Thio Wax Ester Biosynthesis Utilizing the Unspecific Bifunctional Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase of Acinetobacter sp. Strain ADP1
| Autor: | Tim Stöveken, Stefan Uthoff, Rainer Kalscheuer, Erika Klein, Alexander Steinbüchel, N. Weber, Klaus Vosmann |
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| Rok vydání: | 2005 |
| Předmět: |
Drug Industry
Thio Thioester Applied Microbiology and Biotechnology Gas Chromatography-Mass Spectrometry Substrate Specificity Palmitic acid chemistry.chemical_compound Biosynthesis Escherichia coli Diacylglycerol O-Acyltransferase Sulfhydryl Compounds Chromatography High Pressure Liquid chemistry.chemical_classification Wax Acinetobacter Ecology Esters Physiology and Biotechnology Culture Media Wax ester Oleic acid Enzyme Biochemistry chemistry Waxes visual_art visual_art.visual_art_medium lipids (amino acids peptides and proteins) Acyl Coenzyme A Acyltransferases Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 71:790-796 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.71.2.790-796.2005 |
| Popis: | 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′His 6 WS/DGAT comprising an N-terminal His 6 tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a:: 5′His 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 5′His 6 WS/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 ADP1 acr1 ΩKm, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1 acr1 ΩKm 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. |
| Databáze: | OpenAIRE |
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