The principal wine yeast S. cerevisiae is, in addition to its well-known desirable prop­erties (as described above), seemingly the best “platform” choice for lignocellulose — derived substrates because strains are relatively tolerant of the growth inhibitors found in the acid hydrolysates of lignocellulosic biomass.[21] Its biotechnological limitations, on the other hand, derive from its relatively narrow range of fermentable substrates:[22]

• Glucose, fructose, and sucrose are rapidly metabolized, as are galactose and mannose (constituents of plant hemicelluloses) and maltose (a disac­charide breakdown product of starch).

• The disaccharides trehalose and isomaltose are slowly utilized, as are the trisaccharides raffinose and maltotriose (another breakdown product of starch), the pentose sugar ribose, and glucuronic acid (a sugar acid in plant hemicelluloses).

• Cellobiose, lactose, xylose, rhamnose, sorbose, and maltotetraose are nonutilizable.

TABLE 3.1 Ethanol Production by Yeasts on Different Carbon Sources Carbon Temperature Fermentation Maximum Yeast source (°C) time (hr) ethanol (g/l) Reference Saccharomyces Glucose, 30 94 91.8 12 cerevisiae 200 g/l Saccharomyces Sucrose, 28 96 96.7 13 cerevisiae 220 g/l Saccharomyces Galactose, 30 60 40.0 14 cerevisiae 20-150 g/l Saccharomyces Molasses, 30 24 18.4 15 cerevisiae 1.6-5.0 g/l Saccharomyces Glucose, 30 30 21.7 12 pastorianus 50 g/l Saccharomyces Glucose, 30 60 23.0 12 hayanus 50 g/l Kluyveromyces Glucose, 30 192 49.0 12 fragilis 120 g/l Kluyveromyces Glucose, 30 40 24.2 12 marxianus 50 g/l Candida utilis Glucose, 30 80 22.7 12 50 g/l

product of starch) and cellobiose (a degradation product of cellulose).17 This effect is only one of four important O2-related metabolic phenomena, the others being[23]

• The Pasteur effect, that is, the inhibition of sugar consumption rate by O219

• The Crabtree effect, that is, the occurrence (or continuance) of ethanol formation in the presence of O2 at high growth rate or when an excess of sugar is provided20

• The Custers effect, that is, the inhibition of fermentation by the absence of O2 — found only in a small number of yeast species capable of fermenting glucose to ethanol under fully aerobic conditions21

For efficient ethanol producers, the probable optimum combination of phenotypes is

1. Pasteur-positive, (that is, with efficient use of glucose and other readily uti — lizable sugars for growth when O2 levels are relatively high)

2. Crabtree-positive, for high rates of ethanol production when supplied with abundant fermentable sugar from as soon as possible in the fermentation

3. Custers-negative, that is, insensitive to fluctuating, sometimes very low, O2 levels

4. Kluyver-negative, for the widest range of fermentable substrates

Fermentation of Galactose, Five Disaccharides, and Two Trisaccharides by Yeasts

Yeast	Galactose	Maltose	Sucrose	Trehalose	Melibiose	Lactose	Cellobiose	Melezitose	Raffinose
Ambrosiozyma monospora	—	—	—	—	—	—	—	—	—
Candida chilensis	К	К	К	К	—	К	К	К	—
Candida salmanticensis	+	+	+	+	+	—	+	+	+
Candida silvicultrix	+	—	+	—	+	—	+	к	+
Candida shehatae	+	+	—	+	—	К	К	к	—
Kluyveromyces marxianus	+	+	+	+	—	+	к	к	к
Pachysolen tannophilus	К	—	—	—	—	—	к	—	—
Pichia hampshirensis	—	К	К	К	—	—	к	к	—
Pichia stipitis	+	+	к	+	—	к	к	к	—
Pichia subpelliculosa	к	+	+	к	—	—	к	к	+
Saccharomyces bayanus	+	+	+	+	+	—	—	к	+
Saccharomyces cerevisiae	+	+	+	к	+	—	—	+	+
Saccharomyces kluyveri	+	к	+	к	+	—	к	—	+
Saccharomyces pastorianus	+	+	+	к	+	—	—	—	+
Schizosaccharomyces pombe	—	+	+	—	+	—	—	—	+
Zygosaccharomyces fermentati	+	+	+	+	+	—	к	+	+

TABLE 3.2

K: exhibits aerobic respiratory growth but no fermentation (Kluyver effect); may include delayed use (after 7 days) Source: Data from Barnett et al.17

Not all of these effects can be demonstrated with common wine yeasts (or only under special environmental or laboratory conditions), but they are all of relevance when considering the use of novel (or nonconventional) yeasts or when adapting the growth and fermentative capacities of yeast ethanologens to unstable fermentation conditions (e. g., low O2 supply, intermittent sugar inflow) for optimum ethanol production rates. Nevertheless, S. cerevisiae, with many other yeast species, faces the serious meta­bolic challenges posed by the use of mixtures of monosaccharides, disaccharides, and oligosaccharides as carbon sources (figure 3.1). Potential biochemical “bottle­necks” arise from the conflicting demands of growth, cell division, the synthesis of cellular constituents in a relatively fixed set of ratios, and the requirement to balance redox cofactors with an inconsistent supply of both sugar substrates and O2:

• Oligosaccharide hydrolysis

• Disaccharide hydrolysis and uptake

• Hexose transport into the cells

• Conversion of hexoses to glucose 6-phosphate

• The glycolytic pathway for glucose 6-phosphate catabolism

• Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH)

• The tricarboxylic (Krebs) cycle for aerobic respiration and the provision of precursors for cell growth

• Mitochondrial respiration

In the concrete circumstances represented by an individual yeast species in a partic­ular nutrient medium and growing under known physical conditions, specific combi­nations of these parameters may prove crucial for limiting growth and fermentative ability. For example, during the >60 years since its discovery, various factors have been hypothesized to influence the Kluyver effect, but a straightforward product inhibition by ethanol could be the root cause. In aerobic cultures, ethanol, suppresses the utilizability of those disaccharides that cannot be fermented, the rate of their catabolism being “tuned” to the yeast culture’s respiratory capacity.22 The physi­ological basis for this preference is that Kluyver-positive yeasts lack high-capacity transporter systems for some sugars to support the high substrate transport into cells necessary for fermentative growth, whereas energy-efficient respiratory growth sim­ply does not require a high rate of sugar uptake.23,24

The function of O2 in limiting fermentative capacity is complex; in excess, it blocks fermentation in many yeasts, but a limited O2 supply enhances fermentation in other species.10,20 Detailed metabolic analyses have shown that the basic pathways of carbon metabolism in ethanologenic yeasts are highly flexible on a quantitative basis of expression, with major shifts in how pathways function to direct the “traf­fic flow” of glucose-derived metabolites into growth and oxidative or fermentative sugar catabolism.25 26 The Crabtree effect, that is, alcoholic fermentation despite aerobic conditions, can be viewed as the existing biochemical networks adapting to consume as much of the readily available sugar (a high-value carbon source for microorganisms) as possible — and always with the possibility of being able to reuse the accumulated ethanol as a carbon source when the carbohydrate supply eventually becomes depleted.22 2728

oligosaccharides MEDIUM

A

( ^ » disaccharides hexose FIGURE 3.1 Biochemical outline of the uptake and metabolism of oligosaccharides and hexoses by yeasts. Indicated steps: A, glycosidases; B, sugar transport and uptake; C, entry into glucose-phosphate pool; D, glycolysis; E, pyruvate decarboxylase; F, alcohol dehydro­genase; G, pyruvate dehydrogenase; H, tricarboxylic acid cycle (mitochondrial); I, electron transport (mitochondrial).

Even when glucose fermentation occurs under anaerobic or microaerobic conditions, the fermentation of xylose (and other sugars) may still require O2. For example, when xylose metabolism commences by its reduction to xylitol (catalyzed by NADPH-dependent xylose reductase), the subsequent step is carried out under the control of an NAD-dependent xylitol dehydrogenase, thus resulting in a dis­turbed redox balance of reduced and oxidized cofactors if O2 is not present, because NADH cannot then be reoxidized, and fermentation soon ceases (figure 3.2).2930

FIGURE 3.2 Metabolic pathways of D-xylose and L-arabinose utilization by bacteria and yeasts: interconnections with oxidative and nonoxidative pen­tose phosphate pathways (for clarity, sugar structures are drawn without hydroxyl groups and both H atoms and C-H bonds on the sugar backbones).

It is precisely this biochemical complexity in yeasts that makes accurate control of an ethanol fermentation difficult and that has attracted researchers to fermenta­tive bacteria where metabolic regulation is more straightforward and where the full benefits of advances in biochemical engineering hardware and software can be more readily exploited. The remarkably high growth rate attainable by S. cerevisiae at very low levels of dissolved O2 and its efficient transformation of glucose to ethanol that made it originally so attractive for alcohol production maintain, however, its A-list status in the rankings of biologically useful organisms (figure 3.3).31 Whole genome sequencing has shown that the highly desirable evolution of the modern S. cerevisiae yeast ethanologen has occurred over more than 150 million years, result­ing in a Crabtree-positive species that can readily generate respiratory-deficient, high alcohol-producing “petite” cells immune to the Pasteur effect in a readily acquired and efficient fermentative lifestyle.32,33