Characterization of Neurospora crassa mutants isolated following repeat-induced point mutation of the beta subunit of fatty acid synthase.

Neurospora crassa cel-2 mutants were isolated following repeat-induced point mutation using part of the gene encoding beta-fatty acid synthase. These mutants are phenotypically less leaky than cel-1, which has a defective alpha-fatty acid synthase. The cel-2 mutant had a strict fatty acid (16:0) requirement for growth, and synthesized less fatty acid de novo than cel-1. Unlike cel-1, cel-2 has impaired fertility, and homozygous crosses are infertile, suggesting a low but strict requirement for fatty acid synthesis during sexual development. Like cel-1, cel-2 synthesized unusually high levels of the polyunsaturate 18:3(Delta9,12,15), and elongated 18:2(Delta9,12 )and 18:3(Delta9,12,15 )to 20:2(Delta11,14) and 20:3(Delta11,14,17), respectively. These fatty acids are not synthesized by wild-type, except following treatment with cerulenin (a fatty acid synthase inhibitor), demonstrating that inhibition of fatty acid biosynthesis results in a relative increase in both fatty acid desaturation and elongation activity.

Conversion of palmitate to unsaturated fatty acids differs in a Neurospora crassa mutant with impaired fatty acid synthase activity.

The Neurospora crassa cel (fatty acid chain elongation) mutant has impaired fatty acid synthase activity. The cel mutant requires exogenous 16:0 for growth and converts 16:0 to other fatty acids. In contrast to wild-type N. crassa, which converted only 42% of the exogenous [7,7,8,8-(2)H4]16:0 that was incorporated into cell lipids to unsaturated fatty acids, cel converted 72%. In addition, cel contains higher levels of 18:3(delta 9,12,15) than wild-type, and synthesizes two fatty acids, 20:2(delta 11,14 and 20:3(delta 11,14,17, found at only trace levels in wild-type. Thus, the delta 15-desaturase activity and elongation activity on 18-carbon polyunsaturated fatty acids are higher for cel than wild-type. This altered metabolism of exogenous 16:0 may be directly due to impaired flux through the endogenous fatty acid biosynthetic pathway, or may result from altered regulation of the synthesis of unsaturated fatty acids in the mutant.

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase.

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[14C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[14C]ricinoleoyl-sn-glycero-3-phosphocholine, 14C-labeled 2-linoleoyl-phosphatidylcholine (PC), and 14C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A2 strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[14C]ricinoleoyl-PC and [14C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC-->2-ricinoleoyl-PC-->ricinoleate-->triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.

Pathways for fatty acid elongation and desaturation in Neurospora crassa.

Neurospora crassa incorporated exogenous deuterated palmitate (16:0) and 14C-labeled oleate (18:1 delta 9) into cell lipids. Of the exogenous 18:1 delta 9 incorporated, 59% was desaturated to 18:2 delta 9,12 and 18:3 delta 9,12,15. Of the exogenous 16:0 incorporated, 20% was elongated to 18:0, while 37% was elongated and desaturated into 18:1 delta 9, 18:2 delta 9,12, and 18:3 delta 9,12,15. The mass of unsaturated fatty acids in phospholipid and triacylglycerol is 12 times greater than the mass of 18:0. Deuterium label incorporation in unsaturated fatty acids is only twofold greater than in 18:0, indicating a sixfold preferential use of 16:0 for saturated fatty acid synthesis. These results indicate that the release of 16:0 from fatty acid synthase is a key control point that influences fatty acid composition in Neurospora.

Metabolism of ricinoleate by Neurospora crassa.

Neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. One such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. To develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). We therefore examined effects of ricinoleate on lipid biosynthesis and growth of N. crassa. Ricinoleate inhibited growth and reduced levels of phospholipids and 2-hydroxy fatty acids in glycolipids, but led to increased lipid accumulation on a mass basis. To evaluate incorporation and metabolism of ricinoleate, we followed the fate 14 microM-3mM [1-14C]ricinoleate. The fate of the [14C]ricinoleate was concentration-dependent. At higher concentrations, ricinoleate was principally incorporated into triacylglycerols. At lower concentrations, ricinoleate was principally metabolized to other compounds. Thus, N. crassa transformants expressing the hydroxylase gene can be detected if the level of hydroxylase expression allows both growth and ricinoleate accumulation.

Characterization of oleoyl-12-hydroxylase in castor microsomes using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine.

We have characterized the oleoyl-12-hydroxylase in the microsomal fraction of immature castor bean using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine (2-oleoyl-PC). Previous characterizations of this enzyme used oleoyl-CoA as substrate and relied on the enzyme transferring oleate from oleoyl-CoA to lysophosphatidylcholine to form 2-oleoyl-PC (acyl-CoA:lysophosphatidylcholine acyltransferase) in addition to oleoyl-12-hydroxylase. The present assay system and characterization use 2-oleoyl-PC as substrate (oleoyl-12-hydroxylase alone). Use of the actual substrate for assay purposes is important for the eventual purification of the oleoyl-12-hydroxylase. Ricinoleate (product of oleoyl-12-hydroxylase) and linoleate (product of oleoyl-12-desaturase) were identified as metabolites of oleate of 2-oleoyl-PC by high-performance liquid chromatography and gas chromatography/mass spectrometry. The activity of oleoyl-12-hydroxylase in the microsomal fraction reached a peak about 44 d after anthesis of castor, while the activity of oleoyl-12-desaturase reached a peak about 23 d after anthesis. The optimal temperature for the oleoyl-12-hydroxylase was about 22.5 degrees C, and the optimal pH was 6.3. Catalase stimulated oleoyl-12-hydroxylase while bovine serum albumin and CoA did not activate oleoyl-12-hydroxylase. The phosphatidylcholine analogue, oleoyloxyethyl phosphocholine, inhibited the activity of oleoyl-12-hydroxylase. These results further support the hypothesis that the actual substrate of oleoyl-12-hydroxylase is 2-oleoyl-PC.

The plant growth regulator methyl jasmonate inhibits aflatoxin production by Aspergillus flavus.

Aflatoxins are highly toxic and carcinogenic compounds produced by certain Aspergillus species on agricultural commodities. The presence of fatty acid hydroperoxides, which can form in plant material either preharvest under stress or postharvest under improper storage conditions, correlates with high levels of aflatoxin production. Effects on fungal growth and aflatoxin production are known for only a few of the numerous plant metabolites of fatty acid hydroperoxides. Jasmonic acid (JA), a plant growth regulator, is a metabolite of 13-hydroperoxylinolenic acid, derived from alpha-linolenic acid. The volatile methyl ester of JA, methyl jasmonate (MeJA), is also a plant growth regulator. In this study we report the effect of MeJA on aflatoxin production and growth of Aspergillus flavus. MeJA at concentrations of 10(-3)-10(-8) M in the growth medium inhibited aflatoxin production, by as much as 96%. Exposure of cultures to MeJA vapour similarly inhibited aflatoxin production. The amount of aflatoxin produced depended on the timing of the exposure. MeJA treatment also delayed spore germination and inhibited the production of a mycelial pigment. These fungal responses resemble plant jasmonate responses.

Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.