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

Abstract

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid-and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/ 20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta. Keywords Marinobacter aquaeolei VT8 Á Neutral lipids Á Seed oil Á Storage lipids Á Wax ester Á Wax synthase/acyl CoA:diacylglycerol O-acyltransferase Lipids (2020) 55: 479-494. Abbreviations A. baylyi Acinetobacter baylyi A. calcoaceticus Acinetobacter calcoaceticus, nowadays: Acinetobacter baylyi A. thaliana Arabidopsis thaliana aa amino acids AbWSD1 Acinetobacter baylyi WSD1, Ac1, AtfA ACP acyl carrier protein ATP adenosine triphosphate C. sativa Camelina sativa Supporting information Additional supporting information may be found online in the Supporting Information section at the end of the article.

Plastidial Wax Ester Biosynthesis As A Tool To Synthesize Shorter And More Saturated Wax Esters

“…20 independent lines were screened subsequently for high WE content in T2 seeds by WE extraction and thin layer chromatography (TLC) (Iven et al, 2013). For MaFAR/MaWSD5, ten new lines were screened in addition to the ones published by Vollheyde et al (2020). In case of MaFAR/MaWSD2, clMaFAR/clMaWSD2, MaFAR/MaWSD5 and clMaFAR/clMaWSD5 plants, 50 % to 80 % of the screened lines showed WE formation in T2 seeds.…”

“…As signi cant WE amounts were obtained in T2 seeds of plants expressing the four constructs MaFAR/MaWSD2, clMaFAR/clMaWSD2, MaFAR/MaWSD5 and clMaFAR/clMaWSD5, three independent lines per construct with high WE levels were chosen for further analyses regarding protein expression, WE content and generated WE species (Table 1). For a comparison with results published for MaFAR/MaWSD5, three additional lines were analyzed in detail as the ve already published ones were only analyzed by nanoelectrospray ionization tandem mass spectrometry (nanoESI-MS/MS) (Vollheyde et al, 2020).…”

“…To compare plastidial and cytosolic WE production, we designed constructs following our recently published analysis of transgenic A. thaliana MaFAR/MaWSD5 plants (Vollheyde et al, 2020) and generated nine additional constructs consisting of MaFAR combined with either MaWSD2 or MaWSD5 with and without plastidial transit peptide (Fig. 1, Table 1).…”

“…Especially WE derived from monounsaturated long chain substrates are requested in industry due to excellent lubrication properties (Heilmann et al, 2012). Which and how much WE species are synthesized in planta, depends on one hand on the activities and substrate speci cities of the expressed FAR and WS enzymes, on the other hand on the availability of acyl-CoA/ACP substrates: Whereas the expression of mouse FAR and mouse WS led to the formation of WE with mainly polyunsaturated 18 carbon acyl moieties (Heilmann et al, 2012;Iven et al, 2016), enzyme combinations with Marinobacter aquaeolei FAR (MaFAR) and jojoba WS (ScWS), Acinetobacter baylyi WSD1 (AbWSD1) or M. aquaeolei WSD5 (MaWSD5) produced WE with mainly monounsaturated 18 and 20 carbon acyl and alcohol moieties (Iven et al, 2016;Vollheyde et al, 2020;Yu et al, 2018). Interestingly, expression of different enzyme combinations in the high oleic acid mutant fad2 fae1 (Kunst et al, 1992;Okuley et al, 1994) resulted in more than 60 mol% 18:1/18:1 (alcohol moiety/acyl moiety) WE (Heilmann et al, 2012;Iven et al, 2016;Yu et al, 2018).…”

Randy Weselake: Celebrating a Gentleman Scholar Contributing across a Wide Spectrum of Lipid Biochemistry

“…Wax esters are a critically important component of industrial lubricants, but currently the production of wax esters is dependent upon petroleum. In this original article from Ivo Feussner's group, they report the isolation and characterization of a newly identified wax ester synthase (MaWSD5) from Marinobacter aquaeolei VT8 (Vollheyde et al, 2020). Although this family of enzymes is normally considered bifunctional enzymes that synthesize wax esters and TAG, MaWSD5 only catalyzes the synthesis of wax esters.…”