Mixed acid fermentation

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The mixed acid fermentation pathway in E. coli. End products are highlighted in blue. Mixed Acid Fermentation in E. coli.jpg
The mixed acid fermentation pathway in E. coli. End products are highlighted in blue.

In biochemistry, mixed acid fermentation is the metabolic process by which a six-carbon sugar (e.g. glucose, C6H12O6) is converted into a complex and variable mixture of acids. It is an anaerobic (non-oxygen-requiring) fermentation reaction that is common in bacteria. It is characteristic for members of the Enterobacteriaceae, a large family of Gram-negative bacteria that includes E. coli . [3]

Contents

The mixture of end products produced by mixed acid fermentation includes lactate, acetate, succinate, formate, ethanol and the gases H2 and CO2. The formation of these end products depends on the presence of certain key enzymes in the bacterium. The proportion in which they are formed varies between different bacterial species. [4] The mixed acid fermentation pathway differs from other fermentation pathways, which produce fewer end products in fixed amounts. The end products of mixed acid fermentation can have many useful applications in biotechnology and industry. For instance, ethanol is widely used as a biofuel. [5] Therefore, multiple bacterial strains have been metabolically engineered in the laboratory to increase the individual yields of certain end products. [2] This research has been carried out primarily in E. coli and is ongoing. Variations of mixed acid fermentation occur in a number of bacterial species, including bacterial pathogens such as Haemophilus influenzae where mostly acetate and succinate are produced and lactate can serve as a growth substrate. [6]

Mixed acid fermentation in E. coli

E. coli use fermentation pathways as a final option for energy metabolism, as they produce very little energy in comparison to respiration. [7] Mixed acid fermentation in E. coli occurs in two stages. These stages are outlined by the biological database for E. coli, EcoCyc. [1]

The first of these two stages is a glycolysis reaction. Under anaerobic conditions, a glycolysis reaction takes place where glucose is converted into pyruvate:

    glucose → 2 pyruvate

There is a net production of 2 ATP and 2 NADH molecules per molecule of glucose converted. ATP is generated by substrate-level phosphorylation. NADH is formed from the reduction of NAD.

In the second stage, pyruvate produced by glycolysis is converted to one or more end products via the following reactions. In each case, both of the NADH molecules generated by glycolysis are reoxidized to NAD+. Each alternative pathway requires a different key enzyme in E. coli. After the variable amounts of different end products are formed by these pathways, they are secreted from the cell. [1]

The conversion of pyruvate to lactate is catalysed by the enzyme lactate dehydrogenase. Reaction catalyzed by lactate dehydrogenase.png
The conversion of pyruvate to lactate is catalysed by the enzyme lactate dehydrogenase.

Lactate formation

Pyruvate produced by glycolysis is converted to lactate. This reaction is catalysed by the enzyme lactate dehydrogenase (LDHA). [1]

    pyruvate + NADH + H+ → lactate + NAD+

Acetate formation

Pyruvate is converted into acetyl-coenzyme A (acetyl-CoA) by the enzyme pyruvate dehydrogenase. This acetyl-CoA is then converted into acetate in E. coli, whilst producing ATP by substrate-level phosphorylation. Acetate formation requires two enzymes: phosphate acetyltransferase and acetate kinase. [1]

The mixed acid fermentation pathway is characteristic of the family Enterobacteriaceae, which includes E. coli E. coli Bacteria (7316101966).jpg
The mixed acid fermentation pathway is characteristic of the family Enterobacteriaceae, which includes E. coli

    acetyl-CoA + phosphate → acetyl-phosphate + CoA

    acetyl-phosphate + ADP → acetate + ATP

Ethanol formation

Ethanol is formed in E. coli by the reduction of acetyl coenzyme A using NADH. This two-step reaction requires the enzyme alcohol dehydrogenase (ADHE). [1]

    acetyl-CoA + NADH + H+ → acetaldehyde + NAD+ + CoA

    acetaldehyde + NADH + H+ → ethanol + NAD+

Formate formation

Formate is produced by the cleavage of pyruvate. This reaction is catalysed by the enzyme pyruvate-formate lyase (PFL), which plays an important role in regulating anaerobic fermentation in E. coli. [8]

    pyruvate + CoA → acetyl-CoA + formate

Succinate formation

Skeletal structure of succinate Bernsteinsaure2.svg
Skeletal structure of succinate

Succinate is formed in E. coli in several steps.

Phosphoenolpyruvate (PEP), a glycolysis pathway intermediate, is carboxylated by the enzyme PEP carboxylase to form oxaloacetate. [9] This is followed by the conversion of oxaloacetate to malate by the enzyme malate dehydrogenase. Fumarate hydratase then catalyses the dehydration of malate to produce fumarate. [10]

    phosphoenolpyruvate + HCO3 → oxaloacetate + phosphate

    oxaloacetate + NADH + H+ → malate + NAD+

    malate → fumarate + H2O

The final reaction in the formation of succinate is the reduction of fumarate. It is catalysed by the enzyme fumarate reductase.

    fumarate + NADH + H+ → succinate + NAD+

This reduction is an anaerobic respiration reaction in E. coli, as it uses electrons associated with NADH dehydrogenase and the electron transport chain. ATP is generated by using an electrochemical gradient and ATP synthase. This is the only case in the mixed acid fermentation pathway where ATP is not produced via substrate-level phosphorylation. [1] [2]

Vitamin K2, also known as menaquinone, is very important for electron transport to fumarate in E. coli. [11]

Hydrogen and carbon dioxide formation

Formate can be converted to hydrogen gas and carbon dioxide in E. coli. This reaction requires the enzyme formate-hydrogen lyase. It can be used to prevent the conditions inside the cell becoming too acidic. [1]

    formate → H2 and CO2

Methyl red test

Methyl red test: The test tube on the left shows a positive result as acidic end products are formed by mixed acid fermentation in E. coli. The test tube on the right shows a negative result as no acidic products are formed by fermentation. Methylrot Probe methyl red test.jpg
Methyl red test: The test tube on the left shows a positive result as acidic end products are formed by mixed acid fermentation in E. coli. The test tube on the right shows a negative result as no acidic products are formed by fermentation.

The methyl red (MR) test can detect whether the mixed acid fermentation pathway occurs in microbes when given glucose. A pH indicator is used that turns the test solution red if the pH drops below 4.4. [12] If the fermentation pathway has taken place, the mixture of acids it has produced will make the solution very acidic and cause a red colour change.

The methyl red test belongs to a group known as the IMViC tests.

Metabolic engineering

Multiple bacterial strains have been metabolically engineered to increase the individual yields of end products formed by mixed acid fermentation. For instance, strains for the increased production of ethanol, lactate, succinate and acetate have been developed due to the usefulness of these products in biotechnology. [2] The major limiting factor for this engineering is the need to maintain a redox balance in the mixture of acids produced by the fermentation pathway. [13]

For ethanol production

Ethanol is the most commonly used biofuel and can be produced on large scale via fermentation. The maximum theoretical yield for the production of ethanol was achieved around 20 years. [14] [15] A plasmid that carried the pyruvate decarboxylase and alcohol dehydrogenase genes from the bacteria Z. mobilis was used by scientists. This was inserted into E. coli and resulted in an increased yield of ethanol. The genome of this E. coli strain, KO11, has more recently been sequenced and mapped. [16]

The skeletal formula of polylactic acid Polylactid sceletal.svg
The skeletal formula of polylactic acid
Teabags made from polylactic acid (PLA) Teebeutel Polylactid 2009.jpg
Teabags made from polylactic acid (PLA)

For acetate production

The E. coli strain W3110 was genetically engineered to generate 2 moles of acetate for every 1 mole of glucose that undergoes fermentation. This is known as a homoacetate pathway. [17]

For lactate production

Lactate can be used to produce a bioplastic called polylactic acid (PLA). The properties of PLA depend on the ratio of the two optical isomers of lactate (D-lactate and L-lactate). D-lactate is produced by mixed acid fermentation in E. coli. [18] Early experiments engineered the E. coli strain RR1 to produce either one of the two optical isomers of lactate. [19]

Later experiments modified the E. coli strain KO11, originally developed to enhance ethanol production. Scientists were able to increase the yield of D-lactate from fermentation by performing several deletions. [20]

For succinate production

Increasing the yield of succinate from mixed acid fermentation was first done by overexpressing the enzyme PEP carboxylase. [21] This produced a succinate yield that was approximately 3 times greater than normal. Several experiments using a similar approach have followed.

Alternative approaches have altered the redox and ATP balance to optimize the succinate yield. [22]

There are a number of other fermentation pathways that occur in microbes. [4] All these pathways begin by converting pyruvate, but their end products and the key enzymes they require are different. These pathways include:

Related Research Articles

<span class="mw-page-title-main">Citric acid cycle</span> Interconnected biochemical reactions releasing energy

The citric acid cycle—also known as the Krebs cycle, Szent-Györgyi-Krebs cycle or the TCA cycle (tricarboxylic acid cycle)—is a series of biochemical reactions to release the energy stored in nutrients through the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins. The chemical energy released is available under the form of ATP. The Krebs cycle is used by organisms that respire (as opposed to organisms that ferment) to generate energy, either by anaerobic respiration or aerobic respiration. In addition, the cycle provides precursors of certain amino acids, as well as the reducing agent NADH, that are used in numerous other reactions. Its central importance to many biochemical pathways suggests that it was one of the earliest components of metabolism. Even though it is branded as a 'cycle', it is not necessary for metabolites to follow only one specific route; at least three alternative segments of the citric acid cycle have been recognized.

<span class="mw-page-title-main">Glycolysis</span> Series of interconnected biochemical reactions

Glycolysis is the metabolic pathway that converts glucose into pyruvate and, in most organisms, occurs in the liquid part of cells. The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH). Glycolysis is a sequence of ten reactions catalyzed by enzymes.

Pyruvic acid (IUPAC name: 2-oxopropanoic acid, also called acetoic acid) (CH3COCOOH) is the simplest of the alpha-keto acids, with a carboxylic acid and a ketone functional group. Pyruvate, the conjugate base, CH3COCOO−, is an intermediate in several metabolic pathways throughout the cell.

<span class="mw-page-title-main">Cellular respiration</span> Process to convert glucose to ATP in cells

Cellular respiration is the process by which biological fuels are oxidized in the presence of an inorganic electron acceptor, such as oxygen, to drive the bulk production of adenosine triphosphate (ATP), which contains energy. Cellular respiration may be described as a set of metabolic reactions and processes that take place in the cells of organisms to convert chemical energy from nutrients into ATP, and then release waste products.

<span class="mw-page-title-main">Acetyl-CoA</span> Chemical compound

Acetyl-CoA is a molecule that participates in many biochemical reactions in protein, carbohydrate and lipid metabolism. Its main function is to deliver the acetyl group to the citric acid cycle to be oxidized for energy production.

Digestion is the breakdown of carbohydrates to yield an energy-rich compound called ATP. The production of ATP is achieved through the oxidation of glucose molecules. In oxidation, the electrons are stripped from a glucose molecule to reduce NAD+ and FAD. NAD+ and FAD possess a high energy potential to drive the production of ATP in the electron transport chain. ATP production occurs in the mitochondria of the cell. There are two methods of producing ATP: aerobic and anaerobic. In aerobic respiration, oxygen is required. Using oxygen increases ATP production from 4 ATP molecules to about 30 ATP molecules. In anaerobic respiration, oxygen is not required. When oxygen is absent, the generation of ATP continues through fermentation. There are two types of fermentation: alcohol fermentation and lactic acid fermentation.

<span class="mw-page-title-main">Biological carbon fixation</span> Series of interconnected biochemical reactions

Biological carbon fixation or сarbon assimilation is the process by which inorganic carbon is converted to organic compounds by living organisms. The compounds are then used to store energy and as structure for other biomolecules. Carbon is primarily fixed through photosynthesis, but some organisms use a process called chemosynthesis in the absence of sunlight.

<span class="mw-page-title-main">Ethanol fermentation</span> Biological process that produces ethanol and carbon dioxide as by-products

Ethanol fermentation, also called alcoholic fermentation, is a biological process which converts sugars such as glucose, fructose, and sucrose into cellular energy, producing ethanol and carbon dioxide as by-products. Because yeasts perform this conversion in the absence of oxygen, alcoholic fermentation is considered an anaerobic process. It also takes place in some species of fish where it provides energy when oxygen is scarce.

<span class="mw-page-title-main">Oxaloacetic acid</span> Organic compound

Oxaloacetic acid (also known as oxalacetic acid or OAA) is a crystalline organic compound with the chemical formula HO2CC(O)CH2CO2H. Oxaloacetic acid, in the form of its conjugate base oxaloacetate, is a metabolic intermediate in many processes that occur in animals. It takes part in gluconeogenesis, the urea cycle, the glyoxylate cycle, amino acid synthesis, fatty acid synthesis and the citric acid cycle.

<span class="mw-page-title-main">Mitochondrial matrix</span> Space within the inner membrane of the mitochondrion

In the mitochondrion, the matrix is the space within the inner membrane. The word "matrix" stems from the fact that this space is viscous, compared to the relatively aqueous cytoplasm. The mitochondrial matrix contains the mitochondrial DNA, ribosomes, soluble enzymes, small organic molecules, nucleotide cofactors, and inorganic ions.[1] The enzymes in the matrix facilitate reactions responsible for the production of ATP, such as the citric acid cycle, oxidative phosphorylation, oxidation of pyruvate, and the beta oxidation of fatty acids.

The Pasteur effect describes how available oxygen inhibits ethanol fermentation, driving yeast to switch toward aerobic respiration for increased generation of the energy carrier adenosine triphosphate (ATP). More generally, in the medical literature, the Pasteur effect refers to how the cellular presence of oxygen causes in cells a decrease in the rate of glycolysis and also a suppression of lactate accumulation. The effect occurs in animal tissues, as well as in microorganisms belonging to the fungal kingdom.

Acetyl-CoA synthetase (ACS) or Acetate—CoA ligase is an enzyme involved in metabolism of acetate. It is in the ligase class of enzymes, meaning that it catalyzes the formation of a new chemical bond between two large molecules.

<span class="mw-page-title-main">Isocitrate lyase</span>

Isocitrate lyase, or ICL, is an enzyme in the glyoxylate cycle that catalyzes the cleavage of isocitrate to succinate and glyoxylate. Together with malate synthase, it bypasses the two decarboxylation steps of the tricarboxylic acid cycle and is used by bacteria, fungi, and plants.

The enzyme methylglyoxal synthase catalyzes the chemical reaction

<span class="mw-page-title-main">Lactate dehydrogenase</span> Class of enzymes

Lactate dehydrogenase (LDH or LD) is an enzyme found in nearly all living cells. LDH catalyzes the conversion of pyruvate to lactate and back, as it converts NAD+ to NADH and back. A dehydrogenase is an enzyme that transfers a hydride from one molecule to another.

Glutaminolysis (glutamine + -lysis) is a series of biochemical reactions by which the amino acid glutamine is lysed to glutamate, aspartate, CO2, pyruvate, lactate, alanine and citrate.

The Arc system is a two-component system found in some bacteria that regulates gene expression in faculatative anaerobes such as Escheria coli. Two-component system means that it has a sensor molecule and a response regulator. Arc is an abbreviation for Anoxic Redox Control system. Arc systems are instrumental in maintaining energy metabolism during transcription of bacteria. The ArcA response regulator looks at growth conditions and expresses genes to best suit the bacteria. The Arc B sensor kinase, which is a tripartite protein, is membrane bound and can autophosphorylate.

<span class="mw-page-title-main">Cofactor engineering</span> Modification of use and function of cofactors in an organisms metabolic pathways

Cofactor engineering, a subset of metabolic engineering, is defined as the manipulation of the use of cofactors in an organism’s metabolic pathways. In cofactor engineering, the concentrations of cofactors are changed in order to maximize or minimize metabolic fluxes. This type of engineering can be used to optimize the production of a metabolite product or to increase the efficiency of a metabolic network. The use of engineering single celled organisms to create lucrative chemicals from cheap raw materials is growing, and cofactor engineering can play a crucial role in maximizing production. The field has gained more popularity in the past decade and has several practical applications in chemical manufacturing, bioengineering and pharmaceutical industries.

Pseudohypoxia refers to a condition that mimics hypoxia, by having sufficient oxygen yet impaired mitochondrial respiration due to a deficiency of necessary co-enzymes, such as NAD+ and TPP. The increased cytosolic ratio of free NADH/NAD+ in cells (more NADH than NAD+) can be caused by diabetic hyperglycemia and by excessive alcohol consumption. Low levels of TPP results from thiamine deficiency.

Aerobic fermentation or aerobic glycolysis is a metabolic process by which cells metabolize sugars via fermentation in the presence of oxygen and occurs through the repression of normal respiratory metabolism. Preference of aerobic fermentation over aerobic respiration is referred to as the Crabtree effect in yeast, and is part of the Warburg effect in tumor cells. While aerobic fermentation does not produce adenosine triphosphate (ATP) in high yield, it allows proliferating cells to convert nutrients such as glucose and glutamine more efficiently into biomass by avoiding unnecessary catabolic oxidation of such nutrients into carbon dioxide, preserving carbon-carbon bonds and promoting anabolism.

References

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  2. 1 2 3 4 Förster, Andreas H. & Johannes Gescher (2014). "Metabolic engineering of Escherichia coli for production of mixed-acid fermentation end products". Frontiers in Bioengineering and Biotechnology . 2: 506–508. doi: 10.3389/fbioe.2014.00016 . PMC   4126452 . PMID   25152889.
  3. ↑ M.Magidan & J. Martinko (2006). "Brock's Biology of Microorganisms, NJ, Pearson Prentice Hall". 11: 352.{{cite journal}}: Cite journal requires |journal= (help)
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  5. ↑ Farrell, Alexander E.; et al. (2006). "Ethanol can contribute to energy and environmental goals". Science . 311 (5760): 506–508. Bibcode:2006Sci...311..506F. doi:10.1126/science.1121416. PMID   16439656. S2CID   16061891.
  6. ↑ Hosmer, Jennifer; Nasreen, Marufa; Dhouib, Rabeb; Essilfie, Ama-Tawiah; Schirra, Horst Joachim; Henningham, Anna; Fantino, Emmanuelle; Sly, Peter; McEwan, Alastair G.; Kappler, Ulrike (2022-01-27). "Access to highly specialized growth substrates and production of epithelial immunomodulatory metabolites determine survival of Haemophilus influenzae in human airway epithelial cells". PLOS Pathogens. 18 (1): e1010209. doi: 10.1371/journal.ppat.1010209 . ISSN   1553-7374. PMC   8794153 . PMID   35085362.
  7. ↑ Sawers, R. Gary; Blokesch, Melanie; Böck, August (2004). "Anaerobic formate and hydrogen metabolism". EcoSal Plus . 1 (1). doi:10.1128/ecosalplus.3.5.4. PMID   26443350.
  8. ↑ Knappe, Joachim & Gary Sawers (1990). "A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli". FEMS Microbiology Reviews . 6 (4): 383–398. doi: 10.1111/j.1574-6968.1990.tb04108.x . PMID   2248795.
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  12. ↑ H. T. Clarke; W. R. Kirner (1922). "Methyl Red". Org. Synth. 2: 47. doi:10.15227/orgsyn.002.0047.
  13. ↑ van Hoek; Milan JA & Roeland MH Merks (2012). "Redox balance is key to explaining full vs. partial switching to low-yield metabolism". BMC Systems Biology . 6 (1): 22. doi: 10.1186/1752-0509-6-22 . PMC   3384451 . PMID   22443685.
  14. ↑ Ingram, L. O.; et al. (1987). "Genetic engineering of ethanol production in Escherichia coli". Applied and Environmental Microbiology . 53 (10): 2420–2425. Bibcode:1987ApEnM..53.2420I. doi:10.1128/aem.53.10.2420-2425.1987. PMC   204123 . PMID   3322191.
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