Improving the nutrient composition of Chlamydomonas reinhardtii to enhance the production of photosynthetic active metabolites: a molecular study on the Photosystem II complex
Author(s)
Antonacci, Amina
Date Issued
February 26, 2010
Type
Doctoral Thesis
Abstract
This thesis has been supported by the Nutra-Snack project, aimed to the production of “Ready to eat food for breakfast and sport activity with high content of nutraceutics reducing disease risk and promoting public health”. In this context, the unicellular green alga Chlamydomonas reinhardtii has been used to characterize particular features of the photosynthetic process and to test its capability to over-produce photosynthesis-derived pigments, to be used as supplements in healthy diets. Nowadays, photosynthesis-derived pigments, such as carotenes and xantophylls, are of great interest for health and human nutrition; they are normally synthesized by several bacteria, algae and plants, but not by humans that should take them from the diet.
In particular, we exploited chlamydomonas strains carrying mutation in the D1 protein and in Non Photochemical Quenching (NPQ) process, and their relative wild types. According to the literature, the physiological characterization of the NPQ mutants revealed no important differences between mutants and wild type concerning the photosynthetic parameters, such as oxygen evolution and photosynthetic efficiency. On the contrary, the physiological characterization of D1 mutants revealed a remarkably reduction of the electron transfer efficiency between the primary (QA) and the secondary (QB) quinone acceptors in photosystem II (PSII), and a lower oxygen evolution capacity compared to the reference strain. In addition, the D1 mutants displayed a different Chla/cell ratio compared to the reference strains, which is indicative of a reduced peripheral antenna cross section, hence of a reduced light absorption capacity. Also the photosynthetic pigment quantification was different among D1 strains, mainly in the content of the total xanthophylls, e.g. violaxanthin, anteraxanthin, zeaxanthin and lutein, which are located in the peripheral antennae proteins. These evidences suggested that a single aminoacid substitution in the D1 protein located in the QB binding site could affect the overall PSII molecular structure. Molecular dynamic simulations could help to clarify this issue.
Comparison of the pigment profiles of all the analysed strains showed a notably higher pigments content in the NPQ compared to the D1 strains, indicating possible good candidate for biological farms developing.
In order to enhance the content of antioxidant photosynthetic pigments in C. reinhardtii D1 cultures, we applied high light and high temperature conditions and carried out time course analyses over a 90 minutes period. This treatment determined a strong reduction in photosynthetic efficiency, without affecting the total chlorophyll content in all the analysed D1 strains. Pigment profile highlighted the noteworthy response of the reference strain in comparison with mutants. In fact, a higher lutein and zeaxanthin accumulation in reference strain was observed, as well as the activation of xanthophylls cycle.
Real-time RT-PCR analyses on these samples were also performed on 11 genes involved in the biosynthesis of carotenoids, plastoquinones synthesis, D1 and D2 PSII reaction centre proteins. Time course analyses suggested that the first algal necessity in stressful condition was the induction of carotenoids biosynthesis genes, and that these data strongly correlated to pigment profile. Subsequently, also the plastoquinone genes were induced, probably because the requirement of oxidised PQ synthesis is important not only for the electron transport, but also for the activity of some enzymes involved in the initial carotenoids biosynthetic pathway.
In order to exploit the unicellular microalgae as natural source for valuable compounds, a second set of elicitation experiments was performed on the reference strain of D1 strains and included also the NPQ double mutant, which accumulates very high levels of zeaxanthin as a consequence of its mutation. In this way, we attempted to achieve bigger accumulation of antioxidant compounds in a shorter time, using very extreme high light and high temperature conditions (2000 µmol of photons/m2/s and 50°C). The ontained results showed that the reference strain of D1 set is the most responsive to the stress, for its rapidly capability to modulate its metabolism for zeaxanthin production. Instead, the NPQ double mutant accumulated a major zeaxanthin and β-carotene content, but it was less susceptible to metabolism modification. It is possible that xanthophylls accumulation levels exceeding a particular threshold could became dangerous for the cells that as a consequence, regulates its biosynthesis. As a whole these data indicated that the NPQ double mutant was the best producer of antioxidants pigments. However, the identification and characterization of the reference strain of D1 mutants revealed a genotype having a very flexible metabolism that is a desiderable culture threat for any nutraceutical application.
Future work will be dedicated to further clarify the involvement of PQ redox state in carotenogenesis pathway. In addition, taking into consideration the relevance of the lipid environment in the light and heat stress response, we will determine the lipid profile of different chlamydomonas mutants in these stressful conditions, in order to highlight and correlate structural and compositional modifications to the tolerance mechanisms.
For nutraceutical purposes, chlamydomonas growth conditions will be set up for large scale production of biomass with high nutraceutics content, and a safe protocol extraction will be developed. In addition, as C. reinhardtii belongs to the category of organism having a GRAS status (Generally Regarded As Safe, granted by Food and Drug Administration), a feasibility study will be carried out in an attempt to use it directly as food ingredient, obviously only after application of EU directives on novel food.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
File(s)![Thumbnail Image]()
Name
aantonacci_tesid.pdf
Size
2.82 MB
Format
Adobe PDF
Checksum (MD5)
1c055987cb46694e9bfbaafd8dea81e0