Utilizing inhibitors and genetic engineering to study the biosynthesis of unsaturated fatty acids and lipids in two thraustochytrid strains
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Thraustochytrids are oleaginous heterotrophic marine eukaryotic microbes known to produce large amounts of docosahexaenoic acid (DHA)-containing triacylglycerols (TAGs), and they also synthesize terpenoids like squalene. These compounds have great economic value and are in high demand. However, DHA produced from thraustochytrids is still more costly than from fish oil whose availability is limited and unstable. Hence, a deeper understanding of the lipid metabolism and genetic manipulation tools in thraustochytrids is vital to developing high productive strains for profitable biomanufacturing. In thraustochytrids, since the fatty acid synthase (FAS) pathway and the DHA synthesizing polyketide synthase-like pathway (PKS) share the same precursors, the first part of this thesis characterized the fatty acid and lipid accumulation between Aurantiochytrium sp. T66 (T66) and Aurantiochytrium limacinum SR21 (SR21) and studied how it is affected by FAS inhibition. The result showed that the DHA proportion, but not the DHA productivity, was greater under FAS inhibition in the two strains. This suggests that precursor availability for the PKS pathway is not the rate-limiting factor for DHA synthesis. FAS inhibition could also enrich DHA-rich TAG TG(22:6/22:6/22:6) production. Moreover, the conversion of DG(22:6/22:6) to TAGs is potentially a bottleneck for DHA-rich TAGs synthesis. This work then reviewed the genetic engineering methods employed for different thraustochytrid strains and the relevant experience in other organisms to support research on strains that have yet to be successfully transformed. The variables being considered are the transformation methods, genomic integration of DNAs, the elements that regulate gene expression and transformant selection. Genetic engineering was then utilized to characterize genes potentially related to fatty acid (FA) and lipid metabolism, including one Δ12-desaturase-like gene, T66des9, and two type-2 Acyl-CoA:diacylglycerol acyltransferases (DGAT2)-like genes of T66, T66ASATa and T66ASATb, and their homologs in SR21, AlASATa and AlASATb. The expression of T66des9 via genomic knockin in SR21 produced palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7), while both FAs were not detected in the control strains. This indicates that T66Des9 is a Δ9-desaturase accepting palmitic acid (C16:0) as a substrate. Genomic knockout of AlASATa and AlASATb or knockin of the four genes in SR21 showed that the expression of AlASATb and T66ASATb, and in less extent, AlASATa and T66ASATa, elevated the accumulation of total steryl esters (SEs), the SEs of C16:0, SE(16:0), and DHA, SE(22:6), and, on the other hand, did not significantly change the level of TAGs or other lipid classes. The results suggest that the four genes encoded proteins possessing acyl-CoA:sterol acyltransferase (ASAT) activity. Furthermore, the expression and overexpression of T66ASATb and AlASATb enhanced squalene production in SR21. The functional discoveries of the above-mentioned genes pave the way to enhance C16:1 n-7, C18:1 n-7, and squalene production in thraustochytrids through metabolic engineering, and highlight the functional diversity of desaturases and acyl-CoA acyltransferases, which are important to consider when attempting to deduce the function of these proteins solely based on their sequence.
Has partsPaper 1: Utilizing lipidomics and fatty acid synthase inhibitors to explore lipid accumulation in two Aurantiochytrium species
Paper 2: Rau, E-Ming; Ertesvåg, Helga. Method Development Progress in Genetic Engineering of Thraustochytrids. Marine Drugs 2021 ;Volum 19.(9) https://doi.org/10.3390/md19090515 This is an open access article distributed under the Creative Commons Attribution License (CC BY 4.0)
Paper 3: Rau, E-Ming; Aasen, Inga Marie; Ertesvåg, Helga. A non-canonical Δ9-desaturase synthesizing palmitoleic acid identified in the thraustochytrid Aurantiochytrium sp. T66.. Applied Microbiology and Biotechnology 2021 ;Volum 105. s. 5931-5941 https://doi.org/10.1007/s00253-021-11425-5 https://doi.org/10.1007/s00253-021-11425-5 This article is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0)
Paper 4: Aurantiochytrium species encode two acyl-CoA:diacylglycerol acyltransferase 2 like acyl-CoA:sterol acyltransferases whose overexpression facilitate squalene accumulation.