5. Major Metabolic Pathways

A major challenge in bioprocess development is to select an organism that can efficiently make a desired product. Before about 1980 only naturally occurring organisms were available. With the advent of genetic engineering, it is possible to remove and add genes to an organism to alter its metabolic functions in a predetermined manner (metabolic engineering). Further, with mixed cultures of multiple species for food fermentations and waste treatment, metabolic cross-feeding may be critical to the behavior of such populations, particularly biodegradation of xenobiotics. In any case, the bioprocess developer must understand the metabolic capabilities of natural organisms either to use them directly or to know how to metabolically engineer them to make a desired, perhaps novel, product. Consequently, we turn our focus toward learning about some essential metabolic pathways.

Differences in microbial metabolism can be attributed partly to genetic differences and/or to differences in their responses to changes in their environment. Even the same species may produce different products when grown under different nutritional and environmental conditions. The control of metabolic pathways by nutritional and environmental regulation has become an important consideration in bioprocess engineering. For example, Saccharomyces cerevisiae (baker’s yeast) produces ethanol when grown under anaerobic conditions. However, the major product is yeast cells (baker’s yeast) when growth conditions are aerobic. Moreover, even under aerobic conditions, at high glucose concentrations some ethanol formation is observed, which indicates metabolic regulation not only by oxygen but also by glucose. This effect is known as the Crabtree effect. Ethanol formation during baker’s yeast fermentation may be reduced or eliminated with intermittent addition of glucose. The major metabolic pathways and products of various microorganisms are briefly covered in this chapter. Metabolic pathways are subgrouped as aerobic and anaerobic metabolism.

There are two key concepts in our discussion. Catabolism is the intracellular process of degrading a compound into smaller and simpler products (e.g., glucose to CO2 and H2O). Catabolism produces energy for the cell. Anabolism is involved in the synthesis of more complex compounds (e.g., glucose to glycogen) and requires energy.

5.1. Bioenergetics

Living cells require energy for biosynthesis, transport of nutrients, motility, and maintenance. This energy is obtained from the catabolism of carbon compounds, mainly carbohydrates. Carbohydrates are synthesized from CO2 and H2O in the presence of light by photosynthesis. The sun is the ultimate energy source for the life processes on earth.

An exception is near some thermal vents at the bottom of the ocean, where nonphotosynthetic ecosystems exist independently of sunlight.

Metabolic reactions and resulting networks are fairly complicated and vary from one organism to another. However, these reactions can be classified in three major categories. A schematic diagram of these reactions is presented in Figure 5.1. The major categories are (I) degradation of nutrients, (II) biosynthesis of small molecules (amino acids, nucleotides), and (III) biosynthesis of large molecules. These reactions take place in the cell simultaneously. As a result of metabolic reactions, end products are formed and released from the cells. These end products (organic acids, amino acids, antibiotics) are often valuable products for human and animal consumption.


Figure 5.1. Schematic diagram of reactions in a bacterial cell.

Energy in biological systems is primarily stored and transferred via adenosine triphosphate (ATP), which contains high-energy phosphate bonds.

The active form of ATP is complexed with Mg2+. The standard free-energy change for the hydrolysis of ATP is 7.3 kcal/mol. The actual free-energy release in the cell may be substantially higher because the concentration of ATP is often much greater than that for adenosine diphosphate (ADP):


Biological energy is stored in ATP by reversing this reaction to form ATP from ADP and Pi (inorganic phosphate). Similarly, ADP dissociates to release energy:


Analog compounds of ATP, such as guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP), also store and transfer high-energy phosphate bonds but not to the extent of ATP. High-energy phosphate compounds produced during metabolism, such as phosphoenol pyruvate and 1,3-diphosphoglycerate, transfer their ~P group into ATP. Energy stored in ATP is later transferred to lower-energy phosphate compounds such as glucose-6-phosphate and glycerol-3-phosphate, as depicted in Figure 5.2.


Figure 5.2. Transfer of biological energy from high-energy to low-energy compounds via adenosine triphosphate (ATP). Phosphoenolpyruvate and 1,3-diphosphoglycerate are high-energy phosphate compounds and act as phosphate donors. Low-energy compounds such as glucose 6-phosphate and glycerol 3-phosphate are phosphate acceptors.

Hydrogen atoms released in biological oxidation–reduction reactions are carried by nucleotide derivatives, especially by nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) (see Figure 5.3). The oxidation–reduction reaction described is readily reversible. Reduced form of NAD (NADH) can donate electrons to certain compounds and accept from others, depending on the oxidation–reduction potential of the compounds. NADH has two major functions in biological systems:


Figure 5.3. Structure of NAD (nicotinamide adenine dinucleotide). (R. Y. Stainer, J. L. Ingraham, M. L. Wheelis, P. R. Painter, The Microbial World, 5th ed., © 1986. Reprinted and electronically reproduced by permission of Pearson Education, Inc., New York, NY.)

Reducing power: NADH and reduced form of NADP (NADPH) supply hydrogen in biosynthetic reactions, such as CO2 fixation by autotrophic organisms:


ATP formation in respiratory metabolism: The electrons (or H atoms) carried by NADH are transferred to oxygen via a series of intermediate compounds (respiratory chain). The energy released from this electron transport results in the formation of up to three ATP molecules. ATP can be formed from the reducing power in NADH in the absence of oxygen if an alternative electron acceptor is available (e.g., Image).

Some research suggests that the theoretical limit may be 2.5 ATP, rather than 3 ATP, for each NADH.

5.2. Glucose Metabolism: Glycolysis and the TCA Cycle

Glucose is a major carbon and energy source for many organisms. Several different metabolic pathways are used by different organisms for the catabolism of glucose. The catabolism of glucose by glycolysis, or the Embden–Meyerhof–Parnas (EMP) pathway, is the primary pathway in many organisms. Other pathways, such as the hexose monophosphate (HMP) and Entner–Doudoroff (ED) pathways are covered later in this chapter.

Aerobic catabolism of organic compounds such as glucose may be considered in three phases:

• EMP pathway for fermentation of glucose to pyruvate.

Krebs, tricarboxylic acid (TCA), or citric acid cycle for conversion of pyruvate to CO2 and NADH.

• Respiratory or electron transport chain for formation of ATP by transferring electrons from NADH to an electron acceptor.

The final phase, respiration, changes reducing power into a biologically useful energy form (ATP). Respiration may be aerobic or anaerobic, depending on the final electron acceptor. If oxygen is used as the final electron acceptor, the respiration is called aerobic respiration. When other electron acceptors, such as Image, Image, Fe3+, Cu2+, and S0, are used, the respiration is termed anaerobic respiration.

Glycolysis, or the EMP pathway, results in the breakdown of glucose to two pyruvate molecules. The enzymatic reaction sequence involved in glycolysis is presented in Figure 5.4. The first step in glycolysis is phosphorylation of glucose to glucose-6-phosphate (G-6P) by hexokinase. Phosphorylated glucose can be kept inside the cell. Glucose-6-phosphate is converted to fructose-6-phosphate (F-6P) by phosphoglucose isomerase, which is converted to fructose 1,6-diphosphate by phosphofructokinase. The first and the third reactions are the only two ATP-consuming reactions in glycolysis. They are irreversible. The breakdown of fructose-1,6-diphosphate into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA-3P) by aldolase is one of the key steps in glycolysis (e.g., C6 to 2 C3). DHAP and GA-3P are in equilibrium. As GA-3P is utilized in glycolysis, DHAP is continuously converted to GA-3P. GA-3P is first oxidized with the addition of inorganic phosphate (Pi) to 1,3-diphosphoglycerate (1,3-dP-GA) by GA-3P dehydrogenase. 1,3-dP-GA releases one phosphate group to form ATP from ADP and is converted to 3-phosphoglycerate (3P-GA) by 3-phosphoglycerate kinase. 3P-GA is further converted to 2-phosphoglycerate (2P-GA) by phosphoglyceromutase. Dehydration of 2P-GA to phosphoenolpyruvate (PEP) by enolase is the next step. PEP is further dephosphorylated to pyruvate (Pyr) by pyruvate kinase, with the formation of an ATP. Reactions after DHAP and GA-3P formation repeat twice during glycolysis.


Figure 5.4. The Embden–Meyerhol (glycolytic) pathway of conversion of glucose to pyruvic acid. (R. Y. Stainer, J. L. Ingraham, M. L. Wheelis, P. R. Painter, The Microbial World, 5th ed., © 1986. Reprinted and electronically reproduced by permission of Pearson Education, Inc., New York, NY.)

The end-product pyruvate is a key metabolite in metabolism. Under anaerobic conditions, pyruvate may be converted to lactic acid, ethanol, or other products, such as acetone, butanol, and acetic acid. Anaerobic conversion of glucose to the aforementioned compounds used to be known as fermentation. However, that term today covers a whole range of enzymatic and microbial conversions. Under aerobic conditions, pyruvate is converted to CO2 and NADH through the TCA cycle.

The overall reaction in glycolysis follows:


The net ATP yield in glycolysis is 2 mol ATP/glucose under anaerobic conditions.

Pyruvate produced in the EMP pathway transfers its reducing power to NAD+ via the Krebs cycle. Glycolysis takes place in cytoplasm, whereas the site for the Krebs cycle is the matrix of mitochondria in eucaryotes. In procaryotes, these reactions are associated with membrane-bound enzymes. Entry into the Krebs cycle is provided by the acylation of coenzyme-A by pyruvate:


Acetyl coenzyme A (CoA) is transferred through mitochondrial membrane at the expense of the conversion of the two NADHs produced in glycolysis to two FADH (flavin adenine dinucleotide). Acetyl-CoA is a key intermediate in the metabolism of amino acids and fatty acids.

The reactions involved in the TCA cycle are presented in Figure 5.5. Condensation of acetyl-CoA with oxaloacetic acid results in citric acid, which is further converted to isocitric acid and then to α-ketoglutaric acid (α-KGA) with a release of CO2. α-KGA is decarboxylated and oxidized to succinic acid (SA), which is further oxidized to fumaric acid (FA). Hydration of fumaric acid to malic acid (MA) and oxidation of malic acid to oxaloacetic acid (OAA) are the two last steps of the TCA cycle. For each pyruvate molecule entering the cycle, three CO2, four NADH + H+, and one FADH2 are produced. The succinate and α-ketoglutarate produced during the TCA cycle are used as precursors for the synthesis of certain amino acids. The reducing power (NADH + H+ and FADH2) produced is used either for biosynthetic pathways or for ATP generation through the electron transport chain.


Figure 5.5. The tricarboxylic acid (TCA) cycle by which acetyl-CoA is oxidized. (R. Y. Stainer, J. L. Ingraham, M. L. Wheelis, P. R. Painter, The Microbial World, 5th ed., © 1986. Reprinted and electronically reproduced by permission of Pearson Education, Inc., New York, NY.)

The overall reaction of the TCA cycle follows:


Note from Figure 5.5 that GTP can be converted easily into ATP; some descriptions of the TCA cycle show directly the conversion of ADP plus inorganic phosphate into ATP, as succinyl-CoA is converted to succinate.

The major roles of the TCA cycle are to provide electrons (NADH) for the electron transport chain and biosynthesis, to supply C skeletons for amino acid synthesis, and to generate energy.

Many of the intermediates in the TCA cycle are used extensively in biosynthesis. Removal of these intermediates “short-circuits” the TCA cycle. To maintain a functioning TCA cycle, the cell can fix CO2 (heterotrophic CO2 fixation). In some microbes, PEP can be combined with CO2 to yield oxaloacetate. Three enzymes that can catalyze such a conversion have been found (PEP carboxylase, PEP carboxykinase, and PEP carboxytransphosphorylase). Pyruvate can be combined with CO2 to yield oxaloacetate, with the expenditure of one ATP, using the enzyme pyruvate carboxylase. Malate dehydrogenase promotes the reversible formation of malate from pyruvate and CO2, using the reducing power of one molecule of NADH + H+.

Heterotrophic CO2 fixation can be an important factor in culturing microbes. When a culture is initiated at low density (i.e., few cells per unit volume) with little accumulation of intracellular CO2, or when a gas sparge rate into a fermentation tank is high, then growth may be limited by the rate of CO2 fixation to maintain the TCA cycle.

5.3. Respiration

The respiration reaction sequence is also known as the electron transport chain. The process of forming ATP from the electron transport chain is known as oxidative phosphorylation. Electrons carried by NADH + H+ and FADH2 are transferred to oxygen via a series of electron carriers, and ATPs are formed. Three ATPs are formed from each NADH + H+ and two ATPs for each FADH2 in eucaryotes. The details of the respiratory (cytochrome) chain are depicted in Figure 5.6. The major role of the electron transport chain is to regenerate NADs for glycolysis and ATPs for biosynthesis. The term P/O ratio is used to indicate the number of phosphate bonds made (ADP + Pi → ATP) for each oxygen atom used as an electron acceptor (e.g., ImageO2 + NADH + H+ → H2O + NAD+).


Figure 5.6. Electron transport and electron transport phosphorylation. Top: Oxidation of NADH and the flow of electrons through the electron transport system, leading to the transfer of protons (H+) from the inside to the outside of the membrane. The tendency of protons to return to the inside is called proton-motive force. Bottom: ATP synthesis occurs as protons reenter the cell. An ATPase enzyme uses the proton-motive force for the synthesis of ATP. The proton-motive force is discussed in Section 4.6. (Brock, Thomas D., Brock, Katherine M., Ward, David M., Basic Microbiology with Applications, 3rd Ed., ©1986. Reprinted and electronically reproduced by permission of Pearson Education, Inc., New York, NY.)

Formation of NADH + H+, FADH2, and ATP at different stages of the aerobic catabolism of glucose is summarized in Table 5.1. The overall reaction (assuming 3 ATP/NADH) of aerobic glucose catabolism in eucaryotes is as follows:

The yield is about 30 ATP if we assume 2.5 ATP/NADH, which may be more representative of actual metabolism.


TABLE 5.1. Summary of NADH, FADH2, and ATP Formation during Aerobic Catabolism of Glucose


The energy deposited in 36 mol of ATP is 263 kcal/mol glucose. The free-energy change in the direct oxidation of glucose is 686 kcal/mol glucose. Therefore, the energy efficiency of glycolysis is 38% under standard conditions. With the correction for nonstandard conditions, this efficiency is estimated to be greater than 60%, which is significantly higher than the efficiency of man-made machines. The remaining energy stored in glucose is dissipated as heat. However, in procaryotes the conversion of the reducing power to ATP is less efficient. The number of ATP generated from NADH + H+ is usually ≤2, and only one ATP may be generated from FADH2. Thus, in procaryotes a single glucose molecule will yield less than 24 ATP, and the P/O ratio is generally between 1 and 2.

5.4. Control Sites in Aerobic Glucose Metabolism

Several enzymes involved in glycolysis are regulated by feedback inhibition. The major control site in glycolysis is the phosphorylation of fructose-6-phosphate by phosphofructokinase:


The enzyme phosphofructokinase is an allosteric enzyme activated by ADP and Pi but inactivated by ATP:


At high ATP/ADP ratios, this enzyme is inactivated, resulting in a reduced rate of glycolysis and reduced ATP synthesis.

The concentration of dissolved oxygen or oxygen partial pressure has a regulatory effect on rate of glycolysis, known as the Pasteur effect. The rate of glycolysis under anaerobic conditions is higher than that under aerobic conditions. In the presence of oxygen, ATP yield is high, since the TCA cycle and electron transport chain are operating. As a result of high levels of ATP, ADP and Pi become limiting, and phosphofructokinase becomes inhibited. Also, some enzymes of glycolysis with — SH groups are inhibited by high levels of oxygen. A high NADH/NAD+ ratio also reduces the rate of glycolysis.

Certain enzymes of the Krebs cycle are also regulated by feedback inhibition. Pyruvate dehydrogenase is inhibited by ATP, NADH, and acetyl-CoA and activated by ADP, AMP, and NAD+. Similarly, citrate synthase is inactivated by ATP and activated by ADP and AMP; succinyl CoA synthetase is inhibited by NAD+. In general, high ATP/ADP and NADH/NAD+ ratios reduce the processing rate of the TCA cycle.

Several steps in the electron transport chain are inhibited by cyanide, azide, carbon monoxide, and certain antibiotics, such as amytal. Such inhibitions are important because of their potential to alter cellular metabolism.

5.5. Metabolism of Nitrogenous Compounds

Most of the organic nitrogen compounds have an oxidation level between carbohydrates and lipids. Consequently, organic nitrogenous compounds can be used as a nitrogen, carbon, and energy source. Proteins are hydrolyzed to peptides and further to amino acids by proteases. Amino acids are first converted to organic acids by deamination (removal of amino group). Deamination reaction may be oxidative, reductive, or dehydrative, depending on the enzyme systems involved. A typical oxidative deamination reaction can be represented as follows:


Ammonia released from deamination is utilized in protein and nucleic acid synthesis as a nitrogen source, and organic acids can be further oxidized for energy production (ATP).

Transamination is another mechanism for conversion of amino acids to organic acids and other amino acids. The amino group is exchanged for the keto group of α-KGA. Following is a typical transamination reaction:


Nucleic acids can also be utilized by many organisms as a carbon, nitrogen, and energy source. The first step in nucleic acid utilization is enzymatic hydrolysis by specific nucleases hydrolyzing RNA and DNA. Nucleases with different specificities hydrolyze different bonds in nucleic acid structure, producing ribose/deoxyribose, phosphoric acid, and purines/pyrimidines. Sugar molecules are metabolized by glycolysis and the TCA cycle, producing CO2 and H2O under aerobic conditions. Phosphoric acids are used in ATP, phospholipid, and nucleic acid synthesis.

Purines/pyrimidines are degraded into urea and acetic acid and then to ammonia and CO2. For example, the hydrolysis of adenine and uracil can be represented as follows:


5.6. Nitrogen Fixation

Certain microorganisms fix atmospheric nitrogen to form ammonia under reductive or microaerophilic conditions. Organisms capable of fixing nitrogen under aerobic conditions include Azotobacter, Azotomonas, Azotococcus, and Beijerinckia. Nitrogen fixation is catalyzed by the enzyme nitrogenase, which is inhibited by oxygen. Typically, these aerobic organisms sequester nitrogenase in compartments that are protected from oxygen. Equation 5.14 summarizes the nitrogen fixation process:


Azotobacter species present in soil provide ammonium for plants by fixing atmospheric nitrogen, and some form associations with plant roots. Some facultative anaerobes, such as Bacillus, Klebsiella, Rhodopseudomonas, and Rhodospirillum, fix nitrogen under strict anaerobic conditions, and strict anaerobes such as Clostridia can also fix nitrogen under anaerobic conditions. Certain cyanobacteria, such as Anabaena sp., fix nitrogen under aerobic conditions. The lichens are associations of cyanobacteria and fungi. Cyanobacteria provide nitrogen to fungi by fixing atmospheric nitrogen. Rhizobium sp. are heterotrophic organisms growing in the roots of leguminous plants. Rhizobium fix atmospheric nitrogen under low oxygen pressure and provide ammonium to plants. Rhizobium and Azospirillum are widely used for agricultural purposes and are bioprocess products.

5.7. Metabolism of Hydrocarbons

The metabolism of aliphatic hydrocarbons is important in some bioprocesses and often critical in applications such as bioremediation. Such metabolism requires oxygen, and only few organisms (e.g., Pseudomonas, Mycobacteria, certain yeasts and molds) can metabolize hydrocarbons. The low solubility of hydrocarbons in water is a barrier to rapid metabolism.

The first step in metabolism of aliphatic hydrocarbons is oxygenation by oxygenases. Hydrocarbon molecules are converted to an alcohol by incorporation of oxygen into the end of the carbon skeleton. The alcohol molecule is further oxidized to aldehyde and then to an organic acid, which is further converted to acetyl-CoA, which is metabolized by the TCA cycle.

Oxidation of aromatic hydrocarbons takes place by the action of oxygenases and proceeds more slowly than those of aliphatic hydrocarbons. Catechol is the key intermediate in this oxidation sequence and can be further broken down ultimately to acetyl-CoA or TCA cycle intermediates. Following is a depiction of aerobic metabolism of benzene:


Anaerobic metabolism of hydrocarbons is more difficult. Only a few organisms can metabolize hydrocarbons under anaerobic conditions. The C–C bonds are cleaved and saturated with hydrogen to yield methane.

5.8. Biodegradation of Xenobiotics

Xenobiotics encompass toxic aromatic compounds, including nitroaromatic compounds (NACs), halogenated aromatic compounds (HACs), and polyaromatic hydrocarbons (PAH). Herbicides; pesticides; insecticides used for agricultural activities; and synthetic chemicals such as plasticizers, dyes, pigments, solvents, and pharmaceuticals, which are toxic, refractory compounds, all are considered xenobiotics. Biodegradation of xenobiotics is especially important for soil bioremediation. Biodegradation mechanisms of xenobiotics vary depending on the type of bacteria and environmental conditions. Aerobic and anaerobic bacteria use different pathways for biodegradation of xenobiotics. Aerobic mechanisms are usually oxidative, leading to a TCA cycle. Anaerobic metabolism is reductive, leading to formation of amino or carboxylic groups followed by ring cleavage and methane formation. Pseudomonas, Rhodococcus, Nocardia, Alcaligenes, Mycobacterium, and Arthrobacter spp. are some of the aerobic bacteria capable of degrading xenobiotic compounds.

In aerobic degradation of nitroaromatics, the first step is removal of the attached nitro groups. Formation of hydroxyl groups by oxygenation, ring cleavage, and formation of carboxyl groups are the next steps in which the carboxylated organic compounds enter the TCA cycle for further degradation. As an example, the major steps involved in oxidative biodegradation of p-nitrophenol are as follows:


The first step is nitrite removal, followed by ketone, alcohol, and organic acid formation along with the ring cleavage.

Biodegradation of PAHs such as naphthalene takes place by carboxylation and ring cleavage. Carboxylated organics enter the TCA cycle for further degradation. Biodegradability of PAHs decreases with the increasing number of rings and the attached groups. Two major mechanisms are identified for biodegradation of halogenated aromatics:

• Halogen groups are removed as the first step in biodegradation by oxidative, reductive, or hydrolytic mechanisms.

• Cleavage of the aromatic ring takes place first, followed by dehalogenation from an aliphatic intermediate.

Cleavage of the halogen–carbon bond is the critical step in biodegradation of halogenated aromatics. The rate of biodegradation varies depending on the number, type, and the position of halogen groups on the aromatic ring. Biodegradation may take place under oxic (O2 as the electron acceptor), anoxic (nitrate or sulfate as the electron acceptor), or anaerobic (no O2) conditions. Monochlorinated and dichlorinated phenols are degraded by aerobic bacteria through chlorinated catechol. Usually, chlorophenols are oxidized to chlorocatechols by phenol monooxygenase and then degraded by a ring cleavage. Dechlorination takes place after the cleavage of the aromatic ring.

Biodegradation of xenobiotics may result in formation of even more toxic intermediates. Complete biodegradation (mineralization) of xenobiotics to CO2, H2O, and HCl (or HNO3) may be accomplished by using a consortium of bacteria with different metabolic capabilities.

5.9. Overview of Biosynthesis

The TCA cycle and glycolysis are critical catabolic pathways and also provide important precursors for the biosynthesis of amino acids, nucleic acids, lipids, and polysaccharides. Although many additional pathways exist, we describe only two more, one in the context of biosynthesis and the other under anaerobic metabolism.

The first is the pentose–phosphate pathway or hexose–monophosphate (HMP) pathway (see Figure 5.7). Although this pathway produces significant reducing power, which could be used, in principle, to supply energy to the cell, its primary role is to provide carbon skeletons for biosynthetic reactions and the reducing power necessary to support anabolism. Normally, NADPH is used in biosynthesis, whereas NADH is used in energy production. The HMP pathway provides an array of small, organic compounds with three, four, five, and seven carbon atoms. These compounds are particularly important for the synthesis of ribose, purines, coenzymes, and the aromatic amino acids. The GA-3P formed can be oxidized to yield energy through conversion to pyruvate and further oxidation of pyruvate in the TCA cycle.


Figure 5.7. The pentose–phosphate pathway of glucose oxidation. (R. Y. Stainer, J. L. Ingraham, M. L. Wheelis, P. R. Painter, The Microbial World, 5th ed., © 1986. Reprinted and electronically reproduced by permission of Pearson Education, Inc., New York, NY.)

A vital component of biosynthesis, which consumes a large amount of cellular building material, is the production of amino acids. Many amino acids are also important commercial products, and the alteration of pathways to induce overproduction (see Chapter 4, “How Cells Work”) is critical to commercial success. The 20 amino acids can be grouped into various families. Figure 5.8 summarizes these families and the compounds from which they are derived. The amino acid histidine is not included in Figure 5.8. Its biosynthesis is fairly complicated and cannot be easily grouped with the others. However, ribose-5-phosphate from HMP is a key precursor in its synthesis.


Figure 5.8. Summary of the amino acid families and their synthesis from intermediates in the Embden–Meyerhof–Parnas, tricarboxylic acid, and hexose monophosphate pathways. The amino acids are underlined.

In addition to synthesizing amino acids and nucleic acids, the cell must be able to synthesize lipids and polysaccharides. The key precursor is acetyl-CoA (see Figure 5.5 for the TCA cycle). Fatty acid synthesis consists of the stepwise buildup of acetyl-CoA. Also, CO2 is an essential component in fatty acid biosynthesis. Acetyl-CoA and CO2 produce malonyl-CoA, which is a three-carbon-containing intermediate in fatty acid synthesis. This requirement for CO2 can lengthen the start-up phase (or lag phase; see Chapter 6, “How Cells Grow”) for commercial fermentations if the system is not operated carefully. The requirement for CO2 can be eliminated if the medium is formulated to supply key lipids, such as oleic acid.

The synthesis of most of the polysaccharides from glucose or other hexoses is readily accomplished in most organisms. However, if the carbon energy source has less than six carbons, special reactions need to be used. Essentially, the EMP pathway needs to be operated in reverse to produce glucose. The production of glucose is called gluconeogenesis. Since several of the key steps in the EMP pathway are irreversible, the cell must circumvent these irreversible reactions with energy-consuming reactions. Since pyruvate can be synthesized from a wide variety of pathways, it is the starting point. However, in glycolysis the final step to convert PEP into pyruvate is irreversible. In gluconeogenesis, PEP is produced from pyruvate as follows:




These two equations can be summarized as follows:


The reactions in glycolysis are reversible (under appropriate conditions) up to the formation of fructose-1,6-diphosphate. To complete glucogenesis, two enzymes (fructose-1,6-diphosphatase and glucose-6-phosphatase) not in the EMP pathway are required. Thus, an organism with these two enzymes and the ability to complete the reaction shown in equation 5.19 should be able to grow a wide variety of nonhexose carbon–energy sources.

So far, we have concentrated on aerobic metabolism. Many of the reactions we have described would be operable under anaerobic conditions. The primary feature of anaerobic metabolism is energy production in the absence of oxygen and in most cases the absence of other external electron acceptors. The cell must also balance its generation and consumption of reducing power. In the next section, we show how the pathways we have discussed can be adapted to the constraints of anaerobic metabolism.

5.10. Overview of Anaerobic Metabolism

The production of energy in the absence of oxygen can be accomplished by anaerobic respiration (see Section 5.1). The same pathways as employed in aerobic metabolism can be used; the primary difference is the use of an alternative electron acceptor. One excellent example is the use of nitrate Image which can act as an electron acceptor. Its product, nitrous oxide (N2O), is also an acceptor leading to the formation of dinitrogen (N2). This process, known as denitrification, is important environmentally. Many advanced biological waste-treatment systems are operated to promote denitrification.

Many organisms grow without using the electron transport chain. The generation of energy without the electron transport chain is called fermentation. This definition is the exact and original meaning of the term fermentation, although currently it is often used in a broader context. Since no electron transport is used, the organic substrate must undergo a balanced series of oxidative and reductive reactions. This constraint requires that the rates of conversion of NAD+ and NADP+ to NADH and NADPH must equal the rates of conversion of NADH and NADPH to NAD+ and NADP+. For example, with the EMP pathway, the 2 mol of NAD reduced in this pathway in the production of pyruvate are reoxidized by oxidation of pyruvate to other products. Two prime examples are lactic acid and ethanol production (see Figure 5.9). Both lactic acid and ethanol are important commercial products from bioprocesses. Some other commercially important products of fermentation are acetone–butanol, propionic acid, acetic acid (for vinegar), 2,3-butanediol, isopropanol, and glycerol.


Figure 5.9. Comparison between (a) lactic acid and (b) alcoholic fermentations. (R. Y. Stainer, J. L. Ingraham, M. L. Wheelis, P. R. Painter, The Microbial World, 5th ed., © 1986. Reprinted and electronically reproduced by permission of Pearson Education, Inc., New York, NY.)

Figure 5.10 summarizes common routes to some of these fermentation end products. Pyruvate is a key metabolite in these pathways. In most cases, pyruvate is formed through glycolysis. However, alternative pathways to form pyruvate exist. The most common of these is the Entner–Doudoroff pathway (see Figure 5.11). This pathway is important in the fermentation of glucose by the bacterium Zymomonas. The use of Zymomonas to convert glucose into ethanol is of potential commercial interest, because the use of the Entner–Doudoroff pathway produces only 1 mol of ATP per mole of glucose. This low energy yield forces more glucose into ethanol and less into cell mass, yielding a higher specific rate of ethanol formation as compared to ethanol fermentations by yeasts.


Figure 5.10. Derivations of some major end products of the bacterial fermentations of sugars from pyruvic acid. The end products are shown in boldface type. (R. Y. Stainer, J. L. Ingraham, M. L. Wheelis, P. R. Painter, The Microbial World, 5th ed., © 1986. Reprinted and electronically reproduced by permission of Pearson Education, Inc., New York, NY.)


Figure 5.11. Entner–Doudoroff pathway. (R. Y. Stainer, J. L. Ingraham, M. L. Wheelis, P. R. Painter, The Microbial World, 5th ed., © 1986. Reprinted and electronically reproduced by permission of Pearson Education, Inc., New York, NY.)

No one organism alone makes all the products indicated in Figure 5.10. Different organisms contain different combinations of pathways. Thus, it is important to screen a wide variety of organisms to select one that maximizes the yield of a desired product while minimizing the formation of by-products.

5.11. Overview of Autotrophic Metabolism

So far we have been concerned primarily with heterotrophic growth (e.g., organic molecules serve as carbon–energy sources). However, autotrophs obtain nearly all their carbon from CO2. Most autotrophs (either photoautotrophs or chemoautotrophs) fix or capture CO2 by a reaction catalyzed by the enzyme ribulose bisphosphate carboxylase, which converts ribulose-1,5-diphosphate plus CO2 and H2O into two molecules of glyceric acid-3-phosphate. This is the key step in the Calvin cycle (or Calvin–Benson cycle). This cycle is summarized in Figure 5.12 and provides the building blocks for autotrophic growth.


Figure 5.12. Schematic representation of the Calvin–Benson cycle, illustrating its three phases: CO2 fixation, reduction of fixed CO2, and regeneration of the CO2 acceptor. (R. Y. Stainer, J. L. Ingraham, M. L. Wheelis, P. R. Painter, The Microbial World, 5th ed., © 1986. Reprinted and electronically reproduced by permission of Pearson Education, Inc., New York, NY.)

Energy for autotrophic growth can be supplied by light (photoautotroph) or chemicals (chemoautotroph). Here we consider the special case of photoautotrophic growth.

Photosynthesis takes place in two phases. The overall reaction follows:


The first phase of photosynthesis is known as the light phase. Light energy is captured and converted into biochemical energy in the form of ATP and reducing agents, such as NADPH. In this process, hydrogen atoms are removed from water molecules and are used to reduce NADP, leaving behind molecular oxygen. Simultaneously, ADP is phosphorylated to ATP. Following is the light-phase reaction of photosynthesis:


In the second phase, the energy-rich products of the first phase, NADPH and ATP, are used as the sources of energy to reduce the CO2 to yield glucose (see Figure 5.12). Simultaneously, NADPH is reoxidized to NADP+, and the ATP is converted into ADP and phosphate. This dark phase is described by the following reaction:


Both procaryotic and eucaryotic cells can fix CO2 by photosynthesis. In procaryotes (e.g., cyanobacteria), photosynthesis takes place in stacked membranes, whereas in eucaryotes an organelle called the chloroplast conducts photosynthesis. Both systems contain chlorophyll to absorb light. Light absorption by chlorophyll molecules results in an electronic excitation. The excited chlorophyll molecule returns to the normal state by emitting light quanta in a process known as fluorescence. The excited chlorophyll donates an electron to a sequence of enzymes, and ATP is produced as the electrons travel through the chain. This ATP-generation process is called photophosphorylation. Electron carriers in this process are ferredoxin and several cytochromes.

The light phase of photosynthesis consists of two photosystems. Photosystem I (PS I) can be excited by light of wavelength shorter than 700 nm and generates NADPH. Photosystem II (PS II) requires light of wavelength shorter than 680 nm and splits H2O into ImageO2 + 2H+. ATPs are formed as electrons flow from PS II to PS I.

5.12. Summary

Cellular metabolism is concerned with two primary functions: catabolism and anabolism. Catabolism involves the degradation of a substrate to more highly oxidized end products for the purpose of generating energy and reducing power. Anabolism is the biosynthesis of more complex compounds from simpler compounds, usually with the consumption of energy and reducing power. The key compound to store and release energy is adenosine triphosphate, or ATP. Reducing power is stored by nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH).

Three of the most important pathways in the cell are the Embden–Meyerhof–Parnas (EMP) pathway, or glycolysis, which converts glucose into pyruvate; the tricarboxylic acid (TCA) cycle, which can oxidize pyruvate through acetyl-CoA into CO2 and H2O; and the pentose–phosphate or hexose–monophosphate (HMP) pathway, which converts glucose-6-phosphate into a variety of carbon skeletons (C3, C4, C5, C6, and C7), with glyceraldehyde-3-phosphate as the end product. Although all three pathways can have catabolic and anabolic roles, the EMP pathway and TCA cycle are the primary means for energy generation, and HMP plays a key role in supplying carbon skeletons and reducing power for direct use in biosynthesis. In this chapter, we briefly considered the relationship of these pathways to amino acid, fatty acid, and polysaccharide biosynthesis. The conversion of pyruvate to glucose, necessary for polysaccharide biosynthesis when the carbon source does not have six carbons, is called glucogenesis.

Reducing power can be used to generate ATP through the electron transport chain. If oxygen is the final electron acceptor for this reducing power, the process is called aerobic respiration. If another electron acceptor is used in conjunction with the electron transport chain, then the process is called anaerobic respiration. Cells that obtain energy without using the electron transport chain use fermentation. Substrate-level phosphorylation supplies ATP. The end products of fermentative metabolism (e.g., ethanol, acetone–butanol, and lactic acid) are important commercially and are formed in response to the cell’s need to balance consumption and the production of reducing power.

Autotrophic organisms use CO2 as their carbon source and rely on the Calvin (or Calvin–Benson) cycle to incorporate (or fix) carbon from CO2 into cellular material. Energy is obtained either through light (photoautotroph) or through oxidation of inorganic chemicals (chemoautotroph). Figure 5.13 summarizes the major metabolic pathways and their interrelationship. Metabolic pathways can be altered with the use of modern biochemical tools such as genetic engineering. We discuss later in the book metabolic engineering, which combines a knowledge of the existing metabolic pathways with the modern tolls of genetic and epigenetic manipulations.


Figure 5.13. Interrelationship of major metabolic pathways in Escherichia coli. (Adapted from J. D. Watson, Molecular Biology of the Gene, 2d ed., W. A. Benjamin, Inc., New York, 1970.)

Suggestions for Further Reading

General Information on Metabolic Pathways

ALBERTS, B. D., D. BRAY, K. HOPKIN, A. D. JOHNSON, J. LEWIS, M. RAFF, K. ROBERTS, AND P. WALTER, Essential Cell Biology, 4th ed., Garland Science, New York, 2013.

BLACK, J. G., AND L. J. BLACK, Microbiology: Principles and Applications, 9th ed., Wiley, Hoboken, NJ, 2014.

JAIN, R. K., M. KAPUR, S. LABANA, B. LAL, P. M. SARMA, D. BHATTACHARYA, AND I. S. THAKUR, Microbial Diversity: Application of Microorganisms for Biodegradation of Xenobiotics, Curr. Sci. 89(1): 101–112, 2005.

MADIGAN, M. T., J. M. MARTINKO, K. S. BINDER, D. H. BUCKLEY, AND D. A. STAHL, Brock Biology of Microorganisms, 14th ed., Pearson Education, New York, 2015.

Specific Information on Interaction of Metabolism and Product Formation

CRUEGER, W., AND A. CRUEGER, Biotechnology. A Textbook of Industrial Microbiology (T. D. Brock, ed., English edition), 2d ed., Sinauer Associates, Sunderland, MA, 1990.

WAITES, M. J., N. L. MORGAN, J. S. ROCKEY, AND G. HIGTON, Industrial Microbiology: An Introduction, Blackwell Science, Malden, MA, 2001.


5.1. Cite the ATP-consuming and ATP-generating steps in glycolysis.

5.2. Briefly specify major functions of the TCA cycle.

5.3. What are the major control sites in glycolysis?

5.4. What is the Pasteur effect? Explain in terms of regulation of metabolic flow into a pathway.

5.5. How is glucose synthesized from pyruvate?

5.6. Explain the major functions of the dark and light reaction phases in photosynthesis.

5.7. What are the major differences between microbial and plant photosynthesis?

5.8. What is transamination? Provide an example.

5.9. Briefly explain the Crabtree effect.

5.10. What are the major steps in aerobic metabolism of hydrocarbons? What are the end products?

5.11. What is nitrogen fixation? Compare the aerobic and anaerobic nitrogen fixation mechanisms.

5.12. What are the major mechanisms for aerobic biodegradation of halogenated aromatics?

5.13. What are the major steps involved in degradation of nitroaromatics?

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