Wax ester production from n-alkanes by Acinetobacter sp. strain M-1: ultrastructure of cellular inclusions and role of acyl coenzyme A reductase

Abstract

Acinetobacter sp. strain M-1 accumulated a large amount of wax esters from an n-alkane under nitrogen-limiting conditions. Under the optimized conditions with n-hexadecane as the substrate, the amount of hexadecyl hexadecanoate in the cells reached 0.17 g/g of cells (dry weight). Electron microscopic analysis revealed that multilayered disk-shaped intracellular inclusions were formed concomitant with wax ester formation. The contribution of acyl-CoA reductase to wax ester synthesis was evaluated by gene disruption analysis.

FIG. 1.

The proposed carbon flow from n-alkanes to wax esters in Acinetobacter spp. Dotted lines indicate alternative ways to wax ester synthesis. Enzymes: 1, alkane hydroxylase complex; 2, NAD(P)-dependent alcohol dehydrogenase; 3, NAD(P)-dependent aldehyde dehydrogenase; 4, acyl-CoA synthetase; 5, acyl-CoA reductase; 6, aldehyde reductase; 7, acyl-CoA: alcohol transacylase; 8, acyl-CoA dehydrogenase.

FIG. 2.

Genetic organization of the cloned region including the acrM gene and that of the disruptant. (A) ORFs and restriction map of the cloned BamHI-HindIII 4.0-kb cloned fragment. (B) Organization of the disruption vector, pDAR1, derived from pBM4. The hatched box represents the kanamycin resistance gene. (C) Genomic Southern analysis of DraI-digested total DNA (5.0 μg per each lane) from the wild-type strain (lane 1) and the acrM-KO strain (lane 2), using the ³²P-labeled acrM gene fragment as a probe.

FIG. 3.

Electron microscopy of accumulated wax ester in Acinetobacter sp. strain M-1. Thin sections of cells grown on nutritious medium (A) and of cells after a shift to nitrogen-limited medium containing 0.5% n-hexadecane and incubation for 72 h at 30°C (B and C). (D and E) Images obtained by the quick-freezing replica method for cells prepared under the same conditions as in panel B.

FIG. 4.

Time course of wax ester accumulation in Acinetobacter sp. strain M-1. Cells were precultured in 2× YT medium (4), shifted to nitrogen-limited medium containing 0.5% n-hexadecane for the indicated incubation periods, and then subjected to electron microscopy.