Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme A reductase

Abstract

Acinetobacter calcoaceticus BD413 accumulates wax esters and triacylglycerol under conditions of mineral nutrient limitation. Nitrosoguanidine-induced mutants of strain BD413 were isolated that failed to accumulate wax esters under nitrogen-limited growth conditions. One of the mutants, Wow15 (without wax), accumulated wax when grown in the presence of cis-11-hexadecenal and hexadecanol but not hexadecane or hexadecanoic acid. This suggested that the mutation may have inactivated a gene encoding either an acyl-acyl carrier protein or acyl-coenzyme A (CoA) reductase. The Wow15 mutant was complemented with a cosmid genomic library prepared from wild-type A. calcoaceticus BD413. The complementary region was localized to a single gene (acr1) encoding a protein of 32,468 Da that is 44% identical over a region of 264 amino acids to a product of unknown function encoded by an open reading frame associated with mycolic acid synthesis in Mycobacterium tuberculosis H37Ra. Extracts of Escherichia coli cells expressing the acr1 gene catalyzed the reduction of acyl-CoA to the corresponding fatty aldehyde, indicating that the gene encodes a novel fatty acyl-CoA reductase.