-aminolevulinate synthetase of the reorganization bacterium that contains the hemA gene is 13.8nmol
-1Min
-1Mg of protein
-1(1 unit of enzyme is defined as and generates the required enzyme amount of 1nmol ALA in the 1min; The ratio vigor of enzyme is every milligram of unit of enzyme that albumen is contained), and do not contain the activity of e. coli bl21 (DE3) detection of recon less than 5-aminolevulinate synthetase.
Sequence table
SEQ?ID?NO.1
Length: 1086bp
Type: thymus nucleic acid
Chain number: two strands
Geometry: linearity
Source: pcr amplification from the agrobacterium radiobacter genome
Feature: the nucleotide sequence of coding 5-aminolevulinate synthetase
ATGgacttcg?ggcattttt?tacgacggaa?ctgcagagcc?tgcattctga?gggacgctat 60
Cgcgtttttg?cggatatcgaa?cgccggcagg?gcaattttcc?ccgcgcgaca?cggtacaac?120
Gccaatggcg?agcgcaagga?cgtaaccgtc?tggtgttcca?acgattatct?gggcatgggc?180
Cagaacccca?aagtcatcga?agccatgaaa?gccgccatcg?atcactgtgg?cgcgggtgcg?240
Ggaggcaccc?ggaacatttc?tgggaccaac?catcatcacg?tcctgctgga?acaggaactt?300
Gccgatctgc?acggcaagga?atcggcgctg?atcttcacgt?cgggttacgt?ttccaactgg?360
Gcgacactcg?gtacacttgg?ccagaaaatt?ccgggcctca?tcattttctc?cgatgcgctc?420
Aaccatgcct?cgatgatcga?aggcatccgt?tacggccgtt?gcgagcgggt?gatctggaaa?480
Cacaatgatc?tcgaagatct?cgaggcaaag?cttcaaggca?gccgatccat?gtacggaccg?540
Cgcggcggcg?gtattgccga?gcgcgaaggc?ctgatggatc?gcctgacgat?catcgaggga?600
Acgctcggca?aggctttcgg?cgtgatgggc?ggttatattg?ccgggtccac?ggcggtctgt?660
Gattttatcc?gttctttcgc?ctccggtttc?atcttcacga?cggccctgcc?gccgtcgctc?720
Gctgccggcg?caatcgcctc?gatccagcat?ttgaaggcaa?gcccctttga?gcgcgcccgc?780
Catcaggacc?gggtgcgcaa?gctgcggggg?cttctggatg?cacgcggcat?tccgcatatg?840
Gacaatccca?gccatatcgt?accggtcatg?gtgggcgatg?ccgccaagtg?caaatggatt?900
Tcggatatcc?tgctcgacaa?tcacggcgtc?tatgtccagc?cgatcaacta?tccgaccgtg?960
Ccgcgcaaga?ccgagcgtct?gcgcatcacc?ccgacaccgc?tgcacaccga?tgccgacatc?1020
Gaacagttgg?tcggcgcgtt?gcaccagctc?tggtcgcatt?gtgcgctggc?gcgtgcggtg?1080
GcgTGA 1086
SEQ?ID?NO.2
Length: 362 amino-acid residues
Type: peptide chain
Chain number: strand
Geometry: linearity
The source: the 5-aminolevulinate synthetase gene deduction of agrobacterium radiobacter obtains
Feature: have the 5-aminolevulinate synthetase activity
MDFEAFFTTELQSLH 15
SEGRYRVFADIERRQ 30
GNFPRATRYNANGER 45
KDVTVWCSNDYLGM 60
GQNPKVIEAMKAAID 75
HCGAGAGGTRNISGT 90
NHHHVLLEQELADLH 105
GKESALIFTSGYVSN 120
WATLGTLGQKIPGLI 135
IFSDALNHASMIEGI 150
RYGRCERVIWKHNDL 165
EDLEAKLQGSRSMYG 180
PRGGGIAEREGLMDR 195
LTIIEGTLGKAFGVM 210
GGYIAGSTAVCDFIR 225
SFASGFIFTTALPPS 240
LAAGAIASIQHLKAS 255
PFERARHQDRVRKLR 270
GLLDARGIPHMDNPS 285
HIVPVMVGDAAKCKW 300
ISDILLDNHGVYVQP 315
INYPTVPRKTERLRI 330
TPTPLHTDADIEQLV 345
GALHQLWSHCALARA 360
VA 362
Claims (5)
1. engineering bacteria of producing the 5-amino-laevulic acid, it is characterized in that this bacterium is the engineering bacteria that contains the 5-aminolevulinate synthetase gene of agrobacterium radiobacter, Chinese colon bacillus by name, the Latin formal name used at school is BL21 (DE3) pET28a-A.R-hemA, in the common micro-organisms center preservation of China Committee for Culture Collection of Microorganisms of specified depositary institution of Patent Office of the People's Republic of China, deposit number is: CGMCC No.1332.
2. the engineering bacteria of production 5-amino-laevulic acid according to claim 1 is characterized in that the nucleotides sequence of said 5-aminolevulinate synthetase gene is classified the nucleotide sequence shown in the SEQ ID NO.1 as.
3. the engineering bacteria of production according to claim 1 5-amino-laevulic acid, the aminoacid sequence that it is characterized in that said 5-aminolevulinate synthetase be the aminoacid sequence shown in the SEQ ID NO.2 or reduce, substitute or increase an amino-acid residue on its basis have an active aminoacid sequence of 5-aminolevulinate synthetase.
4. the construction process of the engineering bacteria of production 5-amino-laevulic acid according to claim 1 is characterized in that may further comprise the steps:
1) from the bacterium liquid of agrobacterium radiobacter, extracts total genomic dna;
2) go out the 5-aminolevulinate synthetase gene with PCR amplification;
3) the 5-aminolevulinate synthetase gene that amplifies is connected with cloning vector pGEM-T, carries out dna sequencing, obtain the nucleotide sequence shown in the SEQ ID NO.1;
4) the goal gene segment after will checking order is connected with expression vector pET28a or pTRC99a, constructs recon pET28a-A.R-hemA or pTRC99a-A.R-hemA;
5) recon pET28a-A.R-hemA or pTRC99a-A.R-hemA are converted in the host bacterium, get final product.
5. the construction process of the engineering bacteria of production 5-amino-laevulic acid according to claim 4 is characterized in that said host bacterium is e. coli bl21 (DE3).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012129748A1 (en) * | 2011-03-31 | 2012-10-04 | 山东大学 | Recombinant e.coli and use thereof in producing 5-aminolevulinic acid |
| CN106434514A (en) * | 2016-11-09 | 2017-02-22 | 天津大学 | Corynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid |
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| CN101063105B (en) * | 2007-04-20 | 2012-03-07 | 浙江大学 | Engineering bacterium producing 5-glycyl ethylformic acid and construction and application method thereof |
| CN102816717B (en) * | 2012-08-06 | 2014-01-08 | 福建省农业科学院 | Staphylococcus cohnii and method for preparing 5-aminolevulinic acid by using staphylococcus cohnii |
| CN103468736A (en) * | 2013-09-27 | 2013-12-25 | 浙江大学 | Intracellular high-activity expressing method of capsule red bacterium hemA gene |
| CN104004701B (en) * | 2014-06-18 | 2017-02-15 | 江南大学 | Method for building high-yield 5-aminolevulinic acid escherichia coli engineering strains |
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| CN106434514A (en) * | 2016-11-09 | 2017-02-22 | 天津大学 | Corynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid |
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