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In vitro and in vivo biosynthesis of wax diesters by an unspecific bifunctional wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase from Acinetobacter calcoaceticus ADP1
Author(s) -
Kalscheuer Rainer,
Uthoff Stefan,
Luftmann Heinrich,
Steinbüchel Alexander
Publication year - 2003
Publication title -
european journal of lipid science and technology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.614
H-Index - 94
eISSN - 1438-9312
pISSN - 1438-7697
DOI - 10.1002/ejlt.200300840
Subject(s) - wax , biosynthesis , biochemistry , acinetobacter calcoaceticus , acyltransferase , acylation , chemistry , palmitic acid , wax ester , glycerophospholipids , enzyme , fatty acid , phospholipid , acinetobacter , catalysis , antibiotics , membrane
Abstract Acinetobacter calcoaceticus ADP1 possesses a bifunctional wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase (WS/DGAT) catalyzing the biosynthesis of wax esters and triacylglycerols. The unspecificity of WS/DGAT was used for in vitro and in vivo biosynthesis of wax diesters consisting of 1, 16‐hexadecanediol esterified with longchain fatty acids. An in vitro assay employing the membrane fraction of recombinant Escherichia coli XL1‐Blue expressing wax/dgat coding for WS/DGAT and using 1, 16‐hexadecanediol and 1 ‐14 C‐palmitoyl‐CoA as substrates resulted in the production of 2 radiolabeled substances as revealed by autoradiography suggesting the acylation of one or both hydroxy groups of 1, 16‐hexadecanediol by WS/DGAT. For in vivo biosynthesis of wax diesters, the knock‐out mutant A. calcoaceticus ADP1 acr1 | Km was generated by disruption of acr1 coding for acyl‐CoA reductase which caused the inability to synthesize fatty alcohols and, thus in consequence, wax esters. Co‐cultivation of A. calcoaceticus ADP1 acr1 | Km on gluconate and 1, 16‐hexadecanediol in nitrogen‐limited mineral salts medium resulted in the accumulation of a mixture of wax diesters of 1, 16‐hexadecanediol esterified with palmitic and oleic acid as revealed by electron impact ionization mass spectrometry. 1‐Monopalmitoylglycerol could also be utilized as an alternative acyl acceptor by the unspecific WS/DGAT in vitro resulting in the synthesis of 1, 2‐ and 1, 3‐dipalmitoylglycerol, whereas 1‐oleoylglycerol‐3‐phosphate (lysophosphatidic acid) was not accepted.