Enhancing LC-PUFA production in Thalassiosira pseudonana by overexpressing the endogenous fatty acid elongase genes

Abstract

The health beneficial omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are naturally synthesized by diatoms through consecutive steps of fatty acid elongase and desaturase enzymes. In Thalassiosira pseudonana, these fatty acids constitute about 10-20 % of the total fatty acids, with EPA accumulation being five to ten times higher than DHA. In order to identify the subcellular localization of enzymes in the pathway of LC-PUFA biosynthesis in T. pseudonana and to manipulate the production of EPA and DHA, we generated constructs for overexpressing each of the T. pseudonana long-chain fatty acid elongase genes. Full-length proteins were fused to GFP, and transgenic lines were generated. In addition, overexpressed native proteins with no GFP fusion were tested. The subcellular localization of each elongase protein was determined. We then examined the total amount of lipids and analyzed the fatty acid profile in each of the transgenic lines compared to wild type. Lines with overexpressed elongases showed an increase of up to 1.4-fold in EPA and up to 4.5-fold in DHA, and the type of fatty acid that was increased (EPA vs. DHA) depended on the type of elongase that was overexpressed. This data informs future metabolic engineering approaches to further improve EPA and DHA content in diatoms.

Characterization and manipulation of a DGAT2 from the diatom Thalassiosira pseudonana: Improved TAG accumulation without detriment to growth, and implications for chloroplast TAG accumulation

“…S2). In T. pseudonana, we have demonstrated an increase in DHA and EPA resulting from silicon-limitation [52], when TAG accumulates. Perhaps any trigger that stimulates TAG accumulation, such as Thaps3_25595 overexpression, shifts the FA composition similarly.…”

Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype

“…Although C. cryptica contained fewer copies of type 1 desaturases, it had all the types including Δ5, Δ6, Δ8, Δ9, Δ11 and Δ12. Both diatoms contain a single copy of a Δ4 desaturase that catalyzes the addition of the sixth double bond to docosapentaenoic acid (22:5) chain to make DHA (C22:6) [20]. The existence of the same types of elongases and desaturases in both diatoms suggest that the greater ability of C. cryptica to produce EPA compared to T. pseudonana is not due to an increase in isozymes in the genome and could be more dependent on the flux of carbon from upstream processes into this pathway.…”

“…Both random mutagenesis and directed genetic manipulation can be used to accomplish this. The latter requires knowledge of an organism’s genome sequence, and such information has successfully been used to identify targets for genetic manipulation in diatoms to improve lipid productivity [14, 17, 20, 21]. To identify appropriate gene targets for manipulation, a thorough understanding of the enzymes involved in central carbon metabolism is required, including the number of isozymes that catalyze each chemical reaction and the compartment-specific localization of enzymes or enzymatic processes within the cell.…”

Editing of the urease gene by CRISPR-Cas in the diatomThalassiosira pseudonana

“…It is a model centric diatom with a sequenced genome (first eukaryotic marine phytoplankton to be sequenced [2]) and wellestablished transformation systems [19,20]. The genus has multiple biotechnology applications [8,21,22], and although gene silencing has been established, a method to easily and efficiently knock-out and edit genes and the entire genome would be highly advantageous. The genus Thalassiosira is among the top 10 genera of diatoms in the World's Ocean in terms of ribotype (V9 of 18S) diversity and abundance [23] and the species T. pseudonana is a model for understanding the mechanisms behind silicification [24][25][26].…”