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

Abstract

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.

Thraustochytrids of Mangrove Habitats from Andaman Islands: Species Diversity, PUFA Profiles and Biotechnological Potential

“…Among the strains, ANVKK-06 showed the highest growth (OD value 3.32) and ANVKK-08 displayed the least growth (OD value 2.22). In general, the growth behavior of thraustochytrids varies with strains/species to strains/species and the results obtained in this study were similar to earlier work on thraustochytrids [ 35 , 50 , 52 ]. Generally, the growth of thraustochytrids depends on the availability of nutrients.…”

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

“…Aurantiochytrium limacinum SR21 (ATCC MYA-1381) and Aurantiochytrium sp. T66 (ATCC PRA-276) and strains generated in this study ( Supplementary Table 1 ) were cultivated and stored as described earlier ( Rau et al, 2021 ).…”

“…Molecular cloning procedures and materials were performed as described earlier ( Rau et al, 2021 ). NEBuilder ® HiFi DNA Assembly Master Mix (New England Biolabs, United States) was used for Gibson assembly, Plasmids, primers and gene accession numbers for the cloned genes are described in Supplementary Tables 2 – 4 , respectively.…”

“…The electrotransformation protocol of A. limacinum SR21 followed the procedures described earlier ( Rau et al, 2021 ). Briefly, subcultured cells with OD 600 ∼3 were collected and washed once by BSS (10 mM KCl, 10 mM NaCl, 3 mM CaCl 2 ) and twice by 50 mM sucrose.…”

“…RNA isolation, cDNAs synthesis and RT-PCR were performed as described earlier ( Rau et al, 2021 ) using the β-tubulin gene cDNA as a reference gene ( Zhang K. et al, 2017 ). PCR was performed to amplify parts of AlASATa cDNA by the primer pair 137698RT_f and 137698RT_r, AlASATb cDNA by the primer pair 43553RT_f and 43553RT_r, and β-tubulin gene cDNA by the primer pair SR21tubF2 and SR21tubR2 ( Supplementary Table 3 ).…”