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

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

doi:10.3389/fmicb.2022.822254

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.

Highlights

Fatty acids can be stored in lipid bodies as neutral triacylglycerol (TAG) or steryl ester (SE) and serve as energy or membrane material reservoirs (Liu et al, 2012; Korber et al, 2017)
Membrane-bound diacylglycerol acyltransferase (DGAT)-related enzymes can be divided into five clearly separated groups (Xu et al, 2021), and from Figure 2 it is seen that the DGAT2-like protein cluster becomes very diverse when the microalgae and thraustochytrid sequences are included
The results from the present study suggest that AlASATa, T66ASATa, AlASATb, and T66ASATb display acyl-CoA:sterol acyltransferase (ASAT) activity and contribute to the formation of SEs

Summary

Introduction

Fatty acids can be stored in lipid bodies as neutral triacylglycerol (TAG) or steryl ester (SE) and serve as energy or membrane material reservoirs (Liu et al, 2012; Korber et al, 2017). TAG can be synthesized by the sn-glycerol-3-phosphate (G3P) pathway starting from G3P as the backbone, followed by steps including three acylations with the acyl groups provided by fatty acyl-CoA (Figure 1). The final acylation of diacylglycerol (DAG) generates TAG, usually catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). It has been shown that DAG can be produced from monoacylglycerol (MAG) by acyl-CoA:monoacylglycerol acyltransferase (MGAT) (Liu et al, 2012). The acyl-groups may be donated by phosphatidylcholine, in reactions catalyzed by a phospholipid: diacylglycerol acyl transferase (PDAT). Acetyl-CoA serves as a precursor for both fatty acids (FAs) and sterols (Figure 1)

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Conclusion