1. Anaerobe prokaryotes oxidize NADH and other electron carriers → anaerobic respiration (nitrate, sulfate or fumaratee NADH (other electron carriers) is oxidize by metabolites produced by the pathway → occurs in cytosol → ATP is produced via substrate level phosphorylation Fermentationoccursonlyamong prokaryotes. In yeast O 2 is necessary, unless the medium is supplemented with sterols and unsaturated fatty acids. Certain animal cells are capable of fermentation, such as muscle cells & human red blood cells 1 Fermentations are named after the major end products they generate. Example : yeasts carry out an as the electron acceptor) or fermentation ethanol fermentation; muscle cells Fermentation: is a pathway in which carry out a lactic acid fermentation. Carbohydratesfermentation can be grouped into six classes : lactic, ethanol, butyric acid, mixed acid, propionic and homoacetic

2. 2 Electron Sinks The fermentation pathway must produce the electron acceptors The electron acceptors are called as “electron sinks”, because they excrete the reduced products into the medium → large quantities of reduced organic compounds (alcohol, organic acids and solvents, frequently hydrogen gas) Propionate fermentation using the acrylate pathway Clostridium propionicum : 3 lactates → 2 propionates + 1 acetate + 1 CO 2 + 1ATP ATP is derived via substrate-level phosphorylation, during the conversion of acetyl-P to acetate (acetate kinase). One acetate is made per 3 lactates used → 1/3 ATP /pathway Important! In glycolisis, acetate is associated with the production of 2 ATPs Transfer of Coenzyme A from one organic acid compound to the others (CoA transferase)

3. (2H) 1 (2H) CO 2 2 CoASH 3 O CH 3 C  SCoA acetyl- CoA O CH 3 C O  P acetyl-P ADP 4 ATP O C  SCoA lactyl-CoA CH 3 CHOH CoASH 6 O C  SCoA acrylyl-CoA H 2 O CH 2 CH 2(H) CH 3 CHOH COOH lactate 5 O CH 3 C COOH pyruvate CH 3 COOH acetate CH 3 CH 2 O C  SCoA lactyl-CoA COOH propionate 7 X2 Enzymes : (1)lactatedehydrogenase,(2)pyruvate-ferredoxin oxidoreductase, (3) phosphotransacetylase, (4) acetate kinase, (5) CoA transferase, (7) dehydrogenase. Reaction 6 is not sufficiently characterized. 3 Succinate-Propionate Pathway Propionibacterium : Gram +, anaerobic, nonmotile, non-sporulating, pleomorphic rod, normal flora in the rumen of herbivores, human skin and dairy products, used in the making of swiss cheese Yields more ATP than the acrylate pathway per mole propionate formed The Transcarboxylase Reaction Spares an ATP Pyruvate carboxylation reaction uses pyruvate carboxylase, CO2 and ATP Propionibacterium:methyl-malonyl-CoA pyruvat transcarboxylase → transfers a carboxyl group from methylmalonyl-CoA to pyruvate without using ATP

4. fumarate 4 O 6(H) 1 O (2H) CO 2 2 CoASH 3 CH 3 C  SCoA acetyl- CoA O CH 3 C O  P acetyl-P ADP 4 4(H) 3CH 3 CHOH COOH lactate 3CH 3 C COOH pyruvate 5 COOH C O CH 2 oxaloacetate 2COOH 6 COOH CH 3 CH 2 propionate 2COOH CH 2 C HOH COOH malate 7 2COOH C H C COOH 2H 2 O H COOH 8 ADP+Pi ATP 2COOH C H 2 succinate 9 C H 2 C  SCoA O succinyl-CoA 2CH 3 CH 2 C  SCoA O 2COOH C H CH 3 C  SCoA propionyl-CoA mehtyl malonyl-CoA O 10 COOH 4(H) 2(CoAS) ATP CH 3 COOH acetat e enzyme: (1) lactate dehydrogenase (a flavoprotein), (2) pyruvate dehydrogenase (enzyme NAD + ) (3) phosphotransacetylase, (4) acetate kinase, (5) mehtylmalonyl-CoA- pyruvate transcarboxylase, (6) malate dehydrogenase, (7) Fumarase, (8) fumarate reductase, (9)CoA transferase (10) mehtylmalonyl-CoA- racemase. Propionibacteria can produce succinate and propionate as an end-product of fermentation when growing on glucose that enter the glycolytic pathway → having an enzyme to carboxylate C3 to C4. PEP is carboxylated to oxaloacetat and then reduced to succinate. During carboxylation, PPi is formed. PPi is used to phosphorylate Fru-6P to andtoserineFru 1,6 bisP phosphoserine

5. 5 The pathway from PEP to succinate: 1. fumarate is an electron sinks enabling NADH to be oxidized 2. fumarate reductase is a coupling site 3.succinate can be converted to succinyl-CoA, which is required forthebiosynthesisof tetrapyrroles,diaminopimelic acid, lysin, and methionine andButanediolMixed Acid Fermentation The enteric bacteria are facultative anaerobes → the changes according to the anaerobic environment: 1.The terminal reductases replace the oxidases in the electron transport chain 2.TCA becomes a reductive pathway, succinate dehydrogenase is replaced by fumarate reductase 3.Pyruvate-formate lyase is substituted for pyruvate dehydrogenase 4.Carry out a mixed acid or butanediol fermentations

6. 6 Themixedacidandbutanediol fermentations are similar in that both produce a mixture of organic acids, CO 2 and NADH. Butanediolfermenters(Serratia, Erwinia andEnterobacter) produce large amounts of2,3 butanediol, acetoin, CO 2 and ethanol Mixed acid fermentation: Escherichia, Salmonella, Shigella Mixed acid fermentation The reactants : PEP + CO 2 or pyruvate The products: succinate, lactate, acetate, ethanol, formate, CO 2, H 2. Formate is oxidized to CO 2 and H 2 by the enzyme system formate-hidrogen liase which contains of formate dehydrogenase and hydrogenase. Formate dehydrogenase oxidizes formate to CO 2 and reduces hydrogenase that will transfer electron to 2 moles of proton to form H 2. Shigella and Erwinia do not have formate- hydrogen lyase → no gas

7. OAA 11 2(H) malate H2OH2O 12 2H 1/2 glucose ATP 1 ADP ADP + Pi ATP pyruvate PEP ADP 2 ATP 3 CoASH lactate 4 Pi CO 2 10 succinate fumarate 13 2(H) acetyl-P ADP ATP 9 acetate acetyl-CoA + Pi 2(H) 8 6 formate CoASH acetaldehyde 7 ethanol H2H2 CO 2 5 (1) glycolytic enzyme, (2) pyruvate kinase, (3) pyruvate-formate liase, (4) lactate dehydrogenase, (5) formate-hidrogen lyase, (6) acetaldehyde dehydrogenase, (7) alcohol dehydrogenase, (8) phosphotransacetylase, (9) acetate kinase, (10) PEP carboxylase, (11) malate dehydrogenase, (12) fumarase, (13) fumarate reductase. 7 How the pathways are balanced by gene expression? ☼ Fnr (Fumarate nitrate reduction) protein plays an overarching role as a global transcriptional regulator to ensure effective use of pathways for fermentation and/or anaerobic respiration ☼ Except for the presence of a cysteine-rich N-terminal extension, Fnr is highly similar to Crp (cyclic enzymes : AMP receptor Protein)

8. 8 ☼ Fnr box contain a 5 bp inverted repeat TTGAT ….. ATCAA, whereas Crp box: TGTGA …… TCACA ☼ Fnr sense the redox condition of the environment through the iron atom bound to a cluster of four cysteine residues that are highly conserved among all bacterial Fnr protein. ☼ When the iron is in the Fe 2+ state, the protein is functionally active as a transcriptional regulator; when the iron oxidized to the Fe 3+ state, the protein is altered in conformation and becomes nonfunctional. D-Lactate formation  The activity of NAD + -linked D- lactate oxidoreductase is elevated during fermentative growth at low pH. Cleavage of Pyruvate Pyruvat-formate lyase cleaves pyruvat to acetyl CoA and formate and encoded by the plf operon. Transcription of this gen is activated by Fnr in concert with ArcA Expression of pfl under anoxic condition can be increased further by exogenous pyruvat

9. Ethanol production Ethanol is formed by two consecutive reductions at the expense of two NADH molecules The enzyme called ethanol dehydrogenase or alcohol dehydrogenase encoded by adhE. Trancription is regulated by two different mechanisms: 1. Enhancement of transcription is associated with a high NADH/NAD + ratio 2. repression of transcription is exerted by the NarX/NarL system 9 Under anoxic condition, acetyl CoA will converts to acetyl phosphate with the help of phosphotransacetylase. The phosphoryl group can generate ATP from ADP with the help of acetyl kinase. Acetat exits the cell, probably via a H + - symport system

10. Regulation of Formate  Format-hydrogen lyase comprising formate dehydrogenase H (encoded by fdhF) and hydrogenase 3 (encoded by hycE)  They convert formate to CO2 and H2, or formate will be excreted in a manner similar to that of acetate  An interesting feature of formate dehydrogenase H is the presence of a selenocystein at position 140 from the N-terminal end  The catalytic activity of Format- hydrogen lyase requires a molybdenum cofactor, Ni and Fe 10  The activity level of the lyase are strongly diminished during aerobic growth  The fdhF and hyc and hyp operon are activated during anaerob growth by the FhlA regulatory protein  The FhlA protein shows partial homology to the response regulators of the two-component system and respond to formate as a signal

11. 2H 2 Fd ox Fd red 4H butiryl-CoA Butyrate And Butanol-Aceton Fermentation Occurs in Clostridium acetobutylicum. Butyrate, acetate, CO 2 and H 2 are produced in log phase (acidogenic phase). Organic acids are converted to butanol, aceton and ethanol when entering the stationary phase (solventogenic phase). via NADH:ferredoxin oxidoreductase enabling the bacteria to produce more acetate → larger amount of ATP rather than reduce acetyl CoA to butyrate, yet energetically unfavorable and inhibited by the accumulation of H 2 glucose ATP ADP glucose-6-P pentose ATP ADP pentose-P 2NADH + 2H + The ability to move the electron to hydrogen ATP ADP Fruktosa-6-P ATP ADP 2-phosphogliceraldehyde 4ADP + 2Pi NADH + H + 4ATP 2NAD + NAD + H2H2 16 + 2 pyruvate 17 1 2 acetyl-CoA 2 CoASH Asetoacetyl-CoA NADH + 2H + 3 NAD + hidroxybutiryl-CoA 4 H 2 O Crotonil-CoA 5 Butiraldehyde NADPH + H + 15 NADP + NADH + H + 14 Butanol 11 Pi NAD + CoASH 6 ADP butiryl-P ATP 7 butyrate CO 2 aceton 8 ATP acetate Acetoacetateik 11 CoASH acetaldehyde NADH + 2H + 13 NAD + ethanol 2CO 2 ADP acetyl-P 9 CoASH 10 Pi

12. 12 Fermentation without Substrate-Level Phosphorylation ☺ The fermentation of certain compound yields insufficient energy to synthesize ATP by substrate-level phosphorylation. ☺ In these cases, catabolism of compound is linked to ion pumps that establish a proton motive force or sodium motive force across the cytoplasmic membrane ☺ Propionigenum modestum was first isolated in anoxic enrichment cultures lacking alternative electron acceptors and feed succinate as electron donor ♣ P. modestum catabolize succinate under strictly anoxic condition Succinate 2- + H 2 O → propionate - + HCO3 - ∆G 0 ’ = -20,5 KJ ♣ Energy conservation is linked to the decarboxylation of succinate by membrane bound decarboxylase yielding propionate ♣ This reaction releases sufficient free energy to drive the export of a sodium ion across the cytoplasmic membrane

13. 13 ♣ Oxalobacter formigenes catabolizes oxalate and produces formate Oxalate 2- + H 2 O → formate - + HCO 3- ∆ G o ’ = - 26,7 KJ ♣ The decarboxylation of oxalate is exergonic and forms formate, which is excreted from the cell