Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids

Abstract

We have engineered the diatom Phaeodactylum tricornutum to accumulate the high value omega-3 long chain polyunsaturated fatty acid docosahexaenoic acid (DHA). This was achieved by the generation of transgenic strains in which the Δ5-elongase from the picoalga Ostreococcus tauri was expressed to augment the endogenous fatty acid biosynthetic pathway. Expression of the heterologous elongase resulted in an eight-fold increase in docosahexaenoic acid content, representing a marked and valuable change in the fatty acid profile of this microalga. Importantly, DHA was shown to accumulate in triacylglycerols, with several novel triacylglycerol species being detected in the transgenic strains. In a second iteration, co-expression of an acyl-CoA-dependent Δ6-desaturase with the Δ5-elongase further increased DHA levels. Together, this demonstrates for the first time the potential of using iterative metabolic engineering to optimise omega-3 content in algae.

Keywords: Diatoms; Genetic engineering; Lipids; Microalgae; Omega-3 long chain polyunsaturated fatty acids.

Fig. 1

Pathways for the biosynthesis of LC-PUFAs in P. tricornutum. The predominant route for the biosynthesis of EPA is shown with red arrows and in bold text, along with other minor routes described by Arao and Yamada (1994). The predicted pathways for the biosynthesis of DHA are within the broken lined box and the expected most active route is shown with red broken arrows and with text in grey. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

The effect of heterologous Δ5-elongase expression on P. tricornutum. (A) Cellular growth of Pt_WT and transgenic P. tricornutum expressing the Ostreococcus tauri Δ5 elongase cells were harvested for lipid analysis where indicated. (B) Fatty acid composition of Pt_WT and transgenic Pt_OtElo5 cells during exponential (E) and stationary (S) phases. (C) EPA and 22 acyl carbon product content in Pt_WT and transgenic Pt_OtElo5 cells. Values are the average of three experiments (± standard error). Growth stage was determined by cell counting with reference to growth curves measured for Pt_WT and transgenic cells at 16 °C and 20 °C.

Fig. 3

The effect of heterologous Δ5-elongase expression on the acyl-CoA pool of P. tricornutum. (A) Acyl-CoA profiles of Pt_WT and Pt_ OtElo5 P. tricornutum cells cultures during stationary phase of growth. (B) EPA and selected 22 acyl carbon products from the total acyl-CoA profile. Values are the average of three experiments (± standard error).

Fig. 4

Analysis of triacylglycerol from P. tricornutum expressing an O. tauri Δ5-elongase. (A) The distribution of TAG species from Pt_WT and transgenic Pt_OtElo5 at different stages of cellular growth. (B) The distribution of DHA in TAG molecular species from Pt_WT and Pt_OtElo5 at different stages of cellular growth. Values are the average of three experiments (± standard error).

Fig. 5

Co-expression of two heterologous omega-3 LC-PUFA biosynthetic activities in P. tricornutum. Fatty acid composition of Pt_WT, pPhOS2.1 (expressing OtElo5) and pPhOS2.2 (expressing OtD6Pt and OtElo5) cells during the S phase of growth at 16 °C and 20 °C. Values are the average of three experiments (± standard error).