Biosyntese af en flavonol fra en Flavanone ved at etablere en en-pot Bienzymatisk kaskade
Summary
Afledningen af en flavonol er afgørende for dens anvendelse i sundhedssektoren og fødevareindustrien. Her giver vi en detaljeret protokol for biosyntesen af en flavonol fra en flavanonglucosid og diskuterer de afgørende skridt og dens fordele i forhold til andre tilgange.
Abstract
Flavonoler er en stor under klasse af flavonoider med en række biologiske og farmakologiske aktiviteter. Her, vi giver en metode til in vitro enzymatiske syntese af en flavonol. I denne metode, Atf3h og Atfls1, to vigtige gener i den biosyntetiske vej af flavonoler, er klonet og overudtrykt i Escherichia coli. De rekombinante enzymer renses via en affinitets kolonne, og derefter etableres en bienzymatisk kaskade i en specifik syntetisk buffer. To flavonoler syntetiseres i dette system som eksempler og bestemmes af TLC og HPLC/LC/MS analyser. Metoden viser indlysende fordele i afledningen af flavonoler over andre tilgange. Det er tid-og arbejdsbesparende og meget omkostningseffektiv. Reaktionen er let at blive præcist kontrolleret og dermed opskaleret til masseproduktion. Målproduktet kan renses let på grund af de enkle komponenter i systemet. Men dette system er normalt begrænset til produktion af en flavonol fra en flavanone.
Introduction
Flavonoler er en stor under klasse af plante flavonoider og er involveret i plante udvikling og pigmentering1,2,3. Endnu vigtigere, disse forbindelser besidder en bred vifte af sundheds-gavnlige aktiviteter, såsom anti-cancer4,5, anti-oxidativ6, anti-inflammatorisk7, anti fedme8, anti-hypertensive9 og hukommelse Recall egenskaber10, hvilket fører til et stort antal undersøgelser af disse plante afledte sekundære metabolitter. Traditionelt er disse forbindelser hovedsageligt afledt af plante ekstraktion med organiske opløsningsmidler. Men på grund af deres meget lave indhold i planter11,12,13, produktionsomkostningerne for de fleste flavonoler fortsat høj, hvilket indebærer store begrænsninger på deres anvendelse i sundhedssektoren og fødevarer Industri.
I løbet af de seneste årtier, forskere har udviklet en hel række metoder til at udlede flavonoider14,15. Men, kemisk syntese af disse komplicerede molekyler besidder en række iboende ulemper16. Det kræver ikke kun giftige reagenser og ekstreme reaktionsbetingelser, men også mange skridt til at producere et mål flavonoid sammensatte14,17. Desuden er en anden vigtig udfordring i denne strategi er den chirale syntese af aktive flavonoid molekyler. Derfor er det ikke en ideel strategi at producere flavonoider i kommerciel målestok via kemisk syntese16,17.
For nylig har forskerne udviklet en lovende alternativ strategi for at producere disse komplicerede naturlige forbindelser ved hjælp af tekniske mikrober med en vej for flavonoid biosyntese18,19,20, 21 , 22, som med succes er blevet dechiseret i planter23. F. eks. indførte Duan et al. en biosyntetisk vej ind i den spirende gær Saccharomyces cerevisiae til fremstilling af kaempferol (kmf)24. Malla et al. produceret astragalin, en glykosyleret flavonol, ved at introducereflavanonglucosid 3-hydroxylase (f3h), flavonol syntase (fls1), og UDP-glucose: flavonoid 3-O-glucosyltransferase UGT78K1 gener i Escherichia coliBL21 (DE3)17. Selv om der er en hel del paradigmer, ikke alle genetisk manipulerede mikrober producere produkter af interesse på grund af kompleksiteten af en cellulære platform, uforenelighed mellem kunstigt syntetiserede genetiske elementer og værter, den hæmmende effekt af Target produkter mod værtsceller, og ustabilitet af en manipuleret cellulære system selv16.
En anden lovende alternativ strategi for flavonoid produktion er at etablere en multienzymatisk kaskade in vitro. Cheng et al. har rapporteret, at enterocin polyketides kan med succes syntetiseres ved at samle en komplet enzymatisk pathway i en pot25. Denne celle frie syntetiske strategi omgår begrænsningerne af en mikrobiel produktionsfabrik og er dermed gennemførlig til at producere nogle flavonoider i store mængder16.
For nylig har vi med succes udviklet et bienzym syntetisk system til at konvertere naringenin (NRN) til KMF i en pot16. Her beskriver vi dette system i store detaljer og de metoder, der er involveret i at analysere produkterne. Vi præsenterer også to eksempler, der bruger dette system til at producere KMF fra NRN og quercetin (QRC) fra eriodictyol (ERD). Derudover diskuterer vi vigtige trin i denne metode og fremtidige forskningsretninger i biosyntesen af flavonoider.
Protocol
Representative Results
Discussion
En hel række undersøgelser er fokuseret på derisering af flavonoler på grund af deres potentielle anvendelse i sundhedssektoren og fødevareindustrien. Men traditionel plante ekstraktion med organiske opløsningsmidler og kemisk syntese besidder iboende ulemper, som begrænser deres anvendelse i produktionen af flavonoler. Her rapporterer vi en detaljeret metode til fremstilling af en flavonol fra en flavanonglucosid i en gryde ved at etablere en in vitro bienzymatisk kaskade. De kritiske trin i denne protokol er: 1)…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev finansielt støttet af Yangzhou University Special udnævnt professor start-up fonde, Jiangsu specielt udnævnt professor start-up fonde, seks talent Peaks projekt i Jiangsu provinsen (Grant No. 2014-SWYY-016), og et projekt finansieret af den prioriterede akademiske program udvikling af Jiangsu videregående uddannelsesinstitutioner (veterinærmedicin). Vi takker test Center for Yangzhou University for HPLC og MS analyser af flavonoider.
Materials
| 2× Pfu MasterMix | Beijing CoWin Biotech Co., Ltd | CW0717A | PCR amplification of genes with high fidelity |
| Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system | Agilent Technologies, Inc | N/A | an equipment for analysis of flavonoids by HPLC/MS |
| Agilent MassHunter Workstation (version B.03.01) | Agilent Technologies, Inc | N/A | a software for collection of the data from the Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system |
| dihydrokaempferol | Sigma-Aldrich Co. LLC | 91216 | intermediate product for producing kaempferol from naringenin |
| dihydroquercetin | Sichuan Provincial Standard Substance Center for Chinese Herbal Medicine | PCS0371 | intermediate product for producing quercetin from eriodictyol |
| DNA Clean-up Kit | Beijing CoWin Biotech Co., Ltd | CW2301 | purification of PCR-amplified or gel-purified DNA |
| eriodictyol | Shanghai Yuan Ye Biotechnology Co., Ltd. | B21160 | substrate for producing quercetin |
| Escherichia coli BL21(DE3) | Beijing CoWin Biotech Co., Ltd | CW0809 | bacteria strain for expressing target genes |
| Escherichia coli DH5α | Beijing CoWin Biotech Co., Ltd | CW0808 | bacteria strain for plasmid proliferation |
| FreeZone 1 Liter Benchtop Freeze-Dry System | Labconco Corporation | 7740020 | an equipment for freeze-drying of flavonoids dissolved in organic solvent |
| Gel Extraction Kit | Beijing CoWin Biotech Co., Ltd | CW2302 | purification of a DNA band from an agarose gel |
| Gel Imaging System | Shanghai Tanon Science & Technology Co. Ltd. | Tanon- 2500 |
an equipment for visualization of DNA band on an agarose gel or flavonoid spot on a polyamide TLC plate |
| GenElute Plasmid Miniprep Kit | Sigma-Aldrich Co. LLC | PLN350-1KT | minipreparation of plasmids |
| kaempferol | Sigma-Aldrich Co. LLC | 60010 | final reaction product and standard substance |
| MassHunter Quanlitative Analysis (version B.01.04) | Agilent Technologies, Inc | N/A | a software for analysis of HPLC/LC/MS data |
| NanoDrop Microvolume UV-Vis Spectrophotometer | Thermo Fisher Scientific | ND-8000-GL | an equipment for determination of DNA/RNA concentration |
| naringenin | Sigma-Aldrich Co. LLC | N5893 | substrate for producing kaempferol |
| Ni-IDA Agarose Resin | Beijing CoWin Biotech Co., Ltd | CW0010 | purification of His-tagged fusion proteins |
| pET-32a(+) | Novagen | 69015-3 | plasmid for cloning and expressing target genes |
| plasmid sequencing | GENEWIZ Suzhou | N/A | sequencing of recombinant plasmids |
| primer synthesis | GENEWIZ Suzhou | N/A | synthesis of PCR primers |
| quercetin | Shanghai Aladdin Biochemical Technology Co.,Ltd. | Q111273 | final reaction product and standard substance |
| SuperRT cDNA Synthesis Kit | Beijing CoWin Biotech Co., Ltd | CW0741 | synthesis of the first strand of cDNA from total RNA |
| T4 DNA Ligase | Thermo Fisher Scientific | EL0016 | ligation of an insert into a linearized vector DNA |
| Trizol | Thermo Fisher Scientific | 15596018 | isolation of total RNA |
| Vector NTI Advance | Thermo Fisher Scientific | 12605099 | a software for PCR primer design and DNA sequence analysis |
| Xcalibur v2.0.7 | Thermo Fisher Scientific | N/A | a software for analysis of HPLC data |
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