Abstract
Oxygenic photosynthesis allows plants, algae and cyanobacteria to depend primarily on readily available light, carbon dioxide and water, in turn generating the chemical energy required for complex metabolism. This makes photosynthetic organisms ideal hosts for metabolic engineering aimed at sustainable production of high-value and commodity products. Cytochrome P450 enzymes play key roles in the biosynthesis of important natural products. The electron carrier ferredoxin can couple P450s non-natively to photosynthetic electron supply, providing ample reducing power for catalysis. However, photosynthetic reducing power feeds into both central and specialized metabolism, which leads to a fiercely competitive system from which to siphon reductant.
This thesis explores the optimization of light-driven P450 activity, and proposes strategies to overcome the limitations imposed by competition for photosynthetic reducing power. Photosynthetic electron carrier proteins interact with widely different partners because they use relatively non-specific interactions. The mechanistic basis of these interactions and its impact on natural electron transfer complexes is discussed. This particular type of interaction explains the innate ability to interact with non-cognate enzymes such as P450s, and suggests ways to improve the effectiveness of the photosystem I-P450 electron transfer chain. Two strategies to control the flow of electrons from photosynthesis were investigated using the tobacco transient expression system. The first strategy uses variants of ferredoxin and flavodoxin proteins, revealing the carrier redox potentials as key to determine the fate of electrons from photosynthesis. This offers a way to avoid channeling of reducing power into competing metabolism. The second strategy involves fusion between redox carrier protein and P450, and leads to increased rates of electron transfer from photosystem I. Together, the approaches allow optimization of the effectiveness of novel electron transfer chains, a step towards establishing these electron carrier proteins as orthogonal modules useful for metabolic engineering of photosynthetic hosts.
This thesis explores the optimization of light-driven P450 activity, and proposes strategies to overcome the limitations imposed by competition for photosynthetic reducing power. Photosynthetic electron carrier proteins interact with widely different partners because they use relatively non-specific interactions. The mechanistic basis of these interactions and its impact on natural electron transfer complexes is discussed. This particular type of interaction explains the innate ability to interact with non-cognate enzymes such as P450s, and suggests ways to improve the effectiveness of the photosystem I-P450 electron transfer chain. Two strategies to control the flow of electrons from photosynthesis were investigated using the tobacco transient expression system. The first strategy uses variants of ferredoxin and flavodoxin proteins, revealing the carrier redox potentials as key to determine the fate of electrons from photosynthesis. This offers a way to avoid channeling of reducing power into competing metabolism. The second strategy involves fusion between redox carrier protein and P450, and leads to increased rates of electron transfer from photosystem I. Together, the approaches allow optimization of the effectiveness of novel electron transfer chains, a step towards establishing these electron carrier proteins as orthogonal modules useful for metabolic engineering of photosynthetic hosts.
| Original language | English |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Number of pages | 205 |
| Publication status | Published - 2016 |