CN106497858B - A kind of Escherichia coli engineering bacteria producing 5-aminolevulinic acid - Google Patents
A kind of Escherichia coli engineering bacteria producing 5-aminolevulinic acid Download PDFInfo
- Publication number
- CN106497858B CN106497858B CN201611094235.7A CN201611094235A CN106497858B CN 106497858 B CN106497858 B CN 106497858B CN 201611094235 A CN201611094235 A CN 201611094235A CN 106497858 B CN106497858 B CN 106497858B
- Authority
- CN
- China
- Prior art keywords
- gene
- escherichia coli
- coaa
- coad
- dfp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1235—Diphosphotransferases (2.7.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01037—5-Aminolevulinate synthase (2.3.1.37)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01033—Pantothenate kinase (2.7.1.33)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01036—Phosphopantothenoylcysteine decarboxylase (4.1.1.36)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种生产5‑氨基乙酰丙酸的大肠杆菌工程菌，属于代谢工程和微生物发酵领域。本发明在使用载体pRSFDuet‑1‑hemA过量表达5‑氨基乙酰丙酸C4合成途径关键酶5‑氨基乙酰丙酸合酶hemA的基础上，将来源于大肠杆菌CoA合成途径中编码泛酸激酶的的基因coaA、编码磷酸泛酰半胱氨酸合成酶和脱羧酶的基因dfp、编码磷酸CoA焦磷酸化酶的基因coaD，使用pUC19和pETDuet‑1载体共表达三个基因，构建重组菌株。通过摇瓶培养验证，共表达CoA途径基因的重组菌ALA产量均明显提高，其中产量最高为700mg/L，相对于对照提高约46％。The invention discloses an Escherichia coli engineering bacterium for producing 5-aminolevulinic acid, which belongs to the fields of metabolic engineering and microbial fermentation. In the present invention, on the basis of using the carrier pRSFDuet-1-heMA to overexpress 5-aminolevulinic acid C4 synthesis pathway key enzyme 5-aminolevulinic acid synthase heMA, the protein derived from Escherichia coli CoA synthesis pathway encoding pantothenate kinase The gene coaA, the gene dfp encoding phosphopantoylcysteine synthase and decarboxylase, and the gene coaD encoding phospho-CoA pyrophosphorylase were co-expressed with the pUC19 and pETDuet-1 vectors to construct recombinant strains. It was verified by shake flask culture that the ALA yield of the recombinant bacteria co-expressing the CoA pathway gene was significantly increased, and the highest yield was 700 mg/L, which was about 46% higher than that of the control.
Description
Technical field
The present invention relates to a kind of colibacillus engineerings for producing 5-ALA, belong to metabolic engineering and microorganism
Fermentation arts.
Background technique
5-ALA (5-aminolevulinic acid, ALA), molecular formula C5O3NH9, molecular weight is
131.13, fusing point is 149-151 DEG C, it is organism synthesis chlorophyll, ferroheme, vitamin B12Deng critical precursors.
ALA is gradually taken seriously as the photodynamic agents of a kind of safety, selection, good penetrability in medical domain, successfully answers
In diagnosis and optical dynamic therapy for cutaneum carcinoma, bladder cancer, digestive system cancer, lung cancer etc..In addition, ALA is as a kind of environment phase
Capacitive and the very high novel photo-activation pesticide of selectivity, it is very widely used in pesticide field, such as a kind of non-harmful green
Color pesticide, herbicide and plant growth regulator etc..
Currently, ALA synthesis is mainly synthesized using chemical method, the fifties in last century is appeared in earliest, until 90 years 20th century
Generation, correlative study are just largely carried out, and achieve certain achievement.But due to chemosynthesis reaction complex steps, by-product
It is more, the problems such as separating-purifying is difficult, and the yield of ALA is relatively low, and environmental pollution is serious, in recent years, microbial fermentation production
ALA has become the hot spot of research.In nature, the biosynthesis of ALA is there are two approach, and one is C4 approach, by 5- amino
Levulic acid synzyme (ALAS, hemA coding) catalysis succinyl-CoA and glycine generate the step enzymatic reaction composition of ALA,
It is primarily present in some photosynthetic bacterias, fungi and animal body.Other one is C5 approach, and glutamic acid is in glutamy-first
Under tRNA synzyme (GluRS, gltX coding) catalysis, glutamy-tRNA is generated, then, glutamy-tRNA is in glutamy-
TRNA reductase (GluTR, hemA coding) effect is lower to generate glutamic acid -1- semialdehyde (GSA), and last GSA is by glutamic acid -1- half
Aldehyde -2,1- aminopherase (GSA-AM, hemL coding) catalysis generates ALA.The approach is widely present in plant, algae and thin
In bacterium (such as Escherichia coli).
In early days, people screen the photosynthetic bacteria hydrogenlike silicon ion (Rhodobacter sphaeroides) for producing ALA, lead to
It crosses induced mutation breeding method and mutagenesis is carried out to it, screen the superior strain of ALA, and by fermentation optimization etc. the yield of ALA is reached
7.2g/L.But due to the particularity of photosynthetic bacteria, higher cost is not suitable for large-scale industrial production.
Summary of the invention
The invention solves first technical problem be to provide the colibacillus engineering of 5-ALA a kind of
Strain, is with Escherichia coli for host, on the basis of expression C4 pathway key gene hemA (coding 5-Aminolevulinate synthase)
On, the related gene of coexpression Escherichia coli CoA pathway is the gene coaA for encoding Pantothen kinase, coding phosphopan tetheine half
The gene coaD of the gene dfp and coding phosphoric acid CoA pyrophosphorylase of cystine synzyme and decarboxylase.
In one embodiment of the invention, the nucleotide sequence of the gene hemA is as shown in SEQ ID NO.1.
In one embodiment of the invention, the nucleotide sequence of the gene coaA is as shown in SEQ ID NO.2.
In one embodiment of the invention, the nucleotide sequence of the gene dfp is as shown in SEQ ID NO.3.
In one embodiment of the invention, the nucleotide sequence of the gene coaD is as shown in SEQ ID NO.4.
In one embodiment of the invention, the Escherichia coli are e. coli bl21 (DE3).
In one embodiment of the invention, the gene hemA carries out total table by expression vector of pRSFDuet-1
It reaches.
In one embodiment of the invention, described gene coaA, dfp, coaD share an expression vector, the table
It include pUC19 and pETDuet-1 up to carrier.
The invention solves Second Problem be to provide the construction method of the colibacillus engineering strain, the method
It is with Escherichia coli for host, expresses hemA gene by expression vector of pRSFDuet-1.
In one embodiment of the invention, the method is also with pUC19 or pETDuet-1 carrier, and express coaA,
Dfp, coaD gene.
In one embodiment of the invention, the method is that coaA, dfp and coaD gene are passed through homologous recombination side
Method connects carrier pUC19 and pETDuet-1 carrier.
In one embodiment of the invention, the method specifically includes the following steps:
(1) it will be connected to by amplification using methods of homologous recombination from the gene hemA of Rhodobacter capsulatus and equally passed through
It expands in obtained carrier pRSFDuet-1, obtains recombinant expression carrier pRSFDuet-1-hemA；(2) large intestine bar will be derived from
CoaA, dfp, coaD gene of bacterium are connected in carrier pUC19 and pETDuet-1 carrier by methods of homologous recombination, respectively
To expression vector pUC19-coaA-dfp-coaD and pETDuet-1-coaA-dfp-coaD；(3) by the recombinant plasmid of building
PRSFDuet-1-hemA Transformed E .coli BL21 (DE3)；Or by the recombinant plasmid pRSFDuet-1-hemA and pUC19- of building
CoaA-dfp-coaD cotransformation is to E.coli BL21 (DE3)；Or by the recombinant plasmid pRSFDuet-1-hemA of building with
PETDuet-1-coaA-dfp-coaD cotransformation E.coli BL21 (DE3).
The invention solves third technical problem be to provide the method for fermenting and producing 5-ALA a kind of, institute
The method of stating is that the colibacillus engineering strain is seeded in fermentation medium to 37 DEG C, and 200~220r/min cultivates 24-32h.
In one embodiment of the invention, the fermentation medium contains: (NH4)2SO415g/L, KH2PO45.0g/
L, Na2HPO4·12H2O 15g/L, MgSO4·7H2O 1.0g/L, yeast extract 1.0g/L, glucose 20g/L.
The present invention also provides application of the colibacillus engineering in product of the preparation containing 5-ALA.
The utility model has the advantages that the present invention, on the basis of expressing C4 pathway key gene hemA, coexpression Escherichia coli coacetylase is on the way
The related gene of diameter is the gene coaA for encoding Pantothen kinase, the base for encoding phosphopanthothenoylcysteine synthetase and decarboxylase
Because of dfp, the gene coaD of coding phosphoric acid CoA pyrophosphorylase, unexpected technical results have been achieved: coexpression coA approach
E. coli BL21 (DE3) the pRSFDuet-1-hemA pETDuet-1-coaA-dfp-coaD of key gene exists
5-ALA 700mg/L can be accumulated in 250m L shaking flask culture, improves 46% compared to control strain, effectively
The synthesis for promoting 5-ALA using C4 approach realizes microbe fermentation method direct fermentation glucose synthesis 5- amino
Levulic acid shortens fermentation period, reduces production cost.
Detailed description of the invention
Fig. 1 is ALA C4 route of synthesis in Escherichia coli；
Fig. 2 is coacetylase route of synthesis in Escherichia coli；
Fig. 3 is that recombinant plasmid pRSFDuet-1-hemA constructs bacterium colony PCR electrophoretogram；M:DL 5000Marker；
Fig. 4 is that recombinant plasmid pUC19-coaA-dfp-coaD and pETDuet-1-coaA-dfp-coaD construct bacterium colony PCR
Electrophoretogram；M:DL 5000Marker；1-12:pUC19-coaA-dfp-coaD plasmid construction bacterium colony PCR result；13-24:
PETDuet-1-coaA-dfp-coaD plasmid construction bacterium colony PCR result；M:DL 5000Marker；
Fig. 5 is recombinant bacterium shake flask fermentation ALA yield；It is from left to right respectively E.coli BL21 (DE3) pRSFDuet-1-
HemA, E.coli BL21 (DE3) pRSFDuet-1-hemA pUC19-coaA-dfp-coaD, E.coli BL21 (DE3)
pRSFDuet-1-hemA pETDuet-1-coaA-dfp-coaD。
Specific embodiment
ALA analysis method: using the spectrophotometry of Mauzerall and Granick: sample being diluted to 2mL, is added
The acetate buffer of 1mL, the acetylacetone,2,4-pentanedione of 0.5mL, then boils 15min.It is cooled to room temperature, takes the reaction solution of 2mL to new
Then improvement Ehrlich ' the s reagent of 2mL is added in Guan Zhong, 20min is reacted, using detecting under spectrophotometer 554nm.
Culture medium:
Slant medium (g/L): peptone 10, sodium chloride 10, yeast powder 5.0, agar 20, pH 7.0；
Seed culture medium (g/L): peptone 10, sodium chloride 10, yeast powder 5.0, pH 7.0, liquid amount 20mL/250mL；
Fermentation medium (g/L): (NH4)2SO415, KH2PO45.0, Na2HPO4·12H2O 15, MgSO4·7H2O 1.0,
Yeast extract 1.0, glucose 20, pH 7.0, liquid amount 30mL/250mL.
Condition of culture:
Spawn incubation: glycerol tube scribing line, then 37 DEG C of picking single colonie streak plate cultures, as seed source；
Seed culture: plate picking thallus, 37 DEG C, 220r/min, according to requiring addition 100 μ g/mL of kanamycins and ammonia
100 μ g/mL of benzyl mycin cultivates about 12h, fermentation medium of transferring；
Fermented and cultured: with the switching of 2% (by volume) inoculum concentration, 0.1-0.5mM IPTG induced gene table is added when 0h
It reaches, kanamycins (100 μ g/mL) or ammonia benzyl mycin (100 μ g/mL) is added according to the resistance for being transferred to plasmid, 37 DEG C, 220r/min
Culture, period 24-32h.
The building and identification of 1 recombinant plasmid of embodiment
(1) recombinant plasmid pRSFDuet-1-hemA, pUC19-coaA-dfp-coaD, pETDuet-1-coaA-dfp-
The building of coaD.
Primer is as follows:
RSF-F:TGTTTAACTTTAATAAGGAGGAAAATATATGGACTACAATCTCGCGCTCGAC
RSF-R:CTCCTTATTAAAGTTAAACAAAATTATTTCTACAG
hemA-F:TGTTTAACTTTAATAAGGAGGAAAATATATGGACTACAATCTGGCACTCG
hemA-R:CGAGCTCGGCCACGAAGTGCTCAGGCAGAGGCCTCGGCGCGA
pUC19-F:TGATGGCGAAGTTAGCGTAGGTCATAGCTGTTTCCT
pUC19-R:CTCTTTTATACTCATTACGAGCCGGAAGCATAAAG
pET-F:GATGGCGAAGTTAGCGTAGGGCGCGCCTGCAGGTCGACAAGCTTG
pET-R:GTTTGCTCTTTTATACTCATGAATTCGGATCCTGGCTGTGGTG
coaA-19F:TGCTTCCGGCTCGTAATGAGTATAAAAGAGCAAACGTTAAT
coaA-ETF:CACAGCCAGGATCCGAATTCATGAGTATAAAAGAGCAAAC
coaA-R:ACCGGCCAGGCTCATTTATTTGCGTAGTCTGACCTCTTCT
dfp-F:AGACTACGCAAATAAATGAGCCTGGCCGGTAAAAAAATCG
dfp-R:CGCCCGTTTTTGCATTTAACGTCGATTTTTTTCATCATAA
coaD-F:AAAAATCGACGTTAAATGCAAAAACGGGCGATTTATCCGG
coaD-19R:CAGCTATGACCTACGCTAACTTCGCCATCAGCGCC
coaD-ETR:CACCACAGCCAGGATCCGATAACTTCGCCATCAGCGCC
It using pRSFDuet-1 plasmid as template, is expanded with primer RSF-F and RSF-R, it is linear to obtain pRSFDuet-1
Carrier；Using 11166 genome of Rhodobacter capsulatus ATCC as template, hemA gene is expanded with primer hemA-F and hemA-R, is obtained
PRSFDuet-1 linear carrier is reacted with hemA genetic fragment by homologous recombination and is attached by hemA genetic fragment, is obtained
Recombinant plasmid pRSFDuet-1-hemA.
It using pUC19 plasmid as template, is expanded with primer pUC19-F and pUC19-R, obtains pUC19 linear carrier；With
Genome of E.coli is template, expands coaA gene with primer coaA-19F and coaA-R, with primer dfp-F and dfp-R expansion
Increase dfp gene, coaD gene is expanded with primer coaD-F and coaD-19R, obtains coaA-19, dfp, coaD-19 gene respectively
PUC19 linear carrier, coaA-19, dfp, coaD-19 segment are attached by homologous recombination reaction, are recombinated by segment
Plasmid pUC19-coaA-dfp-coaD.
It using pETDuet-1 plasmid as template, is expanded with primer pET-F and pET-R, obtains pETDuet-1 and linearly carry
Body；Using genome of E.coli as template, coaA gene is expanded with primer coaA-ETF and coaA-R, with primer dfp-F and
Dfp-R expands dfp gene, expands coaD gene with primer coaD-F and coaD-ETR, obtains coaA-ET, dfp, coaD- respectively
ET genetic fragment connects pETDuet-1 linear carrier, coaA-ET, dfp, coaD-ET segment by homologous recombination reaction
It connects, obtains recombinant plasmid pETDuet-1-coaA-dfp-coaD.
Reaction condition are as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min/kb (32 circulations)；72 DEG C of 10min into
Row PCR reaction, and pcr amplification product is verified and recycled with 1% agarose gel electrophoresis, by segment (0.04ng/bp) and carrier
(0.02ng/bp) is mixed in same PCR pipe, and 4 μ l recombination enzyme buffer liquid and 2 μ l recombinases are added, add ddH2O polishing to 20 μ l,
In 37 degree of reaction 30min.Reaction product is gone into E.coli JM109, positive clone molecule is selected and extracts plasmid and serve Hai Sheng
Work sequence verification, bacterium colony PCR result electrophoretogram such as Fig. 3 and Fig. 4 (arrow show purpose band).
(2) building of ALA fermentation recombinant bacterial strain
Basic skills is the recombinant plasmid pRSFDuet-1-hemA Transformed E .coli BL21 (DE3) that will be constructed, and is weighed
Group bacterium A1.By recombinant plasmid pRSFDuet-1-hemA, the pUC19-coaA-dfp-coaD cotransformation E.coli BL21 of building
(DE3), recombinant bacterium A2 is obtained.The recombinant plasmid pRSFDuet-1-hemA, pETDuet-1-coaA-dfp-coaD of building is total
Transformed E .coli BL21 (DE3) obtains recombinant bacterium A3.
The verifying of 2 recombinant bacterium shake flask fermentation of embodiment
Bacterial strain:
A1:E.coli BL21(DE3)pRSFDuet-1-hemA
A2:E.coli BL21(DE3)pRSFDuet-1-hemA pUC19-coaA-dfp-coaD
A3:E.coli BL21(DE3)pRSFDuet-1-hemA pETDuet-1-coaA-dfp-coaD
Recombinant bacterium A2 and A3 are subjected to seed culture and fermentation respectively, are not co-express the strains A 1 of CoA related gene
Control, 37 DEG C, 220r/min fermentation 24-32h measures ALA yield.(Fig. 5) as the result is shown passes through high copy number plasmid and middle copy
Plasmid combinations express coaA, dfp, coaD, and compared with control strain A1, ALA yield is improved.CoA phase is expressed using pUC19
The ALA yield of the recombinant bacterium A2 of correlation gene is 654mg/L, improves about 37% than control strains A 1；Use plasmid pETDuet-1
The ALA yield for expressing the recombinant bacterium A3 of CoA related gene is 700mg/L, improves about 46% than control strains A 1.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of colibacillus engineering for producing 5-ALA
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 1230
<212> DNA
<213>artificial sequence
<400> 1
atggactaca atctcgcgct cgacaaagcg atccagaaac tccacgacga gggacgttac 60
cgcacgttca tcgacatcga acgcgagaag ggcgccttcc ccaaggcgca gtggaaccgc 120
cccgatggcg gcaagcagga catcaccgtc tggtgcggca acgactatct gggcatgggc 180
cagcacccgg tcgttctggc cgcgatgcat gaggcgctgg aagcggtcgg ggccggttcg 240
ggcggcaccc gcaacatctc gggcaccacg gcctatcacc gccgtctgga agccgagatc 300
gccgatctgc acggcaagga agcggcgctt gtcttctcct cggcctatat cgccaatgac 360
gcgacgctct cgacgctgcg cgtgcttttc cccggcctga tcatctattc cgacagcctg 420
aaccacgcct cgatgatcga ggggatcaag cgcaatgccg ggccgaagcg gatcttccgt 480
cacaatgacg tcgcccatct gcgcgagctg atcgccgctg atgatccggc cgcgccgaag 540
ctgatcgcct tcgaatcggt ctattcgatg gatggcgact tcggcccgat caaggaaatc 600
tgcgacatcg ccgatgaatt cggcgcgctg acctatatcg acgaagtcca tgccgtcggc 660
atgtatggcc cccgcggcgc gggcgtggcc gagcgtgacg gtctgatgca ccgcatcgac 720
atcttcaacg gcacgctggc gaaagcctat ggcgtcttcg gcggctacat cgccgcttcg 780
gcgaagatgg tcgatgccgt gcgctcctat gcgccgggct tcatcttctc gacctcgctg 840
ccgccggcga tcgccgctgg cgcgcaggcc tcgatcgcgt ttttgaaaac cgccgaaggg 900
cagaagctgc gcgacgcgca acagatgcac gcgaaggtgc tgaaaatgcg gctcaaggcg 960
ctggggatgc cgatcatcga ccatggcagc cacatcgttc cggtggtcat cggtgacccc 1020
gtgcacacca aggcggtgtc ggacatgctc ctgtcggatt acggcgttta cgtgcagccg 1080
atcaacttcc cgacggtgcc gcgcggcacc gaacggctgc gcttcacccc ctcgccggtg 1140
catgacctga aacagatcga cgggctggtt catgccatgg atctgctctg ggcgcgctgt 1200
gcgctgaatc gcgccgaggc ctctgcctga 1230
<210> 2
<211> 951
<212> DNA
<213>artificial sequence
<400> 2
atgagtataa aagagcaaac gttaatgacg ccttacctac agtttgaccg caaccagtgg 60
gcagctctgc gtgattccgt acctatgacg ttatcggaag atgagatcgc ccgtctcaaa 120
ggtattaatg aagatctctc gttagaagaa gttgccgaga tctatttacc tttgtcacgt 180
ttgctgaact tctatataag ctcgaatctg cgccgtcagg cagttctgga acagtttctt 240
ggtaccaacg ggcaacgcat tccttacatt atcagtattg ctggcagtgt cgcggtgggg 300
aaaagtacaa ccgcccgtgt attgcaggcg ctattaagcc gttggccgga acatcgtcgt 360
gttgaactga tcactacaga tggcttcctt caccctaatc aggttctgaa agaacgtggt 420
ctgatgaaga agaaaggctt cccggaatcg tatgatatgc atcgcctggt gaagtttgtt 480
tccgatctca aatccggcgt gccaaacgtt acagcacctg tttactcaca tcttatttat 540
gatgtgatcc cggatggaga taaaacggtt gttcagcctg atattttaat tcttgaaggg 600
ttaaatgtct tacagagcgg gatggattat ccacacgatc cacatcatgt atttgtttct 660
gattttgtcg atttttcgat atatgttgat gcaccggaag acttacttca gacatggtat 720
atcaaccgtt ttctgaaatt ccgcgaaggg gcttttaccg acccggattc ctattttcat 780
aactacgcga aattaactaa agaagaagcg attaagactg ccatgacatt gtggaaagag 840
atcaactggc tgaacttaaa gcaaaatatt ctacctactc gtgagcgcgc cagtttaatc 900
ctgacgaaaa gtgctaatca tgcggtagaa gaggtcagac tacgcaaata a 951
<210> 3
<211> 1221
<212> DNA
<213>artificial sequence
<400> 3
atgagcctgg ccggtaaaaa aatcgttctc ggcgttagcg gcggtattgc tgcctataaa 60
acccctgaac tggtgcgtcg tttgcgcgat cgcggggccg acgtccgcgt agccatgacc 120
gaagcggcaa aagcctttat caccccactt agcttgcagg cggtttctgg ttatcccgtt 180
tccgacagtc tgctggaccc ggcagccgaa gccgctatgg gccatattga gctgggtaaa 240
tgggctgatt tagtgattct cgcccctgcc acggcagatt tgattgcccg tgttgctgcc 300
ggaatggcga atgacctggt atcgacgatt tgtctggcta cacctgcgcc tgtagccgtg 360
ctccccgcca tgaaccagca gatgtaccgt gccgctgcca cgcagcataa tttagaggtg 420
cttgcttccc gtggtttgct catctggggg ccagacagtg gcagtcaggc ttgtggtgat 480
atcggtcctg ggcgaatgct cgatccgtta accattgtgg atatggcggt agcgcatttt 540
tcgcccgtca acgacctgaa acatctgaac attatgatta ccgccggccc gacgcgtgaa 600
ccgctcgatc cggtgcgtta tatctctaat cacagctccg gcaagatggg ttttgctatc 660
gccgccgccg ctgcccgtcg tggcgcgaac gtcacgctgg tatcaggtcc ggtttcacta 720
ccgacgccac cgtttgttaa acgtgttgat gtgatgaccg cgctggaaat ggaagccgcc 780
gtgaatgctt ctgtacagca gcaaaatatt tttatcggct gcgccgccgt ggcggattat 840
cgcgcagcta ccgtggcccc agagaaaatc aaaaagcagg ccacgcaggg tgatgaatta 900
acaataaaaa tggttaaaaa ccccgatatc gtcgcaggcg ttgccgcact aaaagaccat 960
cgaccctacg tcgttggatt tgccgccgaa acaaataatg tggaagaata cgcccggcaa 1020
aaacgtatcc gtaaaaacct tgatctgatc tgcgcgaacg atgtttccca gccaactcaa 1080
ggatttaaca gcgacaacaa cgcattacac cttttctggc aggacggaga taaagtctta 1140
ccgcttgagc gcaaagagct ccttggccaa ttattactcg acgagatcgt gacccgttat 1200
gatgaaaaaa atcgacgtta a 1221
<210> 4
<211> 480
<212> DNA
<213>artificial sequence
<400> 4
atgcaaaaac gggcgattta tccgggtact ttcgatccca ttaccaatgg tcatatcgat 60
atcgtgacgc gcgccacgca gatgttcgat cacgttattc tggcgattgc cgccagcccc 120
agtaaaaaac cgatgtttac cctggaagag cgtgtggcac tggcacagca ggcaaccgcg 180
catctgggga acgtggaagt ggtcgggttt agtgatttaa tggcgaactt cgcccgtaat 240
caacacgcta cggtgctgat tcgtggcctg cgtgcggtgg cagattttga atatgaaatg 300
cagctggcgc atatgaatcg ccacttaatg ccggaactgg aaagtgtgtt tctgatgccg 360
tcgaaagagt ggtcgtttat ctcttcatcg ttggtgaaag aggtggcgcg ccatcagggc 420
gatgtcaccc atttcctgcc ggagaatgtc catcaggcgc tgatggcgaa gttagcgtag 480
<210> 5
<211> 52
<212> DNA
<213>artificial sequence
<400> 5
tgtttaactt taataaggag gaaaatatat ggactacaat ctcgcgctcg ac 52
<210> 6
<211> 35
<212> DNA
<213>artificial sequence
<400> 6
ctccttatta aagttaaaca aaattatttc tacag 35
<210> 7
<211> 50
<212> DNA
<213>artificial sequence
<400> 7
tgtttaactt taataaggag gaaaatatat ggactacaat ctggcactcg 50
<210> 8
<211> 42
<212> DNA
<213>artificial sequence
<400> 8
cgagctcggc cacgaagtgc tcaggcagag gcctcggcgc ga 42
<210> 9
<211> 36
<212> DNA
<213>artificial sequence
<400> 9
tgatggcgaa gttagcgtag gtcatagctg tttcct 36
<210> 10
<211> 35
<212> DNA
<213>artificial sequence
<400> 10
ctcttttata ctcattacga gccggaagca taaag 35
<210> 11
<211> 45
<212> DNA
<213>artificial sequence
<400> 11
gatggcgaag ttagcgtagg gcgcgcctgc aggtcgacaa gcttg 45
<210> 12
<211> 43
<212> DNA
<213>artificial sequence
<400> 12
gtttgctctt ttatactcat gaattcggat cctggctgtg gtg 43
<210> 13
<211> 41
<212> DNA
<213>artificial sequence
<400> 13
tgcttccggc tcgtaatgag tataaaagag caaacgttaa t 41
<210> 14
<211> 40
<212> DNA
<213>artificial sequence
<400> 14
cacagccagg atccgaattc atgagtataa aagagcaaac 40
<210> 15
<211> 40
<212> DNA
<213>artificial sequence
<400> 15
accggccagg ctcatttatt tgcgtagtct gacctcttct 40
<210> 16
<211> 40
<212> DNA
<213>artificial sequence
<400> 16
agactacgca aataaatgag cctggccggt aaaaaaatcg 40
<210> 17
<211> 40
<212> DNA
<213>artificial sequence
<400> 17
cgcccgtttt tgcatttaac gtcgattttt ttcatcataa 40
<210> 18
<211> 40
<212> DNA
<213>artificial sequence
<400> 18
aaaaatcgac gttaaatgca aaaacgggcg atttatccgg 40
<210> 19
<211> 35
<212> DNA
<213>artificial sequence
<400> 19
cagctatgac ctacgctaac ttcgccatca gcgcc 35
<210> 20
<211> 38
<212> DNA
<213>artificial sequence
<400> 20
caccacagcc aggatccgat aacttcgcca tcagcgcc 38
Claims (9)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611094235.7A CN106497858B (en) | 2016-12-02 | 2016-12-02 | A kind of Escherichia coli engineering bacteria producing 5-aminolevulinic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611094235.7A CN106497858B (en) | 2016-12-02 | 2016-12-02 | A kind of Escherichia coli engineering bacteria producing 5-aminolevulinic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106497858A CN106497858A (en) | 2017-03-15 |
| CN106497858B true CN106497858B (en) | 2019-06-21 |
Family
ID=58330348
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611094235.7A Active CN106497858B (en) | 2016-12-02 | 2016-12-02 | A kind of Escherichia coli engineering bacteria producing 5-aminolevulinic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106497858B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531437B (en) * | 2018-04-13 | 2021-12-10 | 北京化工大学 | 5-aminolevulinic acid biosynthesis pathway mediated by glyoxylate aminotransferase |
| CN114150027A (en) * | 2021-12-03 | 2022-03-08 | 河北维达康生物科技有限公司 | Method for synthesizing N-acetyl-5-methoxytryptamine by using 5-hydroxy beta-indolylalanine as substrate through biological method |
| CN114369562B (en) * | 2022-03-21 | 2022-05-31 | 中国农业科学院北京畜牧兽医研究所 | Method for improving expression quantity of 5-aminolevulinic acid |
| CN115747125B (en) * | 2022-08-07 | 2024-06-14 | 中国科学院天津工业生物技术研究所 | Engineering strain for high production of 5-aminolevulinic acid and construction method of 5-aminolevulinic acid high production strain |
| CN116769748B (en) * | 2023-07-07 | 2024-06-28 | 江南大学 | A 5-aminolevulinic acid synthase mutant and Escherichia coli producing B12 precursor ALA |
-
2016
- 2016-12-02 CN CN201611094235.7A patent/CN106497858B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Cloning of two 5-aminolevulinic acid synthase isozymes HemA and HemO from Rhodopseudomonas palustris with favorable characteristics for 5-aminolevulinic acid production.;Lilu Zhang et al.;《Biotechnol Lett》;20130122;第35卷;763-768 |
| Metabolic engineering to improve 5-aminolevulinic acid production.;Zhen Kang et al.;《Bioengineered Bugs》;20111101;第2卷(第6期);1-4 |
| 微生物发酵生产5-氨基乙酰丙酸研究进展;康振 等;《生物工程学报》;20130925;第29卷(第9期);1214-1222 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106497858A (en) | 2017-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106497858B (en) | A kind of Escherichia coli engineering bacteria producing 5-aminolevulinic acid | |
| CN104830748B (en) | A kind of reduction hemB gene expressions improve the method that Escherichia coli synthesize 5 amino-laevulic acids | |
| CN103981203B (en) | 5 amino-laevulic acid superior strains and its preparation method and application | |
| CN104004701A (en) | Method for building high-yield 5-aminolevulinic acid escherichia coli engineering strains | |
| CN112795582B (en) | An Enzyme Gene Suitable for Efficient Synthesis of NAD Derivatives in Microorganisms | |
| CN110387379B (en) | Mixed culture process and application of recombinant escherichia coli for producing glutathione | |
| CN113684164A (en) | Construction method and application of microorganism for high yield of lactoyl-N-neotetraose | |
| CN114381416B (en) | Recombinant escherichia coli strain for high yield of 5-aminolevulinic acid and application thereof | |
| US8778654B2 (en) | Recombinant bacteria for producing deoxyviolacein and uses thereof | |
| WO2022007205A1 (en) | Method for increasing content of intracellular heme in escherichia coli | |
| CN103146694B (en) | Gene and the construction process thereof of 5-ALA C4 biosynthetic pathway is built intestinal bacteria | |
| CN104195190B (en) | Method for producing 5-aminolevulinic acid by carrying out anaerobic fermentation by utilizing recombinant escherichia coli | |
| CN108220264B (en) | Application of a glycosyltransferase in the biosynthesis of salidroside | |
| CN108531437A (en) | A kind of 5-ALA biosynthesis pathway that Glyoxylate transaminase mediates | |
| CN113564190A (en) | High-yield riboflavin escherichia coli engineering strain and construction method thereof | |
| CN110734479A (en) | A kind of Escherichia coli cyclic adenylate receptor protein mutant, genetic engineering bacteria and application | |
| CN104017767B (en) | A kind of method utilizing combinatorial regulation strategy to improve 5-ALA yield | |
| CN105238708B (en) | One plant of bacterium for producing L- hydroxyprolines and its application | |
| CN116042545A (en) | Glutathione bifunctional synthetase mutant S722A with improved enzyme activity and application thereof | |
| CN109234337B (en) | Biosynthesis method of quercetin glycoside | |
| CN110835621A (en) | Genetically engineered bacterium and application thereof in producing xylitol | |
| CN117230099A (en) | Construction method of bacillus capable of efficiently expressing 5-aminolevulinic acid | |
| CN105969751B (en) | A kind of β-glucosidase gene and its application | |
| CN104561158A (en) | A method of adding Fe2+ to improve the synthesis of 5-aminolevulinic acid by Escherichia coli engineering bacteria | |
| CN114854658A (en) | A method for enhancing the utilization of acetic acid and improving the fermentation production of L-arginine by Escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |