Thio Wax Ester Biosynthesis Utilizing the Unspecific Bifunctional Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase of Acinetobacter sp. Strain ADP1

Abstract

The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5His 6 WS/DGAT comprising an N-terminal His 6 tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5His 6 atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein ؊1 min ؊1 was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5His 6 WS/DGAT with 1-hexadecanethiol and palmitoylCoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1⍀Km, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1⍀Km in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.Polyhydroxyalkanoic acids (PHA) in bacteria represent the most abundant group of neutral storage lipids, which serve as intracellular carbon and energy storage compounds (23). In addition, triacylglycerols (TAGs) and wax esters also occur as bacterial storage lipids, though at a much lower frequency than in eukaryotic microorganisms (22). Significant TAG accumulation was found, particularly in species belonging to the class Actinomycetes (1), as was reported for the genera Mycobacterium (3), Nocardia, and Streptomyces (2). TAGs are synthesized by the acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (DGAT) (13), which catalyzes the esterification of diacylglycerol with long-chain acyl-CoA. Wax esters are oxoesters of primary long-chain fatty alcohols and long-chain fatty acids. Occurrence of wax esters has been frequently reported for various Acinetobacter species (6). Under growthlimiting conditions, Acinetobacter calcoaceticus accumulates wax esters intracellularly as insoluble inclusions. The chemical structure of the wax esters synthesized by A. ca...

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