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Applied and Environmental Microbiology, February 2005, p. 790-796, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.790-796.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Stefan Uthoff,1 Tim Stöveken,1 Nikolaus Weber,2 Klaus Vosmann,2 Erika Klein,2 Rainer Kalscheuer,1 and Alexander Steinbüchel1*

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster,1 Institut für Lipidforschung, Bundesforschungsanstalt für Ernährung und Lebensmittel, Münster, Germany2

Received 18 May 2004/ Accepted 1 September 2004

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, 5'His6WS/DGAT comprising an N-terminal His6 tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5'His6atf). 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 5'His6WS/DGAT with 1-hexadecanethiol and palmitoyl-CoA 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{Omega}Km, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1{Omega}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.

* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany. Phone: 49-251-8339821. Fax: 49-251-8338388. E-mail: steinbu{at}uni-muenster.de.

Applied and Environmental Microbiology, February 2005, p. 790-796, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.790-796.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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