Identification, isolation, and heterologous expression of Sunflower wax synthase for the synthesis of tailored wax esters.

Abstract

Wax esters (WE) are neutral lipids formed by condensation of fatty alcohol with fatty acyl-CoA by wax synthases. They serve as carbon and energy reserves and are potential substrates for various commercial applications. Sunflower (Helianthus annuus) an edible oil seed is a source of WE, however, the gene responsible for WE formation has hitherto remained unidentified. Using an in silico approach we identified, isolated putative Sunflower wax synthase (HaWS) gene and investigated it's potential for WE production in yeast. Heterologous expression of HaWS in Saccharomyces cerevisiae H1246 exhibited 57kDa protein which was confirmed by immunoblotting. Recombinant yeast expressing HaWS were fed with combinations of C16, C18 fatty alcohols with 16:0, 18:0 fatty acyl CoA's as potential substrates to validate WE formation in vivo. The yeast cells accumulated C-32 to C-36 WE. Our study reveals identification, isolation, and heterologous functional expression of WS gene from Sunflower for the first time. PRACTICAL APPLICATIONS: Wax synthases (WSs) are critical enzymes for wax ester (WE) biosynthesis. WEs are high value products having several industrial applications. WE serve as substrates for lubricants, food coatings, cosmetics, and pharmaceuticals. There is a demand for alternate renewable resource of WEs. In this study, we have successfully isolated a putative wax synthase gene from Sunflower and submitted its sequence data to the GenBank (Accession number MH460820). Conserved sequence search analysis showed presence of condensation superfamily motif‒HHXXXDG, critical for WE biosynthesis. Heterologous expression ofHaWSin yeast revealed synthesis of C-32 to C-36 WE. Our study demonstrates the efficacy ofHaWSto accumulate specific WE of desired lengths in yeast, and thus represents an alternate source of WE for commercial applications and for biotechnological production of tailored WE in eukaryotic expression systems.