Biosynthesis of triacylglycerol in the filamentous fungus Mucor circinelloides

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Abstract
Lipid metabolism was studied in 2-d-old liquid cultures of Mucor circinelloides grown at 25 C. Under these conditions, oil accumulated to 0.5 g I-1 with a ?-linolenic acid content (?18:3) of 60 mg I-1. The major labelled lipids in cultures incubated with [14C]acetate were triacylglycerol (TAG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The proportion of label declined in the phospholipids and increased in TAG with time. [14C]18:1 and [14C]18:2 rapidly appeared in PC and PE and later accumulated in [14C]?18:3. TAG-synthesizing capacity was greatest in the microsomal membrane fraction, which accumulated high levels of phosphatidic acid in the presence of glycerol 3-phosphate and acyl-CoA substrates at pH 7.0. Further metabolism of phosphatidic acid to diacylglycerol and TAG was achieved by increasing the pH to 8.0. Lysophosphatidic acid:acyl-CoA acyltransferase (LPAAT) activity was particularly high and may have accounted for the rapid accumulation of phosphatidic acid in the membranes. The glycerol-3-phosphate:acyl-CoA acyltransferase (GPAAT) and LPAAT were non-specific for a range of saturated and unsaturated species of acyl-CoA although the GPAAT showed a marked selectivity for palmitoyl-CoA and the LPAAT for oleoyl- and linoleoyl-CoA. ?-Linolenic acid was detected at all three positions of sn-TAG and was particularly enriched at the sn-3 position. The preparation of active in vitro systems (microsomal membranes) capable of the complete biosynthetic pathway for TAG assembly may be valuable in understanding the assembly of oils in future transgenic applications.