22.2
Fatty Acid Metabolism

22.2.1 Introduction

Lipids are a diverse group of biomolecules with the common property of being soluble in non‐polar solvents like chloroform: they represent about 7–15% of yeast biomass by dry weight [1]. While lipids constitute a broad class of compounds, fatty acids are of particular quantitative and sensory importance. In wine yeasts, the majority of fatty acids (>99%) are esterified with glycerol to form mono‐, di‐, and tri‐acylglycerides, or are esterified with glycerophosphate groups to form glycerophospholipids (also called phospholipids, PL), with only a small amount existing as free fatty acids (FFAs) [2] (Figure 22.2.1). Although the mid‐chain fatty acids (MCFAs) (Chapter 3) and their esters (Chapter 7) have the most profound effects on wine organoleptic properties, the majority of fatty acids in yeast PL are long‐chain, particularly the saturated fatty acids (SFAs) palmitate (16 carbons, zero double bonds, designated 16:0) and stearate (18:0), and the unsaturated fatty acid (UFA) oleate (18:1) (Figure 22.2.1). The cell membrane of yeasts and other eukaryotes is composed largely (70%) of PL, which are organized into the well‐known membrane bilayer structure in which the polar “heads” face the aqueous intracellular or extracellular matrices and the hydrophobic tails face each other in the bilayer interior (Figure 22.2.2) [3, 4]. The resulting cell membrane – approximately 5–9 nm thick – is largely impervious to passive diffusion of polar components.

Figure 22.2.1 Structures of representative free fatty acids (FFA) produced by yeast

Figure 22.2.2 (a) Structure and (b) cartoon of a representative phospholipid, showing the effect of saturated (stearic) and unsaturated (oleic) fatty acids on conformation, and the location of the hydrophobic and hydrophilic component, and (c) cartoon of the lipid membrane bilayer showing the separation of must and yeast cytosolic components. The presence of unsaturated fatty acids results in less tight packing of the bilayer.

Adapted from Reference [4]

22.2.2 Long‐chain fatty acid metabolism

A detailed account of long‐chain fatty acid biosynthesis can be found in biochemistry textbooks [5, 6]. The description in this chapter will focus only on key details that eventually impact wine flavor chemistry; the steps of fatty acid biosynthesis critical to an understanding of this chemistry are summarized in Figure 22.2.3, as adapted from elsewhere [7].

Figure 22.2.3 Overview of fatty acid biosynthesis in yeast. (a) Palmitate (16:0) is synthesized in the fatty acid synthase (FAS) complex from acetyl‐CoA and malonyl‐CoA substrates in sequential 2‐carbon elongations steps. (b) Following release from FAS, 16:0 can be elongated or desaturated, and used as substrates for formation of phospholipids (PL) during the growth phase. (c) Under anerobic conditions, desaturation is not possible and PL synthesis stops, resulting in accumulation of saturated fatty acids (SFA), which inhibit the initial acetyl‐CoA formation steps. (d) This arrests activity in the FAS, resulting in release of intermediate mid‐chain fatty acids (MCFA)

22.2.2.1 Initial steps – formation of coenzyme A thioesters

Initial and intermediate steps of fatty acid biosynthesis utilize coenzyme A (CoA), a co‐factor with a reactive sulfhydryl (–SH) group. The sulfhydryl group can be enzymatically esterified with carboxylic acids to yield thioester compounds, including acetyl‐CoA (Figure 22.2.4). Acetyl‐CoA was previously mentioned in the discussion of glycolysis (Chapter 22.1). While acetyl‐CoA can be formed directly from pyruvate, this transformation occurs within the mitochondria and the resulting acetyl‐CoA is not available for fatty acid synthesis in the cytosol under fermentation conditions [8]. Instead, the major source of acetyl‐CoA during alcoholic fermentation is acetic acid, formed by oxidation of acetaldehyde. Acetyl‐CoA can subsequently be transformed to malonyl‐CoA (Figure 22.2.3).

Figure 22.2.4 ATP‐dependent formation pathway for acetyl‐CoA and malonyl‐CoA, the building blocks of fatty acids. Acetyl‐CoA formation is catalyzed by acetyl‐CoA synthase (Acs1p) starting from acetic acid and coenzyme A (CoA). Malonyl‐CoA is subsequently formed from acetyl‐CoA and bicarbonate via acetyl‐CoA carboxylase (Acc1p)

22.2.2.2 Next steps – synthesis of palmitate by fatty acid synthase

Following synthesis of acetyl‐ and malonyl‐CoA, palmitate (16:0) can be formed in the multienzyme fatty acid synthase complex (FAS, Figure 22.2.3). The pathway begins with the enzymatic elongation of acetyl‐CoA to malonyl‐CoA to form a 4‐carbon intermediate (Figure 22.2.4). Analogous elongation steps by additional malonyl‐CoA equivalents result in formation of intermediates that are 6‐carbon, 8‐carbon, etc., before eventual release of palmitate. Because palmitate and other fatty acids are synthesized in sequential steps involving two‐carbon “chunks”, the majority of fatty acids in yeast and other organisms are straight‐chained and even‐numbered. The reaction requires reducing equivalents (NADPH) derived primarily from the pentose phosphate pathway, which is also responsible for formation of nucleic acids [6]. The net reaction for formation of palmitate is

(22.2.1)

22.2.2.3 Final steps – elongation and desaturation

Following palmitate synthesis, long‐chain FFA with >16‐carbons can be formed by elongation outside of the FAS complex and unsaturated fatty acids (particularly 18:1, oleate) can be formed by oxygen‐dependent desaturase enzymes such as Ole1p [9]. Concentrations of unsaturated fatty acids may be close to 70% in commercial dry yeasts produced in (semi) aerobic conditions, but will be at much lower concentrations under anaerobic winemaking conditions [10]. Polyunsaturated fatty acids like linoleate (18:2) or linolenate (18:3) are not produced in significant concentrations by S. cerevisiae (<2% of fatty acids), even when oxygen is not limiting, although significant amounts may be formed by non‐Saccharomyces yeasts [1].

Branched‐chain and odd‐numbered fatty acids can be formed by an analogous pathway to straight‐chain fatty acids by substitution of an appropriate acyl‐CoA group in place of acetyl‐CoA [11]. For example, isovaleryl‐CoA can serve as a starting point for longer branched‐chain fatty acids. This compound can be formed by CoA acylation of isovaleric acid formed via Ehrlich degradation and subsequent oxidation of leucine, in a manner analogous to the formation of higher alcohols (Chapter 22.3). Fatty acids with an odd number of carbons can be formed starting from propionyl‐CoA, likely formed via α‐ketobutyric acid (2‐oxobutanoic acid) as an intermediate in the Ehrlich degradation of threonine [12].

During the growth stage of fermentation, FFA will be converted to their corresponding fatty acid‐CoA thioesters and incorporated into the PL and membrane bilayers of growing yeast cells (Figure 22.2.2). Throughout fermentation, yeast will adapt the FFA composition of their membranes to suit their changing environment [13]. In particular, the optimal functioning of membrane proteins and other cell membrane components is believed to require an appropriately fluid bilayer, with the term “membrane fluidity” referring to the degree of disorder [14]. Membrane fluidity increases roughly with the decreasing melting points of constituent fatty acids (i.e., the higher melting SFA have lower membrane fluidity), and will also increase with greater concentrations of sterols.¹ For a given bilayer composition, lower temperatures will result in a decrease in membrane fluidity. Poikilothermic organisms like yeast (i.e., internal temperature varies with that of the surrounding medium) will attempt to counteract this fluidity loss by modifying their membrane composition to regain the original fluidity properties. A common response of yeasts is to increase the ratio of lower‐melting unsaturated fatty acids (UFA) to higher‐melting saturated fatty acids (SFA),² and under cooler fermentation conditions (13 °C versus 25 °C) the UFA/SFA ratio in S. cerevisiae roughly doubles [10].³ The presence of ethanol results in a similar increase of UFA/SFA in yeast [16], and thus the UFA content will often increase during fermentation [13].

Because of the benefits of UFA to membrane fluidity, the UFA/SFA ratio of commercially purchased S. cerevisiae produced under aerobic conditions may be 2:1 or greater [1]. However, UFA formation requires oxygen, as described above, and under typical fermentation conditions UFA/SFA will be closer to 1:1. The limited availability of UFA may eventually constrain PL biosynthesis and yeast growth (Figure 22.2.3), especially if growth is not limited by other nutritional deficiencies (e.g., insufficient nitrogen). As a result, FFA synthesis and yeast growth will often cease after the first 3–6 days of fermentation, especially under highly anaerobic conditions.

22.2.3 Mid‐chain fatty acids (MCFAs) and ethyl esters

The major products of fatty acid metabolism – long‐chain fatty acids and phospholipids – are only sparingly soluble in aqueous environments, possess low volatility, and are of negligible impact on finished wines. Conversely, mid‐chain fatty acids (MCFAs, 4–12 carbons), which are minor byproducts of fatty acid metabolism, along with their corresponding esters, can have a significant flavor impact on wine due to their greater solubility and volatility.

The accumulation of MCFA during fermentation is hypothesized to be correlated with depletion of UFA and sterols, and the arrest of fatty acid biosynthesis [7], as described above. This will result in accumulation of long‐chain saturated acyl‐CoA compounds, which inhibits the initial stages of fatty acid synthesis. Under these conditions, MCFAs are released from the FAS complex in free form and/or as ethyl esters, which are subsequently excreted from the yeast cell [17].⁴

MCFAs are toxic to yeast and other microorganisms, and at sufficient concentrations can result in stuck or sluggish fermentations [20, 21]. MCFAs can permeate the yeast cell membrane at wine pH and cause intracellular acidification, and may also be incorporated into and adversely affect the properties of the membrane. Addition of UFA or sterols to stuck and sluggish fermentations can often increase the fermentation rate [22], likely by restarting phospholipid biosynthesis and removing MCFA from the fermentation media. In commercial wine production, these effects can be achieved through aeration [23] or by addition of yeast hulls (or yeast “ghosts,” i.e. yeast cell wall material).

22.2.3.1 Ethyl esters of MCFA

The ethyl esters of MCFA are key contributors to “fruity” aromas in wines. As described earlier (Chapter 7), the expected molar ratio of an MCFA and its corresponding ethyl ester in a table wine‐like matrix is approximately 6:1, and this equilibrium will slowly be approached during storage due to acid‐catalyzed esterification and hydrolysis reactions. However, during fermentation, MCFA‐ethyl esters can also be formed enzymatically by condensation of MCFA‐CoA with ethanol [24] (Figure 22.2.5). Esterification reactions involving MCFA‐CoAs are energetically favorable as compared to those involving free MCFA, and as a result the ratios of ethyl esters to free acids at the end of fermentation are frequently in excess of equilibrium predictions (Chapter 7).

Figure 22.2.5 Enzymatic formation of an ethyl ester from fatty acid–CoA and ethanol

Two acyl‐CoA:ethanol O‐acyltransferase enzymes, Eht1p and Eeb1p, encoded by the genes EHT1 and EEB1, respectively, have been characterized in yeast, and strains lacking these genes produce 10‐fold lower concentrations of ethyl hexanoate and related ethyl esters [24]. However, gene overexpression results in only minor increases in ethyl esters (<50%, [25]) or no enhancement at all [24], possibly because Eht1p and Ehb1p also possess ester hydrolysis side activity.

22.2.3.2 Timing of MCFA and MCFA ethyl ester production

Under typical fermentation conditions, MCFA and MCFA ethyl ester production commences with yeast growth, and will peak at the end of the growth phase. Both MCFA and MCFA ethyl esters will decrease during the stationary phase once the majority of sugars are consumed, possibly because cell death results in release of UFA which can restart fatty acid biosynthesis in the FAS complex [7]. Exemplary data, demonstrating the peak and decline for ethyl hexanoate during fermentation, is shown in Figure 22.2.6. The second peak corresponds with the expected start of the decline phase (cell death) and may be indicative of a release in intracellular ethyl hexanoate following yeast cell autolysis.

Figure 22.2.6 Concentration of ethyl hexanoate in a fermentation as a function of fermentation time. Fermentation progress and sugar consumption are indicated by the decrease in fermenter weight. Data from Reference [26]

22.2.4 Increasing MCFA and their ethyl esters in winemaking

Because MCFA ethyl ester concentrations are not highly sensitive to enzymatic activity, their concentrations in wine are largely dependent on MCFA concentrations [7]. This behavior is in contrast to acetate esters, whose formation is highly dependent on enzymatic activity (Atf1p and others) and less dependent on substrate concentration (Chapter 22.3). Winemakers interested in increasing MCFA production during fermentation can achieve this goal by increasing UFA demand and decreasing UFA supply as follows:

• Anaerobic conditions prevent enzymatic formation of UFA and will arrest fatty acid synthesis, resulting in greater MCFA release [27].
• Cooler temperatures increase the demand for UFA, resulting in earlier cessation of long‐chain fatty acid synthesis and greater MCFA release (Figure 22.2.7).
• Ceasing fermentation before dryness can prevent further metabolism of MCFA ethyl esters (Figure 22.2.6), and presumably their corresponding MCFA, too.
• Decreasing sources of UFA or sterols, e.g. clarifying to remove grape solids, since these compounds will restart long‐chain fatty acid synthesis and decrease MCFA release [28].
• Yeast strains can differ by roughly an order of magnitude in MCFA production under similar fermentation conditions, possibly because of differences in UFA requirements (Figure 22.2.7).

Figure 22.2.7 Effects of fermentation temperature (13 °C versus 25 °C) and yeast strain (A versus B) on MCFA concentrations in wine. C4 = butyric acid, C6 = hexanoic acid, C8 = octanoic acid. Data from Reference [10]

These fermentation conditions – lower oxygen, cooler temperatures, clarification to remove grape solids, appropriate yeast strain selection – are the standard tools used in white winemaking for the production of fruitier wines.⁵

The effects of these fermentation parameters on MCFA ethyl esters immediately after fermentation are often complicated by physiochemical factors. For example, it is reported that increasing fermentation temperatures from 14 °C to 26 °C increases ethyl decanoate by a factor greater than 2, although ethyl hexanoate is unaffected, purportedly because diffusion of the more non‐polar ethyl decanoate out of the cell is limited at cooler temperatures [29]. Cooler temperatures will also decrease losses of volatiles during fermentation due to CO₂ entrainment. Due to their low volatility, only negligible amounts of MCFA are expected to be lost through CO₂ entrainment. However, significant losses due to volatilization – over 50% – are possible for ethyl esters of MCFA during fermentation (Chapter 22.1). Cooler fermentation temperatures can have both physiochemical effects (less volatilization) and physiological effects (enhanced yeast MCFA biosynthesis), and the relative importance of each effect is not clear. During storage, the relative ratios of MCFA and MCFA ethyl esters will move towards equilibrium, and factors that affect MCFA production during fermentation are expected to be of greater long‐term importance.

References

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Notes

1 Sterol is an abbreviation of “steroid alcohol” and represents the other major class of lipids found in cell membranes other than phospholipids. Many organisms will produce sterols to modify lipid membrane function, but the specific structures will vary among kingdoms – yeast and other fungi produce ergosterols, plants produce phytosterols, and mammals (including humans) produce cholesterol. As described later in this chapter, yeast can incorporate plant phytosterols into their membranes, but the effects of cholesterol additions on fermentation efficiency have not been evaluated.

2 This difference in melting behavior can also be observed between olive oil (mostly oleic acid – 18 carbons, 1 double bond, m.p. 13 °C) as compared to butter (mostly stearic acid – 18 carbons, no double bonds, m.p. 70 °C).

3 The inverse correlation of the UFA/SFA ratio and decreasing temperature extends to higher animals. Farkas et al. [15] observed that the ratio of stearic to oleic acid in the brain tissue of birds, fish, and mammals was well-correlated with organism body temperature over a range of 5–41 °C.

4 The reason for MCFA release under these conditions is still debated, but may be a strategy for yeast to regenerate CoA. An alternative hypothesis for the release of MCFA is that they could substitute for UFA under conditions that require an increase in membrane fluidity [18]. However, this seems unlikely, as the majority of MCFA are released extracellularly [17], and no increase in MCFA content of yeast cell membranes is observed at cooler temperatures [19].

5 One challenge for winemakers is that cool, low-solids, anaerobic conditions are also more stressful to yeast, and therefore present a greater risk of stuck and sluggish fermentations.