Mixed acid fermentation

E. coli use fermentation pathways as a final option for energy metabolism, as they produce very little energy in comparison to respiration.{{Cite journal|author=Sawers|first1=R. Gary|last2=Blokesch|first2=Melanie|author-link2=Melanie Blokesch|last3=Böck|first3=August|year=2004|title=Anaerobic formate and hydrogen metabolism|journal=EcoSal Plus|volume=1|issue=1|doi=10.1128/ecosalplus.3.5.4|pmid=26443350}}

Mixed acid fermentation in E. coli occurs in two stages. These stages are outlined by the biological database for E. coli, EcoCyc.{{Cite journal | author=Keseler, Ingrid M. | title=EcoCyc: a comprehensive database of Escherichia coli biology | journal=Nucleic Acids Research | volume= 39| issue=Database issue | year=2011 | doi=10.1093/nar/gkq1143|display-authors=etal | pmid=21097882 | pmc=3013716 | pages=D583–D590}}

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.

File:Reaction catalyzed by lactate dehydrogenase.png.]]

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

      pyruvate + NADH + H⁺ → lactate + NAD⁺

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.

File:E. coli Bacteria (7316101966).jpg, which includes E. coli]]

      acetyl-CoA + phosphate → acetyl-phosphate + CoA

      acetyl-phosphate + ADP → acetate + ATP

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).

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

      acetaldehyde + NADH + H⁺ → ethanol + NAD⁺

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.{{Cite journal |author1=Knappe, Joachim |author2=Gary Sawers |name-list-style=amp | title=A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli | journal=FEMS Microbiology Reviews | volume=6 |issue=4 | year=1990 | pages = 383–398 | doi=10.1111/j.1574-6968.1990.tb04108.x|pmid=2248795 | doi-access=free }}

      pyruvate + CoA → acetyl-CoA + formate

File:Bernsteinsäure2.svg]]

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.{{Cite journal | author=Kai, Yasushi, Hiroyoshi Matsumura, and Katsura Izui | title=Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms | journal=Archives of Biochemistry and Biophysics | volume= 414| issue=2 | year=2003 | pages = 170–179 | doi=10.1016/S0003-9861(03)00170-X | pmid=12781768}}

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.{{Cite journal | author=Thakker, Chandresh | title=Succinate production in Escherichia coli | journal=Biotechnology Journal | volume=7 | issue=2 | year=2012 | pages = 213–224 | doi=10.1002/biot.201100061| pmid=21932253 |display-authors=etal| pmc=3517001 }}

      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.

Vitamin K₂, also known as menaquinone, is very important for electron transport to fumarate in E. coli.{{Cite journal | author=Guest, JOHN R | title=Menaquinone biosynthesis: mutants of Escherichia coli K-12 requiring 2-succinylbenzoate | journal=Journal of Bacteriology | volume=130 | issue=3 | year=1977 | pages = 1038–1046| doi=10.1128/jb.130.3.1038-1046.1977 | pmid=324971 | pmc=235325 }}

Formate can be converted to hydrogen gas and carbon dioxide in E. coli. This reaction requires the enzyme [http://biocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=FHLMULTI-CPLX formate-hydrogen lyase]. It can be used to prevent the conditions inside the cell becoming too acidic.

      formate → H₂ and CO₂

File:Methylrot Probe methyl red test.jpg 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.{{Cite journal |author1=H. T. Clarke |author2=W. R. Kirner | title=Methyl Red | journal=Org. Synth. | volume=2 | year=1922 | pages = 47 | doi=10.15227/orgsyn.002.0047}} 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.

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.

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.{{Cite journal |author1=van Hoek |author2=Milan JA |author3=Roeland MH Merks |name-list-style=amp | title=Redox balance is key to explaining full vs. partial switching to low-yield metabolism | journal=BMC Systems Biology | volume=6 |issue=1 | year=2012 | page = 22 | doi=10.1186/1752-0509-6-22|pmid=22443685 |pmc=3384451 |doi-access=free }}

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.{{Cite journal | author= Ingram, L. O. | title= Genetic engineering of ethanol production in Escherichia coli | journal=Applied and Environmental Microbiology | volume=53 | year=1987 | issue= 10 | pages = 2420–2425| doi= 10.1128/aem.53.10.2420-2425.1987 | pmid= 3322191 | pmc= 204123 | bibcode= 1987ApEnM..53.2420I |display-authors=etal}}{{Cite journal | author= Ohta, Kazuyoshi | title= Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II | journal=Applied and Environmental Microbiology | volume=57 | issue= 4 | year=1991 | pages = 893–900| doi= 10.1128/aem.57.4.893-900.1991 | pmid= 2059047 | pmc= 182819 | bibcode= 1991ApEnM..57..893O |display-authors=etal}} 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.{{Cite journal | author=Turner, Peter C. | title=Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes | journal=Journal of Industrial Microbiology & Biotechnology | volume=39 | issue=4 | year=2012 | pages = 629–639|display-authors=etal | doi=10.1007/s10295-011-1052-2| pmid=22075923 | s2cid=15100287 | doi-access=free }}

File:Polylactid sceletal.svg]]

File:Teebeutel Polylactid 2009.jpg

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.{{Cite journal | author=Causey, T. B. | title=Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: homoacetate production | journal=Proceedings of the National Academy of Sciences | volume= 100| issue=3 | year=2003 | pages = 825–832 | doi=10.1073/pnas.0337684100|display-authors=etal | pmid=12556564 | pmc=298686| bibcode=2003PNAS..100..825C | doi-access=free }}

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.{{Cite journal | author= Clark, David P | title= The fermentation pathways of Escherichia coli | journal=FEMS Microbiology Reviews | volume=5 | issue= 3 | year=1989 | pages = 223–234 | doi= 10.1111/j.1574-6968.1989.tb03398.x| pmid= 2698228 | doi-access= free }} Early experiments engineered the E. coli strain RR1 to produce either one of the two optical isomers of lactate.{{Cite journal | author=Chang, Dong-Eun | title=Homofermentative production of d-orl-lactate in metabolically engineered Escherichia coli RR1 | journal=Applied and Environmental Microbiology | volume=65 | issue=4 | year=1999 | pages = 1384–1389 | doi=10.1128/AEM.65.4.1384-1389.1999 | pmid=10103226 | pmc=91196 | bibcode=1999ApEnM..65.1384C |display-authors=etal}}

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.{{Cite journal | author= Zhou, S. | title= Fermentation of 10%(w/v) sugar to D (−)-lactate by engineered Escherichia coli B | journal=Biotechnology Letters | volume=27 | issue= 23–24 | year=2005 | pages = 1891–1896 | doi= 10.1007/s10529-005-3899-7 | pmid= 16328986 | s2cid= 43204090 |display-authors=etal}}

Increasing the yield of succinate from mixed acid fermentation was first done by overexpressing the enzyme PEP carboxylase.{{Cite journal | author=Millard, Cynthia Sanville | title= Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli | journal=Applied and Environmental Microbiology | volume=62 | issue= 5 | year=1996 | pages = 1808–1810| doi= 10.1128/aem.62.5.1808-1810.1996 | pmid= 8633880 | pmc= 167956 | bibcode= 1996ApEnM..62.1808M |display-authors=etal}} 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.{{Cite journal | author=Singh, Amarjeet | title=Manipulating redox and ATP balancing for improved production of succinate in E. coli | journal=Metabolic Engineering | volume=13 | issue=1 | year=2011 | pages = 76–81 | doi=10.1016/j.ymben.2010.10.006| pmid=21040799 |display-authors=etal}}

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

These pathways include:

• Ethanol fermentation
• Lactic acid fermentation
• Propionic acid fermentation
• Butanol fermentation
• Butanediol fermentation

• [http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=FERMENTATION-PWY Mixed acid fermentation]
• [http://ecolistudentportal.org/article_fermentation EcoCyc Summary of Fermentation]

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Category:Anaerobic digestion

Category:Fermentation