New Pathway for Long-Chain n-Alkane Synthesis via 1-Alcohol in Vibrio furnissii M1

Alkane biosynthesis in the bacterium Vibrio furnissii M1 involvesthe synthesis of long-chain alkanes via 1-alcohol. Evidencefor this novel pathway are the following. (i) Both even- andodd-carbon-number n-alkanes were produced from glucose, whileonly even-carbon-number fatty acids were produced in V. furnissiiM1. This result cannot be explained by the decarbonylation pathway.(ii) Pentadecane and hexadecane were produced from 1-hexadecanoicacid by membrane fractions of V. furnissii M1, and radioisotopeprecursor-tracer experiments, in which 1-[1-¹⁴C]hexadecanoicacid was fed, identified the corresponding alcohol, aldehyde,and alkane derivatives. Since all metabolites maintained theradioisotope label at 1-C, they were produced by a pathway inwhich the carbon structure was retained, i.e., a reduction pathway.(iii) n-Hexadecane was produced when 1-hexadecanol was fed tomembrane preparations.

INTRODUCTION

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Introduction

Long-chain alkanes are ubiquitous in living organisms, althoughonly a trace amount of these alkanes are present (9, 16). Ithas long been believed that the decarbonylation of fatty aldehydesis the only pathway for alkane production (3, 6-8). Figure 1Ashows the reactions involved. First, a fatty acid elongase elongateshexadecanoic acid to an even-carbon-number fatty acid (e.g.,C_2n); then, a fatty acid reductase reduces the fatty acids toaldehydes (C_2n); and finally, aldehyde decarbonylase decarbonylatesthe aldehydes to yield alkanes (C_2n–1). It has been reportedthat a much larger proportion of odd-carbon-number alkanes thaneven-carbon-number alkanes was present in higher plants (9,16). Since even-carbon-number fatty acids are produced as themajor fatty acid components in most organisms (9), this pathwayexplains well the predominance of odd-carbon-number n-alkanes.

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FIG. 1. Alkane biosynthesis pathway in eucaryotes (A) and proposed pathway in V. furnissii M1 from the results of this study (B). The thin arrow indicates the pathway elucidated by this study.

In contrast, similar levels of even- and odd-carbon-number alkaneswere found in some bacteria (1), while even-carbon-number fattyacids were predominant in these bacteria (1). The decarbonylationpathway does not adequately explain these facts, and this promptedus to consider that an alternative alkane synthesis pathwaymay exist in the bacteria.

Parker et al. have isolated a Vibrio furnissii M1 strain witha high alkane-producing ability (12) from an environmental sample.The strain produces alkanes of C₁₄ to C₂₂ (Table 1) when usingglucose as the sole carbon source (13). No pattern was apparentin the concentration of even- and odd-carbon number alkanes,while even-carbon-number fatty acids were predominant in theculture (Table 1). This result agreed well with the resultsof the previous report (1).

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TABLE 1. Fatty acids and alkanes produced from glucose by V. furnissii M1

In the present study, I tested the hypothesis that an alternativepathway for alkane synthesis exists in V. furnissii M1 basedon the fact that when 1-[1-¹⁴C]hexadecanoic acid was fed, thecorresponding alcohol, aldehyde, and alkane derivatives wasproduced. This pathway involves alkanes being synthesized via1-alcohol, the reduction of fatty alcohols to alkanes not havingpreviously been reported.

MATERIALS AND METHODS

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Materials and Methods

Culture conditions and preparation of cell extract fraction. V. furnissii M1 (12) was grown with shaking overnight in 200ml of Luria-Bertani medium (15) supplemented with 3% (wt/vol)sodium chloride (2) at 37°C. Cells were harvested by centrifugation(6,000 x g; 10 min) and resuspended in 20 ml of 10 mM sucrose-10mM Tris-HCl (pH 8.0) containing an EDTA-free protease inhibitor(Roche Applied Science) and disrupted by sonication with a BransonSonifier 250 (Branson) at a power setting of 4 and a 40% dutycycle on ice. The resulting homogenate was centrifuged at 8,000x g for 10 min at 4°C to remove the cell debris. The resultingsupernatant was centrifuged at 105,000 x g for 60 min at 4°C.The pellet formed, which is designated as the membrane fraction,was resuspended in 1 ml of the same buffer containing 0.1% (wt/vol)Triton X-100. The protein concentration was determined accordingto method of Bradford (4) by a protein detection kit (Bio-Rad).

Enzyme assay. A 1-mg aliquot of the membrane fraction was suspended in 500µl of 10 mM Tris-HCl (pH 8.0) containing 25 µM NADH,25 µM NADPH, 3.3 mM glutathione, and 330 µM MgCl₂.1-Hexadecanoic acid (25 µM; >99% chemical purity; Sigma)was added to the mixture after adding 0.1% (wt/vol) Triton X-100and was dispersed by sonication (6). 1-Hexadecanoic acid (25µM) and 740-kBq 1-[1-¹⁴C]hexadecanoic acid (99.2% radiochemicalpurity; Amersham Pharmacia) were used as substrates in the radioisotopetracer experiments. Alternatively, 25 µM 1-hexadecanol(>99% chemical purity; Sigma) and 37-kBq 1-[1-¹⁴C]hexadecanol(98% radiochemical purity; Sigma) were used. Each reaction mixturewas incubated for 40 min at 37°C, and the neutral lipidswere then immediately extracted twice with 1 ml of hexane. Afterreducing the amount of solvent under nitrogen reflux, the solutionwas spotted onto a silica gel plate (Merck). Before applyingthe sample, a mixture of authentic cold hexadecanoic acid, hexadecanal,hexadecanol, and hexadecane was spotted onto the origin pointof the plate to prevent any change in R_f value of the alkanesdue to downsizing the run in the radioisotope experiments. Developingand visualization were done in the same manner as that describedin previous reports (12, 13). In brief, the silica gel platewas developed with a solvent system of 80:20:1 (hexane-diethylether-water). The spots were visualized by exposing the plateto iodine vapor.

The alkanes were recovered from the silica gel plate (13), extractedin 1 ml of chloroform, and then analyzed by gas chromatography-massspectrometry (GC-MS) (a 5890A instrument for GC and a 5989Ainstrument for MS; Hewlett Packard). Each sample was analyzedtwice in the GC-MS assay with electron ionization and positivechemical ionization MS. Electron ionization MS yielded a largerfragment ion peak for n-alkane, while a larger parent ion peakwas obtained by positive chemical ionization MS.

The spots corresponding to hexadecanoic acid, hexadecanal, hexadecanol,and hexadecane were extracted from the silica gel plates, andthe lipids were dissolved in an AL-1 scintillation cocktail(Dojindo) for radioisotope tracer experiments. The cocktailwas directly measured by a liquid scintillation counter as describedelsewhere (3). Carbon monoxide was trapped with RhCl[(C₆H₆)₃P]₃and measured as described elsewhere (8). Negative controls werecarried out with membrane fractions boiled for 10 min and thenquickly cooled on ice for 2 min. Another control without membranefractions was also run in all experiments. The activity to convert1-hexadecanoic acid to alkane or to convert 1-hexadecanol toalkane was localized in the membrane fraction.

RESULTS AND DISCUSSION

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Results and Discussion

Synthesis of two alkanes from 1-hexadecanoic acid. The alkanes produced from 1-hexadecanoic acid in the presenceof the membrane fraction of V. furnissii M1 were analyzed byGC-MS, by using simultaneous electron ionization and positivechemical ionization MS. GC chromatograms revealed peaks correspondingto the retention times of authentic n-pentadecane (11.6 min)and n-hexadecane (12.9 min) (Fig. 2). The first GC fractioncorresponding to n-pentadecane gave mass spectra at m/z 57 (C₄H₉⁺),71 (C₅H₁₁⁺), 85 (C₆H₁₃⁺), 99 (C₇H₁₅⁺), 113 (C₈H₁₇⁺), 127 (C₉H₁₉⁺),141 (C₁₀H₂₁⁺), 155 (C₁₁H₂₃⁺), 169 (C₁₂H₂₆⁺), 183 (C₁₃H₂₇⁺),and 212 (M⁺: C₁₅H₃₂) in the positive chemical ionization mode(Fig. 2). The same fraction gave mass spectra at m/z 57 (C₄H₉⁺),71 (C₅H₁₁⁺), 85 (C₆H₁₃⁺), 99 (C₇H₁₅⁺), 113 (C₈H₁₇⁺), 127 (C₉H₁₉⁺),141 (C₁₀H₂₁⁺), 155 (C₁₁H₂₃⁺), 169 (C₁₂H₂₆⁺), 183 (C₁₃H₂₇⁺),197 (C₁₄H₂₉⁺), 211 (M-1), and 212 m/z (M⁺) in the electron ionizationmode, the peak height decreasing with increasing mass. ThisGC fraction was therefore confirmed as n-pentadecane. The secondGC fraction corresponding to n-hexadecane gave mass spectraat m/z 57 (C₄H₉⁺), 71 (C₅H₁₁⁺), 85 (C₆H₁₃⁺), 99 (C₇H₁₅⁺), 113(C₈H₁₇⁺), 127 (C₉H₁₉⁺), 141 (C₁₀H₂₁⁺), 155 (C₁₁H₂₃⁺), 169 (C₁₂H₂₆⁺),183 (C₁₃H₂₇⁺), 197 (C₁₄H₂₉⁺), 211 (C₁₅H₃₁⁺), and 226 (M⁺: C₁₆H₃₄)in the positive chemical ionization mode. The same fractiongave mass spectra at m/z 57 (C₄H₉⁺), 71 (C₅H₁₁⁺), 85 (C₆H₁₃⁺),99 (C₇H₁₅⁺), 113 (C₈H₁₇⁺), 127 (C₉H₁₉⁺), 141 (C₁₀H₂₁⁺), 155(C₁₁H₂₃⁺), 169 (C₁₂H₂₆⁺), 183 (C₁₃H₂₇⁺), 197 (C₁₄H₂₉⁺), 211(C₁₅H₃₁⁺), 225 (M-1), and 226 (M⁺) in the electron ionizationmode, the peak height decreasing with increasing mass. ThisGC fraction was therefore confirmed as hexadecane. No alkaneswere detected in the negative control runs when using denaturedprotein or without adding protein (results not shown).

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FIG. 2. GC-MS data for the alkanes produced from 1-hexadecanoic acid in the presence of the membrane fraction from V. furnissii M1. nsrsid87594\delrsid87594 *, background noise, not alkane.

The formation of pentadecane can be explained by decarbonylationof the aldehyde formed by the reduction of 1-hexadecanoic acidwhich involves the alkane biosynthesis pathway known in higherplants and a microalga, which has not been reported in bacteriayet. On the other hand, the formation of hexadecane from 1-hexadecanoicacid cannot be explained by this decarbonylation route; therefore,the existence of an alternative pathway that does not subtractthe carbon of 1-hexadecanoic acid is suggested

Precursors in the alkane synthesis from 1-[1-¹⁴C]hexadecanoic acid. Alcohol as well as aldehyde and alkane with radioisotope labelswere produced from 1-[1-¹⁴C]hexadecanoic acid in the presenceof the membrane fraction of V. furnissii M1 as shown in Table2. Since only the 1-C site was labeled with the radioisotopein this experiment, these labeled chemicals retaining the 1-Cstructure respectively corresponded to 1-hexadecanol, 1-hexadecanal,and hexadecane. Carbon monoxide with the radioisotope labelwas also detected, confirming that decarbonylation had occurred.

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TABLE 2. Radioactivity in bacterial fractions produced from 1-[1-¹⁴C]hexadecanoic acid

Since 1-hexadecanal and 1-hexadecanol were produced from 1-hexadecanoicacid in this experiment, a plausible pathway for alkane synthesiswas the successive reduction of the fatty acid. These experimentalresults support the existence of two pathways for biosynthesisof alkanes.

Alkane synthesis from 1-[1-¹⁴C]hexadecanol. Since alcohol was found as a potential precursor in alkane synthesis,synthesis of hexadecane from 1-[1-¹⁴C]hexadecanol was assayedin the presence of the membrane fraction of V. furnissii M1.High amounts of hexadecane (11,000 ± 2,000 cpm) weredetected while no radioactive alkane was detected in the negativecontrol assays.

Our results show that alkane synthesis in V. furnissii M1 canproceed as shown in Fig. 1B. First, 1-hexadecanal is producedfrom 1-hexadecanoic acid; then, the aldehyde is oxidized to1-hexadecanol, or alternatively, pentadecane and carbon monoxideare produced from 1-hexadecanal; and then, hexadecane is producedfrom 1-hexadecanol.

No precedent so far has been detected for the reduction of analcohol to aldehyde in living organisms for alkane biosynthesis.On the other hand, the reverse pathway, in which an alcoholis produced from an alkane, has been reported for several bacteria(5, 10, 11, 14).

The results of this study offer new insight about the occurrenceof even-carbon-number n-alkane biosynthesis in microbes andsupport the occurrence of even-carbon-number n-alkanes in microorganisms.

ACKNOWLEDGMENTS

I am grateful to K. Matsumoto and K. Nanba of Shionogi and Co.Ltd. for technical advice on the GC-MS analysis and to ShigeakiHarayama, Norihide Kurano, Hiroyasu Nagase, Kazumasa Hirata,and Kazuhisa Miyamoto for useful comments.

FOOTNOTES

* Mailing address: Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0041, Japan. Phone: 81 19326 6544. Fax: 81 19326 6592. E-mail: park.myong-ok{at}mbio.jp.

REFERENCES

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