Structural and Biochemical Studies of a Biocatalyst for the Enzymatic Production of Wax Esters

Abstract

Wax esters are high-value products whose enzymatic synthesis is of increasing biotechnological interest. The fabrication of cell factories that mass-produce wax esters may provide a facile route towards a sustainable, and environment-friendly approach to a large-scale process for this commodity chemical. An expedient route for wax-ester biocatalysis may be facilitated by the action of enzymes termed wax ester synthases/diacylglycerol acyltransferases (WS/DGAT), which produce wax esters using fatty acids and alcohols as a precursor. In this work, we report the structure for a member of the WS/DGAT superfamily. The structural data in conjunction with bioinformatics and mutational analyses allowed us to identify the substrate binding pockets, and residues that may be important for catalysis. Using this information as a guide, we generated a mutant with preference towards shorter acyl-substrates. This study demonstrates the efficacy of a structure-guided engineering effort towards a WS/DGAT variant with preference towards wax esters of desired lengths.

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

“…As average values from five independent plant lines were summed up, no error bars are shown this study. The result is in line with other publications describing MaWSD1 and MhWS1 as bifunctional WSD (Barney et al, 2012;Holtzapple and Schmidt-Dannert, 2007;Petronikolou and Nair, 2018). MaWSD3 did not show any activity when expressed in S. cerevisiae H1246.…”

“…MaWSD5-his 6 had a higher WS activity with 18:1 OH compared to 18:0 OH and no WS activity was observed for saturated fatty alcohols of 20 carbons chain length or longer. This observation might be either explained by a different solubility of saturated and desaturated fatty alcohols which can affect the accessibility of the substrate for the enzyme or by spatial limitations of the fatty alcohol binding site (Barney et al, 2013(Barney et al, , 2015Petronikolou and Nair, 2018). The binding site might allow only the binding of fatty alcohols with a certain chain length.…”

“…To identify new WSD enzymes in the bacterium M. aquaeolei VT8, a BLAST homology search on the genome of M. aquaeolei VT8 using the amino acid sequence of AbWSD1 was performed. In addition to the four already known and partly characterized WSD MaWSD1, MaWSD2, MaWSD3, and MaWSD4 (Barney et al, 2012(Barney et al, , 2013(Barney et al, , 2015Petronikolou and Nair, 2018;Röttig et al, 2016;Villa et al, 2014), a putative fifth WSD (MaWSD5, Maqu_3411) was identified. While characterizing MaWSD5, the existence of the protein was also mentioned in a book chapter (Knutson et al, 2017).…”

Uncovering the role of a positive selection site of wax ester synthase/diacylglycerol acyltransferase in two closely related Stipa species in wax ester synthesis under drought stress

“…In addition, mutational analyses of bacterial WSD (Ma1; WP_011783747) from M. aquaeolei VT8 also showed that mutations of 25G, 144 A, 356L, 360A, and 405M sites could alter substrate selectivity to synthesize wax esters of different lengths ( Barney et al, 2013 , 2015 ; Petronikolou et al , 2018 ). However, 259T and 403A sites of SpWSD1 do not have corresponding positions in the Ma1 sequence due to absence of the corresponding structure Ma1 based on the pairwise alignment of the two gene sequences ( Supplementary Fig.…”

“…Recombinant expression analyses of WSD1 in yeast and Escherichia coli revealed predominant wax synthase functions, and showed that it could synthesize different wax ester products, including C34, C40, and C44, using different substrate combinations of primary alcohols and primary acids ( Li et al , 2008 ). The substrate selectivity of WS/DGAT enzymes from Marinobacter aquaeolei can be altered through mutations in specific residues, resulting in the synthesis of different wax ester components ( Barney et al, 2013 , 2015 ; Petronikolou et al , 2018 ).…”