Long-chain aldehyde dehydrogenase that participates in n-alkane utilization and wax ester synthesis in Acinetobacter sp. strain M-1

Abstract

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C(4) to C(14)), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Delta) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Delta strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.

FIG. 1

Genetic organization of the cloned region including ald1 and that of disrupted ald1. (A) Restriction map of the 5.7-kb HindIII cloned fragment. (B) The ORFs within the sequenced region of the 3.5-kb PstI fragment. ORF1 showed homology with a hypothetical 47.3-kDa protein in the thcA 5′ region (ORF3) from Rhodococcus sp. strain NI86/21 (GenBank accession number U17129) (15), and ORF2 showed homology with ethanolamine permease from Rhodococcus sp. strain NI86/21 (GenBank accession number L24492) (4). (C) Construction of the gene disruption vector pDALD1, derived from pSH6. The hatched box represents the kanamycin resistance gene. (D) Genomic Southern analysis of EcoRV-KpnI double-digested total DNAs (5.0 μg each) from the wild-type strain (lane 1) and the ald1Δ strain (lane 2), with the ³²P-labeled ald1 fragment as a probe.

FIG. 2

Substrate specificity of rAld1 purified from E. coli(pCM1). Enzyme activity was measured under standard conditions. One hundred percent corresponds to 10.9 U/mg of protein. Numbers on the x axis indicate n-alkanals as follows: 2, ethanal (100 mM); 3, propanal (100 mM); 4, butanal (20 mM); 5, pentanal (10 mM); 6, hexanal (5 mM); 7, heptanal (5 mM); 8, octanal (2 mM); 9, nonanal (2 mM); 10, decanal (2 mM); 11, undecanal (1 mM); 12, dodecanal (1 mM); 13, tridecanal (1 mM); 14, tetradecanal (1 mM).

FIG. 3

(A) Northern analysis of the ald1 gene. A 20-μg portion of total RNA was loaded on each lane, and ald1 transcription was detected by hybridization with the ³²P-labeled ald1 fragment as a probe. RNA was prepared from Acinetobacter sp. strain M-1 cells grown on a salt medium containing 1% (wt/vol) sodium acetate (NaAc) or 0.5% (vol/vol) n-alkane, the carbon length of which is indicated. (B) Relative enzyme activity of aldehyde dehydrogenase in cells grown on each substrate. The enzyme activity was measured under standard conditions using tetradecanal as a substrate. One hundred percent corresponds to 0.76 U/mg of protein.

FIG. 4

Degradation of intracellular wax esters in the wild-type and ald1Δ strains. The cells, which were grown on a nitrogen-limiting medium containing 0.5% (vol/vol) n-hexadecane, were shifted to a salt medium without a carbon source, and then the amount of intracellular wax esters was measured at intervals. Solid bars, wild-type strain; open bars, ald1Δ strain.

FIG. 5

Growth of the wild-type and ald1Δ strains on n-hexadecane. Cells were cultured in a salt medium containing 0.5% (vol/vol) n-hexadecane, and growth was measured as described in Materials and Methods. Open circles, wild-type strain; solid circles, ald1Δ strain.

FIG. 6

Proposed carbon flow of n-alkanes in Acinetobacter sp. strain M-1. Enzymes: 1, aldehyde dehydrogenase; 2, acyl-CoA synthetase; 3, acyl-CoA:ACP transacylase; 4, acyl-CoA dehydrogenase; 5, acyl-CoA reductase; 6, aldehyde reductase; 7, acyl-CoA:alcohol transacylase. R, acyl group; ACP, acyl carrier protein.