|Title||Metabolomics of carotenoid accumulation in Dunaliella salina|
|Source||University. Promotor(en): Rene Wijffels; Raoul Bino, co-promotor(en): Marcel Janssen; Ric de Vos. - [S.l. : S.n. - ISBN 9789085858522 - 173|
PRI BIOS Applied Metabolic Systems
|Publication type||Dissertation, internally prepared|
|Keyword(s)||dunaliella - carotenoïden - metabolomica - industriële microbiologie - carotenoids - metabolomics - industrial microbiology|
|Abstract||Innovative production processes based on renewable resources are required to stop the exhaustion of our natural resources. Microalgae are one of the most promising feedstocks for such sustainable processes, since they can produce valuable biochemicals at high productivity using sunlight, water, carbon dioxide and a few other nutrients, without the need for arable land. This high productivity is attained in (semi)controlled photobioreactors designed to prevent any limitation but light.
Currently, the high costs for investment and operation of such systems only allow for economic production of high-value molecules such as carotenoids and omega-3 fatty acids. Though economically sound, these production processes typically require large amounts of energy and thus cannot be termed sustainable. Nevertheless, they do provide the essential know-how to develop sustainable and cost-effective production of microalgal low-value bulk chemicals and biofuels. To this end, breakthroughs are needed in all areas of microalgal biotechnology, including cell physiology.
One of the most important objectives of physiological studies is the improved understanding of how microalgal metabolism is affected by cultivation conditions. Design of optimal production process conditions can be enabled if the metabolism is fully understood and controlled.
A good example of the interaction of cultivation conditions on microalgal metabolism is the stress-induced accumulation of β-carotene in the unicellular green microalga Dunaliella salina. β-Carotene is a lipid-soluble orange pigment and an antioxidant, that is used in cosmetics and as a colorant for feed and food. This metabolite is accumulated by D. salina up to 10% of the cellular dry weight when the alga is exposed to extreme environmental conditions. However, the knowledge about the regulatory mechanisms that underlie β-carotene accumulation in D. salina is limited.
Therefore, the aim of this thesis was to gain more insight in how D. salina senses and responds to changes in process conditions with the ultimate goal of defining optimal strategies for β-carotene production, and possibly also for other stress-inducible metabolites in other microalgae.
In Chapter 2, the cell-factory potential of D. salina was reviewed and the general ideas concerning mechanisms related to β-carotene accumulation were discussed. D. salina accumulates β-carotene when exposed to a high light intensity or to conditions that lead to a reduced growth rate, such as high salinity, nutrient deprivation or extreme temperatures. In addition, the levels of β-carotene in D. salina correlate positively with the overall amount of irradiation perceived during the cell division cycle. This positive correlation suggests the involvement of a sensing mechanism that is responsive to the imbalance between energy input, i.e. light harvesting in the chloroplasts, and energy demand, i.e. requirement for cell growth.
Singlet oxygen, an excited oxygen species that can be produced upon excess of electron transfer in the chloroplast, seems a likely candidate, as it can induce β-carotene accumulation and is known to be released under the conditions that favor β-carotene accumulation. Another potential sensing mechanism involves the redox state of the plastoquinone pool of the photosynthetic electron transport chain. Since oxidation of the reduced form of plastoquinone is generally assumed to be the rate-limiting step in this electron transport chain, an imbalance between the supply and demand of photosynthetic products may be reflected in the redox-state of the plastoquinone pool. Upon excess of electron production by the photosynthetic machinery, the plastoquinone redox state will shift towards a more reduced form.
However, the way these stress-induced changes in singlet oxygen or plastoquinone redox state are transduced and finally stimulate β-carotene accumulation is still largely unknown. Transcriptional and translational activity have been found essential for β-carotene accumulation, although a clear understanding of the involvement of structural genes that encode enzymes of the carotenoid biosynthetic pathway is still lacking.
Finally, β-carotene accumulation may also be driven by the formation of β-carotene-sequestering lipid globules. In this case, β-carotene accumulation does not require upregulation of genes encoding enzymes of the carotenoid biosynthetic pathway, but relies on activity-enhancement of these enzymes through removal of their end-product (i.e. β-carotene) by newly formed oil globules.
In Chapters 3 and 4, the effects of either a sudden light increase or nitrogen starvation on the carotenoid metabolism of D. salina were described. Since high-light is one of the most potent inducers of β-carotene accumulation, the experiments were done in flat panel photobioreactors that were run in turbidostat mode to ensure a constant light regime throughout the entire duration of the experiments.
A 7-fold increase in light intensity proved more powerful in inducing β-carotene accumulation than complete nitrogen starvation, as was demonstrated by a 2 times higher maximum productivity (37 mg per liter reactor volume per day as compared to 18.5 mg per liter reactor volume per day). However, nitrogen depletion appears more efficient with regard to energy usage, since 7 times more light energy was used in the high-light experiment.
Interestingly, in our experimental turbidostat systems the maximal productivities found for both stresses were about an order of magnitude larger than the average productivity reported for a commercial β-carotene production facility, which indicates a vast potential for improvement in the latter.
Apart from β-carotene accumulation, the growth characteristics of the D. salina cells were also influenced by the stress treatments. Induction by either type of stress resulted in cell swelling and an increase in the cell-specific density. The increased amount of β-carotene could only partially explain the increase in cell-specific density, which suggests that other metabolites accumulated as well.
The initial cell division rate was differentially affected, with high-light causing a temporary cell cycle arrest and nitrogen starvation leading to a transient increase in the cell division rate. Nevertheless, the volumetric biomass productivity increased temporarily for both nitrogen starvation (2-fold) and high-light stress (6-fold).
These results implied that the 7-fold increase in incident light caused a decrease in the biomass yield on absorbed light energy, whereas nitrogen depletion led to a transient increase in the yield caused by the accumulation of non-nitrogenous biomass. This finding points towards the potential of nitrogen-limitation strategies for permanent improvement of lipid or carbohydrate yield on light in large scale microalgal production systems.
Since D. salina accumulates β-carotene in lipid globules and it has been suggested that fatty acid biosynthesis determines the amount of accumulating β-carotene, we determined the fatty acid content and composition during both stress treatments and correlated the exact time course of fatty acid levels to that of β-carotene production. The intracellular concentration of the total fatty acid pool did not change significantly during nitrogen starvation and decreased following treatment with high-light.
These results indicated that β-carotene and total fatty acid accumulation were unrelated, at least in our turbidostat experiments. Nevertheless, carotenoid overproduction was associated with oil globule formation and a decrease in the degree of fatty acid unsaturation. The accumulation of β-carotene appeared to correlate positively with oleic acid production, suggesting that oleic acid may be a key component of the lipid globule-localized triacylglycerols and thereby in β-carotene accumulation.
In Chapter 5, the cellular mechanisms that could be related with stress-induced β-carotene accumulation in D. salina were investigated. Samples were taken from the aforementioned high-light and nitrogen-starvation experiments, both before and during stress induction. Subsequent untargeted GC-TOF-MS-based analysis of derivatized polar extracts, mainly containing primary metabolites, in combination with unbiased peak picking and clustering of signals into metabolite mass spectra, yielded 87 unique polar metabolites of which 31 were in common between both stress experiments.
By combining these polar metabolite profiles with the previously determined levels of carotenoids, chlorophylls and fatty acids, it was found that D. salina cells exhibit essentially similar overall responses towards both types of stress, with the principal stress-specific variation being caused by only 3 out of the 44 polar and apolar metabolites.
Furthermore, we observed accumulation of various metabolites that are usually linked with energy storage, both during high-light stress and during nitrogen depletion. Because both stress treatments are known to cause an imbalance in absorbed and required light energy, it was suggested that β-carotene accumulation and an increased production of energy storage molecules reflect a uniform and concerted effort of D. salina cells to cope with such damaging imbalances.
In Chapter 6, data of preliminary experiments towards elucidating the mechanisms potentially involved in the sensing of imbalances in the ratio between energy supply and energy consumption in D. salina are presented. In these experiments we used specific inhibitors of the reduction (DCMU) and the oxidation (DBMIB) of the plastoquinone pool in the photosynthetic electron transport chain, under both inducing and non-inducing conditions. It was found that DCMU inhibited both growth and β-carotene accumulation under otherwise carotenogenic conditions, whereas DBMIB inhibited only the growth.
These results pointed towards a possible role of the plastoquinone redox state in the regulation of β-carotene accumulation in D. salina. In addition, these findings suggested that singlet oxygen did not serve as a signaling agent for stress-induced β-carotene accumulation, since this accumulation was inhibited in the presence of DCMU, despite this inhibitor being a well-known stimulator of singlet oxygen release.
Next to performing these photosynthesis inhibitor experiments, we also showed the importance of metabolic profiling techniques for a better understanding of microalgal metabolism. We discussed the relations between β-carotene accumulation and the observed temporal changes in polar and apolar metabolites. This enabled evaluation of several hypotheses concerning the mechanisms involved in β-carotene accumulation, with upregulation of the entire β-carotene biosynthetic pathway proving a likely element of such a mechanism.
Furthermore, because light intensity and growth-reducing stress conditions act together upon the accumulation of secondary metabolites in microalgae, proper physiological studies, as well as optimal commercial production strategies, require cultivation systems that enable tight control of both light and nutrient supply. We therefore advocated the use of turbidostats, both for research on and for commercial production of stress-induced accumulation of microalgal metabolites.
In conclusion, this thesis illustrates the potential of combining sophisticated cultivation techniques with broad-scale metabolite profiling approaches for the detailed study of microalgal metabolism. Hence, similar studies should be applied for gaining the understanding that is needed for the development of optimal and sustainable production processes that will ultimately put a stop to the exhaustion of our natural resources.