BIOCHEMISTRY OF YEAST FERMENTATION Fatty Acid and Ester Metabolism

Fatty acids are long-chain, unbranched carboxylic acids. Most naturally occurring fatty acids contain an even number of carbons because they are synthesized from acetate. Yeasts produce small amounts (1 to 15 mg.L-1) of C6, C8 and C10 fatty acids in wines. These acids may become toxic to Saccharomyces cerevisiae and contribute to stuck fermentations. However they also form esters which are responsible for the pleasant fermentation aroma of wines. These acids are probably formed thru the anabolic pathways in yeast, but they may arise via -oxidation of higher fatty acids.

Anabolism of Fatty Acids

Fatty acid biosynthesis begins in the cytosol with acetyl-CoA (formed from pyruvate) being carboxylated to produce malonyl-CoA. The enzyme is acetyl-CoA carboxylase and it requires biotin (vitamin H), manganese (II) and magnesium (II).
O S CoA ATP + HCO3 ADP Acetyl-SCoA carboxylase O O O S Malonyl-CoA CoA

Acetyl-CoA

The mechanism for this reaction is covered in Organic Chemistry, Bruice (3rd Edition) page 1010. The first step is the formation of activated bicarbonate. The role of Mg2+ is to decrease the negative charge on ATP.
O O HO O O P O Mg bicarbonate ATP + Mg
2

O O P O O

O P O adenosine O

O O

O P O O Mg ADP

activated bicarbonate

Nucleophilic attack by biotin on activated bicarbonate forms carboxybiotin. Because the oxygen of the amide is not nucleophilic, it is likely that the active form of biotin has an enolate-like structure. See Figure 1. All biotin-requiring enzymes follow the same three steps: activation of bicarbonate by ATP, reaction of the activated bicarbonate with biotin to form carboxybiotin, and transfer of the carboxyl group from carboxybiotin to the substrate. Before fatty acid biosynthesis can occur, the acyl groups of acetyl-CoA and malonyl-CoA are transferred to separate molecules of acyl carrier protein (ACP) forming malonyl-ACP (a thioester) and acetyl-ACP (a thioester). ACP reacts as a thiol.
O S Acetyl-CoA O O O S Malonyl-CoA CoA HS ACP O S CoA HS ACP S O ACP HSCoA

O ACP HSCoA

Mechanism for the Carboxylation of Acetyl-CoA with Biotin to form Malonyl-CoA.

O O O Mg O P O O N O S activated bicarbonate (CH2)4CONH(CH2)4Enzyme NH MgO3PO O S (CH2)4CONH(CH2)4Enzyme O N O NH

enolate-like structure of enzyme bound biotin

O O O SCoA N

O O NH O S (CH2)4CONH(CH2)4Enzyme N

O NH

(CH2)4CONH(CH2)4Enzyme

carboxybiotin O S NH CoA S CoA

OH O O O O S Malonyl-CoA S (CH2)4CONH(CH2)4Enzyme CoA N

Acetyl-CoA

FIGURE 1 The elongation of fatty acids begins with a Claisen condensation between malonyl-ACP and acetyl-ACP. The mechanism for this reaction is covered in Organic Chemistry, Bruice (3rd Edition) page 864 and in Organic Chemistry, Vollhardt and Schore (4th Edition) page 1030. A Claisen condensation is the reaction of an enolate of an ester reacting with another molecule of ester. A strong base is required to remove the -proton of the ester. For a biological reaction at pH 7, the -proton is effectively removed by decarboxylation. The product of the Claisen undergos a reduction, a dehydration, followed by a second reduction to form a four-carbon thioester.
O S Acetyl-ACP dehydration reduction ACP O O O S Malonyl-ACP ACP CO2 S ACP O O S ACP S O O ACP

O S ACP

reduction

O S ACP

dehydration

OH

O S ACP

The four carbon thioester undergoes a Claisen condensation with another molecule of malonyl thioester. Again the product of the condensation undergoes a reduction, dehydration and a second reduction to form a six carbon thioester. This sequence of reactions is repeated and each time two more carbons are added to the chain. See Figure 2. Once a thioester with the appropriate number of carbon atoms is obtained, it can undergo a transesterification reaction with water to produce the fatty acid or with ethanol, a higher alcohol or a reduced fatty acid to yield a variety of esters important to the fermentation aroma of wine.

Summary of Fatty Acid Biosynthesis: C4, C6, C8 and C10

[ACP]acetyltransferase CoA ATP ADP Claisen Condensation 3-oxoacyl-[ACP] synthase O S Acetyl-ACP ACP CO2 ACPSH ATP + HCO3 Acetyl-CoA carboxylase ADP O O Reduction 3-oxoacyl-[ACP] reductase ACP NADPH NADP

O S Acetyl-CoA

OH

S Acetoacetyl-ACP

ACP S -Hydroxybutanoyl-ACP

3-Hydroxyacyl-[ACP] dehydrase Dehydration O S Crotonyl-ACP

H2O

ACP

O O

O CoA Enoyl-[ACP] reductase Reduction

NADPH NADP

S Malonyl-CoA

ACPSH [ACP]malonyltransferase CoASH

O S ACP

Butanoyl-ACP Claisen Condensation S Malonyl-ACP ACP CO2 ACPSH O O S ACP

O O

Reduction Dehydration

Reduction O S hexanoyl-ACP Claisen Condensation ACP

CO2 ACPSH

Reduction Dehydration

Reduction CO2 ACPSH O S decanoyl-ACP ACP Reduction Dehydration Reduction Claisen octanoyl-ACP O S ACP

FIGURE 2

In many organisms, the -oxidation of fatty acids is the chief source of energy in the catabolism of lipids. For each cycle one mole of acetyl-CoA, NADH and FADH2 are produced. The acetyl-CoA enters the citric acid cycle to generate energy (ATP), while the NADH and FADH2 enter the electron transport chain (oxidative phosphorylation).
O R S CoA FAD FADH2 R O S CoA H2O Acyl-CoA dehydrogenase Acetyl-CoA O R S CoA Thiolase R HSCoA O O S CoA -Hydroxyacyl-CoA hydratase NAD NADH Enoyl-CoA hydratase R OH O S CoA

-Oxidation of Fatty Acids

Whether -oxidation plays a role in fermentation is uncertain. -Oxidation requires the presence of oxygen and consequently -oxidation could only take place in the early stages of fermentation when the oxygen concentration is still high. However -oxidation is the major source of energy for grape seeds and the seed stores lipids for this purpose.

Esters
The volatile esters contribute important floral and fruity sensory properties to wine. These esters are of two types: ethyl esters of

fatty acids, and acetate esters and both types are formed enzymatically during fermention. Fatty acid ethyl esters (e.g. ethyl butanoate, ethyl hexanoate and ethyl octanoate) are obtained by ethanolysis of the acylCoA that is formed during fatty acid synthesis or degradation. Fatty acids with more than 12 carbons are not found in wine.
O OH butanoic acid H2O O S butanoyl-CoA CoA Ethanol O O ethyl butanoate

Acetate esters (e.g. isoamyl acetate, hexyl acetate) are the result of the reaction of acetylCoA with the higher alcohols. The higher alcohols are formed from the degradation of amino acids, while hexanol probably arises from the oxidation of linoleic acid that occurs on crushing (see section under grapes).
Acetyl-CoA OH hexanol hexyl acetate HSCoA O O

Both groups of esters reach a maximum concentration during fermentation; hexyl acetate and the fatty acid ethyl esters at the midpoint and the higher alcohol acetates towards the end. Enzymatic hydrolysis of esters also occurs during fermentation (via esterases) while chemical hydrolysis occurs during storage and aging. The fatty acid ethyl esters (ethyl butanoate, ethyl hexanoate and ethyl octanoate ) have very pleasant odors of wax and honey which contribute to the aromatic finesse of white wines. The acetates have intense odors; isoamyl acetate has an aroma of banana, hexyl acetate smells of apples. Both groups of esters contribute to the fruitiness of wines and their concentrations slowly decline due to nonenzymic hydrolysis during conservation and aging of the wine. See Table 1.

Table 1. Ester concentrations (mol.L-1) with the age of a wine at 25C Compounds Isobutyl acetate Hexyl acetate Isoamyl acetate Phenyl acetate Ethyl hexanoate Ethyl octanoate Ethyl decanoate Time (months) 0 2 5 0.7 0.4 0 1.9 1.2 0 36.6 13.3 3.1 11.0 2.4 0.5 12.2 8.7 6.4 9.3 9.0 7.4 2.7 3.4 3.1

29 0 0 0.4 0.5 4.3 6.4 2.0

Lactones (internal esters) -Butyrolactone is frequently found among the wine volatiles at concentrations of a few mg.L-1. It does not appear to play a major role in the organoleptic characteristics of wine. It is formed by the Ehrlich reaction (formation of higher alcohols from amino acids) of glutamic acid to form 4-hydroxybutanoic acid which then undergoes an internal esterification to form the lactone, -butyrolactone.
O HO NH2 Glutamic acid 4-Hydroxybutanoic acid O Ehrlich Rxn OH HO O OH HO O O O O

-Butyrolactone

Infection of grapes by Botrytis cinerea probably produces sotolon (4,5-dimethyl-3-hydroxy-2-furanone), involved in the toasty aroma characteristic of wines made from grapes with noble rot. The concentrations (about 5 .L-1) are above the perception threshold. A nonenzymic condensation between 2-oxobutanoic acid and ethanal yields sotolon.
O CH3CHO OH O 2-oxobutanoic acid O O O sotolon O O OH

A mechanism for this reaction may be:

B H O OH O 2-oxobutanoic acid O enolate O H OH ethanal B H OH O O aldol product O OH O dehydration O OH sotolon OH lactonization O O