AUTHOR=Rau E-Ming , Bartosova Zdenka , Kristiansen Kåre Andre , Aasen Inga Marie , Bruheim Per , Ertesvåg Helga 

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

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.822254

DOI=10.3389/fmicb.2022.822254

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Aurantiochytrium</italic> sp. T66, <italic>T66ASATa</italic>, and <italic>T66ASATb</italic>, and their homologs in <italic>Aurantiochytrium limacinum</italic> SR21, <italic>AlASATa</italic> and <italic>AlASATb</italic>, were characterized. In <italic>A. limacinum</italic> SR21, genomic knockout of <italic>AlASATb</italic> reduced the amount of the steryl esters of palmitic acid, SE (16:0), and docosahexaenoic acid, SE (22:6). The double mutant of <italic>AlASATa</italic> and <italic>AlASATb</italic> produced even less of these steryl esters. The expression and overexpression of <italic>T66ASATb</italic> and <italic>AlASATb</italic>, respectively, enhanced SE (16:0) and SE (22:6) production more significantly than those of <italic>T66ASATa</italic> and <italic>AlASATa</italic>. 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 <italic>T66ASATb</italic> and <italic>AlASATb</italic> 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.</p>