Engineering the Unicellular Alga Phaeodactylum tricornutum for Enhancing Carotenoid Production

Abstract

Microalgae represent a promising resource for the production of beneficial natural compounds due to their richness in secondary metabolites and easy cultivation. Carotenoids feature among distinctive compounds of many microalgae, including diatoms, which owe their golden color to the xanthophyll fucoxanthin. Carotenoids have antioxidant, anti-obesity and anti-inflammatory properties, and there is a considerable market demand for these compounds. Here, with the aim to increase the carotenoid content in the model diatom Phaeodactylum tricornutum, we exploited genetic transformation to overexpress genes involved in the carotenoid biosynthetic pathway. We produced transgenic lines over-expressing simultaneously one, two or three carotenoid biosynthetic genes, and evaluated changes in pigment content with high-performance liquid chromatography. Two triple transformants over-expressing the genes Violaxanthin de-epoxidase (Vde), Vde-related (Vdr) and Zeaxanthin epoxidase 3 (Zep3) showed an accumulation of carotenoids, with an increase in the fucoxanthin content up to four fold. Vde, Vdr and Zep3 mRNA and protein levels in the triple transformants were coherently increased. The exact role of these enzymes in the diatom carotenoid biosynthetic pathway is not completely elucidated nevertheless our strategy successfully modulated the carotenoid metabolism leading to an accumulation of valuable compounds, leading the way toward improved utilization of microalgae in the field of antioxidants.

Keywords: Phaeodacytlum tricornutum; carotenoids; diatoms; fucoxanthin; genetic engineering.

Figure 1

Schematic representation of the carotenoid biosynthesis pathway in P. tricornutum, according to the hypothesized pathways proposed by Lohr and Wilhelm 1999 [20] and Dambek et al. 2012 [21]. Dashed lines are used for unknown enzymes. Red arrows represent de-epoxidation reactions and green arrows represent epoxidation reactions operating in the two xanthophyll cycles. GGDS: geranylgeranyl diphosphate synthase; PSY: phytoene synthase; PDS: phytoene desaturase; ZDS: ζ-carotene desaturase; CRTISO: carotene cis/trans isomerase, prolycopene isomerase; LCYB: lycopene cyclase b; LUT: lutein deficient-like; ZEP: zeaxanthin epoxidase; VDE: violaxanthin de-epoxidase. Adapted from M. Bertand et al. (2010) and P. Kuczynska et al. (2015) [9,19].

Figure 2

Schematic representation of the construct and position of the primers annealing sites used to screen exogenous gene integration.

Figure 3

Transgene integration and pigments change in P. tricornutum wild type and OE strains T1, T2 and T3. (a); PCR screening for Vdr, using Forward FcpB and reverse VDRint primers, expected fragment of 383 bp. (b); PCR screening for Vde, using Forward FcpB and reverse VDEint primers, expected fragment of 443 bp. (c); PCR screening for Zep3, using Forward FcpB and reverse ZEP3int primers, expected fragment of 848 bp. L; 100 bp ladder. PT WT; P. tricornutum wild type. T1, T2. T3, triple transformants. +, positive control, the vector used for transformation. (d); Fold change in the pigments content normalised for chlorophyll a in transgenic strains with respect to wild type. Data represent mean (n = 3) and SD.

Figure 4

Transcripts and HA tagged proteins relative expression in P. tricornutum wild type and OE strains T1, T2 and T3. (a), Relative gene expression (endogenous and exogenous) as assessed by qPCR. (b), Western blot analysis performed on PT WT, T1, T2 and T3 in order to detect exogenous HA tagged proteins and control protein D2 of PSII. (c), relative exogenous protein expression.

Figure 5

Non-photochemical quenching (NPQ max) developed by the four strains analysed.