The glycerol-3-phosphate acyltransferase PLAT2 functions in the generation of DHA-rich glycerolipids in Aurantiochytrium limacinum F26-b

Abstract

Thraustochytrids possess docosahexaenoic acid (DHA, 22:6n-3) as acyl chain(s) of triacylglycerol (TG) and phosphatidylcholine (PC), some of which contain multiple DHAs. However, little is known about how these DHA-rich glycerolipids are produced in thraustochytrids. In this study, we identified PLAT2 in Aurantiochytrium limacinum F26-b as a glycerol-3-phosphate (G3P) acyltransferase (GPAT) by heterologous expression of the gene in budding yeast. Subsequently, we found that GPAT activity was reduced by disruption of the PLAT2 gene in A. limacinum, resulting in a decrease in DHA-containing lysophosphatidic acid (LPA 22:6). Conversely, overexpression of PLAT2 increased both GPAT activity and LPA 22:6. These results indicate that PLAT2 is a GPAT that transfers DHA to G3P in vivo as well as in vitro. Overexpression of the PLAT2 gene increased the production of a two DHA-containing diacylglycerol (DG 44:12), followed by an increase in the three DHA-containing TG (TG 66:18), two-DHA-containing TG (TG 60:12), and two DHA-containing PC (PC 44:12). However, overexpression of PLAT2 did not increase DHA-free DG (DG32:0), which was preferentially converted to three 16:0-containing TG (TG 48:0) but not two 16:0-containing PC (PC 32:0). Collectively, we revealed that DHA-rich glycerolipids are produced from a precursor, LPA 22:6, which is generated by incorporating DHA to G3P by PLAT2 in the A. limacinum.

The lipid metabolism in thraustochytrids

“…Among non-polar lipids, diacylglycerol (DAG) represents less than 1% of total lipids whereas TAG may fluctuate widely during the growth period and can reach up to 90% of total lipids (Table 1). Mining the available sequencing data of Aurantiochytrium limacinum genome, and using reciprocal blast approach to identify orthologous genes in H. fermentalgiana, all the genes involved in the synthesis of glycerolipids have now been identified [6,75,76], enabling the reconstitution of the pathway (Fig. 5).…”

“…For example, overexpression of a glycerol-3-phosphate acyltransferase, the first enzyme in this pathway, responsible for the attachment of the first FA on the glycerol backbone ( Fig. 5) results in a modest increase of the TAG content [76]. Also, a lysophosphatidyl acyltransferase involved in the transfer of 16:0 to lysoPC and lysoPE was identified [73].…”

Lipid and DHA-production in Aurantiochytrium sp. – Responses to nitrogen starvation and oxygen limitation revealed by analyses of production kinetics and global transcriptomes

“…4c; Supplementary Table S1). An acyltransferase identified in A. limacinum 17 , which adds DHA to glycerol-3-phosphate, was also identified in T66 (GPAT-1, Fig. 4c).…”

Method Development Progress in Genetic Engineering of Thraustochytrids

“… ( a ) The number of successfully engineered thraustochytrid strains by one or more methods (electroporation [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ], biolistic transformation [ 5 , 27 , 53 , 54 , 55 ], and others [ 56 , 57 , 58 , 59 ]) ( Tables S1 and S2 ). The doughnut pie charts indicate the proportion of different genera.…”