CN105754920A - Genetically engineered blue algae and application thereof - Google Patents

Genetically engineered blue algae and application thereof Download PDF

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CN105754920A
CN105754920A CN201610211295.6A CN201610211295A CN105754920A CN 105754920 A CN105754920 A CN 105754920A CN 201610211295 A CN201610211295 A CN 201610211295A CN 105754920 A CN105754920 A CN 105754920A
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cyanophyceae
encoding gene
seqidno
genetic engineering
synthase
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许平
倪俊
陶飞
唐鸿志
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Shanghai Jiaotong University
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Abstract

The invention discloses genetically engineered blue algae. The genetically engineered blue algae provided by the invention can directly utilize CO2 to efficiently produce phenylpropane substances through photosynthesis, so that consumption of carbohydrates and use of an expensive precursor are avoided; and moreover, the yield of an important node compound for improving metabolism of phenylpropane can be increased. The invention further provides an application of the genetically engineered blue algae, especially the application in producing phenylpropane substances, so that a new technology is developed for producing the phenylpropane substances, and the production cost is reduced; and therefore, the application has an important application prospect.

Description

A kind of genetic engineering cyanophyceae and application thereof
Technical field
The invention belongs to genetic engineering field, be specifically related to a kind of genetic engineering cyanophyceae, genetic engineering cyanophyceae provided by the invention can directly utilize CO by photosynthesis2Efficiently produce phenylpropyl alcohol alkanes material.
Background technology
Secondary Metabolism of Plant is the result that ecological environment is adapted to by plant in long-term evolution, its metabolite has the biological function of Various Complex, improving plant to the adaptability of environment, interspecies competition ability and the important role such as enhance disease resistance, Plant Secondary Metabolites also provides valuable raw material for industries such as medicine, chemical industry, food, pigment and pesticide.As natural active matter, Plant Secondary Metabolites is that some diseases being difficult to cure provide an important solution route at present.Phenylpropyl alcohol alkanes material is the Secondary metabolites that a class is important, mainly includes flavonoid, stilbene class and simple phenols etc., and in plant, most of phenylpropyl alcohol alkanes materials are all formed P-coumaric acid and then generation after deaminizating by tyrosine or phenylalanine.Phenylpropyl alcohol alkanes material has stronger physiologically active and pharmacological action, as isoflavone can pre-preventing bone rarefaction, climacteric syndrome, and with kinds cancer that breast carcinoma is representative；Resveratrol can prevent the disease such as cardiovascular disease, arteriosclerosis.Additionally, phenylpropyl alcohol alkanes material and derivant thereof are also used as industrial chemicals, food additive and spice etc. widely, if vanillin is one of natural perfume material of being most widely used of the current world.
Although the mankind are created huge effect by the Secondary metabolites such as phenylpropyl alcohol alkanes, but the content that these natural products are in plant is low, separate and extract complex, and many materials could produce at the specific part of specified plant, the yield making them is extremely low, it is impossible to meet people for the growing demand of Secondary metabolites.Although chemical synthesis can synthesize the relatively simple phenylpropyl alcohol alkanes material of some structures, but day by day highlighting and people's attention for food safety of environmental problem, limit the development of this method.Owing to technique is simple, separate and extract the advantages such as simplicity, safety non-pollution, fermentable is utilized to become the focus of research to synthesize Secondary metabolites.As, 2005, Yan et al. utilizes gene engineering microzyme that P-coumaric acid is converted into 28.3mg/L naringenin (Yan, Y., Kohli, A., Koffas, M.A.BiosynthesisofnaturalflavanonesinSaccharomycescerevi siae.Appl.Environ.Microbiol., 2005,71:5610-5613.)；2012, Lin et al. utilizes the recombination bacillus coli of tyrosine high yield to convert glucose into 50.2mg/L caffeic acid (Lin, Y., Yan, Y.BiosynthesisofcaffeicacidinEscherichiacoliusingitsendo genoushydroxylasecomplex.Microb.CellFact.2012,11:42-50.)；Kang et al. imports the related gene of phenylpropyl alcohol alkanes material synthesis in escherichia coli, it is possible to convert glucose into 974mg/L P-coumaric acid, 150mg/L caffeic acid and 196mg/L ferulic acid (Kang, S.Y., Choi, O., Lee, J.K., Hwang, B.Y., Uhm, T.B., Hong, Y.S.Artificialbiosynthesisofphenylpropanoicacidsinatyros ineoverproducingEscherichiacolistrain.Microb.CellFact.20 12,11,153.).Although these production technologies can obtain a certain amount of phenylpropyl alcohol alkanes material, but ATP and the NADPH that general microorganism lacks abundance is supplied to these external source route of synthesis so that yield is absorbed in bottleneck.And production process needs to add the precursor substance of carbohydrate or costliness so that this mode of production is very uneconomical.Additionally, many phenylpropyl alcohol alkanes route of synthesis need the plant enzyme that some general industry microorganisms are beyond expression of words, limit the application of this biological production technology.
Cyanophyceae is to carry out photosynthetic prokaryote, is widely distributed in fresh water, sea water even sewage.Blue algae growth is rapid, and structure and genetic background are simple, it is easy to genetic manipulation, and the most prominent is that it can by solar energy and greenhouse gases CO2It is converted into product, is one of optimal path realizing sustainable production.Additionally, due to can photosynthesis so that cyanophyceae has ATP and the NADPH of abundance；As the ancestors of plant, cyanophyceae can also express the enzyme of most plant origin, and these features make the particularly suitable carrier as the synthesis of phenylpropyl alcohol alkanes of cyanophyceae.But, do not have at present and cyanophyceae is carried out genetic modification so that it is directly utilize CO2With the relevant report that solar energy carrys out synthetic styrene-acrylic alkanes material.
Summary of the invention
Because the drawbacks described above of prior art, the invention provides a kind of genetic engineering cyanophyceae, genetic engineering cyanophyceae provided by the invention can directly convert CO2Produce phenylpropyl alcohol alkanes material.
For solving the problems referred to above, the present invention adopts the technical scheme that: a kind of genetic engineering cyanophyceae, and described genetic engineering cyanophyceae is prepared from by one of them of following methods:
Method one: the genomic DNA that the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase and the encoding gene of tyrosine ammonia lyase that solve feedback suppression import wild cyanophyceae obtains producing the genetic engineering cyanophyceae of P-coumaric acid；
Method two: obtain producing caffeinic genetic engineering cyanophyceae by the genomic DNA solving the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, the encoding gene of tyrosine ammonia lyase and the encoding gene of P-coumaric acid hydroxylase and importing wild cyanophyceae；
Method three: the genomic DNA solving the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, the encoding gene of tyrosine ammonia lyase, the encoding gene of P-coumaric acid hydroxylase and the encoding gene of caffeic acid and importing wild cyanophyceae is obtained producing the genetic engineering cyanophyceae of ferulic acid；
Method four: by solve the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, tyrosine ammonia lyase encoding gene, the encoding gene of coumaroyl A ligase and the encoding gene of stilbene synthase imported the genomic DNA of wild cyanophyceae obtain producing the genetic engineering cyanophyceae of resveratrol；
Method five: by solve the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, tyrosine ammonia lyase encoding gene, the encoding gene of coumaroyl A ligase, the encoding gene of chalcone synthase and the encoding gene of enzyme, namely chalcone isomerase imported the genomic DNA of wild cyanophyceae obtain producing the genetic engineering cyanophyceae of naringenin；
Method six: by solve the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, tyrosine ammonia lyase encoding gene, the encoding gene of coumaroyl A ligase and the encoding gene of curcumin synthase are imported the genetic engineering cyanophyceae producing bisdemethoxycurcumin that the genomic DNA of wild cyanophyceae obtains.
Preferably, described wild cyanophyceae is selected from cyanophyceaes such as Synechococcus genus, synechocystis, hidden ball Trentepohlia, Anabaena, nostoc, Oscillatoria, ball Trentepohlia, Ah lattice's Trentepohlia, Scytonema and whip branch Trentepohlias.
It is highly preferred that described wild cyanophyceae is cytoalgae (Synechocystissp.).
Preferably, exogenous gene imports the genomic DNA of wild cyanophyceae by being connected in plasmid.
It is highly preferred that the exogenous gene structure of expressing in series contain one with in cascaded structure first promoter that gene is operably connected, or on described cascaded structure, each gene is each independent of the promoter being operably connected.
It is highly preferred that described promoter is trc promoter.
Preferably, the encoding gene of the 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of described solution feedback suppression is such as shown in SEQIDNO:1 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:1；
The encoding gene of described tyrosine ammonia lyase is such as shown in SEQIDNO:2 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:2；
The encoding gene of described P-coumaric acid hydroxylase is such as shown in SEQIDNO:3 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:3；
The encoding gene of described caffeic acid is such as shown in SEQIDNO:4 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:4；
The described encoding gene to coumaroyl A ligase is such as shown in SEQIDNO:5 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:5；
The encoding gene of described stilbene synthase is such as shown in SEQIDNO:6 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:6；
The encoding gene of described chalcone synthase is such as shown in SEQIDNO:7 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:7；
The encoding gene of described enzyme, namely chalcone isomerase is such as shown in SEQIDNO:8 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:8；
The encoding gene of described curcumin synthase is such as shown in SEQIDNO:9 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:9.
It is highly preferred that the 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of described solution feedback suppression derives from escherichia coli；
Described tyrosine ammonia lyase derives from saccharothrix；
Described P-coumaric acid hydroxylase derives from arabidopsis；
Described caffeic acid derives from arabidopsis；
Described derives from parsley to coumaroyl A ligase；
Described stilbene synthase derives from Semen arachidis hypogaeae；
Described chalcone synthase derives from petunia；
Described enzyme, namely chalcone isomerase derives from arabidopsis；
Described curcumin synthase derives from Oryza sativa L..
Present invention also offers the genetic engineering cyanophyceae provided by the present invention application in producing phenylpropyl alcohol alkanes material.
The invention have the benefit that cyanophyceae is carried out genetic modification by the present invention, the phenylpropyl alcohol alkanes material route of synthesis of plant reconstructed in cyanophyceae, it is thus achieved that genetic engineering bacterium can by inexhaustible solar energy and greenhouse gases CO2It is converted into specific phenylpropyl alcohol alkyl compound, it is to avoid consuming and the use of expensive precursor of carbon water and compound.In a particular embodiment of the present invention, through cultivating, genetic engineering cyanophyceae can produce 128.2mg/l P-coumaric acid, 4.7mg/l caffeic acid, 6.3mg/l ferulic acid, 4.6mg/l resveratrol, 7.1mg/l naringenin and 4.1mg/l bisdemethoxycurcumin respectively.These materials are as the important node compound of phenylpropyl alcohol alkane metabolism, it is possible to synthesize other phenylpropyl alcohol alkyl compounds many, and this is that the production of phenylpropyl alcohol alkanes material opens new technology, has prospects for commercial application widely.The present invention provides application process to reduce substrate cost, it is also possible to reduce CO2Discharge, have important practical significance and industrial application value.
Below with reference to accompanying drawing, the technique effect of the design of the present invention, concrete structure and generation is described further, to be fully understood from the purpose of the present invention, feature and effect.
Accompanying drawing explanation
Fig. 1 is that the embodiment of the present invention 5 utilizes the concentration of P-coumaric acid in genetic engineering cyanophyceae S-COU production P-coumaric acid to scheme over time.
The LC-ESI-MS that Fig. 2 is P-coumaric acid in the embodiment of the present invention 5 detects collection of illustrative plates.
Caffeinic LC-ESI-MS is detected collection of illustrative plates in the embodiment of the present invention 6 by Fig. 3.
The LC-ESI-MS of ferulic acid is detected collection of illustrative plates in the embodiment of the present invention 7 by Fig. 4.
The LC-ESI-MS of resveratrol is detected collection of illustrative plates in the embodiment of the present invention 8 by Fig. 5.
The LC-ESI-MS of naringenin is detected collection of illustrative plates in the embodiment of the present invention 9 by Fig. 6.
The LC-ESI-MS of bisdemethoxycurcumin is detected collection of illustrative plates in the embodiment of the present invention 10 by Fig. 7.
Detailed description of the invention
Below in conjunction with embodiment, the technology contents of the present invention is described further: following embodiment is illustrative, is not determinate, it is impossible to limit protection scope of the present invention with following embodiment.The test method used in following embodiment if no special instructions, is conventional method.Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
Bacterial strain and growth conditions that the present invention uses are as follows:
Cloning host DH5 α purchased from Invitrogen company, all escherichia coli in the LB culture medium containing 100mg/l spectinomycin, 37 DEG C of cultivations.
Cytoalgae (Synechocystissp.) PCC7942 purchased from American Type culture collection warehousing (Americantypeculturecollection).Cytoalgae PCC7942 grows in BG11 fluid medium, and solid medium adds the agar powder of 1.5% in BG11 fluid medium, adds 20mg/l spectinomycin for the recombinant bacterium after converting.Condition of culture: 30 DEG C, light intensity 100 μ E s-1·m-2, and pass into 5% volume ratio CO continuously2Gas.
Wherein, described LB culture medium prescription is: peptone 10g/L, yeast extract 5g/L, NaCl10g/L, pH7.0；
Described BG11 liquid culture based formulas is: 10mlBG11 mother solution, 1ml ferric citrate ammonium salt solution (6g/L), 1mlNa2CO3Solution (20g/L), 1mlK2HPO4Solution (30.5g/L), water is settled to 1L；
The formula of above-mentioned BG11 mother solution is: 149.6gNaNO3, 7.5gMgSO4·7H2O, 3.6gCaCl2·2H2O, 0.6g citric acid, 1.12mlNaEDTA solution (PH8.0, concentration 0.25M), 100ml trace element solution, water is settled to 1L；
The formula of above-mentioned BG11 trace element solution is: 2.86gH3BO3, 1.81gMnCl2 7H2O, 0.22gZnSO4·7H2O, 0.39gNa2MoO4·2H2O, 0.079gCuSO4·5H2O, 0.049gCo (NO3)2·6H2O, water is settled to 1L；
All plasmids are PAM2991 plasmid, express genes of interest for being incorporated in cytoalgae PCC7942 genome by homologous recombination.
Embodiment 1
Construction expression plasmid pAM-aroGfbr-sam8:
(1) pAM2991 plasmid is extracted:
By the bacillus coli DH 5 alpha containing pAM2991 plasmid according to 1% inoculum concentration, it is seeded in 5mLLB fluid medium, 37 DEG C of incubators are cultivated 10h.The little extraction reagent kit of ordinary plasmids (Tian Gen biochemical technology company limited) is utilized to extract plasmid the thalline after cultivating.
(2) amplification Ptrc promoter:
Utilizing pAM2991 plasmid for template, adopt conventional method to carry out pcr amplification, this process is referred to the method described in " Molecular Cloning: A Laboratory guide (third edition) ".
PCR primer: Ptrc promoter forward primer Ptrc-F:5 '-catgagatctCagcttatcatcgactgcacg-3 ', downstream primer Ptrc-R:5 '-catgctcgaggtctgtttcctgtgtgaaattgtt-3′.The base sequence that wherein underscore indicates is the restriction enzyme site of BglII and XhoI respectively.
DNA fragmentation is cut glue by the AxyPrepDNAGelExtractionKit of the PCR primer Axygen company obtained reclaim.
(3) restricted enzyme BglII and the XhoI that the pAM2991 plasmid obtained in the Ptrc promoter obtained in step (2) and step (1) is utilized respectively NEB carries out double digestion.Ptrc promoter gene fragment after enzyme action and pAM2991 plasmid are reclaimed with the AxyPrepDNAGelExtractionKit of Axygen company respectively, it is attached with T4DNA ligase (buying from NewEnglandBiolabs company), being connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain the recombiant plasmid pAM2 after connecting.
(4) amplification solves the 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase (aroG of feedback suppressionfbr):
Adopting conventional method to prepare the genomic DNA of e. coli k12, this process is referred in " the fine works Molecular Biology " that Science Press publishes the miniprep procedure of bacterial genomes；
Utilizing e. coli k12 genome for template, adopt conventional method to carry out pcr amplification, this process is referred to the method described in " Molecular Cloning: A Laboratory guide (third edition) ".
PCR primer: aroG upstream region of gene primer aroG-F:
5′-catgcttaagAtgaattatcagaacgacgatttacg-3 ', downstream primer aroG-R:
5′-catgagatctTtacccgcgacgcgct-3 ', the base sequence that wherein underscore indicates is the restriction enzyme site of AflII and BglII respectively.
Obtain above-mentioned product, through the method for recombinant PCR, the C bases of 539 are replaced with T, thus the serine by the 180th replaces with phenylalanine, obtain encoding the sequence of the 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase solving feedback suppression.DNA fragmentation is cut glue by the AxyPrepDNAGelExtractionKit of the PCR primer Axygen company obtained reclaim.
(5) aroG that will obtain in step (4)fbrThe pAM2 plasmid obtained in genetic fragment and step (3) is utilized respectively restriction enzyme A flII and the BglII of NEB and carries out double digestion.By the aroG after enzyme actionfbrGenetic fragment and pAM2991 plasmid reclaim with reference to the method described in description with the AxyPrepDNAGelExtractionKit of Axygen company respectively, it is attached with T4DNA ligase (buying from NewEnglandBiolabs company), connection procedure is with reference to the method in description, being connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain the recombiant plasmid pAM-aroG after connectingfbr。
(6) amplification tyrosine ammonia lyase gene (sam8):
Adopting conventional method to prepare the genomic DNA of Spain's saccharothrix, this process is referred in " the fine works Molecular Biology " that Science Press publishes the miniprep procedure of bacterial genomes；
Utilizing Spain's saccharothrix genome is template, adopts conventional method to carry out pcr amplification, and the method that this process is referred to described in " Molecular Cloning: A Laboratory guide (third edition) " carries out.
PCR primer: sam8 upstream region of gene primer sam8-F:
5′-catgctcgagGcatgacgcaggtcgtggaacg-3 ', downstream primer sam8-R:
5′-catgggatccttatccgaaatccttcccgt-3′.The base sequence that wherein underscore indicates is the restriction enzyme site of XhoI and BamHI respectively.
DNA fragmentation is cut glue by the AxyPrepDNAGelExtractionKit of the PCR primer Axygen company obtained reclaim.
(7) pAM-aroG that will obtain in the sam8 genetic fragment obtained in step (6) and step (5)fbrPlasmid is utilized respectively restricted enzyme XhoI and the BamHI of NEB and carries out double digestion.By the sam8 genetic fragment after enzyme action and pAM-aroGfbrPlasmid reclaims with reference to the method described in description with the AxyPrepDNAGelExtractionKit of Axygen company respectively, it is attached with T4DNA ligase (buying from NewEnglandBiolabs company), being connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain the recombiant plasmid pAM-aroG after connectingfbr-sam8。
Embodiment 2
Construction expression plasmid pAM-sts-4cl-sam8-aroGfbr、pAM-chs-chi-4cl-sam8-aroGfbrAnd pAM-cus-4cl-sam8-aroGfbr:
(1) synthesis containing trc promoter to coumaroyl A ligase (4cl) fragment:
Utilize online software JCat, 4cl gene in parsley (Petroselinumcrispum) is carried out codon optimized by the codon preference according to cytoalgae PCC7942, and in front end plus trc promoter sequence and EcoRI restriction enzyme site, end adds BamHI restriction enzyme site.Utilize online software DNAWorks to design primer, carry out gene chemical synthesis by the method for recombinant PCR, and with the AxyPrepDNAGelExtractionKit of Axygen company, DNA fragmentation is cut glue and reclaim.
(2) synthesis is to coumaroyl A ligase (4cl) fragment:
Utilizing the genetic fragment obtained in step (1) is template, adopts conventional method to carry out pcr amplification, and the method that this process is referred to described in " Molecular Cloning: A Laboratory guide (third edition) " carries out.
PCR primer: 4cl upstream region of gene primer 4cl-F:5 '-catgctcgagAtgggtgactgcgttgccc-3 ', downstream primer 4cl-R:5 '-catgggatccttacttcggcaggtcgccg-3′.The base sequence that wherein underscore indicates is the restriction enzyme site of XhoI and BamHI respectively.
DNA fragmentation is cut glue by the AxyPrepDNAGelExtractionKit of the PCR primer Axygen company obtained reclaim.
(3) restricted enzyme XhoI and the BamHI that the 4cl genetic fragment in the pAM2 plasmid obtained in embodiment 1 and step (2) is utilized respectively NEB carries out double digestion.4cl genetic fragment after enzyme action and pAM2 plasmid are reclaimed with the AxyPrepDNAGelExtractionKit of Axygen company respectively, it is attached with T4DNA ligase (buying from NewEnglandBiolabs company), being connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain the recombiant plasmid pAM-4cl after connecting.
(4) synthesis stilbene synthase gene (sts) fragment, chalcone synthase gene (chs) fragment and curcumin synthase gene (cus) fragment:
Utilize online software JCat, chs gene in sts gene in Semen arachidis hypogaeae (Arachishypogaea), petunia (Petuniahybrida) and the cus gene in Oryza sativa L. (Oryzasativa) are carried out codon optimized by the codon preference according to cytoalgae PCC7942, and in front end plus EcoRI restriction enzyme site, end adds AflII restriction enzyme site.Utilize online software DNAWorks to design primer, carry out gene chemical synthesis by the method for recombinant PCR, and with the AxyPrepDNAGelExtractionKit of Axygen company, DNA fragmentation is cut glue and reclaim.
(5) by sts and the cus genetic fragment obtained in step (4) with utilize restricted enzyme EcoRI and AflII of NEB to carry out double digestion with the pAM-4cl plasmid in step (3) respectively.Sts, cus genetic fragment after enzyme action and pAM-4cl plasmid are reclaimed with the AxyPrepDNAGelExtractionKit of Axygen company respectively, it is attached with T4DNA ligase (buying from NewEnglandBiolabs company) respectively, being connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain recombiant plasmid pAM-sts-4cl and the pAM-cus-4cl after connecting.
(6) synthesizing chalcone isomerase gene (chi) fragment:
Utilize online software JCat, according to the codon preference of cytoalgae PCC7942, the chi gene in arabidopsis (Arabidopsisthaliana) is carried out codon optimized, and in front end plus XhoI restriction enzyme site, end adds EcoRI restriction enzyme site.Utilize online software DNAWorks to design primer, carry out gene chemical synthesis by the method for recombinant PCR, and with the AxyPrepDNAGelExtractionKit of Axygen company, DNA fragmentation is cut glue and reclaim.
(7) restricted enzyme EcoRI and the AflII that the chs genetic fragment in the pAM2 plasmid obtained in embodiment 1 and step (4) is utilized respectively NEB carries out double digestion.Chs genetic fragment after enzyme action and pAM2 plasmid are reclaimed with the AxyPrepDNAGelExtractionKit of Axygen company respectively, it is attached with T4DNA ligase (buying from NewEnglandBiolabs company), being connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain the recombiant plasmid pAM-chs after connecting.
(8) restricted enzyme EcoRI and the AflII of NEB is utilized to carry out double digestion the pAM-chs plasmid in step (7).Restricted enzyme EcoRI and the BamHI that genetic fragment in step (1) utilizes NEB is carried out double digestion.Restricted enzyme XhoI and the EcoRI that chi genetic fragment in step (6) utilizes NEB is carried out double digestion.Two genetic fragments after enzyme action and pAM-chs plasmid are reclaimed with the AxyPrepDNAGelExtractionKit of Axygen company respectively, it is attached with T4DNA ligase (buying from NewEnglandBiolabs company), being connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain the recombiant plasmid pAM-chs-chi-4cl after connecting.
(9) pAM-aroG in embodiment 1 is utilizedfbr-sam8 plasmid is template, adopts conventional method to carry out pcr amplification two containing different the Ptrc promoter of restriction enzyme site, aroGfbrWith sam8 genetic fragment, the method that this process is referred to described in " Molecular Cloning: A Laboratory guide (third edition) " carries out
The PCR primer of promoter Ptrc1: forward primer Ptrc1-F:
5′-catgcttaagCagcttatcatcgactgcacg-3 ', downstream primer Ptrc1-R:
5′-catggtcgacctctgtttcctgtgtgaaattgtt-3′.The base sequence that wherein underscore indicates is respectively
The restriction enzyme site of AflII and SalI.
The PCR primer of promoter Ptrc2: forward primer Ptrc2-F:
5′-catgaagcttCagcttatcatcgactgcacg-3 ', downstream primer Ptrc2-R:
5′-catgctcgagctctgtttcctgtgtgaaattgtt-3′.The base sequence that wherein underscore indicates is respectively
The restriction enzyme site of AflII and SalI.
Fragment aroGfbrPCR primer: forward primer aroGfbr-F:
5′-catggtcgacAtgaattatcagaacgatttacg-3 ', downstream primer aroGfbr-R:
5′-catgaagcttacccgcgacgcgct-3′.The base sequence that wherein underscore indicates be respectively SalI and
The restriction enzyme site of HindIII.
The PCR primer of fragment sam8: forward primer sam8-F:
5′-catgctcgagAtgacgcaggtcgtggaacg-3 ', downstream primer sam8-R:
5′-catgagatctttatccgaaatccttcccgt-3′.The base sequence that wherein underscore indicates is the restriction enzyme site of XhoI and BglII respectively.
DNA fragmentation is cut glue by the AxyPrepDNAGelExtractionKit of the PCR primer Axygen company obtained reclaim.
(10) restriction enzyme A flII and the BglII of NEB is utilized to carry out double digestion the pAM-chs-chi-4cl plasmid in pAM-sts-4cl and the pAM-cus-4cl plasmid in step (5) and step (8).Restriction enzyme A flII and the SalI that Ptrc1 genetic fragment in step (9) utilizes NEB is carried out double digestion.Restriction enzyme A flII and the SalI that Ptrc2 genetic fragment in step (9) utilizes NEB is carried out double digestion.By the aroG in step (9)fbrGenetic fragment utilizes restricted enzyme SalI and the HindIII of NEB to carry out double digestion.Restricted enzyme XhoI and the BglII that sam8 genetic fragment in step (9) utilizes NEB is carried out double digestion.The AxyPrepDNAGelExtractionKit of the genetic fragment after enzyme action and plasmid Axygen company is reclaimed.Four genetic fragments after reclaiming being attached with three plasmid T4DNA ligases (buying from NewEnglandBiolabs company) respectively, be connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain the recombiant plasmid pAM-sts-4cl-sam8-aroG after connectingfbr、pAM-chs-chi-4cl-sam8-aroGfbrAnd pAM-cus-4cl-sam8-aroGfbr。
Embodiment 3
Construction expression plasmid pAM-sam8-aroGfbr-ref8 and pAM-sam8-aroGfbr-ref8-comt:
(1) synthesis P-coumaric acid hydroxylase (ref8) fragment:
Utilize online software JCat, ref8 gene in arabidopsis (Arabidopsisthaliana) is carried out codon optimized by the codon preference according to cytoalgae PCC7942, and in front end plus EcoRI restriction enzyme site, end adds AflII restriction enzyme site.Utilize online software DNAWorks to design primer, carry out gene chemical synthesis by the method for recombinant PCR, and with the AxyPrepDNAGelExtractionKit of Axygen company, with reference to the method described in description, DNA fragmentation is cut glue and reclaim.
(2) restricted enzyme EcoRI and the AflII that the ref8 genetic fragment in the pAM2 plasmid obtained in embodiment 1 and step (2) is utilized respectively NEB carries out double digestion.Ref8 genetic fragment after enzyme action and pAM2 plasmid are reclaimed with reference to the method described in description with the AxyPrepDNAGelExtractionKit of Axygen company respectively, it is attached with T4DNA ligase (buying from NewEnglandBiolabs company), connection procedure is with reference to the method in description, being connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain the recombiant plasmid pAM-ref8 after connecting.
(3) pAM-sts-4cl-sam8-aroG obtained in embodiment 1 is utilizedfbrPlasmid is template, adopts conventional method to carry out the Ptrc1-aroG of pcr amplification series connectionfbr-Ptrc2-sam8 genetic fragment, this process is referred to the method described in " Molecular Cloning: A Laboratory guide (third edition) ".
PCR primer: upstream region of gene primer Ptrc1-F:5 '-catgcttaagCagcttatcatcgactgcacg-3 ', downstream primer sam8-R:5 '-catgagatctttatccgaaatccttcccgt-3′.The base sequence that wherein underscore indicates is the restriction enzyme site of AflII and BglII respectively.
DNA fragmentation is cut glue by the AxyPrepDNAGelExtractionKit of the PCR primer Axygen company obtained reclaim.
(4) Ptrc1-aroG in the pAM-ref8 plasmid (2) in step obtained and step (3)fbr-Ptrc2-sam8 genetic fragment is utilized respectively restriction enzyme A flII and the BglII of NEB and carries out double digestion.By the Ptrc1-aroG after enzyme actionfbr-Ptrc2-sam8 genetic fragment and pAM-ref8 plasmid reclaim with the AxyPrepDNAGelExtractionKit of Axygen company respectively, it is attached with T4DNA ligase (buying from NewEnglandBiolabs company), being connected in 16 DEG C of water-baths and carry out, the response time is 10h.Obtain the recombiant plasmid pAM-sam8-aroG after connectingfbr-sam5。
(5) synthesis caffeic acid gene (comt) fragment:
Utilize online software JCat, according to the codon preference of cytoalgae PCC7942, the comt gene in arabidopsis (Arabidopsisthaliana) is carried out codon optimized, and in front end plus XhoI restriction enzyme site, end adds BamHI restriction enzyme site.Utilize online software DNAWorks to design primer, carry out gene chemical synthesis by the method for recombinant PCR, and with the AxyPrepDNAGelExtractionKit of Axygen company, DNA fragmentation is cut glue and reclaim.
(6) pAM-sam8-aroG that (4) in step are obtainedfbrComt genetic fragment in-sam5 plasmid and step (5) is utilized respectively restriction enzyme A flII and the BglII of NEB and carries out double digestion.By the comt genetic fragment after enzyme action and pAM-sam8-aroGfbr-sam5 plasmid reclaims with the AxyPrepDNAGelExtractionKit of Axygen company respectively, is attached with T4DNA ligase (buying from NewEnglandBiolabs company), is connected in 16 DEG C of water-baths and carries out, and the response time is 10h.Obtain the recombiant plasmid pAM-sam8-aroG after connectingfbr-ref8-comt。
Embodiment 4
The conversion of cyanophyceae and the acquisition of genetic engineering cyanophyceae:
(1) cyanophyceae bacterial strain converts:
Take and be in exponential phase (OD730To 0.4-0.8) Cells of Blue-green Algae 10ml, collect thalline after centrifugal 5 minutes for 5000 revs/min, with aseptic 10mMNaCl washing once, then by BG11 culture medium fresh for 5ml, thalline is resuspended.Taking 500ul bacterium solution to EP pipe, add the recombiant plasmid of final concentration of 100ng/ml, after mixing, 30 DEG C of lucifuges are cultivated 12-18 hour.The mixture of Cells of Blue-green Algae Yu recombinant plasmid dna is coated BG11 solid medium (containing 20 μ g/mL spectinomycins), is placed in 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 10-14 days, single bacterium colony occurs to solid medium.
(2) acquisition of genetic engineering cyanophyceae:
Picking transformant from the solid medium after step (1) described conversion, accesses the fresh BG11 fluid medium (containing 20 μ g/mL spectinomycins) of 5ml, is placed in 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 7-10 days.Adopting conventional method to prepare the genomic DNA of genetic engineering cyanophyceae, this process is referred in " the fine works Molecular Biology " that Science Press publishes the miniprep procedure of bacterial genomes；Utilizing genetic engineering cyanophyceae genome is template, to proceed to the amplimer of exogenous gene on recombiant plasmid, adopts conventional method to carry out pcr amplification checking.
(3) preservation of genetic engineering cyanophyceae:
The genetic engineering cyanophyceae that step (2) obtains is inoculated in the BG11 fluid medium containing 20 μ g/mL spectinomycins, is placed in 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 7-10 days；Under sterile working, take in the overnight culture 1mL 1.5mL centrifuge tube joining sterilizing, 5000 revs/min of centrifugal 3min.Abandoning supernatant, with 15% glycerite of sterilizing, bacterial sediment is resuspended, preparation becomes glycerol stocks pipe, can preserve half a year to 1 year in-20 DEG C of refrigerators.Every half a year, take out the genetic engineering cyanophyceae preserved in glycerol stocks pipe and activate, and again preserve glycerol stocks pipe.
Embodiment 5
Genetic engineering cyanophyceae S-COU is utilized to produce P-coumaric acid:
(1) solid culture: genetic engineering cyanophyceae S-COU is inoculated on the BG11 solid medium containing 20 μ g/mL spectinomycins, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 10-15 days；
(2) seed culture: bacterial strain S-COU step (1) cultivated, is inoculated in 5mLBG11 fluid medium (containing 20 μ g/mL spectinomycins) with inoculating loop and carries out bacterial strain activation, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 7-10 days, prepare seed；
(3) convert and cultivate: according to 1% (volume ratio) inoculum concentration, the seed obtained in step (2) is inoculated into amplification culture in 50mLBG11 fluid medium (containing 20 μ g/mL spectinomycins), cultivate 3-5 days to OD for 30 DEG C730Reach 0.5-0.6, add 1mMIPTG, 30 DEG C of inducing culture 10-15 days, obtain the conversional solution containing P-coumaric acid；
(4) extraction of P-coumaric acid: the conversional solution salt acid for adjusting pH to less than 2 that will prepare in step (3), add equal-volume butyl acetate, rear chamber is gentle and quiet puts 1.5-2.5 hour in mixing, supernatant liquid is rotated evaporation after centrifugal 15 minutes by 5000 revs/min, and the powder obtained is P-coumaric acid；
(5) sample detection: P-coumaric acid powder step (4) prepared, identifies sample structure with LC-ESI-MS, and qualification result is as shown in Figure 2, it is determined that gained sample is P-coumaric acid.Detecting with HPLC, Fig. 1 schemes over time for converting P-coumaric acid concentration in cultivation, and the maximum output of P-coumaric acid is 128.2mg/l；
HPLC method uses Agilent1200 chromatograph of liquid, chromatographic column is EclipseXDB-C18 post (4.6 × 150mm), mobile phase A is water (containing 1% trifluoroacetic acid), Mobile phase B is acetonitrile (containing 1% trifluoroacetic acid), flow velocity is 1mL/min, gradient elution program is: 0 minute, 95% mobile phase A+5% Mobile phase B；8 minutes, 20% mobile phase A+80% Mobile phase B；10 minutes, 80% mobile phase A+20% Mobile phase B；11 minutes, 95% mobile phase A+5% Mobile phase B.UV-detector wavelength is 310nm, and column temperature is 30 DEG C, and P-coumaric acid appearance time is 5.50 minutes.
The LC-ESI-MS of P-coumaric acid prepared by this embodiment detects collection of illustrative plates as shown in Figure 2.
Embodiment 6
Genetic engineering cyanophyceae S-CAF is utilized to produce caffeic acid:
(1) solid culture: genetic engineering cyanophyceae S-CAF is inoculated on the BG11 solid medium containing 20 μ g/mL spectinomycins, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 10-15 days；
(2) seed culture: bacterial strain S-CAF step (1) cultivated, is inoculated in 5mLBG11 fluid medium (containing 20 μ g/mL spectinomycins) with inoculating loop and carries out bacterial strain activation, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 7-10 days, prepare seed；
(3) convert and cultivate: according to 1% (volume ratio) inoculum concentration, the seed obtained in step (2) is inoculated into amplification culture in 50mLBG11 fluid medium (containing 20 μ g/mL spectinomycins), cultivate 3-5 days to OD for 30 DEG C730Reach 0.5-0.6, add 1mMIPTG, 30 DEG C of inducing culture 10-15 days, obtain containing caffeinic conversional solution；
(4) caffeinic extraction: by conversional solution salt acid for adjusting pH prepared in step (3) to less than 2, add equal-volume butyl acetate, rear chamber is gentle and quiet puts 1.5-2.5 hour in mixing, supernatant liquid is rotated evaporation after centrifugal 15 minutes by 5000 revs/min, and the powder obtained is caffeic acid；
(5) sample detection: caffeic acid powder step (4) prepared, identifies sample structure with LC-ESI-MS, and qualification result is as shown in Figure 3, it is determined that gained sample is caffeic acid.Detecting with HPLC, caffeinic maximum output is 4.7mg/l；
HPLC method uses Agilent1200 chromatograph of liquid, chromatographic column is EclipseXDB-C18 post (4.6 × 150mm), mobile phase A is water (containing 1% trifluoroacetic acid), Mobile phase B is acetonitrile (containing 1% trifluoroacetic acid), flow velocity is 1mL/min, gradient elution program is: 0 minute, 95% mobile phase A+5% Mobile phase B；8 minutes, 20% mobile phase A+80% Mobile phase B；10 minutes, 80% mobile phase A+20% Mobile phase B；11 minutes, 95% mobile phase A+5% Mobile phase B.UV-detector wavelength is 310nm, and column temperature is 30 DEG C, and caffeic acid appearance time is 4.89 minutes.
Prepared by this embodiment detects collection of illustrative plates as shown in Figure 3 to caffeinic LC-ESI-MS.
Embodiment 7
Genetic engineering cyanophyceae S-FER is utilized to produce ferulic acid:
(1) solid culture: genetic engineering cyanophyceae S-FER is inoculated on the BG11 solid medium containing 20 μ g/mL spectinomycins, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 10-15 days；
(2) seed culture: bacterial strain S-FER step (1) cultivated, is inoculated in 5mLBG11 fluid medium (containing 20 μ g/mL spectinomycins) with inoculating loop and carries out bacterial strain activation, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 7-10 days, prepare seed；
(3) convert and cultivate: according to 1% (volume ratio) inoculum concentration, the seed obtained in step (2) is inoculated into amplification culture in 50mLBG11 fluid medium (containing 20 μ g/mL spectinomycins), cultivate 3-5 days to OD for 30 DEG C730Reach 0.5-0.6, add 1mMIPTG, 30 DEG C of inducing culture 10-15 days, obtain the conversional solution containing ferulic acid；
(4) extraction of ferulic acid: the conversional solution salt acid for adjusting pH to less than 2 that will prepare in step (3), add equal-volume butyl acetate, rear chamber is gentle and quiet puts 1.5-2.5 hour in mixing, supernatant liquid is rotated evaporation after centrifugal 15 minutes by 5000 revs/min, and the powder obtained is ferulic acid；
(5) sample detection: ferulic acid powder step (4) prepared, identifies sample structure with LC-ESI-MS, and qualification result is as shown in Figure 4, it is determined that gained sample is ferulic acid.Detecting with HPLC, the maximum output of ferulic acid is 6.3mg/l；
HPLC method uses Agilent1200 chromatograph of liquid, chromatographic column is EclipseXDB-C18 post (4.6 × 150mm), mobile phase A is water (containing 1% trifluoroacetic acid), Mobile phase B is acetonitrile (containing 1% trifluoroacetic acid), flow velocity is 1mL/min, gradient elution program is: 0 minute, 95% mobile phase A+5% Mobile phase B；8 minutes, 20% mobile phase A+80% Mobile phase B；10 minutes, 80% mobile phase A+20% Mobile phase B；11 minutes, 95% mobile phase A+5% Mobile phase B.UV-detector wavelength is 310nm, and column temperature is 30 DEG C, and ferulic acid appearance time is 5.67 minutes.
LC-ESI-MS to ferulic acid prepared by this embodiment detects collection of illustrative plates as shown in Figure 4.
Embodiment 8
Genetic engineering cyanophyceae S-REV is utilized to produce resveratrol:
(1) solid culture: genetic engineering cyanophyceae S-REV is inoculated on the BG11 solid medium containing 20 μ g/mL spectinomycins, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 10-15 days；
(2) seed culture: bacterial strain S-NAR step (1) cultivated, is inoculated in 5mLBG11 fluid medium (containing 20 μ g/mL spectinomycins) with inoculating loop and carries out bacterial strain activation, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 7-10 days, prepare seed；
(3) convert and cultivate: according to 1% (volume ratio) inoculum concentration, the seed obtained in step (2) is inoculated into amplification culture in 50mLBG11 fluid medium (containing 20 μ g/mL spectinomycins), cultivate 3-5 days to OD for 30 DEG C730Reach 0.5-0.6, add 1mMIPTG, 30 DEG C of inducing culture 10-15 days, obtain the conversional solution containing resveratrol；
(4) extraction of resveratrol: the conversional solution salt acid for adjusting pH to less than 2 that will prepare in step (3), add equal-volume butyl acetate, rear chamber is gentle and quiet puts 1.5-2.5 hour in mixing, supernatant liquid is rotated evaporation after centrifugal 15 minutes by 5000 revs/min, and the powder obtained is resveratrol；
(5) sample detection: resveratrol powder step (4) prepared, identifies sample structure with LC-ESI-MS, and qualification result is as shown in Figure 5, it is determined that gained sample is resveratrol.Detecting with HPLC, the maximum output of resveratrol is 4.6mg/l；
HPLC method uses Agilent1200 chromatograph of liquid, chromatographic column is EclipseXDB-C18 post (4.6 × 150mm), mobile phase A is water (containing 1% trifluoroacetic acid), Mobile phase B is acetonitrile (containing 1% trifluoroacetic acid), flow velocity is 1mL/min, gradient elution program is: 0 minute, 95% mobile phase A+5% Mobile phase B；8 minutes, 20% mobile phase A+80% Mobile phase B；10 minutes, 80% mobile phase A+20% Mobile phase B；11 minutes, 95% mobile phase A+5% Mobile phase B.UV-detector wavelength is 310nm, and column temperature is 30 DEG C, and resveratrol appearance time is 6.34 minutes.
LC-ESI-MS to resveratrol prepared by this embodiment detects collection of illustrative plates as shown in Figure 5.
Embodiment 9
Genetic engineering cyanophyceae S-NAR is utilized to produce naringenin:
(1) solid culture: genetic engineering cyanophyceae S-NAR is inoculated on the BG11 solid medium containing 20 μ g/mL spectinomycins, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 10-15 days；
(2) seed culture: bacterial strain S-NAR step (1) cultivated, is inoculated in 5mLBG11 fluid medium (containing 20 μ g/mL spectinomycins) with inoculating loop and carries out bacterial strain activation, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 7-10 days, prepare seed；
(3) convert and cultivate: according to 1% (volume ratio) inoculum concentration, the seed obtained in step (2) is inoculated into amplification culture in 50mLBG11 fluid medium (containing 20 μ g/mL spectinomycins), cultivate 3-5 days to OD for 30 DEG C730Reach 0.5-0.6, add 1mMIPTG, 30 DEG C of inducing culture 10-15 days, obtain the conversional solution containing naringenin；
(4) extraction of naringenin: the conversional solution salt acid for adjusting pH to less than 2 that will prepare in step (3), add equal-volume butyl acetate, rear chamber is gentle and quiet puts 1.5-2.5 hour in mixing, supernatant liquid is rotated evaporation after centrifugal 15 minutes by 5000 revs/min, and the powder obtained is naringenin；
(5) sample detection: naringenin powder step (4) prepared, identifies sample structure with LC-ESI-MS, and qualification result is as shown in Figure 6, it is determined that gained sample is naringenin.Detecting with HPLC, the maximum output of naringenin is 7.1mg/l；
HPLC method uses Agilent1200 chromatograph of liquid, chromatographic column is EclipseXDB-C18 post (4.6 × 150mm), mobile phase A is water (containing 1% trifluoroacetic acid), Mobile phase B is acetonitrile (containing 1% trifluoroacetic acid), flow velocity is 1mL/min, gradient elution program is: 0 minute, 95% mobile phase A+5% Mobile phase B；8 minutes, 20% mobile phase A+80% Mobile phase B；10 minutes, 80% mobile phase A+20% Mobile phase B；11 minutes, 95% mobile phase A+5% Mobile phase B.UV-detector wavelength is 310nm, and column temperature is 30 DEG C, and naringenin appearance time is 7.18 minutes.
LC-ESI-MS to naringenin prepared by this embodiment detects collection of illustrative plates as shown in Figure 6.
Embodiment 10
Genetic engineering cyanophyceae S-DC is utilized to produce bisdemethoxycurcumin:
(1) solid culture: genetic engineering cyanophyceae S-DC is inoculated on the BG11 solid medium containing 20 μ g/mL spectinomycins, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 10-15 days；
(2) seed culture: bacterial strain S-DC step (1) cultivated, is inoculated in 5mLBG11 fluid medium (containing 20 μ g/mL spectinomycins) with inoculating loop and carries out bacterial strain activation, 30 DEG C, 100 μ E s-1·m-2Light intensity under continuous illumination cultivate 7-10 days, prepare seed；
(3) convert and cultivate: according to 1% (volume ratio) inoculum concentration, the seed obtained in step (2) is inoculated into amplification culture in 50mLBG11 fluid medium (containing 20 μ g/mL spectinomycins), cultivate 3-5 days to OD for 30 DEG C730Reach 0.5-0.6, add 1mMIPTG, 30 DEG C of inducing culture 10-15 days, obtain the conversional solution containing bisdemethoxycurcumin；
(4) extraction of bisdemethoxycurcumin: the conversional solution salt acid for adjusting pH to less than 2 that will prepare in step (3), add equal-volume butyl acetate, rear chamber is gentle and quiet puts 1.5-2.5 hour in mixing, supernatant liquid is rotated evaporation after centrifugal 15 minutes by 5000 revs/min, and the powder obtained is bisdemethoxycurcumin；
(5) sample detection: bisdemethoxycurcumin powder step (4) prepared, identifies sample structure with LC-ESI-MS, and qualification result is as shown in Figure 7, it is determined that gained sample is bisdemethoxycurcumin.Detecting with HPLC, the maximum output of bisdemethoxycurcumin is 4.1mg/l；
HPLC method uses Agilent1200 chromatograph of liquid, chromatographic column is EclipseXDB-C18 post (4.6 × 150mm), mobile phase A is water (containing 1% trifluoroacetic acid), Mobile phase B is acetonitrile (containing 1% trifluoroacetic acid), flow velocity is 1mL/min, gradient elution program is: 0 minute, 95% mobile phase A+5% Mobile phase B；8 minutes, 20% mobile phase A+80% Mobile phase B；10 minutes, 80% mobile phase A+20% Mobile phase B；11 minutes, 95% mobile phase A+5% Mobile phase B.UV-detector wavelength is 310nm, and column temperature is 30 DEG C, and bisdemethoxycurcumin appearance time is 8.47 minutes.
LC-ESI-MS to bisdemethoxycurcumin prepared by this embodiment detects collection of illustrative plates as shown in Figure 7.
The preferred embodiment of the present invention described in detail above.Should be appreciated that the ordinary skill of this area just can make many modifications and variations according to the design of the present invention without creative work.Therefore, all technical staff in the art, all should in the protection domain being defined in the patent claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.

Claims (9)

1. a genetic engineering cyanophyceae, it is characterised in that described genetic engineering cyanophyceae is prepared from by one of them of following methods:
Method one: the genomic DNA that the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase and the encoding gene of tyrosine ammonia lyase that solve feedback suppression import wild cyanophyceae obtains producing the genetic engineering cyanophyceae of P-coumaric acid；
Method two: obtain producing caffeinic genetic engineering cyanophyceae by the genomic DNA solving the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, the encoding gene of tyrosine ammonia lyase and the encoding gene of P-coumaric acid hydroxylase and importing wild cyanophyceae；
Method three: the genomic DNA solving the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, the encoding gene of tyrosine ammonia lyase, the encoding gene of P-coumaric acid hydroxylase and the encoding gene of caffeic acid and importing wild cyanophyceae is obtained producing the genetic engineering cyanophyceae of ferulic acid；
Method four: by solve the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, tyrosine ammonia lyase encoding gene, the encoding gene of coumaroyl A ligase and the encoding gene of stilbene synthase imported the genomic DNA of wild cyanophyceae obtain producing the genetic engineering cyanophyceae of resveratrol；
Method five: by solve the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, tyrosine ammonia lyase encoding gene, the encoding gene of coumaroyl A ligase, the encoding gene of chalcone synthase and the encoding gene of enzyme, namely chalcone isomerase imported the genomic DNA of wild cyanophyceae obtain producing the genetic engineering cyanophyceae of naringenin；
Method six: by solve the encoding gene of 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of feedback suppression, tyrosine ammonia lyase encoding gene, the encoding gene of coumaroyl A ligase and the encoding gene of curcumin synthase are imported the genetic engineering cyanophyceae producing bisdemethoxycurcumin that the genomic DNA of wild cyanophyceae obtains.
2. genetic engineering cyanophyceae as claimed in claim 1, it is characterised in that described wild cyanophyceae is selected from cyanophyceaes such as Synechococcus genus, synechocystis, hidden ball Trentepohlia, Anabaena, nostoc, Oscillatoria, ball Trentepohlia, Ah lattice's Trentepohlia, Scytonema and whip branch Trentepohlias.
3. genetic engineering cyanophyceae as claimed in claim 2, it is characterised in that described wild cyanophyceae is cytoalgae (Synechocystissp.).
4. genetic engineering cyanophyceae as claimed in claim 1, it is characterised in that exogenous gene imports the genomic DNA of wild cyanophyceae by being connected in plasmid.
5. genetic engineering cyanophyceae as claimed in claim 4, it is characterized in that, the exogenous gene structure of expressing in series contain one with in cascaded structure first promoter that gene is operably connected, or on described cascaded structure, each gene is each independent of the promoter being operably connected.
6. genetic engineering cyanophyceae as claimed in claim 5, it is characterised in that described promoter is trc promoter.
7. genetic engineering cyanophyceae as claimed in claim 1, it is characterised in that
The encoding gene of the 3-deoxidation-2-Arab ketoheptose-7-phosphate synthase of described solution feedback suppression is such as shown in SEQIDNO:1 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:1；
The encoding gene of described tyrosine ammonia lyase is such as shown in SEQIDNO:2 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:2；
The encoding gene of described P-coumaric acid hydroxylase is such as shown in SEQIDNO:3 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:3；
The encoding gene of described caffeic acid is such as shown in SEQIDNO:4 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:4；
The described encoding gene to coumaroyl A ligase is such as shown in SEQIDNO:5 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:5；
The encoding gene of described stilbene synthase is such as shown in SEQIDNO:6 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:6；
The encoding gene of described chalcone synthase is such as shown in SEQIDNO:7 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:7；
The encoding gene of described enzyme, namely chalcone isomerase is such as shown in SEQIDNO:8 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:8；
The encoding gene of described curcumin synthase is such as shown in SEQIDNO:9 or can with the complementary strand thereof of the nucleotide sequence shown in SEQIDNO:9.
8. genetic engineering cyanophyceae as claimed in claim 7, it is characterised in that
3-deoxidation-2-Arab ketoheptose-7-the phosphate synthase of described solution feedback suppression derives from escherichia coli；
Described tyrosine ammonia lyase derives from saccharothrix；
Described P-coumaric acid hydroxylase derives from arabidopsis；
Described caffeic acid derives from arabidopsis；
Described derives from parsley to coumaroyl A ligase；
Described stilbene synthase derives from Semen arachidis hypogaeae；
Described chalcone synthase derives from petunia；
Described enzyme, namely chalcone isomerase derives from arabidopsis；
Described curcumin synthase derives from Oryza sativa L..
9. the application in producing phenylpropyl alcohol alkanes material of the genetic engineering cyanophyceae as described in arbitrary in claim 1-8.
CN201610211295.6A 2016-04-06 2016-04-06 Genetically engineered blue algae and application thereof Pending CN105754920A (en)

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