Wax Ester Production from n-Alkanes by Acinetobacter sp. Strain M-1: Ultrastructure of Cellular Inclusions and Role of Acyl Coenzyme A Reductase

Acinetobacter sp. strain M-1 accumulated a large amount of waxesters 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 0.17g/g of cells (dry weight). Electron microscopic analysis revealedthat multilayered disk-shaped intracellular inclusions wereformed concomitant with wax ester formation. The contributionof acyl-CoA reductase to wax ester synthesis was evaluated bygene disruption analysis.

INTRODUCTION

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Introduction

Various types of wax ester are widely used in the manufactureof fine chemicals, i.e., cosmetics, candles, printing inks,lubricants, coating stuffs, etc. Acinetobacter spp. are knownto accumulate wax ester as a storage material (10). The pathwayfor wax ester synthesis of Acinetobacter spp. is assumed tostart from acyl coenzyme A (acyl-CoA), which is reduced to thecorresponding alcohol via acyl-CoA reductase (6) and aldehydereductase (12) (Fig. 1). The microbial production of wax esterhas advantages over other biological sources since the wax estercomposition can be controlled by the choice of starting materialor growth conditions (1).

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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.

Acinetobacter sp. strain M-1 has characteristic properties asto n-alkane metabolism, such as the ability to grow on a broadrange of n-alkanes (7, 8), enzymatic diversity of related enzymes(12), and a notable level of wax ester accumulation. Based onthe obtained results, we report here the novel ultrastructureof intracellular inclusion bodies and a metabolic pathway forwax ester synthesis from n-alkanes.

MATERIALS AND METHODS

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Materials and Methods

Chemicals and enzymes.
Restriction enzymes, alkaline phosphatase (calf intestine),T4 DNA ligase, and Ex Taq DNA polymerase were products of TakaraShuzo Co. (Kyoto, Japan). A Thermo Sequence fluorescence-labeledprimer cycle sequencing kit with 7-deaza-dGTP was purchasedfrom Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire,England), and [ ${alpha}$ -³²P]dATP was from Amersham Corp. (ArlingtonHeights, Ill.).

Organism, culture conditions, and vectors.
Acinetobacter sp. strain M-1 (7) was used throughout this work.To induce wax ester accumulation, n-hexadecane or the oxidizedcompounds were added at a concentration of 0.5% (vol/vol) tothe nitrogen-limited medium (2). Growth condition of Escherichiacoli JM109 and its transformants and vectors for gene cloningand expression were principally the same as described previously(4).

Assay.
Determination of protein concentration and sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) were performed as described previously(4). Acyl-CoA reductase activity was assayed by a previouslydescribed method (6). Wax ester was determined by gas chromatography(1).

Electron microscopy.
For the preparation of thin sections, the bacterial cells werefixed with glutaraldehyde as described previously (9). The ultrathinsections were observed under an electron microscope (JEM-1220;JEOL Ltd., Tokyo, Japan). For the quick-freezing replica method,the washed cells were quickly frozen using the technique ofHeuser (3). The replicas, which were obtained by rotary shadowingwith Pt and C, were examined under a Hitachi H-7000 electronmicroscope (Hitachi, Japan) operating at 100 kV.

Cloning of acrM from the genome of Acinetobacter sp. strain M-1.
To amplify the fragment DNA encoding acyl-CoA reductase (AcrM)from the chromosomal DNA of Acinetobacter sp. strain M-1, thefollowing primers were synthesized based on the acr1 gene fromAcinetobacter calcoaceticus BD413: BDN, 5'-GTGAACAAAAAACTTGAAGCTCTC-3',and BDC, 5'-TTACCAGTGTTCGCCTGGGAA-3'. The PCR product was approximately2.8 kb long and contained an open reading frame (ORF), whichshowed high homology with the original gene, acr1. A new primer,BDN2, 5'-AGAGAAGTTAGATGAGGTC-3', was also synthesized to amplifyonly a portion of the objective ORF. PCR with the BDC and BDN2primers produced a gene of 0.7 kb, and the resulting fragmentwas used as a probe. Through Southern hybridization (11) andcolony hybridization, one positive clone which comprised a 4.0-kbBamHI-HindIII fragment in pBluescript II SK(+) was isolated.The whole nucleotide sequence of this plasmid (pBM4) was determined.A restriction map of pBM4 is shown in Fig. 2A.

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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.

Expression of the acrM gene in E. coli JM109.
The following primers were designed for overexpression of theacrM gene in a heterologous host, E. coli JM109: acrM-N, 5'-GAATTCTAAGGAGGTTTTTATATGAATGCAAAACTCAAAAAACTT-3',and acrM-C, 5'-AAGCTTTTACCAGTGTTCGCCTGGG-3'. acrM-N containedan EcoRI site and the ribosomal binding sequence, and acrM-Ccontained a HindIII site. The PCR product was ligated with thepT7Blue vector and then with pUC118. A pUC118-derived plasmid,named pMR1, was introduced into E. coli JM109. Expression wasconfirmed by activity measurement and the protein band on SDS-PAGE.

Construction of the acrM disruptant.
The kanamycin resistance gene (Km) was amplified with the primersdescribed previously (4), except for the flanking site of SalI.The amplified Km gene was inserted at the SalI site in the acrMgene of pBM4, yielding pDAR1 (Fig. 2B). The acrM gene disruptionplasmid, pDAR1, was linearized, dephosphorylated, and then introducedinto Acinetobacter sp. strain M-1 by electroporation. The kanamycinresistance clone was selected, and the gene disruption was confirmedby Southern analysis (Fig. 2C).

Nucleotide sequence accession number.
The nucleotide sequence data reported in this paper have beendeposited in the DDBJ/EMBL/GenBank nucleotide sequence databasesunder accession number AB070446.

RESULTS AND DISCUSSION

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Results and Discussion

Wax ester production.
The production of wax ester was optimized with respect to cellnumber, substrate concentration, and incubation time. As a result,using 1.85 x 10¹⁰ cells/ml with 3% n-hexadecane as the substrate,hexadecyl hexadecanoate was accumulated up to the amount of0.17 g/g of cells (dry weight) in a 10-h reaction. The producedwax ester comprised hexadecyl hexadecanoate (98%) and hexadecylmyristate (2%). The by-product was two carbon atoms shorterthan the main product. This suggested that a part of palmitoyl-CoAwas converted to myristoyl-CoA through the ß-oxidationcycle, which was then utilized by acyl-CoA:alcohol transacylase.The level of by-product formation depends on the substrate usedfor the reaction. When n-hexadecanol was used instead of n-hexadecane,9.2% of hexadecyl myristate was formed as a by-product. Fromn-octadecane, octadecyl octadecanoate and octadecyl hexadecanoatewere accumulated in the ratio of 67:33, and a negligible amountof wax ester with 32 carbon atoms was also detected. One ofthe major determinants for by-product formation may be the substratespecificities of the enzymes that participate in wax ester synthesis.

Ultrastructure of intracellular wax ester.
Thin sections of Acinetobacter sp. strain M-1 cells, which hadbeen incubated in the nitrogen-limited medium with 0.5% n-hexadecane,contained multiple, long and narrow, electron-transparent, intracellularinclusion bodies (Fig. 3B). Figure 3C shows cells grown underthe same conditions as in Fig. 3B, but the cells were slicedin another direction. On the other hand, cells grown on 2x yeastextract-tryptone (YT) medium (4) showed no characteristic intracellularstructures (Fig. 3A). Quick-freezing replica microscopy showedthe clearer structure of the inclusion bodies, which were disk-shaped,had a smooth surface, and grew to almost the same diameter asthe cells (Fig. 3D). No intracytoplasmic membrane structuresor limiting membranes surrounding these inclusions were observed.These ultrastructures were considerably distinct from thosein the case of Acinetobacter sp. strain HO1-N, which accumulatedrectangular inclusion bodies surrounded by a phospholipid-richmembrane. The wax ester accumulation proceeded with the sequentialformation of disks, i.e., the completion of one disk led toanother one (Fig. 4). We suggested that the inclusion bodiesrepresent the accumulated form of wax ester based on the resultsthat the intracellular amount of wax ester was exactly proportionalto the volume of the inclusion bodies.

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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.

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

Cloning of the acyl-CoA reductase gene from Acinetobacter sp. strain M-1.
acrM comprised 888 bp, corresponding to 295 amino acid residueswith a predicted molecular mass of 32,375 Da. Comparison ofAcrM with Acr1 of A. calcoaceticus BD413 (6) showed homology,i.e., identity of 69.8% at the gene level and 78.3% at the aminoacid level, although their initiation codons are different,AcrM (ATG) and Acr1 (GTG).

Expression of the acrM gene in E. coli JM109.
Since the amount of acyl-CoA reductase in Acinetobacter sp.strain M-1 was too low, the acrM gene was overexpressed in E.coli JM109 to study its enzymatic properties. The expressedprotein was recognized as a protein with an apparent molecularmass of ca. 33 kDa on SDS-PAGE. The protein corresponding torecombinant AcrM was exclusively localized in the membrane fractionthat was obtained on ultracentrifugation (105,000 x g for 1h) of the cell extract, and the specific activity of this fractiontoward palmitoyl-CoA was 0.95 U/mg of protein. This was in goodaccordance with the hydrophobic prediction of cloned AcrM andindicated that the enzyme existed on the membranes in the parentalstrain. Recombinant AcrM of the membrane fraction showed thehighest activity toward myristoyl-CoA (1.32 U/mg of protein),and the following activity toward the other substrates: decyl-CoA(2.5% of the activity toward myristoyl-CoA), lauroyl-CoA (33%),palmitoyl-CoA (90%), stearoyl-CoA (44%), and arachidoyl-CoA(29%). The substrates with carbon chain lengths shorter than8 did not show any activity, and ones with chains longer than22 were not tested.

Gene disruption experiments.
Accumulation of wax ester was compared between the wild typeand the acrM disruptant (acrM-KO) strain. As shown in Table1, there was no significant difference in the amount of accumulatedintracellular wax ester between the two strains when n-hexadecaneor n-hexadecanol was used as the substrate. In contrast, whenhexadecanoic acid was used as the substrate, no wax ester formationwas detected for the acrM-KO strain. This indicated the criticalfunction of acyl-CoA reductase in wax ester synthesis from carboxylicacid. An acyl-CoA reductase-deficient mutant of A. calcoaceticusBD413 produced wax ester from neither n-hexadecane nor hexadecanoicacid (6). Judging from recent results, both A. calcoaceticusBD413 and Acinetobacter sp. strain M-1 have principally thesame pathway for n-alkane oxidation (Fig. 1), whose first stepis catalyzed by membrane-bound alkane monooxygenase (5, 12).The high level of production of wax ester from n-alkanes byAcinetobacter sp. strain M-1 can be explained by the fact thatthe alcohol supply is fairly higher in strain M-1 than in otherAcinetobacter strains.

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TABLE 1. Accumulation of wax ester from various carbon sources

Acinetobacter sp. strain M-1 is a good candidate for the commercialproduction of wax ester based on the high productivity fromdiverse substrates, especially n-alkanes. Furthermore, the alcoholand carboxylic acid compositions of wax ester can possibly becontrolled more precisely by alteration of the substrates inthe acrM-KO strain, because the alcohol moiety can be completelyfixed to the substrate carbon source in the disruptant strain.

ACKNOWLEDGMENTS

We thank Yuriko Ikeda and Takako Seki for excellent technicalassistance with the quick-freezing-replica electron microscopy.

FOOTNOTES

* Corresponding author. Mailing address: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. Phone and fax: 81-75-753-6385. E-mail: nkato{at}kais.kyoto-u.ac.jp.

REFERENCES

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