In vitro and in vivo biosynthesis of wax diesters by an unspecific bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase from Acinetobacter calcoaceticus ADP1

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 long-chain 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-C-14-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.