7545 CURRENT MICROBIOLOGY Vol. 32 (1996). pp. 336-342 Current Microbiology An International Journal © Springer-Verlag New York Inc. 1996 Oleyl Oleate and Homologous Wax Esters Synthesized Coordinately from Oleic Acid by Acinetobacter and Coryneform Strains T. Kaneshiro,1 L.K. Nakamura,Z ].1. Nicholson,3 M.O. Bagbyl JOil Chemical Research. National Center for Agricultural Utilization Research. Agricultural Research Service. U.S. Department of Agriculture. 1815 North University Street. Peoria. IL 61604. USA "Microbial Properties Research. National Center for Agricultural Utilization Research. Agricultural Research Service. U.S. Department of Agriculture. 1815 North University Street. Peoria. IL 61604. USA 3Biopolymer Research. National Center for Agricultural Utilization Research. Agricultural Research Service. U.S. Department of Agriculture. 1815 North University Street. Peoria. Illinois 61604. USA Abstract. Newly isolated Acinetobacter (NRRL B-14920, B-14921, B-14923) and coryneform (NRRL B-14922) strains accumulated oleyl oleate and homologous liquid wax esters (C30rC36:z) in culture broths. Diunsaturated oleyl oleate preponderated in 75 mg liquid wax esters (280 mg lipid extract) recovered from 100-ml cultures ofAcinetobacter B-14920 supplemented with 810 mg oleic acid-oleyl alcohol. With soybean oil instead of oleic acid, wax esters (260 mg) were increased to approximately 50% of the lipid extract. Production of wax esters by cultures supplemented with combined fatty (CH-C lH ) alcohols and acids suggests a coordinated synthesis whereby the exog­ enous alcohol remains unaltered, and the fatty acid is partially oxidized with removal of Cz units before esterification. Consequently, CH-C lH primary alcohols control chain lengths of the wax esters. Exogenous fatty acids are presumed to enter an intracellular oxidation pool from which is produced a homologous series of liquid wax esters. ]ojoba (Simmondsia califomica) seeds are a source of A previous survey of cultures selected from liquid waxes, which are monoesterified mixtures of composted manure [14] revealed that 27 of 165 unsaturated fatty acids and unsaturated alcohols [18]. bacterial isolates converted exogenous oleic acid to Wax esters from jojoba contain an even number of solvent-extractable neutral products resembling tri­ carbon atoms from C34 to C4H [12, 18, 19]. This acylglycerols on thin-layer chromatograms (TLC). substitute for sperm whale oil is used in such diverse The present study examines chemical structures and commercial products as lubricants, cosmetics, solid probable biosynthetic paths of the oleate conversion wax coatings, and biofuel additives [1, 18, 20]. Wax products of four bacterial (identified as Acinetobacter esters containing CrCzo alcohols [10] or C l4-ClH spp. and a Gram-positive coryneform) and two yeast alcohols [8, 26] produced by Acinetobacter strains may isolates. afford alternative sources of waxes [3, 11, 22]. How­ Triacylglycerol is well established as a major ever, these C3o-C36 wax esters [4, 26] are mainly storage product of yeast cells and, consequently, may saturated and stored in cells grown on nitrogen have a similar function in bacteria [22]. Wax esters (N)-limited media containing succinate, hexadecane, may have a storage function also. Our oleic acid­ or hexadecanol as carbon sources [8]. containing medium [14] is confirmed to facilitate selection of acinetobacters and other bacterial types that accumulate liquid wax esters containing unsatu­ Names are necessary to report factually on available data; however. rated, even-numbered C3o-C36 chains, which re­ the USDA neither guarantees nor warrants the standard of the semble those in jojoba seeds [18]. The coordinated product. and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. conversion of oleic acid to homologous liquid waxes is deduced from the composition of solvent-extractable Correspondence 10: T. Kaneshiro products formed when cultures are supplemented T. Kaneshiro et al.: Ester Synthesis by Acinetobacter 337 with exogenous long-chained fatty alcohols and acids GLC and MS analyses. Wax esters extracted and concentrated in as well as with soybean oil. methanol-ethyl acetate were detected by capillary GLC isother­ mally at 260°e. All samples described above were chromato­ graphed on a SPB-l column (5 m x 0.32 mm id), and emergent Materials and Methods peaks were identified according to retention times relative to Microbial cultures. Microscopic examinations of isolates from standard compounds. The wax esters were analyzed further by composted manure [14] revealed that strains NRRL Y-17806 and GLC-MS (70 eV) after temperature-programmed separations at o Y-17807 were yeasts; and NRRL B-14920, B-14921, B-14922, and 160 -250°e. Wax esters gave characteristic molecular mass ion and B-14923 were bacteria. The four bacterial isolates were identified mass ion fragments (m/z) stemming from their fatty acid and further on the bases of utilization of 96 substrates (Biolog System) alcohol moieties [8.19,26]. and fatty acid profiles (MIDI System). Estimation ofwax esters (mg per 100 ml culture) was based on Cultures were maintained on tryptone-glucose-yeast extract extractable lipids recovered from duplicate 35-ml cultures. Each agar slants and stored at 4°e. For determination of oleic acid or extract was dissolved in 4.0 ml solvent before injection (1 fLl lipid conversions, cultures were transferred from the slants to a aliquot) into a capillary GLC column. In the absence of an nutrient-limited growth medium [27] which yielded less than adequate internal standard, a mean value of54 x 103 area units per optimal turbidimetric (suboptimal) growth and which contained fLg was used to estimate wax esters in normalized (100 ml) samples. The mean value was calculated from oleyl oleate and palmityl (per L): 5.0 g yeast extract, 4.0 g glucose, 4.0 g K2HP04, 250 mg oleate standards, which gave recorded GLC responses of 49 x 103 MgS04 ' 7H20, and 10 mg FeS04 . 7H20. The medium was ad­ justed to pH 7.3 before autoclaving. These inoculum cultures, and 59 x 103 area units/fLg, respectively. which were fully turbid by 24 h at 25°C (shaken at 200 rpm), were in turn transferred (I%, vol/vol) to 35 ml fresh medium in 125-ml Erlenmeyer flasks and incubated overnight for 16-17 h. To these Results early stationary-phase cultures were added oleic acid or other lipid substrates (0.30 ml or 0.27 g altogether). Although optimal condi­ Classification ofisolates. Strains capable oftransform­ tions were not determined. preliminary studies indicated that the ing oleic acid to triacylglycerol-like products were yields of wax esters were not enhanced significantly beyond 24 h. However, wax esters were increased appreciably by use of this isolated from composted manure [14]; these were turbidity-limiting growth medium [14]. designated NRRL accessions B-14920, B-14921, B-14922, B-14923, Y-17806, and Y-17807. Bacterial Biochemicals. Oleic acid, oleyl alcohol, palmitic acid, and palmi­ toleic acid were purchased from Sigma Chemical Co. (S1. Louis, strains B-14920, B-14921, and B-14923 were strictly Missouri). These and all other biochemicals were used without aerobic, oxidase negative, nonmotile, Gram-negative, further purification. Soybean oil was obtained from Riceland very short rods or cocci. Polyhydroxybutyrate storage Foods (Stuttgart, Arkansas). bodies were not observed in these strains. Strains Extraction, separation, and saponification. After 24 h, the cultures B-14920 and B-14921 were identified as Acinetobacter containing lipid supplements were acidified to pH < 3 and johnsonii, and strain B-14923 asAcinetobacter calcoace­ extracted twice with one volume of 1:9 (vol/vol) methanol-ethyl ticus. All three acinetobacters did not oxidize such acetate [14]. Subsequently, the solvents were removed in a rotary common sugars as D-fructose and D-glucose [2] and evaporator from the combined extracts. A standard wax ester, palmityl oleate, was fully recoverable by such an extraction regi­ were differentiated by differences in noncarbohy­ men. Before saponification, a neutral fraction was separated from drate utilization of either amino acids, organic acids, the lipid extract by column chromatography on Silica Gel 60 or fatty derivatives (Biolog). Profiles of two acineto­ (230-400 mesh; EM Science, Gibbstown, New Jersey). The neutral bacters gave 23-33% CI6:10 17-20% CI6:0, and 17-24% fraction was eluted by sequential treatment with 8 column-volumes C : fatty acids (MIDI). Strain B-14922, a strictly each of petroleum ether. 10:90 (vol/vol) ethyl acetate-petroleum IS I ether, 50:50 ethyl acetate-petroleum ether, and ethyl acetate. Each aerobic Gram-positive coryneform, was not identified fraction was monitored for neutral lipids by TLC Silica Gel 60 plate further. Yeast strains NRRL Y-17806 and Y-17807 (EC Science, Cherry Hill. New Jersey) chromatography as devel­ gave luxuriant growth on yeast extract-malt extract­ oped with a solvent mixture of hexane-ethyl acetate-acetic acid peptone-glucose agar medium at pH 6.5 and were (50:50:1, vol/vol), and the components were made visible by means identified by cursory, microscopic examinations only. of iodine vapor and vanillin spray. The respective Rfs of standard monoolein. oleic acid. and neutral triolein were 0.20. 0.55. and 0.80. Triacylglycerol of yeasts. The two yeast strains pro­ For mild saponification of triacylglycerols. the samples (10 mg duced a mean of 95 mg recovered lipid product when of neutral lipid fraction) were mixed with 1 ml of 2 N NaOH and heated at 65°C for 1 h. Saponified samples were acidified before approximately 810 mg oleic acid was added exog­ extraction with hexane (2 x 5 ml). and the fatty acids were then enously to 100-ml cultures. Separation of the lipid esterified with diazomethane for analysis by capillary GLC isother­ product over a silica gel 60 column yielded 30-50% mally at 185°C [14]. neutral lipid material that resembled triacylglycerols To saponify wax esters, samples (10 mg) dissolved in 0.5 ml by TLC analysis (R 0.80).