Overexpression of Two New Acyl-CoA:Diacylglycerol Acyltransferase 2-Like Acyl-CoA:Sterol Acyltransferases Enhanced Squalene Accumulation in Aurantiochytrium limacinum

Abstract

Thraustochytrids are heterotrophic marine eukaryotes known to accumulate large amounts of triacylglycerols, and they also synthesize terpenoids like carotenoids and squalene, which all have an increasing market demand. However, a more extensive knowledge of the lipid metabolism is needed to develop thraustochytrids for profitable biomanufacturing. In this study, two putative type-2 Acyl-CoA:diacylglycerol acyltransferases (DGAT2) genes of Aurantiochytrium sp. T66, T66ASATa, and T66ASATb, and their homologs in Aurantiochytrium limacinum SR21, AlASATa and AlASATb, were characterized. In A. limacinum SR21, genomic knockout of AlASATb reduced the amount of the steryl esters of palmitic acid, SE (16:0), and docosahexaenoic acid, SE (22:6). The double mutant of AlASATa and AlASATb produced even less of these steryl esters. The expression and overexpression of T66ASATb and AlASATb, respectively, enhanced SE (16:0) and SE (22:6) production more significantly than those of T66ASATa and AlASATa. In contrast, these mutations did not significantly change the level of triacylglycerols or other lipid classes. The results suggest that the four genes encoded proteins possessing acyl-CoA:sterol acyltransferase (ASAT) activity synthesizing both SE (16:0) and SE (22:6), but with the contribution from AlASATb and T66ASATb being more important than that of AlASATa and T66ASATa. Furthermore, the expression and overexpression of T66ASATb and AlASATb enhanced squalene accumulation in SR21 by up to 88%. The discovery highlights the functional diversity of DGAT2-like proteins and provides valuable information on steryl ester and squalene synthesis in thraustochytrids, paving the way to enhance squalene production through metabolic engineering.

The Biosynthesis of Astaxanthin Esters in Schizochytrium sp. is Mediated by a Bifunctional Diacylglycerol Acyltransferase

“…SD116 and T66ASATb from Aurantiochytrium sp. T66 with protein identities of 68.4% and 77.6%, respectively. Further investigations are needed to determine whether ScDGAT2–1 has more functions.…”

Characterization of thraustochytrid-specific sterol O -acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186

“…Rau et al reported that dysfunction of AlASATb, which was found as a DGAT2 homolog, significantly reduced the intracellular SE level in A. limacinum SR21 and suggested that AlASATb was an acyl-CoA:sterol- O -acyltransferase (ASAT) ( 27 ). To further understand the functions of DGAT2 homologs, the C-terminal FLAG-tagged dgat2c and dgat2d were expressed in S. cerevisiae , and their expressions were examined by Western blotting using an anti-FLAG-tag antibody.…”

Microbial genetic engineering approach to replace shark livering for squalene

“…This suggests that the control of this pathway in thraustochytrids (SR21) differs from that in other species. These results suggest a more complex relationship between the squalene and carotenoid biosynthetic pathways than merely competition for the same precursors [47].…”

“…The flavoprotein squalene epoxidase catalyzes the first oxygenation reaction and rate-limiting step in this pathway. Downregulation or knockout of the genes responsible for the conversion of squalene to sterols and further to steryl esters has been shown to change the overall titer of squalene in yeast and the thraustochytrid Aurantiochytrium limacinum SR21 [47,48]. Steryl esters are synthesized by acyl-CoA:sterol acyltransferase (ASAT) through acylation of the hydroxyl group at the C3-position of sterols [48].…”

“…Disruption of acyl-CoA:sterol acyltransferase (ASAT) was associated with an increased titer of squalene in yeast [49]. By contrast, the thraustochytrid Aurantiochytrium limacinum SR21 showed a different behavior in which the expression of AlASATb and T66ASATb probably reduced the quantity of their sterol substrates and subsequently increased the amount of squalene [47], whereas inactivating both AlASATa and AlASATb reduced squalene levels. This suggests that the control of this pathway in thraustochytrids (SR21) differs from that in other species.…”