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首页> 外文期刊>Applied and Environmental Microbiology >Thio Wax Ester Biosynthesis Utilizing the Unspecific Bifunctional Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase of Acinetobacter sp. Strain ADP1
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Thio Wax Ester Biosynthesis Utilizing the Unspecific Bifunctional Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase of Acinetobacter sp. Strain ADP1

机译：利用非特异性双功能蜡酯合酶/酰基辅酶A：不动杆菌属的二酰基甘油酰基转移酶进行硫蜡酯的生物合成。菌株ADP1

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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 ADP1acr1ΩKm, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1ΩKm 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.
机译：来自不动杆菌属的双功能蜡酯合酶/酰基辅酶A（酰基-CoA）：二酰基甘油酰基转移酶（WS / DGAT）。介导蜡酯和三酰基甘油的生物合成的菌株ADP1（以前称为钙乙酸不动杆菌ADP1）被用于体内和体外的硫代蜡酯和二硫代蜡酯的生物合成。为了进行体外生物合成，从大肠杆菌Rosetta（DE3）pLysS（pET23a :: 5'His6atf）的可溶性蛋白级分中纯化出包含N-端His6标签的5'His6WS / DGAT。通过使用SP-Sepharose高压和Ni-tritrilotriacetic快蛋白液相色谱，使用1-十六烷醇和棕榈酰-氨基苯甲酸酯可实现19倍富集，最终比活度为165.2 nmol mg蛋白质？1 min？1。 CoA作为底物。将纯化的5'His6WS / DGAT与1-十六烷硫醇和棕榈酰-CoA作为底物进行孵育导致形成棕榈酸十六烷基硫酯（1-十六烷醇对照的相对比活性为10.4％）。利用1,8-辛二硫醇和棕榈酰-CoA作为底物导致形成1-S-单棕榈酰泛金丝桃酸二硫酚和少量的1,8-S-二棕榈酰泛金丝桃酸二硫酚（1-十六烷醇对照的相对比活性为59.3％）。当将1-S-单棕榈酰泛金刚烷二硫醇和棕榈酰-CoA用作底物时，可以有效地生产后一种二硫蜡酯（相对于1-十六烷醇对照的比活性为13.4％）。对于体内生物合成的硫代蜡酯，敲除突变体不动杆菌属。使用不能产生脂肪醇的菌株ADP1acr1ΩKm。不动杆菌属的培养。在氮有限的矿物盐培养基中存在葡萄糖酸酯，1-十六烷硫醇和油酸的情况下，菌株ADP1acr1ΩKm导致不寻常的硫代蜡酯积聚，占细胞干重的1.19％（wt / wt），由气相色谱-质谱法显示，主要是油酸十六烷基硫酯。

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