2.21).
Through transamination reactions, glutamate then serves as an amino group donor in the biosynthesis of different amino acids. Pyridoxal phosphate (PLP) is the transaminase cofactor (Figure 2.22); it is derived from pyridoxine (vitamin B6).
The carbon skeleton of amino acids originates from intermediates of glycolysis (pyruvate, 3‐phosphoglycerate, and phosphoenolpyruvate), the citric acid cycle (α‐ketoglutarate and oxaloacetate), or the pentose phosphate cycle (ribose 5‐phosphate and erythrose 4‐phosphate). Some of these reactions are very simple, such as the formation of aspartate or alanine by transamination of glutamate into oxaloacetate or pyruvate:
Other biosynthetic pathways are more complex, but still occur in yeasts as in the rest of the living world. The amino acids can be classified into six biosynthetic families, depending on their nature and their carbon precursor (Figure 2.23):
1 In addition to glutamate and glutamine, proline and arginine are formed from α‐ketoglutarate.
2 Asparagine, methionine, lysine, threonine, and isoleucine are derived from aspartate, which comes from oxaloacetate. ATP can activate methionine to formS‐adenosylmethionine, which can be demethylated to form S‐adenosylhomocysteine, the hydrolysis of which liberates adenine to produce homocysteine.FIGURE 2.21 Amidation of glutamate into glutamine by glutamine synthetase (GS).FIGURE 2.22 Pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP).FIGURE 2.23 General biosynthesis pathways of amino acids.
3 Pyruvate is the starting point for the synthesis of alanine, valine, and leucine.
4 3‐Phosphoglycerate leads to the formation of serine and glycine. The condensation of homocysteine and serine produces cystathionine, a precursor of cysteine.
5 The imidazole ring of histidine is formed from ribose 5‐phosphate and adenine coming from ATP.
6 The amino acids possessing an aromatic ring (tyrosine, phenylalanine, and tryptophan) are derived from erythrose 4‐phosphate and phosphoenolpyruvate. These two compounds are intermediates of the pentose cycle and glycolysis, respectively. Their condensation forms shikimate. The condensation of this compound with another molecule of phosphoenolpyruvate produces chorismate, aprecursor of aromatic amino acids.
2.4.2 Assimilation Mechanisms of Ammonium and Amino Acids
The penetration of ammonium and amino acids into the yeast cell activates numerous membrane protein transporters or permeases (Section 1.3.2). Saccharomyces cerevisiae has at least two specific ammonium ion transporters (Dubois and Grenson, 1979). Their activity is inhibited by several amino acids, in a noncompetitive manner.
Two distinct categories of transporters ensure amino acid transport:
1 A general amino acid permease (GAP) transports all of the amino acids. The ammonium ion inhibits and represses GAP. GAP therefore appears to be active during winemaking only when the must no longer contains ammonium, i.e. after the end of the cell growth phase. It acts as a “nitrogen scavenger” toward amino acids (Cartwright et al., 1989).
2 Saccharomyces cerevisiae also has many specific GAPs (at least 11). Each one ensures the transport of one or more amino acids.
The transport of nitrogen sources is subject to complex regulations depending on the nitrogen content of the medium, by means of a system called nitrogen catabolite repression (NCR) (Beltran et al., 2004).
Toward the end of fermentation, yeasts excrete significant but variable amounts of different amino acids. During fermentation, yeasts assimilate between 1 and 2 g/l of amino acids. Finally, at the end of alcoholic fermentation, a few hundred milligrams of amino acids per liter remain; proline generally represents half of this amount.
Contrary to must hexoses that penetrate the cell by facilitated diffusion, ammonium and amino acids require active transport. Their concentration in the cell is generally higher than in the external medium. The permeases involved couple the transport of an amino acid molecule (or ammonium ion) with the transport of a hydrogen ion. The hydrogen ion moves in the direction of the concentration gradient: the concentration of protons in the must is higher than in the cytoplasm. The amino acid and the proton are linked to the same transport protein and penetrate the cell simultaneously. This concerted transport of two substances in the same direction is called symport. Obviously, the proton that penetrates the cell must then be exported to avoid acidification of the cytoplasm. This movement is made against the concentration gradient and requires energy. The membrane ATPase ensures the excretion of the hydrogen ion across the plasma membrane, acting as a proton pump (Figure 2.24).
Ethanol strongly limits amino acid transport. In an alcohol medium, it modifies the composition and the properties of the phospholipids of the plasma membrane. The membrane becomes more permeable to H+ ions in the medium, and these ions massively penetrate the interior of the cell by simple diffusion. The membrane ATPase must increase its operation to control the intracellular pH. As soon as this task monopolizes the ATPase, the symport of the amino acids no longer functions. In other words, at the beginning of fermentation, and for as long as the ethanol concentration in the must is low, yeasts can rapidly assimilate amino acids and concentrate them in the vacuoles for later use, according to their biosynthesis needs.
2.4.3 Catabolism of Amino Acids
The ammonium ion is essential for the synthesis of amino acids necessary for building proteins, but yeasts cannot always find sufficient quantities in their environment. Fortunately, they can obtain ammonium from available amino acids through various reactions.
FIGURE 2.24 Active amino acid transfer mechanisms in the yeast plasma membrane. P, protein playing the role of an amino acid “symporter.”
FIGURE 2.25 Oxidative deamination of an amino acid, catalyzed by a transaminase and glutamate dehydrogenase.
The most common pathway is the transfer of an α‐amino group, originating from one of many different amino acids, onto α‐ketoglutaric acid to form glutamate. Aminotransferases or transaminases catalyze this reaction, whose prosthetic group is PLP. Glutamate is then deaminated by an oxidative pathway to form NH4+
