Abstract
Oxygenic photosynthesis provides plants, algae and cyanobacteria the ability to convert solar energy and CO2 into complex organic molecules. Thus tapping into photosynthesis for synthetic biology efforts has a huge potential for the industrial production of fuels and high value bioactive compounds in a sustainable way. The dhurrin pathway typically resides in the ER of Sorghum bicolor and involves a dynamic metabolon formed by two P450s, a UDP-glucosyltransferase and a P450 oxidoreductase. This pathway has been relocated to the chloroplast where photosystem I and ferredoxin serve as an efficient electron donor to the P450s, bypassing the involvement of the P450 oxidoreductase. Nevertheless, translocation of the pathway from the ER to the chloroplast creates complications, such as intermediate diversion into other pathways. This thesis focuses on challenges related to substrate channeling that arise when relocating a biosynthetic pathway into the chloroplast, as well as possible solutions.
Paper I compares the potential of chloroplasts and cyanobacteria as sustainable biosynthetic compartments and hosts for metabolic engineering. An estimation of the percentage of photosynthetic electrons that can be redirected into the biosynthesis of products is given. The availability of photosynthetic reducing power appears to be non-limiting whereas the redirection of the fixed carbon seems to be main challenge for achieving higher product yields. On paper II a novel strategy where the membrane anchors of the dhurrin pathway enzymes were exchanged with components of the Tat system, in the chloroplast of Nicotiana benthamiana is described. The aim was to co-localize the pathway enzymes and improve substrate channeling. This strategy led to a 4-fold increase in dhurrin titers and to a reduction of intermediate and side-products. Paper III describes a similar approach in Synechocystis sp. PCC 6803. The Tat domains of the fusion proteins appeared to be cleaved of and no substrate channeling was observed. However, several physiological and morphological differences where observed in the dhurrin producing strains, which displayed light-sensitive and iron-deficient like phenotypes.
Paper I compares the potential of chloroplasts and cyanobacteria as sustainable biosynthetic compartments and hosts for metabolic engineering. An estimation of the percentage of photosynthetic electrons that can be redirected into the biosynthesis of products is given. The availability of photosynthetic reducing power appears to be non-limiting whereas the redirection of the fixed carbon seems to be main challenge for achieving higher product yields. On paper II a novel strategy where the membrane anchors of the dhurrin pathway enzymes were exchanged with components of the Tat system, in the chloroplast of Nicotiana benthamiana is described. The aim was to co-localize the pathway enzymes and improve substrate channeling. This strategy led to a 4-fold increase in dhurrin titers and to a reduction of intermediate and side-products. Paper III describes a similar approach in Synechocystis sp. PCC 6803. The Tat domains of the fusion proteins appeared to be cleaved of and no substrate channeling was observed. However, several physiological and morphological differences where observed in the dhurrin producing strains, which displayed light-sensitive and iron-deficient like phenotypes.
| Original language | English |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Publication status | Published - 2017 |