In vitro characterization of five bacterial WS/DGAT acyltransferases regarding the synthesis of biotechnologically relevant short‐chain‐length esters

For the biotechnical production of biofuels, oleo‐, or fine chemicals bacterial wax ester synthase/acyl‐Coenzyme A:diacylglycerol acyltransferases (WS/DGAT) are discussed as interesting candidates for in vivo esterification reactions. In this study, the suitability of selected acyltransferases for the conversion of non‐physiological substrates like short‐chain‐length alcohols and short‐chain length or even branched acyl‐CoAs has been investigated. In vitro analyzes of purified AtfA and AtfA(G355I) from Acinetobacter baylyi , Ma1(A360I) from Marinobacter aquaeolei , WS2 from Marinobacter hydrocarbonoclasticus , and AtfA1 from Alcanivorax borkumensis were conducted to evaluate the specific activities of these enzymes toward n‐hexadecanol (C 16 ), n‐dodecanol (C 12 ), ethanol (C 2 ), and methanol (C 1 ), palmitoyl‐CoA (C 16 ), butyryl‐CoA (C 4 ) as well as toward branched 3‐hydroxybutyryl‐CoA and 2‐hydroxyisobutyryl‐CoA. Athough long‐ and medium‐chain‐length substrates were preferred by all five enzymes, WS2 and AtfA showed the highest relative activities with ethanol or methanol when compared to n‐hexadecanol, whereas residual activities toward short or branched acyl‐CoAs could only be measured with AtfA and AtfA1. Practical applications: Bacterial WS/DGATs can be used for in vivo and in vitro approaches to synthesize custom‐made lipids, such as triglycerides or wax esters. However, due to their broad substrate ranges these enzymes are also promising candidates for the synthesis of other, industrially valuable oleo‐ and fine chemicals. Short‐chain‐length esters are important intermediates and building blocks for many production processes and, at present, there is a great demand for enzymes which are able to catalyze their synthesis. The physiological substrates of bacterial WS/DGAT enzymes are medium‐ to long‐chain length acyl‐CoAs and fatty alcohols to synthesize medium‐ to long‐chain length wax esters. In this study, we investigated five different bacterial WS/DGATs for their ability to synthesize short‐chain length esters, which represent biotechnologically interesting compounds. The enzymes AtfA, AtfA(G355I), Ma1(A360I), WS2, and AtfA1 were selected, purified, and characterized in vitro. In addition to the reference substrates, hexadecanol and palmitoyl‐CoA, their activity with dodecanol, ethanol, or methanol, on the one hand, and lauryl‐CoA, butyryl‐CoA, 3‐hydroxybutyryl‐CoA, or 2‐hydroxyisobutyryl‐CoA, on the other hand, were studied and compared.