Wax ester production by bacteria Takeru Ishige, Akio Tani, Yasuyoshi Sakai and Nobuo Kato The enzymological and genetic aspects of microbial metabolism of hydrocarbons have been extensively revealed. Such molecular information is useful for understanding the bioremediation of oil spill environments and production of hydrocarbon-specific fine chemicals. Addresses Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan e-mail: [email protected]
Current Opinion in Microbiology 2003, 6:244–250 This review comes from a themed issue on Ecology and industrial microbiology Edited by Bernard Witholt and Eugene Rosenberg
amounts from n-alkanes or long-chain alkanols under nitrogen-limited conditions . In several eukaryotes, wax esters have diverse and important biological functions, protecting living cells from desiccation, ultraviolet light and pathogens; they are also a typical energy store of plants. Various types of wax ester from biological sources are widely used in the manufacture of commercial products (i.e. cosmetics, candles, printing inks, lubricants and coating stuffs). In this review, we cover recent progress in the metabolic regulation of bacterial degradation of n-alkanes and synthesis of wax esters from n-alkanes by Acinetobacter spp. We also briefly discuss ester accumulation in some hydrocarbon-degrading Gram-positive bacteria.
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Metabolic pathway for n-alkanes and its regulation in bacteria
The bacterial oxidation of n-alkanes has been extensively characterized using Pseudomonas putida GPo1 (now commonly referred to as Pseudomonas oleovorans GPo1), which uses medium-chain n-alkanes (C5–C12) for growth [15,16,17,18]. The alkane hydroxylase system that is responsible for the total oxidation of an n-alkane to 1-alkanol (RCH3 þ NADH þ Hþ þ O2 ! RCH2OH þ NADþ þ H2O) consists of three components: alkane hydroxylase (AlkB), rubredoxin (AlkG) and rubredoxin reductase (AlkT). AlkB is a non-heme iron integral membrane protein that catalyzes the hydroxylation reaction. AlkG transfers electrons from the NADH-dependent flavoprotein rubredoxin reductase to AlkB. The resultant 1-alkanol is oxidized to 1-alkanoate by a membrane-bound alcohol dehydrogenase (AlkJ) and cytosolic aldehyde dehydrogenase (AlkH). 1-alkanoate is incorporated through b-oxidation via the acyl–CoA synthetase (AlkK) reaction. The gene clusters of alkBFGHJKL and alkST encode these proteins and are located on the OCT plasmid of P. putida GPo1.
Introduction Many microorganisms are able to use petroleum hydrocarbons as growth substrates. Using specific microorganisms, a wide variety of processes for microbial production of chemicals from purified hydrocarbons have been established. During the 1970s, hydrocarbons were commercially attractive resources for bulk biological products, including single-cell protein, amino acids, nucleotides, organic acids and vitamins. More recently, renewable resources, such as agricultural products, are of general interest from the viewpoint of sustainable industrial production, and the use of petroleum hydrocarbons as microbial nutrients for bulk production is declining. However, much attention is being paid to the microbial degradation of hydrocarbons from the viewpoint of bioremediation of areas contaminated with petroleum hydrocarbons [1,2]. Through intensive investigations on microbiological degradation, a wide variety of microbial processes for hydrocarbons have been re-examined. Examples include, unique enzymes related to hydrocarbon degradation [3,4], which can be used in the biotransformation of useful compounds; a variety of bio-emulsifiers [5,6,7], which are produced during microbial assimilation of hydrocarbons; and some cell-reserve materials of hydrocarbon-utilizers [8,9,10,11,12,13]. Bacteria differ in the type of reserve material that they accumulate under restricted conditions; examples include glycogen, polyhydroxyalkanoates, triacylglycerols and polyphosphate . Uniquely, some strains of Acinetobacter produce and accumulate wax esters in enormous Current Opinion in Microbiology 2003, 6:244–250
Other Gram-negative n-alkane degraders belonging to Acinetobacter grow on longer-chain n-alkanes. Although the reactions for alkane oxidation of Acinetobacter sp. strain ADP1, which uses n-alkanes C12–C18, are principally analogous to those of P. putida GPo1, the organization of the genes involved in n-alkane oxidation is different [19–21]. In Acinetobacter sp. strain ADP1, five chromosomal genes (alkM, rubA, rubB, alkR and xcpR) in at least three different loci are required for n-alkane degradation. Acinetobacter sp. strain M-1 is characterized by its ability to use much longer-chain n-alkanes (C20–C44), and can degrade n-alkanes up to C60 when grown on a ‘paraffin www.current-opinion.com
Bacterial wax esters Ishige et al. 245
wax’ mixture. This strain possesses two chromosomal genes (alkMa and alkMb) that encode alkane hydroxylases, which are located in two different loci on the chromosomal DNA . AlkMa is highly similar to AlkM of Acinetobacter sp. ADP1 (84% amino acid sequence identity), whereas AlkMa and AlkMb show 61% amino acid sequence identity with each other . The amino acid sequence identity between AlkMRa and AlkMRb, which are transcriptional regulators for AlkMa and AlkMb, respectively, is only 5% (i.e. they are only very distantly related). This also implies that alkMa and alkMb might be regulated differently in this strain. From the results of genetic and biochemical studies, it is concluded that alkMa expression is induced by very-long-chain alkanes (>C22), which are solid at ambient temperatures, and alkMb expression is induced preferentially by shorter-chain n-alkanes. However, the genes for rubredoxin (RubA) and rubredoxin reductase (RubB) are encoded on the rubAB operon in a different locus on the chromosomal DNA from those of alkMa and alkMb. From these facts, we propose a model for the regulation of the alkane hydroxylase system of Acinetobacter sp. strain M-1 (Figure 1). According to this model, the organism controls alkane hydroxylase activity in response to the chain-length of
the substrate, by switching the alkane hydroxylase component, AlkMa or AlkMb, without changing other components of the other complex, RubA and RubB. Several types of pyridine-nucleotide-dependent dehydrogenases for alkanol and alkanal oxidation have been found in the cytosolic fractions of n-alkane-grown Acinetobacter spp. In strain M-1, the genes for NADPþ-dependent alcohol dehydrogenase (AlrA)  and NADþdependent aldehyde dehydrogenase (AldI)  are expressed inductively with n-alkanes. These enzymes have important roles in wax ester synthesis, rather than in the principal oxidation of n-alkanes. In addition, Acinetobacter sp. M-1 possesses two genes encoding acyl–CoA dehydrogenases, acdA and acdB, which are arranged in tandem on the chromosomal DNA . AcdA and AcdB are active toward medium-chain and long-chain acyl– CoAs, respectively. Characterization of n-alkane metabolism in Acinetobacter spp has revealed a parallel pathway, as well as enzymes with overlapping specificities in a single pathway. Such physiological complexity seems to be due to the fact that the n-alkane oxidation pathway is used for different purposes, such as growth on n-alkanes and synthesis of wax esters as cell reserves [23,24].
Outer membrane Periplasm Cytoplasmic membrane
Inducible expression Constitutive expression
rubB Current Opinion in Microbiology
A model for the regulation of the alkane hydroxylase system of Acinetobacter sp. strain M-1. alkMa and alkMb encode alkane hydroxylase in Acinetobacter sp. strain M-1. alkMa and alkMb expression is induced by very-long-chain n-alkenes (>C22) and long-chain n-alkanes (
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Apart from their physiological significance, some enzymes have attractive features as biological catalysts. AlrA, which is a thermostable alcohol dehydrogenase, exhibits notably high reductive activity toward medium-chain alkanals; this substrate specificity being evidently different from those of other enzymes (for example, the specific
activity for 1-hexadecanal is about 100 times higher than that of horse liver alcohol dehydrogenase) . AldI is also a thermostable enzyme, whose activity becomes higher with increasing substrate carbon-chain length, the most preferable substrate tested being 1-tetradecanal, which is the longest alkanal commercially available .
200 nm (c)
200 nm (d)
200 nm 200 nm (e)
(f) Disk 1
200 nm Current Opinion in Microbiology
Electron microscopy of accumulated wax ester in Acinetobacter sp. strain M-1. Thin sections of cells were grown on (a) yeast extract-tryptone medium (2 YT medium) and (b) after a shift to nitrogen-limited medium containing 0.5% n-hexadecane incubated for 2 h (b,c) and 10 h (d) at 308C. Cells in (b and c) were incubated under the same conditions but sliced differently. (e,f) Images were obtained by the quick-freezing replica method of cells prepared under the same conditions as in (c). When cells grown on 2 x YT medium (a) were transferred into the medium containing 0.5% n-hexadecane (b), characteristic intracellular inclusion bodies containing wax esters were formed. The number of inclusion bodies increased during the incubation period (b,d). The inclusion bodies of wax esters were disk-shaped, had a smooth surface and grew to almost the same diameter as the cells (c,e,f). Current Opinion in Microbiology 2003, 6:244–250
Bacterial wax esters Ishige et al. 247
High GþC- and mycolic-acid-containing Gram-positive bacteria, such as Rhodococcus, Mycobacterium, Corynebacterium, Gordonia, Nocardia are known to degrade n-alkanes with a wide range of carbon-chain lengths [26–29]. This has been confirmed by molecular screening for alk-like genes in the microbial populations found in oil-contaminated environments [30,31]. Interestingly, two newly isolated strains of Rhodococcus contain at least four alkane hydroxylase homologs (alkB1, alkB2, alkB3 and alkB4), the function of one of which (alkB2) has been confirmed [18,32]. Such Gram-positive bacteria are also of considerable interest with regard to their hydrocarbon-specific activities applicable to biotechnological processes.
bated with 3% n-hexadecane for 10 h at 308C. The produced wax ester comprised hexadecyl hexadecanoate (98%) and hexadecyl myristate (2%). Wax ester production from various n-alkanes by Acinetobacter sp. strain M-1 is shown in Figure 3. When n-alkanes with longer-carbon chains were used, wax esters containing alkanoates with shorter-carbon chains than that of the substrate increase, whereas the alkanol moieties were principally the same as those of the substrate n-alkanes in chain length. This implies that the chain length of some acyl–CoAs, which are shortened through the b-oxidation cycle, are also used for the final step of wax ester synthesis.
Wax ester production by Acinetobacter spp
The accumulation of a large amount of wax esters derived from n-alkanes could be a notable feature of strain M-1; other strains of Acinetobacter might accumulate wax esters derived from n-alkyl alcohols. This can be explained by the fact that the alkanol supply from n-alkanes is higher in strain M-1 than in other strains. The proposed pathway for wax ester synthesis by Acinetobacter spp is shown in Figure 4. The final step of wax ester synthesis is catalyzed by an acyl–CoA:alcohol transferase, which was recently characterized as a novel bifunctional wax ester synthase/acyl–CoA:diacylglycerol acyltransferase of A. carcoaceticus ADP1 by Kalscheuer and Steinbu¨ chel . In Acinetobacter sp. strain M-1, there are two possible routes for supplying alcohol to the final step. One route is conversion of acyl–CoA to alcohol, through the sequential reactions of acyl–CoA reductase and aldehyde reductase; the latter is possibly NADPþ-dependent alcohol dehydrogenase (ArlA) .
Several species of Acinetobacter are known to accumulate wax esters when they are transferred to a nitrogenlimited medium containing n-alkanes or 1-alkanols . Thin sections of Acinetobacter sp. strain M-1 cells reveal electron-transparent intracellular inclusion bodies (Figure 2), which consist of wax esters. Quickfreezing replica microscopy more clearly shows the structure of the inclusion bodies, which are disk-shaped, have a smooth surface, and grow to almost the same diameter as the cells (Figure 2). No intracytoplasmic membrane structures or limiting membranes surrounding these inclusions have been observed. The wax ester accumulation proceeds with the sequential formation of disks that are 30–50 nm in depth, (i.e. the completion of one disk leads to that of another). Wax esters were accumulated optimally up to 0.17 g/g of cells (dry weight) when cells (1.85 1010 cells/ml) were incu-
Composition of wax ester (R1–CO–OR2)
Wax ester accumulated (mg/mg cell)
0.11 C22–C18 C22–C16
C24–C22 C24–C20 C24–C18 Wax ester composition and production from various n-alkanes by Acinetobacter sp. strain M-1. The cells grown on yeast extract-tryptone medium were transferred to nitrogen-limiting media containing n-alkanes (C16–C24) and then incubated at 308C for 10 h. www.current-opinion.com
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n-alkane (a) alkanol
(b) alkanal (c) alkanoate
β-oxidation Cell constituents
The proposed carbon flow from n-alkanes to wax esters in Acinetobacter spp (a) alkane monooxygenase, (b) alcohol dehydrogenase, (c) aldehyde dehydrogenase, (d) acyl–CoA synthetase, (e) acyl–CoA reductase, (f) aldehyde reductase (alcohol dehydrogenase) and (g) acyl–CoA:alcohol transferase.
The second route is direct introduction from 1-alkanols or 1-alkanals, each of which is an intermediate of n-alkane oxidation. Alternatively, the alkanoate moiety of wax esters are exclusively derived from acyl–CoA. In acyl–CoA reductase deficient mutants (e.g. acrM-KO of strain M-1  and Wpw15 of strain ADP1 ), no wax ester was formed from 1-hexadecanoate. Using the acrM-KO strain, the alkanol and alkanoate compositions of wax ester could be controlled precisely by alteration of the substrate, as the alkanol moiety is fixed to the substrate n-alkanes and a desirable carboxylic acid moiety is directly introduced from the substrate added.
als, growth conditions and the mutant strain. Acinetobacter sp. strain M-1 could be a good candidate for this purpose, judging from its ability to use a broad range of n-alkanes. The accumulation of wax esters, as well as triacylglycerols, by Gram-positive bacteria occurs specifically during the degradation of hydrocarbons under nitrogen-limited conditions. Such restricted conditions normally predominate in a natural environment. Understanding metabolic regulation under restricted conditions could be important not only for wax ester production but also for bioremediation.
References and recommended reading Wax ester of Rhodococcus sp. Rhodococcus opacus PD630 accumulates a unique wax ester from phenyldecane during cultivation under nitrogenlimited conditions . The wax ester, phenyldecyl phenyldecanoate, is assumed to be produced through the condensation of the oxidation intermediates phenyldecanol and penyldecanoic acid . The organism also accumulates novel triacylglycerols, one fatty acid of which is replaced by a phenylcarboxylic acid derived from the growth substrate, phenyldecane . In this context, several Gram-positive bacteria are also able to produce and accumulate triacylglycerols from different carbon sources, including hydrocarbons [37–39].
Conclusions The microbial production of wax esters has advantages over other biological processes, as the wax ester composition can be controlled by the choice of starting materiCurrent Opinion in Microbiology 2003, 6:244–250
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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10. Kessler B, Witholt B: Factors involved in the regulatory network of polyhydroxyalkanoate metabolism. J Bacteriol 2001, 86:97-104. Polyhydroxyalkanoates (PHAs) are bacterial storage materials, and promising biotechnological products from a variety of carbon sources including n-alkanes. This paper points out the diversity of regulatory mechanisms involved in PHA metabolism. 11. Jung K, Hazenberg W, Prieto M, Witholt B: Two-stage continuous process development for the production of medium-chainlength poly(3-hydroxyalkanoates). Biotechnol Bioeng 2001, 72:19-24. 12. Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbuchel A: A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 1999, 171:73-80. 13. Ishige T, Tani A, Takabe K, Kawasaki K, Sakai Y, Kato N: Wax ester production from n-alkanes by Acinetobacter sp. strain M-1: ultrastructure of cellular inclusions and role of acyl coenzyme A reductase. Appl Environ Microbiol 2002, 68:1192-1195. The authors report on wax ester accumulation from n-alkanes by Acinetobacter sp. strain M-1. They reveal the fine structure of the inclusion bodies containing wax ester and show that the composition of wax ester might be controlled precisely by alteration of the substrates in the acrM disruptant strains. 14. Finnarty WR: Biopolymers, production and uses of. In Encyclopedia of Microbiology, vol 1. Edited by Lederberg J. NY: Academic Press; 2000. 15. Staijen IE, Van Beilen JB, Witholt B: Expression, stability and performance of the three-component alkane mono-oxygenase of Pseudomonas oleovorans in Escherichia coli. Eur J Biochem 2000, 267:1957-1965. 16. van Beilen JB, Panke S, Lucchini S, Franchini AG, Rothlisberger M, Witholt B: Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. Microbiology 2001, 147:1621-1630. This is a relevant paper for understanding the regulation of the n-alkane oxidation pathway in Pseudomonas putida, and also describes the functions of genes flanking the alk gene clusters located on the OCT plasmid. 17. van Beilen JB, Neuenschwander M, Smits TH, Roth C, Balada SB, Witholt B: Rubredoxins involved in alkane oxidation. J Bacteriol 2002, 184:1722-1732. 18. Smits TH, Balada SB, Witholt B, van Beilen JB: Functional analysis of alkane hydroxylases from Gram-negative and Gram-positive bacteria. J Bacteriol 2002, 184:1733-1742. 19. Ratajczak A, Geissdorfer W, Hillen W: Expression of alkane hydroxylase from Acinetobacter sp. Strain ADP1 is induced by a broad range of n-alkanes and requires the transcriptional activator AlkR. J Bacteriol 1998, 180:5822-5827. www.current-opinion.com
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