Li, Mingji

Resveratrol er et naturligt og meget potent antioxidant, som har flere gavnlige effekter på menneskers helbred. Derfor bruges resveratrol i vid udstrækning i medicin, fødevarer og kosmetik. I min ph.d.-afhandling beskriver jeg videnskabeligt, hvordan gærceller (Saccharomyces cerevisiae) kan genmodificeres til at producere resveratrol ved at fermentere billige kulstof-kilder. Jeg brugte rationel metabolisk genmodificering og rationelle designstrategier, syntetiske biologi-teknologier og en systembiologisk tilgang i designet og i analysen af gærcelle-fabrikkerne. Resveratrol kan dannes gennem to biosyntetiske veje, som begynder med aminosyren tyrosin eller fenylalanin, og som henholdsvis benævnes TAL og PAL-vejene i denne afhandling. I forhold til TAL-vejen arbejdede jeg sammen med Christian Bille Jendresen og Steen Gustav Stahlhut for at screene 14 forskellige heterologe udgaver af tyrosin ammoniak lyase (TALs) for deres aktivitet i gær og E. coli. Jeg udtrykte de 14 TALs i S. cerevisiae og analyserede cellelinjerne for produktion af p-cumarsyre (p-coumaric acid). To af de screenede TALs – en fra Herpetosiphon aurantiacus og en fra Flavobacterium johnsoniae – var yderst aktive og selektive i den forstand, at de ikke omdannede fenylalanin til uønsket kanelsyre (cinnamic acid). Disse resultater er blevet publiceret i Applied and Environmental Microbiology (Jendresen et al., 2015). Efterfølgende designede jeg TAL-vejen til at producere resveratrol via de to TAL-gener kombineret med 4-coumaryl-CoA ligase (4CL) fra Arabidopsis thaliana og resveratrol syntase (VST) fra Vitis vinifera i S. cerevisiae. Den vi mest effektive kombination resulterede i 11.66±0.57 mg l-1 og 2.74±0.05 mg l-1 resveratrol på minimal medie henholdsvist med eller uden 5 mM tyrosin. For at optimere resveratrol-produktionen modificerede jeg cellerne, så der kom større flux i retning af flere forstadier til tyrosyn. Dette blev gjort ved at deregulere biosyntese-vejen for aromatiske aminosyrer (gennemoverekspression af feedback-inhiberings-resistente versioner af Aro4pfbr andAro7pfbr). Desuden forøgede jeg tilførslen af forstadier til malonyl-CoA vedat deregulere acetyl-CoA carboxylase (overexpression af Acc1pS659A, S1157A)for at undgå dens inaktivering ved den globale regulator Snf1p. Den første strategi resultetede i 78 % større koncentration af resveratrol, den anden gav en 31 % forbedring. Ved at kombinere de to strategier opnåedes 130% forbedring af resveratrol koncentration (6.40±0.03 mg l-1 i medie uden tyrosin). Min hypotese var, at aktiviteten af resveratrol biosynteseenzymerne begrænsede fluxen hen mod resveratrol. For at teste hypotesen integrerede jeg generne fra resveratrol-vejen i Ty-4 retrotransposonregionerne, hvilket resulterede i integration af op til otte kopier af generne. Det viste sig, at hypotesen stemte overens med virkeligheden, idet denne strageti boostede resveratrol produktionen med en faktor 36 og således gav 235.57±77.00 mg l-1 i fermenteringssuppen. Jeg fermenterede den endelige cellelinje i kontrollerede fed-batch reaktorer med glukose eller etanol som kulstofskilde, og på denne måde opnåedes en resveratrol-koncentration på henholdsvis 416 mg l-1 og 531 mg l-1. Resultaterne blev publiseret i Metabolic Engineering (Li et al., 2015).Jeg genmodificerede også PAL-vejen for at øge resveratrolproduktionen. Dette gjordes ved at introducere PAL2-kodende fenylalanin ammoniak lyase, C4H (som koder for cinnamate-4-hydroxylase) og 4CL2 fra A. thaliana og VST1 fra V. vinifera i S. cerevisiae. For at øge aktiviteten af C4H – et almindeligt kendt cytokrom P450 enzym – overudtrykte jeg NADPHcytokrom P450 reduktase (ATR2) fra A. thaliana og S. cerevisiae cytokrom b5 (CYB5). Ty-4 element-medieret fler-integration of PAL vejen og overudtryk af ARO4fbr og ARO7fbr sammen med ACC1S659A, S1157A resulterede i 201.72±7.91 mg l-1 resveratrol. For yderligere at forbedre tilførslen af forstadier til resveratrol, lavede jeg et knock-out af ARO10, der koder for fenylpyruvat dekarboxylase og overudtrykte en post-translationel, ureguleret variant af acetyl-CoA syntetase (ACS1L641P) fra Salmonella enterica, hvilket forbedrede resveratrol produktionen til 272.64±1.34 mg l-1. Til sidst lykkedes det at opnå en koncentration på henholdsvis 811.50 mg l-1 og 754.70 mg l-1 resveratrol i fed-batch reaktorer med henholdsvis glukose eller ethanol som kulstofskilde. Desuden integrerede jeg heterologe versioner af metyltransferaser ind i resveratrol- platforms-cellelinjerne og på den måde demonstrerede jeg for første gang de novo biosyntesen af to afledninger af resveratrol kaldet pinostilben og pterostilben, som har en bedsre stabilitet og optages bedre i menneskekroppen sammenlignet med resveratrol.Resveratrol is a natural potent antioxidant with multiple beneficial effects on human health and is therefore used in medical, food, and cosmetic areas. In my PhD thesis I describe how I engineered yeast cell factory Saccharomyces cerevisiae for production of resveratrol by fermentation of cheap carbon sources. I adopted rational metabolic engineering rational design strategies, synthetic biology techniques and system biology approaches to engineer and analyse yeast cell factories. There are two biosynthesis pathways of resveratrol, starting from tyrosine or phenylalanine, which in this thesis are defined as the TAL pathway and PAL pathway respectively. For the TAL pathway, I collaborated with Christian Bille Jendresen, Steen Gustav Stahlhut to screen fourteen diverse heterologous tyrosine ammonia lyases (TALs) for their activity in yeast and E. coli. I expressed the 14 TALs in S. cerevisiae and analysed the resulting strains for production of p-coumaric acid. Two of the screened TALs, one from Herpetosiphon aurantiacus and another from Flavobacterium johnsoniae, were highly active and selective, i.e., they did not have a side reaction of converting phenylalanine to cinnamic acid. These results were published in Applied and Environmental Microbiology (Jendresen et al., 2015). I then constructed the TAL pathway to resveratrol by expressing the two TAL genes in combination with 4-coumaryl-CoA ligase (4CL) from Arabidopsis thaliana and resveratrol synthase (VST) from Vitis vinifera in S. cerevisiae; the best combination resulted in 11.66±0.57 mg l-1 and 2.74±0.05 mg l-1 resveratrol on minimum medium in the presence or absence of 5 mM tyrosine respectively. In order to improve resveratrol production, I increased the flux towards tyrosine precursor by de-regulating the aromatic amino acids biosynthesis pathway (overexpression of feedback-inhibition resistant versions of Aro4pfbr and Aro7pfbr) and the supply of malonyl-CoA precursor by de-regulating acetyl-CoA carboxylase (overexpression of Acc1pS659A, S1157A) to avoid inactivation by the global regulator Snf1p. The first strategy resulted in 78% improvement of resveratrol titer and the second in 31% improvement. Combining the two strategies further improved resveratrol titer to 6.40±0.03 mg l-1. I hypothesized that the activity of resveratrol biosynthetic enzymes was limiting the flux towards resveratrol. To test this hypothesis I integrated the resveratrol pathway genes into Ty-4 retrotransposon regions, which resulted in integration of up to 8 copies of the pathway genes. Indeed my hypothesis was correct as this strategy boosted the elements was revealed to be the key limiting factor for resveratrol biosynthesis and sharply improved resveratrol production 36-fold and gave 235.57±77.00 mg l-1 resveratrol in batch fermentation. I fermented the final strain in controlled fed-batch reactors with glucose or ethanol feed and obtained 415.65 and 531.41 mg l-1 of resveratrol respectively. The results were published in Metabolic Engineering (Li et al., 2015). I also engineered the PAL pathway for resveratrol production by introducing PAL2 encoding phenylalanine ammonia lyases, C4H encoding cinnamate-4-hydroxylase, and 4CL2 from A. thaliana and VST1 from V. vinifera in S. cerevisiae. To enhance the activity of C4H, a notorious cytochrome P450 enzyme, I overexpressed NADPH-cytochrome P450 reductase (ATR2) from A. thaliana and S. cerevisiae cytochrome b5 (CYB5). Ty-4 element-mediated multiple integration of PAL pathway and overexpression of ARO4fbr and ARO7fbr together with ACC1S659A, S1157A resulted in 201.72±7.91 mg l-1 resveratrol. In order to further improve the precursor supply, I knocked out ARO10 encoding phenylpyruvate decarboxylase and overexpressed a post-translational unregulated variant of acetyl-CoA synthetase (ACS1L641P) from Salmonella enterica, which improved resveratrol production to 272.64±1.34 mg l -1. Finally, I obtained 811.50 mg l-1 and 754.70 mg l-1 resveratrol in fed-batch fermentation of the engineered strain using glucose or ethanol feed respectively. I further integrated heterologous methyltransferases into the resveratrol platform strain and hereby demonstrated for the first time de novo biosynthesis of two resveratrol derivatives, pinostilbene and pterostilbene, which have better stability and uptake in the human body compared to resveratrol

Online Research Database In Technology

Engineering of <i>Saccharomyces cerevisiae </i>for production of resveratrol and its derivatives

http://orbit.dtu.dk/ws/files/127113778/Engineering_of_Saccharomyces_cerevisiae_for_production_of_resveratrol_and_its_derivatives.pdf

Engineering of <i>Saccharomyces cerevisiae </i>for production of resveratrol and its derivatives

Abstract

Similar works

Full text

Available Versions

Online Research Database In Technology