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
Fermentation occurs only among
prokaryotes. In yeast O2 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 anas the electron acceptor) or fermentation
ethanol fermentation; muscle cellsFermentation: is a pathway in which
carry out a lactic acid fermentation.
Carbohydrates fermentation can be
grouped into six classes : lactic,
ethanol, butyric acid, mixed acid,
propionic and homoacetic
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 theacrylate pathway
Clostridium propionicum :
3 lactates → 2 propionates + 1 acetate +
1 CO2 + 1ATP
ATP is derived via substrate-levelphosphorylation, during the conversionof acetyl-P to acetate (acetate kinase).One acetate is made per 3 lactates used→ 1/3 ATP /pathway
Important!
In glycolisis, acetate is associated withthe production of 2 ATPs
Transfer of Coenzyme A from oneorganic acid compound to the others(CoA transferase)
(2H)
1
(2H) CO2
2
CoASH
3 CoASH
O
CH3 C ∼SCoAacetyl-CoA
O
CH3 C O ∼Pacetyl-P
ADP4
ATP
O
C ∼SCoAlactyl-CoA
CH3 CHOH
CoASH
6
O
C ∼SCoA
acrylyl-CoA
H2O
CH2 CH
2(H)
CH3 CHOH COOHlactate
5
O
CH3 C COOHpyruvate
CH3 COOH
acetateCH3 CH2
CH3 CH2
O
C ∼SCoA
lactyl-CoA
COOH
propionate
7X2
Enzymes :
(1) lactate dehydrogenase, (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
fumarate
4
O6(H)
1
O (2H) CO2
2
CoASH
3
CH3 C ∼SCoAacetyl-CoA
O
CH3 C O ∼Pacetyl-P
ADP4
4(H)
3CH3 CHOH COOHlactate
3CH3 C COOH
pyruvate
5
COOH
C O
CH2 oxaloacetate
2COOH
6
COOHCH3 CH2
propionate
2COOH
CH2
C HOH
COOH malate
7
2COOH
C H
C
COOH
2H2O
H
COOH
8ADP+Pi
ATP
2COOH
C H2
C H2
succinate9C H2
C H2
C ∼SCoA
Osuccinyl-CoA
2CH3
CH2
C ∼SCoA
O
2COOH
C H CH3
C ∼SCoA
propionyl-CoA
mehtyl malonyl-CoA
O
10
COOH
4(H)
2(CoAS)
ATP
CH3 COOH
acetate
enzyme:
(1) lactate dehydrogenase(a flavoprotein),
(2) pyruvate dehydrogenase(enzyme NAD+ )
(3) phosphotransacetylase,
(4) acetate kinase,
(5) mehtylmalonyl-CoA-pyruvatetranscarboxylase,
(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
and toserineFru 1,6 bisP
phosphoserine
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
for the biosynthesis of
tetrapyrroles, diaminopimelic
acid, lysin, and methionine
and ButanediolMixed AcidFermentation
The enteric bacteria are facultativeanaerobes → the changes according tothe anaerobic environment:
1.The terminal reductases replace theoxidases in the electron transport chain
2.TCA becomes a reductive pathway,succinate dehydrogenase is replaced byfumarate reductase
3.Pyruvate-formate lyase is substituted forpyruvate dehydrogenase
4.Carry out a mixed acid or butanediolfermentations
6
The mixed acid and butanediol
fermentations are similar in that both
produce a mixture of organic acids,
CO2 and NADH.
Butanediol fermenters (Serratia,
Erwinia and Enterobacter) produce
large amounts of 2,3 butanediol,
acetoin, CO2 and ethanol
Mixed acid fermentation: Escherichia,
Salmonella, Shigella
Mixed acid fermentation
The reactants : PEP + CO2 or pyruvate
The products: succinate, lactate, acetate,
ethanol, formate, CO2, H2.
Formate is oxidized to CO2 and H2 by the
enzyme system formate-hidrogen liase
which contains of formate dehydrogenase
and hydrogenase. Formate dehydrogenase
oxidizes formate to CO2 and reduces
hydrogenase that will transfer electron to 2
moles of proton to form H2.
Shigella and Erwinia do not have formate-
hydrogen lyase → no gas
OAA
11 2(H)
malate
H2O12
2H
1/2 glucoseATP
1ADP
ADP + Pi
ATP
pyruvate
PEP
ADP2
ATP
3 CoASH
lactate4
Pi
CO2
10
succinate
fumarate
13 2(H)
acetyl-P
ADP
ATP
9
acetate
acetyl-CoA +
Pi 2(H)8 6
formate
CoASH
acetaldehyde
7
ethanol
H2
CO25
(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 (cyclicenzymes :
AMP receptor Protein)
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 Fe2+ state,
the protein is functionally active as a
transcriptional regulator; when the
iron oxidized to the Fe3+ 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
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
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
2H2Fdox
Fdred 4H
butiryl-CoA
Butyrate And Butanol-Aceton Fermentation
Occurs in Clostridium acetobutylicum.
Butyrate, acetate, CO2 and H2 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 H2
glucoseATP
ADP
glucose-6-Ppentose
ATP
ADP
pentose-P
2NADH + 2H+
The ability to move the electron to hydrogen
ATP
ADP
Fruktosa-6-PATP
ADP
2-phosphogliceraldehyde
4ADP + 2PiNADH + H+
4ATP2NAD+ NAD+
H2
16
+
2 pyruvate17
1
2 acetyl-CoA
2CoASH
Asetoacetyl-CoA
NADH + 2H+
3NAD+
hidroxybutiryl-CoA
4H2O
Crotonil-CoA
5
ButiraldehydeNADPH + H+
15NADP+
NADH + H+
14
Butanol
11
Pi NAD+
CoASH
6ADP
butiryl-P
ATP
7butyrate
CO2aceton
8
ATP
acetate
Acetoacetateik
11
CoASH
acetaldehydeNADH + 2H+
13NAD+
ethanol
2CO2
ADP
acetyl-P9
CoASH
10
Pi
12
Fermentation without Substrate-LevelPhosphorylation
☺ The fermentation of certaincompound yields insufficient energyto synthesize ATP by substrate-levelphosphorylation.
☺ In these cases, catabolism ofcompound is linked to ion pumpsthat establish a proton motive forceor sodium motive force across thecytoplasmic membrane
☺Propionigenum modestum was firstisolated in anoxic enrichmentcultures lacking alternative electronacceptors and feed succinate aselectron donor
♣ P. modestum catabolize succinate
under strictly anoxic condition
Succinate 2- + H2O → propionate -
+ HCO3- ∆G0’ = -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
♣ Oxalobacter formigenes catabolizes
oxalate and produces formate
Oxalate2- + H2O → formate -
+ HCO3- ∆ Go’ = - 26,7 KJ
♣ The decarboxylation of oxalate is
exergonic and forms formate, which
is excreted from the cell