CN103484420A - Genetic engineering strain taking tyrosine as substrate to synthesize naringenin and construction method thereof - Google Patents
Genetic engineering strain taking tyrosine as substrate to synthesize naringenin and construction method thereof Download PDFInfo
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Abstract
The invention discloses a genetic engineering stain taking tyrosine as a substrate to synthesize naringenin and a construction method thereof, belonging to the field of synthetic biology or metabolic engineering. According to the construction method disclosed by the invention, a synthesis route constituted by six genes, namely a gene matB encoding a malonic acid transporting enzyme, a gene matC encoding a malonic acid absorption route, a gene encoding a tyrosine ammonialyase (TAL), the gene encoding a 4-cinnamic acid: coenzyme A ligase (4CL), the gene encoding a chalcone synthase (CHS) and the gene encoding a chalcone isomerase (CHI) is introduced into Escherichia coli BL21 to obtain a recombinant strain capable of taking the tyrosine as the substrate to synthesize the naringenin, the synthesis route is further optimized through a modular transformation theory, the strain is finally fermented in a shaking flask for 72 hours by taking the tyrosine as the substrate, and the yield of the naringenin achieves 90mg/L. A strategy adopted by the construction method disclosed by the invention provides certain reference significance for future production of natural small molecular substances by a microbiological method.
Description
Technical field
The present invention relates to a strain take tyrosine and is genetic engineering bacterium and the construction process thereof of the synthetic naringenin of substrate to belong to synthetic biology or metabolic engineering field.
Background technology
The compound that flavonoid compound (flavonoids) is that a class is present in is natural, have 2-phenyl chromone structure, its many tools of hydroxy derivatives yellow, thereby gain the name.Flavonoid compound has multiple biological activity, mainly comprise cardiovascular systems, antibiotic and antiviral, antitumor, resisting oxidation free radical, anti-inflammatory etc., to easing pain, protect the liver, anti-mutation, hypoglycemic and relieving cough and asthma etc. there is prevention, treatment or adjuvant treatment effect, and human body can not synthesize flavonoid compound, can only from plant food, obtain, therefore in recent years, the market of flavonoid compound is annual to be increased more than 30%, on medicine and nutrient chemistry product field, has broad application prospects.
Flavonoid compound is widely distributed, of a great variety, flavonoid compound mainly comprises that flavonoid is (as Baicalein, yellow Cen glycosides etc.), flavonols is (as Quercetin, 5,7-dihydroxy-2-phenyl-4-chromanone etc.), flavanone is (as hesperetin, liquirtin etc.), the flavanone alcohols is (as silymarin, Silydianin etc.), osajin is (as Dai, puerarin etc.), isoflavanone class (as tubatoxin etc.), chalcones is (as isoliquiritigenin, Corylifolinin etc.), aurones class (as aureusidin etc.), flavanoid (as catechin etc.), anthocyan is (as delphinidin, Cyanidin etc.) and bisflavones (as Ginkgetin, different Ginkgetin etc.).And wherein naringenin is a kind of very important flavones skeletal substance, can obtain other Flavonoid substances of kind more than 8,000 by naringenin being carried out to the modifications such as hydroxylation, reducing, alkylation, oxidation, naringenin itself has multiple biological activity as a kind of flavonoid substance in addition, mainly comprise alleviation neurodynia, prevent the generation of cerebral edema, reduce the occurrence probability of cerebral ischemia etc.
At present, industrial production naringenin mainly adopts the method for plant extract, but how from the extracting solution of plant complexity, to obtain needed high-purity naringenin and remain the insurmountable technical barrier of the mankind, and high pollution, use is poisonous and the effects limit such as complex construction of harmful chemical reagent and naringenin the use of chemical method, just because of this, microbe fermentation method receives increasing concern with the discharge of using cheap free of contamination raw material, lower pollution, the lower advantages such as energy demand.The domestic and international research about the Production by Microorganism Fermentation naringenin is all to take coumaric acid as substrate at present, yet coumaric acid is expensive, and solubleness is lower, is difficult to be present in general substratum; What current fermentation method all adopted in addition is two-step approach production, be that at first bacterial strain grows in nutritious substratum, reach certain bacterium dense in, collect all thalline by the method such as centrifugal and be placed in minimum medium and ferment, obviously these have all suppressed the application of microbial method in large-scale industrial production.
It is to take more cheap tyrosine as substrate that the present invention becomes, and transforms the policy optimization bacterial strain by modularization and make it obtain higher naringenin output in single substratum, for large-scale industrial production has been established certain basis.
Summary of the invention
The technical problem to be solved in the present invention is to provide a strain take tyrosine and is genetic engineering bacterium and the construction process thereof of the synthetic naringenin of substrate, is coexpression 3 plasmid: pCDF-RgTAL-Pc4CL, pET-PhCHS-MsCHI and pACYCD-matB-matC in Escherichia coli (BL21).
Described pCDF-RgTAL-Pc4CL has carried coding tyrosine desaminase TAL and the fragrant cinnamic acid of 4-: the pCDFDuet-1 of the gene of CoA ligase 4CL.Described pET-PhCHS-MsCHI is the pETDuet-1 that has carried the gene of coding chalcone synthase CHS, enzyme, namely chalcone isomerase CHI.Described pACYCD-matB-matC is the pACYCDuet-1 that has carried the gene matC of the propanedioic acid transporter gene matB that encodes, propanedioic acid absorption enzyme.PCDF-RgTAL-Pc4CL adopts promotor Trc, and copy number is 20; PET-PhCHS-MsCHI adopts promotor T7, and copy number is 40; PACYCD-matB-matC adopts promotor T7, and plasmid copy number is 10.
The gene matC nucleotide sequence that the described propanedioic acid of encoding is transported is as shown in SEQ ID NO.1, the described propanedioic acid of encoding absorbs the gene matB nucleotide sequence of enzyme as shown in SEQ ID NO.2, encode the gene nucleotide series of described tyrosine desaminase TAL as shown in SEQ ID NO.3, the fragrant cinnamic acid of the described 4-that encodes: the gene nucleotide series of CoA ligase 4CL is as shown in SEQ ID NO.4, encode the gene nucleotide series of described chalcone synthase CHS as shown in SEQ ID NO.5, encode the gene nucleotide series of described enzyme, namely chalcone isomerase CHI as shown in SEQ ID NO.6.
The scheme of Escherichia coli (BL21) recombinant bacterium that structure contains described route of synthesis mainly comprises following content:
(1) adopt full method for synthesizing gene to obtain needed six genes of approach;
(2) cut the connection structure by enzyme and obtain three co-expression plasmid pCDF-RgTAL-Pc4CL, pET-PhCHS-MsCHI, pACYCD-matB-matC, after sequence verification, by three co-expression plasmids with chemical method Transformed E scherichia coli (BL21) competent cell;
(3) select the ammonia benzyl that 100ug/mL can contained, the paraxin of 35ug/mL, the bacterium colony of growing on four kinds of antibiotic LB flat boards such as the Streptomycin sulphate of 50ug/mL, make it contain on four kinds of above-mentioned antibiotic LB flat boards the three generations that transfers continuously, obtain the recon of inheritance stability, further six pathway genes of PCR checking successfully are transferred in Escherichia coli (BL21).
The method of Escherichia coli (BL21) the recombinant bacterium fermentative production naringenin that utilization contains described route of synthesis:
(1) MOPS substratum (g/L): glucose 5, ammonium chloride 4, dipotassium hydrogen phosphate 0.3, propanedioic acid 2, MOPS83.7, N-{ tri-(carboxymethyl) methyl } glycine 7.1668, ammonium chloride 5.1, ferric sulfate 0.03, vitriolate of tartar 0.5, magnesium chloride 1.1, sodium-chlor 29.2, trace element (formula of ATCC BAA-2326) 10ml, the tap water constant volume; The sterilising temp of MOPS substratum is 115-121 ℃, 15min, and 0.22 μ m membrane filtration degerming after the preparation of the temperature-sensitive materials such as VITAMIN, add before inoculation.
(2) engineering strain structure obtained is inoculated in the MOPS substratum, and 500mL shaking flask dress liquid 25mL cultivates 12-20h to bacterial strain OD under 37 ℃, 250rpm condition
600reach 1.0-2.0; And then add MOPS substratum that 25ml is identical and the IPTG of final concentration 1mM, 30 ℃, under the 250rpm condition, cultivate 60h.
Beneficial effect of the present invention: the present invention is that to take a strain Escherichia coli (BL21) be starting strain, utilize synthetic biology and metabolic engineering means at the bacterial strain construct in vitro one from tyrosine to the naringenin by six route of synthesis that gene forms such as TAL, 4CL, CHS, CHI, matB, matC, total approach has been divided into to three modules, by the metabolic flux of fine setting module, make metabolism stream farthest flow to end product.Recombinant bacterium Escherichia coli (BL21) directly take tyrosine as substrate, after fermentation 72h, the output of naringenin reaches 90mg/L, without adding valuable precursor substance, only in single substratum, just can synthesize naringenin, successfully realize take that tyrosine synthesizes naringenin as substrate.The modularization transformation strategy wherein adopted is produced other natural micromolecular compounds for microbial method from now on certain reference is provided.
The accompanying drawing explanation
Fig. 1 modularization transformation and optimization naringenin output.
Fig. 2 LC-MS analyzes the naringenin that recombinant bacterial strain produces; A1, the LC/ESI-MS collection of illustrative plates of product in fermented liquid; A2, the MS/MS collection of illustrative plates of product in fermented liquid; B1, the LC/ESI-MS collection of illustrative plates of standard substance; B2, the MS/MS collection of illustrative plates of standard substance.
Embodiment
The mensuration of naringenin concentration: high performance liquid chromatography (HPLC)
Instrument: Agilent1100 high performance liquid chromatograph (joining UV-vis detector, differential refraction detector and workstation), chromatographic condition:
Chromatographic column: Aminex HPX-87H ion exchange column
Moving phase: 5mM H
2sO
4
Flow velocity: 0.6mL/min
Column temperature: 35 ℃
Sample size: 5 μ L
UV-detector wavelength: 320nm
Sample preparation: 500 μ L fermented liquid centrifugal 10min under 10,000rpm, get supernatant liquor and move into the content of surveying naringenin in the 1.5mL centrifuge tube.Get 100 μ L supernatant liquors and move in the 5mL volumetric flask, ultrapure water is settled to scale marks, and through 0.45 μ m membrane filtration, filtrate is for liquid-phase chromatographic analysis.
MOPS substratum (g/L): glucose 5, ammonium chloride 4, dipotassium hydrogen phosphate 0.3, propanedioic acid 2, MOPS83.7, N-{ tri-(carboxymethyl) methyl } glycine 7.1668, ammonium chloride 5.1, ferric sulfate 0.03, vitriolate of tartar 0.5, magnesium chloride 1.1, sodium-chlor 29.2, trace element (formula of ATCC BAA-2326) 10ml, tap water is settled to 1L.The sterilising temp of MOPS substratum is 115-121 ℃, 15min.0.22 μ m membrane filtration degerming after the preparation of the temperature-sensitive materials such as VITAMIN, add before inoculation.
The selection of embodiment 1 pathway gene
Phenylalanine deaminase (TAL) extensively is present in higher plant, fungi, yeast and a kind of prokaryotic organism streptomycete, and the mankind also do not find this enzyme so far in eubacterium or animal tissues.Although it is very wide that TAL distributes, only have R.glutinis to be used for doing commercial use, R.glutinis is also high than other kinds to the catalytic activity of tyrosine simultaneously.In nearest research, in nearest research, the 4-coumaric acid of people with P.crispum arranged: the combination of the chalcone synthase (CHS) of CoA ligase (4CL), P.hybrida, the enzyme, namely chalcone isomerase (CHI) of M.sativa obtains various non-natural flavonoid skeletal substances.Therefore the present invention selects TAL, the 4CL of curly parsley (Petroselinum crispum), the CHS of petunia (Petunia X hybrida), the CHI (M91079) of alfalfa (Medicago sativa) of rhodothece rubra (Rhodotorula glutinis).
The development of natural product production platform is subject to the restriction of precursor substance or cofactor usually, because in Host Strains, these amount of substances all can not meet the requirement of large-scale industrial production usually.When forming the flavonoid skeletal substance, the skeletal substance of a molecule of every generation needs the malonyl coenzyme A of 3mol.And intestinal bacteria are in Culture Medium Exponential when growth of having supplemented glucose, wherein acetyl-CoA is main ingredient in the coenzyme A metabolic pool, and malonyl coenzyme A only accounts for wherein a part seldom.Therefore the malonyl coenzyme A molecule come from Bacillus coli cells becomes the restricted precursor molecule.The generation approach of malonyl coenzyme A has two kinds, a kind of is that acetyl-CoA is converted into malonyl coenzyme A under the effect of acetyl-CoA carboxylase (ACC), therefore can improve output by coexpression ACC gene, vitamin H ligase enzyme gene (birA) and acetyl-CoA-synthetase (ACS); Another kind is directly malonyl-and coenzyme A to be converted into to malonyl coenzyme A by the malonyl coenzyme A synthetic enzyme, and gene (matC) gene that the gene (matB) that at this moment can transport by the propanedioic acid of overexpression clover rhizobia (Rhizobium trifolii) and propanedioic acid absorb approach improves output.But front a kind of pathways metabolism is unfavorable to scale operationization because will add vitamin H, therefore select rear kind of pathways metabolism optimization.
The gene matC nucleotide sequence of coding propanedioic acid transhipment is as shown in SEQ ID NO.1, the coding propanedioic acid absorbs the gene matB nucleotide sequence of enzyme as shown in SEQ ID NO.2, the gene nucleotide series of coding tyrosine desaminase TAL is as shown in SEQ ID NO.3, the fragrant cinnamic acid of coding 4-: the gene nucleotide series of CoA ligase 4CL is as shown in SEQ ID NO.4, the gene nucleotide series of coding chalcone synthase CHS is as shown in SEQ ID NO.5, and the gene nucleotide series of coding enzyme, namely chalcone isomerase CHI is as shown in SEQ ID NO.6.
For the route of synthesis built, this research wish is divided into three modules by it, mainly based on following principle: i) the TAL gene of the product coumaric acyl coenzyme A of 4CL/cinnyl coenzyme A in can the inhibition approach, therefore TAL and 4CL are divided into to a module, CHS and CHI are divided into to a module, wish, by the strengthening downstream module, weakens up-stream module and alleviates this feedback inhibition; When ii) thalline allos is synthesized the flavonoid skeletal substance, the content that malonyl coenzyme A is lower has limited endobacillary yield of flavone usually, therefore malonyl coenzyme A route of synthesis (matB and matC) is placed in to a separate modular, by the metabolic flux that changes this module, finds optimum malonyl coenzyme A content.Thus total approach is divided into to three modules respectively: module one is comprised of TAL and 4CL; Module two is that CHS and CHI form; Module three is that matB and matC form.The metabolic flux of each module is decided by the dynamics of plasmid copy number and promotor, four plasmids that native system is used are respectively pACYCDuet-1(Novagen:71430), pCDFDuet-1(Novagen:71330), pETDuet-1(Novagen:71353) and pRSFDuet-1(Novagen:71363), wherein plasmid copy number is respectively 10,20,40 and 100; Promotor is elected T7 and Trc as, and the dynamics that wherein dynamics of T7 is 5, Trc is 1.Thereby change the plasmid copy number of each module and the metabolic flux that the promotor dynamics changes module by system, be tested and appraised the content of intermediate product and final product, determine optimum module combination.
As shown in Figure 1, finally find optimum module combination: module one expression plasmid is pCDFDuet-1, and promotor is Trc; Module two expression plasmids are pETDuet-1, and promotor is T7; Module three expression plasmids are pACYCDuet-1, and promotor is T7.
Embodiment 3 is containing the structure of optimizing the modular engineering bacterium
Adopt full method for synthesizing gene to obtain needed six genes of approach, size is respectively 2.1Kb, 1.7Kb, 1.5Kb, 0.9Kb, 1.5Kb, 1.3Kb.Utilize respectively enzyme to cut interconnection technique by TAL, the 4CL gene with through NcoI and HindIII, NdeI is connected with the plasmid after the BlnI enzyme is cut, by CHS, the CHI gene with through NcoI and HindIII, NdeI is connected with the plasmid after the BlnI enzyme is cut, by matB and matC gene and process EcoRI and HindIII, NdeI is connected with the plasmid after the KpnI enzyme is cut, and obtains thus three co-expression plasmid pCDF-RgTAL-Pc4CL, pET-PhCHS-MsCHI, pACYCD-matB-matC.The recombinant plasmid built is through restriction analysis, and carries out DNA sequencing.The gene sequencing result is consistent with expection, shows that construction of recombinant plasmid is correct.By three co-expression plasmids with chemical method Transformed E scherichia coli (BL21) competent cell.Selecting can be at the ammonia benzyl that contains 100ug/mL, the paraxin of 35ug/mL, and the bacterium colony of growing on three kinds of microbiotic LB flat boards such as the Streptomycin sulphate of 50ug/mL, make it containing the three generations that transfers continuously on three kinds of above-mentioned antibiotic LB flat boards, obtains the recon of inheritance stability.The picking positive recombinant, the sequence verification result, show that six pathway genes successfully are transferred in Escherichia coli (BL21).Wherein:
LB substratum (g/L)
Sodium-chlor 10g, Tryptone10g, Yeast extract5g, solid medium adds 20g agar.
The resistant panel (g/L) of LB+ ammonia benzyl, paraxin, Streptomycin sulphate
LB+100mg ammonia benzyl, 35mg paraxin, 50mg Streptomycin sulphate, solid medium adds 20g agar.
The method of embodiment 4 fermentative production naringenins
Adopting the genetic engineering bacterium that embodiment 3 obtains is starting strain, is inoculated in the MOPS substratum, and 500mL shaking flask dress liquid 25mL cultivates 12-20h to bacterial strain OD under 37 ℃, 250rpm condition
600reach 1.0-2.0; And then add the IPTG that MOPS substratum that 25ml is identical and final concentration are 1mM,, 30 ℃, under the 250rpm condition, cultivate 60h.
Recombinant bacterium is compared with the bacterium that contrasts that transforms empty plasmid, do not detect the output of naringenin in the contrast bacterium, and in recombinant bacterium, the output of naringenin reaches 90mg/L.
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (8)
1. the genetic engineering bacterium of tyrosine as the synthetic naringenin of substrate take in a strain, it is characterized in that, be to have expressed the key enzyme system that the naringenin route of synthesis needs in Escherichia coli (BL21): the fragrant cinnamic acid of tyrosine desaminase TAL, 4-: CoA ligase 4CL, chalcone synthase CHS, enzyme, namely chalcone isomerase CHI, propanedioic acid transhipment enzyme matC and propanedioic acid absorb enzyme matB.
2. genetic engineering bacterium according to claim 1, it is characterized in that, the gene matC nucleotide sequence that the described propanedioic acid of encoding is transported is as shown in SEQ ID NO.1, the described propanedioic acid of encoding absorbs the gene matB nucleotide sequence of enzyme as shown in SEQ ID NO.2, encode the gene nucleotide series of described tyrosine desaminase TAL as shown in SEQ ID NO.3, the fragrant cinnamic acid of the described 4-that encodes: the gene nucleotide series of CoA ligase 4CL is as shown in SEQ ID NO.4, encode the gene nucleotide series of described chalcone synthase CHS as shown in SEQ ID NO.5, encode the gene nucleotide series of described enzyme, namely chalcone isomerase CHI as shown in SEQ ID NO.6.
3. genetic engineering bacterium according to claim 1 and 2, it is characterized in that, described 6 genes carry out coexpression by 3 plasmids, described 3 plasmids are: (1) pCDFDuet-1 carries coding tyrosine desaminase TAL and the fragrant cinnamic acid of 4-: the gene of CoA ligase 4CL, obtain plasmid pCDF-RgTAL-Pc4CL, (2) pETDuet-1 carries coding chalcone synthase CHS, the gene of enzyme, namely chalcone isomerase CHI, obtain plasmid pET-PhCHS-MsCHI, (3) pACYCDuet-1 carries coding propanedioic acid transporter gene matC, propanedioic acid absorbs the gene matB of enzyme, obtain plasmid pACYCD-matB-matC.
4. genetic engineering bacterium according to claim 3, is characterized in that, described pCDF-RgTAL-Pc4CL adopts promotor Trc, and copy number is 20; Described pET-PhCHS-MsCHI adopts promotor T7, and copy number is 40; Described pACYCDuet-1 adopts promotor T7, and plasmid copy number is 10.
5. build the method for the described genetic engineering bacterium of claim 4, it is characterized in that, comprise the following steps:
(1) adopt full method for synthesizing gene to obtain needed six genes of approach;
(2) cut connect to build by enzyme and obtain three co-expression plasmid pCDF-RgTAL-Pc4CL, pET-PhCHS-MsCHI, pACYCD-matB-matC, after sequence verification, by three co-expression plasmids with chemical method Transformed E .coli (BL21) competent cell;
(3) select the ammonia benzyl that 100ug/mL can contained, the paraxin of 35ug/mL, the bacterium colony of growing on four kinds of antibiotic LB flat boards of the Streptomycin sulphate of 50ug/mL, make it contain on four kinds of above-mentioned antibiotic LB flat boards the three generations that transfers continuously, obtain the recon of inheritance stability, further six pathway genes of PCR checking successfully are transferred in E.coli (BL21).
6. utilize the method for the described genetic engineering bacterium fermentative production of claim 3 naringenin, it is characterized in that, be that the engineering strain that structure is obtained is inoculated in the MOPS substratum, 500mL shaking flask dress liquid 25mL cultivates 12-20h to bacterial strain OD under 37 ℃, 250rpm condition
600reach 1.0-2.0; And then add MOPS substratum that 25ml is identical and the IPTG of final concentration 1mM, 30 ℃, under the 250rpm condition, cultivate 60h.
7. according to the described method of claims 6, it is characterized in that, the MOPS substratum consists of (g/L): glucose 5, ammonium chloride 4, dipotassium hydrogen phosphate 0.3, propanedioic acid 2, MOPS83.7, N-{ tri-(carboxymethyl) methyl } glycine 7.1668, ammonium chloride 5.1, ferric sulfate 0.03, vitriolate of tartar 0.5, magnesium chloride 1.1, sodium-chlor 29.2, trace element 10ml, the tap water constant volume.
8. method according to claim 7, is characterized in that, described trace element adopts the formula of ATCC BAA-2326.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865864A (en) * | 2014-03-04 | 2014-06-18 | 江南大学 | Method for producing eriodictyol by reforming escherichia coli in metabolic engineering |
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| CN110283857A (en) * | 2019-06-18 | 2019-09-27 | 南京师范大学 | A method of naringenin is produced based on Phomopsis cell fermentation |
| CN110713962A (en) * | 2019-09-06 | 2020-01-21 | 南京农业大学 | Genetic engineering bacterium for high-yield production of malonyl coenzyme A and construction method and application thereof |
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| CN113528471A (en) * | 2021-07-20 | 2021-10-22 | 扬州大学 | Three-functional enzyme for de novo synthesis of flavanone and synthesis method and application thereof |
| CN113621629A (en) * | 2021-07-30 | 2021-11-09 | 扬州大学 | Naringenin in-vitro enzymatic synthesis method based on malonyl coenzyme A regeneration |
| CN114606279A (en) * | 2022-03-21 | 2022-06-10 | 陕西科技大学 | Method for synthesizing naringenin by taking tyrosine as substrate |
| CN114990086A (en) * | 2022-06-16 | 2022-09-02 | 天津大学佐治亚理工深圳学院 | Enzyme composition and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011140344A1 (en) * | 2010-05-07 | 2011-11-10 | Massachusetts Institute Of Technology | Strains for the production of flavonoids from glucose |
-
2013
- 2013-10-15 CN CN201310482140.2A patent/CN103484420B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011140344A1 (en) * | 2010-05-07 | 2011-11-10 | Massachusetts Institute Of Technology | Strains for the production of flavonoids from glucose |
Non-Patent Citations (3)
| Title |
|---|
| C.N.S. SANTOSETAL.: "Optimization of a heterologous pathway for the production of flavonoids from glucose", 《METABOLIC ENGINEERING》, 12 February 2011 (2011-02-12), pages 392 - 400 * |
| JUNJUN WU，ET AL: "Multivariate modular metabolic engineering of Escherichia coli to produce resveratrol from L-tyrosine", 《JOURNAL OF BIOTECHNOLOGY》, vol. 167, 31 July 2013 (2013-07-31) * |
| LUSSIER FX，ET AL.: "Engineering Microbes for Plant Polyketide Biosynthesis", 《COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL》, 31 October 2012 (2012-10-31), pages 1 - 11, XP055165250, DOI: doi:10.5936/csbj.201210020 * |
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| CN110283857A (en) * | 2019-06-18 | 2019-09-27 | 南京师范大学 | A method of naringenin is produced based on Phomopsis cell fermentation |
| CN110713962A (en) * | 2019-09-06 | 2020-01-21 | 南京农业大学 | Genetic engineering bacterium for high-yield production of malonyl coenzyme A and construction method and application thereof |
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| CN113621629B (en) * | 2021-07-30 | 2023-08-25 | 扬州大学 | Naringenin in-vitro enzymatic synthesis method based on malonyl-CoA regeneration |
| CN113502278A (en) * | 2021-08-10 | 2021-10-15 | 天津大学佐治亚理工深圳学院 | Enzyme composition and application thereof in naringenin biosynthesis |
| CN113502278B (en) * | 2021-08-10 | 2023-10-27 | 天津大学佐治亚理工深圳学院 | Enzyme composition and application thereof in naringenin biosynthesis |
| CN114606279A (en) * | 2022-03-21 | 2022-06-10 | 陕西科技大学 | Method for synthesizing naringenin by taking tyrosine as substrate |
| CN114990086A (en) * | 2022-06-16 | 2022-09-02 | 天津大学佐治亚理工深圳学院 | Enzyme composition and application thereof |
| CN114990086B (en) * | 2022-06-16 | 2024-05-07 | 天津大学佐治亚理工深圳学院 | Enzyme composition and application thereof |
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