Towards a sustainable Dunaliella salina microalgal biorefinery for 9-cis β-carotene production

Valorisation of the efficacy of 9-cis beta-carotene in treating atherosclerosis, psoriasis, and inhibiting atherogenesis and retinitis pigmentosa is becoming increasingly urgent, but supplies of 9-cis beta-carotene are scarce and this compound is difficult to synthesise chemically, unlike the much more common all-trans form. Innovative products, processes and services in an algal biorefinery that rely on renewable biological resources instead of fossil fuel alternatives offer the potential to lower the energy costs of traditional chemical processes and reduce carbon emissions, water usage and waste. In 2013, the European Commission supported development of 4 microalgal biorefinery projects to assess the potential for innovative approaches to tackle the major challenges intrinsic to the development of the algae biorefineries. One of these was the D-Factory (KBBE.2013.3.2-02) which sought to evaluate requirements for sustainable, industrial-scale production of Dunaliella salina and extraction of its carotenoids, especially 9-cis beta-carotene in a CO2 microalgae biorefinery. Here we present findings of the D-Factory project and propose a way forward for industrial-scale production of 9-cis beta-carotene using biotechnology based on Dunaliella salina biomass. Cultivation improvements are able to deliver more than double the current levels of productivity, with increased sustainability, whilst the use of natural hyper-accumulating carotenogenic strains combined with the use of red light to boost production of the beta-carotene pathway, will increase the relative concentration of 9-cis beta-carotene in extracts of carotenoids with consequent improvements in downstream processing. These developments pave the way for acquiring data for a Medicine Licence and prepare the market for entry of novel 9-cis beta-carotene products

Vitamin A-Deficient Diet Accelerated Atherogenesis in Apolipoprotein E−/− Mice and Dietary β-Carotene Prevents This Consequence

Vitamin A is involved in regulation of glucose concentrations, lipid metabolism, and inflammation, which are major risk factors for atherogenesis. However, the effect of vitamin A deficiency on atherogenesis has not been investigated. Therefore, the objective of the current study was to examine whether vitamin A deficiency accelerates atherogenesis in apolipoprotein E-deficient mice (apoE−/−). ApoE−/− mice were allocated into the following groups: control, fed vitamin A-containing chow diet; BC, fed chow diet fortified with Dunaliella powder containing βc isomers; VAD, fed vitamin A-deficient diet; and VAD-BC group, fed vitamin A-deficient diet fortified with a Dunaliella powder. Following 15 weeks of treatment, liver retinol concentration had decreased significantly in the VAD group to about 30% that of control group. Vitamin A-deficient diet significantly increased both plasma cholesterol concentrations and the atherosclerotic lesion area at the aortic sinus (+61%) compared to the control group. Dietary βc fortification inhibited the elevation in plasma cholesterol and retarded atherogenesis in mice fed the vitamin A-deficient diet. The results imply that dietary vitamin A deficiency should be examined as a risk factor for atherosclerosis and that dietary βc, as a sole source of retinoids, can compensate for vitamin A deficiency

The inhibition of macrophage foam cell formation by 9-cis β-carotene is driven by BCMO1 activity.

Atherosclerosis is a major cause of morbidity and mortality in developed societies, and begins when activated endothelial cells recruit monocytes and T-cells from the bloodstream into the arterial wall. Macrophages that accumulate cholesterol and other fatty materials are transformed into foam cells. Several epidemiological studies have demonstrated that a diet rich in carotenoids is associated with a reduced risk of heart disease; while previous work in our laboratory has shown that the 9-cis β-carotene rich alga Dunaliella inhibits atherogenesis in mice. The effect of 9-cis β-carotene on macrophage foam cell formation has not yet been investigated. In the present work, we sought to study whether the 9-cis β-carotene isomer, isolated from the alga Dunaliella, can inhibit macrophage foam cell formation upon its conversion to retinoids. The 9-cis β-carotene and Dunaliella lipid extract inhibited foam cell formation in the RAW264.7 cell line, similar to 9-cis retinoic acid. Furthermore, dietary enrichment with the algal powder in mice resulted in carotenoid accumulation in the peritoneal macrophages and in the inhibition of foam cell formation ex-vivo and in-vivo. We also found that the β-carotene cleavage enzyme β-carotene 15,15'-monooxygenase (BCMO1) is expressed and active in macrophages. Finally, 9-cis β-carotene, as well as the Dunaliella extract, activated the nuclear receptor RXR in hepa1-6 cells. These results indicate that dietary carotenoids, such as 9-cis β-carotene, accumulate in macrophages and can be locally cleaved by endogenous BCMO1 to form 9-cis retinoic acid and other retinoids. Subsequently, these retinoids activate the nuclear receptor RXR that, along with additional nuclear receptors, can affect various metabolic pathways, including those involved in foam cell formation and atherosclerosis

9-cis β-Carotene Increased Cholesterol Efflux to HDL in Macrophages

Cholesterol efflux from macrophages is a key process in reverse cholesterol transport and, therefore, might inhibit atherogenesis. 9-cis-β-carotene (9-cis-βc) is a precursor for 9-cis-retinoic-acid (9-cis-RA), which regulates macrophage cholesterol efflux. Our objective was to assess whether 9-cis-βc increases macrophage cholesterol efflux and induces the expression of cholesterol transporters. Enrichment of a mouse diet with βc from the alga Dunaliella led to βc accumulation in peritoneal macrophages. 9-cis-βc increased the mRNA levels of CYP26B1, an enzyme that regulates RA cellular levels, indicating the formation of RA from βc in RAW264.7 macrophages. Furthermore, 9-cis-βc, as well as all-trans-βc, significantly increased cholesterol efflux to high-density lipoprotein (HDL) by 50% in RAW264.7 macrophages. Likewise, food fortification with 9-cis-βc augmented cholesterol efflux from macrophages ex vivo. 9-cis-βc increased both the mRNA and protein levels of ABCA1 and apolipoprotein E (APOE) and the mRNA level of ABCG1. Our study shows, for the first time, that 9-cis-βc from the diet accumulates in peritoneal macrophages and increases cholesterol efflux to HDL. These effects might be ascribed to transcriptional induction of ABCA1, ABCG1, and APOE. These results highlight the beneficial effect of βc in inhibition of atherosclerosis by improving cholesterol efflux from macrophages

Retinol production in Hepa1-6 cells.

<p>HPLC chromatogram shows retinol production in Hepa1-6 cells after 24-hour incubation with 9-cis β-carotene. Total retinol content after control treatment (A) and after 9-cis β-carotene incubation (B). Separation was conducted on C18 column and detection at 325 nm. </p