Ergosterol

Fig. 3.8 Biosynthesis of ergosterol from squalene (adapted from Parks, 1978).

o rated fatty acids. This led the authors to speculate that in this yeast there might be separate partitioned sterol biosynthetic pathways.

There is an abundance of evidence in the literature suggesting that sterol biosynthesis is controlled at the level of hydroxymethylglutaryl-CoA reductase. The activity of this enzyme is low in anaerobically grown yeast. Supply of oxygen permits sterol synthesis and this is accompanied by a concomitant increase in the activity of HMG-CoA reductase (Berndt et al., 1973). Furthermore, the concentration of free sterol in the cell modulates the activity of the early enzymes of the sterologenic pathway in a feedback control system. Servouse and Karst (1986) concluded that ergosterol was responsible for reducing the activities of acetoacetyl-CoA thiolase and hydroxymethylglutaryl-CoA synthase. The activity of HMG-CoA reductase was relatively unaffected by ergosterol. Casey et al. (1991) investigated the feedback control effect of sterols and indicated that molecules with both a C22 unsaturation and a C24 methyl group were capable of reducing sterol synthesis by 50%.

The extent and control of sterol synthesis in yeast during brewery fermentations is interesting to consider. As discussed already, relatively small amounts of sterol are formed, typically about 1% of the total cell dry weight, or five-fold less than derepressed cells. It seems likely that this modest requirement for sterol synthesis can be satisfied by cyclisation of the pre-existing squalene pool. This suggests that the early part of the sterol synthetic pathway retains sufficient activity throughout the brewing cycle to allow some synthesis of squalene. In this respect it was observed that the latter molecule may function as a scavenger of oxygen radicals (Kohno et al., 1995). However, the conclusion must be that in brewing fermentations, sterol biosynthesis in yeast is regulated solely by dissolved oxygen concentration.

Synthesis of unsaturated fatty acids also requires molecular oxygen. It has been calculated that the synthesis of unsaturated fatty acids and sterols in yeast has an apparent Km for oxygen of 0.5 (iM (Rogers & Stewart, 1973). In yeast cells the predominant unsaturated fatty acids are palmitoleic (16:1) and oleic (18:1). These monounsaturated fatty acids usually have the cis configuration (Schweizer,1989; Rattray, 1989). In eukaryotic cells, de novo synthesis of unsaturated fatty acids involves formation of a saturated fatty acid followed by an oxygen-dependent desaturation reaction. As with sterols, biosynthesis proceeds from acetyl-CoA. The three key enzymes of the pathway are acetyl-CoA carboxylase, the fatty acid synthetase complex and a desaturase (Fig. 3.9).

Acetyl-CoA carboxylase converts acetyl-CoA into malonyl-CoA, which is the immediate precursor of even number fatty acids. The reaction requires ATP and the additional carbon atom is derived from bicarbonate ions. Several steps are involved, one of which utilises biotin as an intermediate carrier of the molecule of C02. Acetyl-CoA carboxylase is considered to be the rate limiting step in fatty acid synthesis and is allosterically regulated in a positive manner by citrate and isocitrate.

Fatty acid synthetase is a multi-enzyme complex which catalyses six separate steps during which an acetyl and a malonyl group are combined to form a C4-fatty acid. The additional carbon atom of the malonyl group is released in the form of C02. This latter carbon atom is the same as that which was fixed in the initial acetyl-CoA carboxylase reaction and, thus, the bicarbonate ion has a catalytic role. In E. coli, the fatty acid synthetase includes an acyl carrier protein (ACP) which contains the same

Acetyl-CoA

HCOa

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