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The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDPdiacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5-to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were derepressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.

The addition of ethanolamine or choline to inositol-containing growth medium of Saccharomyces cerevisiae wild-type cells resulted in a reduction of membrane-associated phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activity in cell extracts. The reduction of activity did not occur when inositol was absent from the growth medium. Under the growth conditions where a reduction of enzyme activity occurred, there was a corresponding qualitative reduction of enzyme subunit as determined by immunoblotting with antiserum raised against purified phosphatidylserine synthase. Watersoluble phospholipid precursors did not effect purified phosphatidylserine synthase activity. Phosphatidylserine synthase (activity and enzyme subunit) was not regulated by the availability of water-soluble phospholipid precursors in S. cerevisiae VAL2C(YEp CHOI) and the opil mutant. VAL2C(YEp CHOI) is a plasmid-bearing strain that overproduces phosphatidylserine synthase activity, and the opil mutant is an inositol biosynthesis regulatory mutant. The results of this study suggest that the regulation of phosphatidylserine synthase by the availability of phospholipid precursors occurs at the level of enzyme formation and not at the enzyme activity level. Furthermore, the regulation of phosphatidylserine synthase is coupled to inositol synthesis.

Phospholipid metabolism in the Saccharomyces cerevisiae opil mutant, which excretes inositol and is constitutive for the biosynthetic enzyme inositol-l-phosphate synthase (M. Greenberg, P. Goldwasser, and S. Henry, Mol. Gen. Genet. 186:157-163, 1982), was examined and compared to that of a wild-type strain. In wild-type S. cerevisiae, the phospholipid composition and the relative rates of synthesis of individual phospholipids change in response to the availability of exogenous supplies of soluble phospholipid precursors, particularly inositol. The opil mutant, in contrast, displays a relatively invariant phospholipid composition, and its pattern of phospholipid synthesis does not change in response to exogenous phospholipid precursors. Phosphatidylinositol synthase was not found to be regulated in either wild-type or opil cells. In wild-type cells, phosphatidylserine synthase and the phospholipid N-methyltransferases are coordinately repressed in response to a combination of inositol and choline. However, in opil cells these activities are expressed constitutively. These results suggest that the gene product of the OPIJ locus participates in the coordinate regulation of phospholipid synthesis.

Three human cytochrome P450s, 3A4, 2C9 and 1A2, were each co-expressed with NADPH-P450 reductase in Escherichia coli and used in the preparative synthesis of drug metabolites. Low dissolved oxygen (DO) concentration (<1%) during expression was found to be critical for producing active P450s. Control of temperature, pH and glycerol supplementation in 10-L fermentations enhanced enzyme expression 31-86%. Additional improvements were obtained by altering media formulations, resulting in bicistronic expression levels of 890, 1,800 and 1,010 nmol/L for 3A4, 2C9 and 1A2, respectively. The P450 titers achieved in fermentors exceeded those in flask fermentations by 3- to 6-fold in this study and up to 10-fold when compared with previously reported literature. Intact cells and isolated membranes obtained from 10-L fermentations were used to establish an efficient bioconversion system for the generation of metabolites. To demonstrate the utility of this approach, known metabolites of the anabolic steroid testosterone, the anti-inflammatory agent diclofenac and the analgesic agent phenacetin, were generated using 3A4, 2C9 and 1A2, respectively. The reaction conditions were optimized for pH, temperature, DO concentration, use of co-solvent and glucose supplementation. Conversion yields of 29-93% were obtained from 1-L reactions, enabling isolation of 59 mg 6beta-hydroxytestosterone, 110 mg 4'-hydroxydiclofenac and 88 mg acetaminophen.

Phospholipid metabolism in the fission yeast Schizosaccharomyces pombe was examined. Three enzymes of phospholipid biosynthesis, cytidine diphosphate diacylglycerol synthase (CDP-DG), phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase, were characterized in extracts of S. pombe cells. Contrary to an earlier report, we were able to demonstrate that Cf)P-DG served as a precursor for PI and PS biosynthesis in S. pombe. S. pombe is naturally auxotrophic for the phospholipid precursor inositol. We found that S. pombe was much more resistant to loss of viability during inositol starvation than artificially generated inositol auxotrophs of Saccharomyces cerevisiae. The phospholipid composition of S. pombe cells grown in inositol-rich medium (50 ,uM) was similar to that of S. cerevisiae cells grown under similar conditions. However, growth of S. pombe at low inositol concentrations (below 30 ,uM) affected the ratio of the anionic phospholipids PI and PS, while the relative proportions of other giycerophospholipids remained unchanged. During inositol starvation, the rate of PI synthesis decreased rapidly, and there was a concomitant increase in the rate of PS synthesis. Phosphatidic acid and CDP-DG, which are precursors to these phospholipids, also increased when PI synthesis was blocked by lack of exogenous inositol. The major product of turnover of inositol-containing phospholipids in S. pombe was found to be free inositol, which accumulated in the medium and could be reused by the cell.The fission yeast Schizosaccharomyces pombe is unusual among eucaryotic cells in that it has an absolute requirement for inositol (15,26). In contrast, bakers' yeast, Saccharomyces cerevisiae, can synthesize inositol from glucose-6-phosphate via the repressible enzyme inositol-1-phosphate synthase (10). In S. cerevisiae, free inositol reacts with cytidine diphosphate diacylglycerol (CDP-DG) in a reaction catalyzed by the membrane-associated enzyme phosphatidylinositol (PI) synthase (8,16,32) to yield PI (see Fig. 1). White and Hawthorne (35), in their studies on phospholipid biosynthesis in S. pombe and S. cerevisiae, suggested that PI was not synthesized via this reaction because they could not demonstrate that the synthesis of PI was dependent on CDP-DG in either yeast. PI synthase activity has since been demonstrated in S. cerevisiae (32), and the enzyme has been purified from microsomes to near homogeneity and characterized (8,16). Phosphatidylserine (PS) synthase has also been purified from S. cerevisiae (3), and CDP-DG synthase has been partially purified (5). Little is known about these three enzymes from S. pombe.Various studies have addressed the importance of inositol in eucaryotic cell function. Mutants of S. cerevisiae (11), Neurospora spp. (34), and Ustilago spp. (21) have been generated which contain a block in the inositol biosynthetic pathway, resulting in a requirement for inositol. Some mammalian cell lines have also been shown to require inositol in culture (14). When inositol auxotrophs of previ...

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The addition of L-serine to inositol-containing growth medium repressed membrane-associated CDPdiacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activities and subunit levels in wild-type Saccharomyces cerevisiae. Enzyme activities and subunit levels were not repressed when inositol was absent from the growth medium. The addition of L-serine to the growth medium did not affect the phospholipid composition of wild-type cells. CDPdiacylglycerol synthase and phosphatidylserine synthase were not regulated in the S. cerevisiae inositol biosynthesis ino2, ino4, and opil regulatory mutants, suggesting that regulation by inositol plus L-serine is coupled to inositol synthesis. Inositol and L-serine did not affect the activities of purified CDPdiacylglycerol synthase and phosphatidylserine synthase. The addition of compounds structurally related to L-serine to the growth medium of wild-type cells also resulted in a repression of CDPdiacylglycerol synthase and phosphatidylserine synthase but only in the presence of inositol. Phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was not regulated by inositol plus L-serine.In Saccharomyces cerevisiae, several phospholipid biosynthetic enzymes in the reaction sequence phosphatidictidylcholine (PC) are regulated in a coordinate fashion. CDP-DG synthase (23), PS synthase (10, 27), PS decarboxylase (11), and the phospholipid N-methyltransferase (35, 36) activities are repressed in wild-type cells grown in medium supplemented with inositol plus choline. CDP-DG synthase (23) and PS synthase (34) activities are also repressed when wild-type cells are grown with inositol plus ethanolamine. PE and PC are synthesized via the CDP-ethanolamine-and CDP-choline-based pathways (25) when the cells are supplemented with ethanolamine and choline (2). Ethanolamine or choline has no effect on CDP-DG synthase (23), PS synthase (27, 34), and phospholipid N-methyltransferase (27, 36) activities in the absence of inositol. The addition of inositol alone to the growth medium causes a partial repression of CDP-DG synthase (23), PS synthase (27, 34), and the phospholipid N-methyltransferase (36) activities. However, phosphatidylinositol (PI) synthase, which uses inositol as a substrate, is not regulated in response to inositol, ethanolamine, and choline (17,27).Inositol-l-phosphate synthase is the cytoplasmicassociated enzyme responsible for inositol synthesis. This enzyme is repressed in wild-type cells by inositol and is derepressed in the absence of inositol (15). Mutants with lesions in genes whose wild-type products exert a positive (i.e., the ino2 and ino4 strains) or a negative (i.e., the opil strain) effect upon inositol-1-phosphate synthase expression (12,14,18,19) exert pleiotropic effects upon the coordinately regulated enzymes involved in the pathway leading to PC (4,23,26,27,32,34 the coordinate regulation of the en...