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

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.

Method Development Progress in Genetic Engineering of Thraustochytrids.

Thraustochytrids are unicellular, heterotrophic marine eukaryotes. Some species are known to store surplus carbon as intracellular lipids, and these also contain the long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA). Most vertebrates are unable to synthesize sufficient amounts of DHA, and this fatty acid is essential for, e.g., marine fish, domesticated animals, and humans. Thraustochytrids may also produce other commercially valuable fatty acids and isoprenoids. Due to the great potential of thraustochytrids as producers of DHA and other lipid-related molecules, a need for more knowledge on this group of organisms is needed. This necessitates the ability to do genetic manipulation of the different strains. Thus far, this has been obtained for a few strains, while it has failed for other strains. Here, we systematically review the genetic transformation methods used for different thraustochytrid strains, with the aim of aiding studies on strains not yet successfully transformed. The designs of transformation cassettes are also described and compared. Moreover, the potential problems when trying to establish transformation protocols in new thraustochytrid species/strains are discussed, along with suggestions utilized in other organisms to overcome similar challenges. The approaches discussed in this review could be a starting point when designing protocols for other non-model organisms.

A non-canonical Δ9-desaturase synthesizing palmitoleic acid identified in the thraustochytrid Aurantiochytrium sp. T66.

Thraustochytrids are oleaginous marine eukaryotic microbes currently used to produce the essential omega-3 fatty acid docosahexaenoic acid (DHA, C22:6 n-3). To improve the production of this essential fatty acid by strain engineering, it is important to deeply understand how thraustochytrids synthesize fatty acids. While DHA is synthesized by a dedicated enzyme complex, other fatty acids are probably synthesized by the fatty acid synthase, followed by desaturases and elongases. Which unsaturated fatty acids are produced differs between different thraustochytrid genera and species; for example, Aurantiochytrium sp. T66, but not Aurantiochytrium limacinum SR21, synthesizes palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7). How strain T66 can produce these fatty acids has not been known, because BLAST analyses suggest that strain T66 does not encode any Δ9-desaturase-like enzyme. However, it does encode one Δ12-desaturase-like enzyme. In this study, the latter enzyme was expressed in A. limacinum SR21, and both C16:1 n-7 and C18:1 n-7 could be detected in the transgenic cells. Our results show that this desaturase, annotated T66Des9, is a Δ9-desaturase accepting C16:0 as a substrate. Phylogenetic studies indicate that the corresponding gene probably has evolved from a Δ12-desaturase-encoding gene. This possibility has not been reported earlier and is important to consider when one tries to deduce the potential a given organism has for producing unsaturated fatty acids based on its genome sequence alone. KEY POINTS: • In thraustochytrids, automatic gene annotation does not always explain the fatty acids produced. • T66Des9 is shown to synthesize palmitoleic acid (C16:1 n-7). • T66des9 has probably evolved from Δ12-desaturase-encoding genes.

Dissection of GTPase-activating proteins reveals functional asymmetry in the COPI coat of budding yeast.

The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggest that each niche preferentially recruits one of the two ArfGAPs known to affect COPI, i.e. Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis through the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the Snf1 kinase complex, the yeast homologue of AMP-activated protein kinase (AMPK), phosphorylates the region of Glo3 that is crucial for this effect and, thereby, regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI.This article has an associated First Person interview with the first author of the paper.