D. salina is one of the recognized natural sources to produce β-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of β-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-β-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 µM fosmidomycin, carotenogenesis and the synthesis of β-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and β-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids OPEN ACCESS Mar. Drugs 2009, 7 46and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.

In Dunaliella salina, we studied the early steps in the isoprenoid pathway for the biosynthesis of carotenoids and beta-carotene and the effects of metabolic inhibitors. When D. salina was grown under carotenogenic and non-carotenogenic conditions, mevinolin did not inhibit growth or the accumulation of carotenoids, beta-carotene or chlorophyll. In contrast, fosmidomycin progressively inhibited cell growth and the biosynthesis of carotenoids, beta-carotene and chlorophyll. In this work, we reported for the first time that in D. salina, beta-carotene biosynthesis does not proceed via the classical acetate/mevalonate pathway but via the novel glyceraldehyde 3-phosphate/pyruvate pathway. This favours the yield of C(5) isoprenoid units for synthesis of isopentenyl diphosphate, the precursor in the biosynthesis of C(20) compounds, including geranylgeranyl diphosphate. Consequently, this pathway promotes carotenogenesis and the biosynthesis of C(40) beta-carotene in D. salina.

Current mixotrophic culture systems for Dunaliella salina have technical limitations to achieve high growth and productivity. The purpose of this study was to optimize the mixotrophic conditions imposed by glycerol, light, and salinity that lead to the highest biomass and β-carotene yields in D. salina. The combination of 12.5 mM glycerol, 3.0 M salinity, and 50 μmol photons m−2 s−1 light intensity enabled significant assimilation of glycerol by D. salina and consequently enhanced growth (2.1 × 106 cell mL−1) and β-carotene accumulation (4.43 pg cell−1). The saline and light shock induced the assimilation of glycerol by this microalga. At last stage of growth, the increase in light intensity (300 μmol photons m−2 s−1) caused the β-carotene to reach values higher than 30 pg cell−1 and tripled the β-carotene values obtained from photoautotrophic cultures using the same light intensity. Increasing the salt concentration from 1.5 to 3.0 M NaCl (non-isosmotic salinity) produced higher growth and microalgal β-carotene than the isosmotic salinity 3.0 M NaCl. The mixotrophic strategy developed in this work is evidenced in the metabolic capability of D. salina to use both photosynthesis and organic carbon, viz., glycerol that leads to higher biomass and β-carotene productivity than that of an either phototrophic or heterotrophic process alone. The findings provide insights into the key role of exogenous glycerol with a strategic combination of salinity and light, which evidenced unknown roles of this polyol other than that in osmoregulation, mainly on the growth, pigment accumulation, and carotenogenesis of D. salina.