Schizochytrium sp. is a marine microalga that has been developed as a commercial source for docosahexaenoic acid (DHA, C22:6 (omega-3), enriched biomass, and oil. Previous work suggested that the DHA, as well as docosapentaenoic acid (DPA, C22:5 omega-6), that accumulate in Schizochytrium are products of a multi-subunit polyunsaturated fatty acid (PUFA) synthase (1). Here we show data to support this view and also provide information on other aspects of fatty acid synthesis in this organism. Three genes encoding subunits of the PUFA synthase were isolated from genomic DNA and expressed in E. coli along with an essential accessory gene encoding a phosphopantetheinyl transferase (PPTase). The resulting transformants accumulated both DHA and DPA. The ratio of DHA to DPA was approximately the same as that observed in Schizochytrium. Treatment of Schizochytrium cells with certain levels of cerulenin resulted in inhibition of 14C acetate incorporation into short chain fatty acids without affecting labeling of PUFAs, indicating distinct biosynthetic pathways. A single large gene encoding the presumed short chain fatty acid synthase (FAS) was cloned and sequenced. Based on sequence homology and domain organization, the Schizochytrium FAS resembles a fusion of fungal FAS beta and alpha subunits.

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Two forms of spinach acyl carrier protein (ACP-I and ACP-II) have recently been characterized and found to be expressed in a tissue-specific manner (JB Ohlrogge, TM Kuo, 1985 J Biol Chem 260: 8032). To examine possible different functions for these ACP isoforms, we have tested purified preparations of spinach leaf ACP-I and ACP-II and Escherichia coli ACP in several in vitro reactions of fatty acid metabolism. Total de novo fatty acid synthesis and malonyl-CoA:ACP transacylase do not appear to discriminate between acyl carrier protein isoforms. In contrast, the K, of oleoyl-ACP thioesterase for oleoyl-ACP-II is 10-fold higher than for oleoyl-ACP-I, whereas the K,,, of acyl-ACP glycerol-3-phosphate acyl transferase is 5-fold higher for oleoyl-ACP-I than for oleoyl-ACP-II. A characterization of these reactions and a possible role for ACP isoforms in regulation of fatty acid metabolism in plants are described. Fatty acid synthesis in higher plant plastids requires the presence of acyl carrier protein. The An additional purpose of this study is to further evaluate the activity of Escherichia coli ACP with plant fatty acid enzymes. Most in vitro studies of plant fatty acid metabolism have used E. coli ACP rather than plant ACP as a cofactor. This compromise has been adopted primarily because E. coli ACP can readily be purified in good yield, whereas plant ACP is much more difficult to obtain. Although ACP is clearly a highly conserved protein, E. coli ACP is only partially cross-reactive with antibodies to spinach ACP (10, 15) and their amino acid sequences are only 40% homologous (1 1). These data suggest that prokaryotic and eukaryotic ACP may have significant structural differences. The validity of using E. coli ACP to study plant enzymes is based primarily on the observations of Simoni et al. (27) 20-20-20

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