No abstract

Embryos derived in vitro from isolated microspores of Brassica napus L. were compared with their zygotic counterparts. Parameters investigated included storage-protein accumulation and gene expression, fattyacid composition, storage-lipid biosynthesis, and the appearance of oil-body proteins. The microspore embryos accumulate storage-protein and show increases in levels of their transcripts during the torpedo stage. These embryos were sensitive to abscisic acid (ABA) with respect to accumulation of storage-protein mRNA and oil-body proteins. Post-transcriptional regulation of cruciferin accumulation is indicated by a disparity between ABA-enhanced transcript accumulation and a less marked effect at the level of protein accumulation. To investigate storage-lipid profiles, two cultivars of Brassica napus, Reston and Topas, were used. The former accumulates major quantities of C20 (11.2%) and C22 (39.9%) fatty acids in its seeds, the latter predominantly C18 fatty acids. The higher-molecular-weight fatty acids (>C18) normally occur only in seeds and were used as biochemical markers for seed-specific metabolism in microspore embryos. Microspore embryos from Reston were found to accumulate C20 (10.6%) and C22 (31.2%) fatty acids after 35 d in culture at levels and proportions comparable to those found in seeds. Similarly, microspore embryos of Topas had a fatty-acid profile similar to that of mature Topas seed. Activities of enzymes involved in the accumulation of storage lipids (erucoyl-CoA synthetase [EC 6.2.1.3], erucoyl-CoA thioesterase [EC 3.1.2.2] and erucoyl-CoA acyltransferase [EC 2.3.1.15 or EC 2.3.1.20]) were detected in torpedostage microspore embryos. Their specific activities were higher than have been reported to date for analogous preparations from zygotic embryos of B. napus. The similarities in storage-lipid and protein composition of these embryos to their zygotic counterparts, along with their sensitivity to ABA, indicate that microspore embryos might be exploited to facilitate studies of biochemistry and gene regulation in oilseeds.

Sterols (sitosterol, cholesterol, stigmasterol, ergosterol, and 7-dehydrocholesterol) and sitostanol have been converted in high to near-quantitative yields to the corresponding long-chain acyl esters via esterification with fatty acids or transesterification with methyl esters of fatty acids or triacylglycerols using lipase from Candida rugosa as biocatalyst in vacuo (20-40 mbar) at 40 degrees C. Neither organic solvent nor water is added in these reactions. Under similar conditions, cholesterol has been converted to cholesteryl butyrate and steroids (5alpha-pregnan-3beta-ol-20-one or 5-pregnen-3beta-ol-20-one) have been converted to their propionic acid esters, both in moderate to high yields, via transesterification with tributyrin and tripropionin, respectively. Reaction parameters studied in esterification include the temperature and the molar ratio of the substrates as well as the amount and reuse properties of the C. rugosa lipase. Lipases from porcine pancreas, Rhizopus arrhizus, and Chromobacterium viscosum are quite ineffective as biocatalysts for the esterification of cholesterol with oleic acid under the above conditions.

The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5His 6 WS/DGAT comprising an N-terminal His 6 tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5His 6 atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein ؊1 min ؊1 was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5His 6 WS/DGAT with 1-hexadecanethiol and palmitoylCoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1⍀Km, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1⍀Km in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.Polyhydroxyalkanoic acids (PHA) in bacteria represent the most abundant group of neutral storage lipids, which serve as intracellular carbon and energy storage compounds (23). In addition, triacylglycerols (TAGs) and wax esters also occur as bacterial storage lipids, though at a much lower frequency than in eukaryotic microorganisms (22). Significant TAG accumulation was found, particularly in species belonging to the class Actinomycetes (1), as was reported for the genera Mycobacterium (3), Nocardia, and Streptomyces (2). TAGs are synthesized by the acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (DGAT) (13), which catalyzes the esterification of diacylglycerol with long-chain acyl-CoA. Wax esters are oxoesters of primary long-chain fatty alcohols and long-chain fatty acids. Occurrence of wax esters has been frequently reported for various Acinetobacter species (6). Under growthlimiting conditions, Acinetobacter calcoaceticus accumulates wax esters intracellularly as insoluble inclusions. The chemical structure of the wax esters synthesized by A. ca...

Biosynthesis of very long chain (>C18) fatty acids (VLCFAs) and the pathway for their incorporation into acyl liipids was studied in microspore-derived (MD) inantly at the sn-3 position, but were also found at the sn-1 position.[14C120:1, but not [14C]22:1, was detected at the sn-2 position. Similar patterns of 14C-labeled VLCFA distribution were obtained in experiments conducted using a 15,000g pellet fraction from 18-day MD embryos. All trends observed in the formation of TAGs containing VLCFAs in the Reston MD embryo system were also confirmed in studies of zygotic embryos of the same cultivar. The data support the biosynthesis of 20:1 and then 22:1 via successive condensations of malonyl-coenzyme A with oleoyl-coenzyme A and, for the first time in B. napus, demonstrate the incorporation of newly synthesized VLCFAs into TAGs via the Kennedy pathway.