|Tipo di tesi||Tesi di laurea magistrale|
|Titolo||Microorganismi come piattaforma per la produzione biotecnologica di prodotti naturali bioattivi – creazione di un sistema modulare per la ricostruzione del pathway dei fenilpropanoidi come primo step per la produzione di calconi/flavonoidi|
|Titolo in inglese||Microorganisms as platform for biotechnological production of natural bioactive products – construction of phenylpropanoid module as a first step towards chalcone/flavonoid production|
|Struttura||Dipartimento di Scienze della Vita|
|Corso di studi||BIOTECNOLOGIE INDUSTRIALI (D.M. 270/04)|
|Data inizio appello||2013-12-10|
|Disponibilità||Accessibile via web (tutti i file della tesi sono accessibili)|
I fenilpropanoidi e i flavonoidi sono dei composti naturali di origine vegetale,noti per le loro proprietà salutistiche nell’uomo, importanti per quanto riguarda i processi fisiologici di diverse piante. La loro via biosintetica è composta da diverse reazioni enzimatiche ed è continuamente oggetto di studio nei lavori di ingegneria metabolica. In questo contesto, l’espressione eterologa di proteine ricombinanti nei microrganismi e la biosintesi combinatoriale rappresentano due interessanti mezzi per aumentare le rese di produzione di questi metaboliti secondari della piante.
Phenylpropanoids and derived flavonoids are natural plant products presenting many positive effects on human health. Besides that, they are important compounds regarding several plant physiological aspects. The biosynthetic pathway is composed of several enzymatic steps and is object of many on-going studies on metabolic engineering. In this context, the heterologous expression in microbes and combinatorial biosynthesis are interesting tools to increase production yields of these plant secondary metabolites. In this project the main goal was to establish a production platform for phloridzin, a bioactive compound found in apple trees, by reconstruction of phenylpropanoid/chalcone pathway as a modular system in a single E. coli cell. For this strategy DUET vectors from Novagen® were used for the co-expression of several target genes. In a first step, a codon optimised L-tyrosine ammonia lyase gene from Rhodobacter sphaeroides (RS_TAL) and 4-coumarate:coenzyme A ligases from Arabidopsis thaliana (AT_4CL) and from Malus x domestica (MD_4CL), respectively, were cloned and functionally analyzed. These enzymes together would facilitate the formation of 4-coumaroyl-CoA from L-tyrosine in a first module. Suitable restriction sites for subcloning in the selected DUET vector were introduced at the 5’ and 3’ ends of RS_TAL, AT_4CL and MD_4CL sequences by PCR. Then, the genes were cloned into pCR™8/GW/TOPO® plasmid. In this way, the target genes could be digested by appropriate enzymes and cloned directly into the final destination DUET vector. In order to functionally proof enzyme activities, RS_TAL, AT_4CL, and MD_4CL genes were expressed in E. coli cells. RS_TAL showed activity and converted L-tyrosine into 4-coumaric acid, whereas for both 4CL proteins no activity was detectable. Phloretin itself could be transformed into phloridzin by several glycosyltransferases. In this study the heterologous expression of UDP-glucose-glycosyltransferase (MD_UGT71K1) in E. coli cells was established and several biotransformation assays were performed. Unfortunately under the selected conditions MD_UGT71K1 showed no activity. SDS-PAGE and in vitro enzyme assays suggested that MD_UGT71K1 protein is expressed in inclusion bodies and thus the expression protocol needs further improvement.