TA918C (1s2024) Aula 3

Prévia do material em texto

Prof. Andreas K. Gombert
TA918C - Microbiologia 
e Fermentações
Aula 3 - 21/03/2024 
ASPECTOS METABÓLICOS RELEVANTES EM BIOPROCESSOS
© 2004 Pearson Education, Inc.
Opções metabólicas para a obtenção de 
energia (todos os seres vivos usam alguma destas formas)
Microrganismos 
industriais
Diversidade no metabolismo 
de microrganismos
   
 
photo litho autotroof
chemo organo heterotroof
vrije energie uit elektronendonor koolstofbron





 


Classificação dos diferentes tipos metabólicos
foto lito autotrófico
quimio organo heterotrófico
energia-livre a partir de doador de elétrons fonte de carbono 
A fonte de E e de C pode ser a mesma molécula!
ΔG < 0
Mapa metabólico de um microrganismo
“zoom” no 
metabolismo de 
carboidratos e 
metabolismo 
energético
- Algumas moléculas, como a 
GLICOSE, podem ser fonte de 
carbono (“material de construção”) 
e fonte de energia simultaneamente. 
- As rotas metabólicas envolvidas 
nestes dois papéis são diferentes!
Gombert & Van Maris, Current Opinion in Biotechnology, 2015
Replacing the Embden–Meyerhof glycolysis, which
yields 2 ATP per hexose, by a heterologous Entner–
Doudoroff pathway that yields 1 ATP per hexose would
decrease the ATP yield on sugar. To investigate this
possibility, Benisch and Boles [5!] constructed a yeast
strain containing 6-phosphogluconate dehydratase and 2-
keto-3-deoxygluconate-6-phosphate (KDPG) aldolase
from Escherichia coli. High activities were shown for
KDPG-aldolase. However, activities of the heterologous
6-phosphogluconate dehydratase were insufficient for
functional replacement of the Embden–Meyerhof glycol-
ysis by the Entner–Doudoroff route, which was attributed
to poor assembly of the [4Fe–4S] iron–sulfur cluster of the
6-phosphogluconate dehydratase in yeast. These findings
illustrate that functional expression of bacterial proteins
containing iron–sulfur clusters remains a challenge in
yeast metabolic engineering [5!,6].
Engineering the stoichiometry of sugar transport provides
another opportunity to decrease the ATP-yield on sugar.
Wild type Saccharomyces cerevisiae strains hydrolyze su-
crose extracellularly and use facilitated diffusion to take
up glucose and fructose. When this mechanism is
replaced by sucrose uptake via proton symport and intra-
cellular hydrolysis, the ATP requirement for subsequent
proton extrusion decreases the anaerobic ATP yield on
sucrose from 4 to 3. Requiring a combination of metabolic
and evolutionary engineering, this strategy resulted in an
11% increase of the ethanol yield on sucrose [7!]
(Table 1). This same strategy can in theory be applied
to replace the facilitated diffusion of the hexose sugars
with transport via proton-symport, resulting in a 50%
decrease in the ATP yield from 2 to 1 mol per mol of
hexose. That this strategy not necessarily requires het-
erologous transporters, was shown by the characteriza-
tion of the fructose/H+ symporter Fsy1 from a wine strain
of S. cerevisiae [8,9].
Whereas the abovementioned strategies all rely on chang-
ing the stoichiometry of ATP formation in sugar metabo-
lism, other strategies apply non-stoichiometric ATP
drains by intervening in ATP or H+ homeostasis. A classic
example of this strategy is introduction of ATP-hydrolyz-
ing futile cycles in yeast through the deregulation of some
gluconeogenic enzymes [10]. A recent attempt to increase
ATP hydrolysis, thereby potentially decreasing growth
and increasing alcoholic fermentation, encompassed the
overexpression of ATPase [11]. Further studies are re-
quired to quantify the impact on the ethanol yield under
industrially relevant conditions. In another study, the
authors claim that overexpression of alkaline phospha-
tase Pho8 increased the ethanol yield on sugar by up to
13%, despite a small impact on intracellular concentra-
tions of ATP [12]. However, the challenge with the
introduction of such non-stoichiometric ATP drains,
especially for industrial implementation, is in the fine
tuning between the positive impact and decreased cel-
lular robustness.
Decreasing formation of glycerol as a by-
product to increase the ethanol yield
Glycerol is the 3rd major by-product of alcoholic fermen-
tation after co-production of CO2 and yeast biomass. It is
estimated that in industrial fermentations approximately
4% of the sugar feedstock ends up as glycerol [13]. In
anaerobic yeast fermentations, formation of glycerol is
essential to re-oxidize surplus NADH resulting from
growth on sugars. Additionally, glycerol is the main com-
patible solute in yeast, produced in response to the high
osmotic pressure that can occur in some process config-
urations. A first approach to minimize the formation of
82 Energy biotechnology
Figure 1
sugar
ethanol + CO2glycerol
yeast biomass
ATP growth
m
ai
nt
en
an
ce
NADH
Robustness & diversity
NADH
Current Opinion in Biotechnology
Schematic representation of the distribution of sugar for ethanol
production, formation of yeast biomass, and formation of glycerol as a
by-product. To achieve a high ethanol yield on sugar, the robustness
of the process and yeast strains are essential.
Table 1
Selected strategies to increase ethanol yield on sugar in first generation fuel ethanol production
Genetic strategies Process
strategies
Breeding/biodiversity Recombinant DNA Evolution Shuffling
Desired traits of different strains into one strain [25!,53]
Decrease glycerol [14!,15] [17,20,21!,24!]
Decrease free-energy conservation [5!,7!,11,12] [7!]
Increase stress tolerance in yeast [35,46,34] [36] [32,33]
Decrease contamination [27,30,31!]
Current Opinion in Biotechnology 2015, 33:81–86 www.sciencedirect.com
15/09/14 10:002702362_1475-2859-8-27-1.png 512×669 pixels
Page 1 of 1http://openi.nlm.nih.gov/imgs/512/23/2702362/2702362_1475-2859-8-27-1.png
GLICOSE como 
fonte de carbono 
(“material de 
construção”): 
as setas azuis 
indicam desvio do 
fluxo de carbono 
para a biossíntese 
(anabolismo) de 
macromoléculas
© 2004 Pearson Education, Inc.
FIGURA 5.24Exemplo: 
Glicose
Exemplos: 
Proteínas 
Ácidos nucléicos 
Polissacarídeos 
Lipídeos
Exemplos: 
CO2 
Etanol 
Ácido Lático
Exemplos: 
Aminoácidos 
Nucleotídeos 
Monossacarídeos 
Ácidos graxos
G
lic
os
e 
co
m
o 
fo
nt
e 
de
 C
Glicose como fonte de energia (ou fonte de é)
10
Catabolism is the degradative phase of metabolism
in which organic nutrient molecules (carbohydrates,
fats, and proteins) are converted into smaller, simpler
end products (such as lactic acid, CO2, NH3). Catabolic
pathways release energy, some of which is conserved in
the formation of ATP and reduced electron carriers
(NADH, NADPH, and FADH2); the rest is lost as heat. In
anabolism, also called biosynthesis, small, simple pre-
cursors are built up into larger and more complex mole-
cules, including lipids, polysaccharides, proteins, and
nucleic acids. Anabolic reactions require an input of en-
ergy, generally in the form of the phosphoryl group
transfer potential of ATP and the reducing power of
NADH, NADPH, and MDH2 (Fig. 3).
Some metabolic pathways are linear, and some are
branched, yielding multiple useful end products from a
single precursor or converting several starting materials
into a single product. In general, catabolic pathways are
conuergent and anabolic pathways di,uergent (Fig. 4).
Some pathways are cyclic: one starting component of
Cell
macromolecules
Proteins
Polysaccharides
Lipids
Nucleic acids
Bioenerget ics and Metabol ism | 487 |
the pathway is regenerated in a series of reactions that
converts another starting component into a product. We
shall see examples of each type of pathway in the follow-
ing chapters.
Most cells have the enzlrnes to carqr out both the
degradation and the synthesis of the important categories
of biomolecules-fatty acids, for example. The simultane-
ous synthesis and degradation of fatty acids would be
wasteful,however, and this is prevented by reciprocally
regulating the anabolic and catabolic reaction sequences:
when one sequence is active, the other is suppressed.
Such regulation could not occur if anabolic and catabolic
pathways were catalyzed by exactly the same set of en-
zyrnes, operatrng in one direction for anabolism, the oppo-
site direction for catabolism: inhibition of an enzyrne
involved in catabolism would also inhibit the reaction se-
quence in the anabolic direction. Catabolic and anabolic
pathways that connect the same two end points ($ucose
-+ -+ plmrvate, and pyruvate -+ --) glucose, for example)
may employ many of the same enz).rnes, but invariably at
least one of the steps is catalyzed by different erzSrmes in
the catabolic and anabolic directions, and these eluyrnes
are the sites of separate regulation. Moreover, for both an-
abolic and catabolic pathways to be essentially irre-
versible, the reactions unique to each direction must
include at least one that is thermodynamically very favor-
able-in other words, a reaction for which the reverse re-
action is very uffavorable. As a further contribution to the
separate regulation of catabolic and anabolic reaction se-
quences, paired catabolic and anabolic pathways com-
monly take place in djfferent cellular compartments: for
example, fatty acid catabolism in mitochondria, fatty acid
s}'nthesis in the cltosol. The concentrations of intermedi-
ates, enzl'tnes, and regulators can be maintained at differ-
ent levels in these different compartments. Because
metabolic pathways are subject to kinetic control by sub-
strate concentration, separate pools of anabolic and cata-
bolic intermediates also contribute to the control of
metabolic rates. Devices that separate anabolic and cata-
boLic processes will be of particular interest in our discus-
sions of metabolism.
Metabolic pathways are regulated at several levels,
from within the cell and from outside. The most immedi
ate regulation is by the availability of substrate; when the
intracellular concentration of an enz)'rne's substrate is
near or below K* (as is commonly the case), the rate of
the reaction depends strongly upon substrate concentra-
tion (see Fig. 6-1 1). A second type of rapid control from
within is allosteric regulation (p.220) by a metabolic in-
termediate or coenzyrne-an amino acid or AIP, for ex-
ample-that signals the cell's internal metabolic state.
Precursor
molecules
Amino acids
Sugars
Fatty acids
Nitrogenous bases
Energy-
containing
nutrients
Carbohydrates
Fats
Proteins
Energy-
depleted
end products
coz
Hzo
NHs
FIGURE 3 Energy relationships between catabolic and anabolic path-
ways. Catabolic pathways deliver chemical energy in the form of ATP,
NADH, NADPH, and FADH2. These energy carr iers are used in ana-
bol ic pathways to convert small precursor molecules into cel lular
macromolecules.
© 2004 Pearson Education, Inc.
Esquema de reações envolvendo o par 
NAD+/NADH
elétrons não sabem nadar! ;-)
© 2004 Pearson Education, Inc.
R
© 2004 Pearson Education, Inc.
Armazenamento de E em ligações químicas
Via glicolítica
Glicólise
• filmes
Variações de energia-livre na 
glicóliseGibbs vrije energieveranderingen in de glycolyse
Rxn#���� Enzyme���� ΔΔΔΔG°' 
(kJ/mol)����
ΔΔΔΔG 
(kJ/mol)����
1� Hexokinase� -16.7� -33.5�
2� Phosphoglucoisomerase� +1.7� -2.5�
3� Phosphofructokinase� -14.2� -22.2�
4� Aldolase� +23.9� -1.3�
5� Triose phosphate isomerase� +7.6� +2.5�
6� Glyceraldehyde-3-phosphate 
dehydrogenase� +12.6� -3.4�
7� Phosphoglycerate kinase� -37.6� +2.6�
8� Phosphoglycerate mutase� +8.8� +1.6�
9� Enolase� +3.4� -6.6�
10� Pyruvate kinase� -62.8� -33.4�
 
Rxn#���� Enzyme���� ΔΔΔΔG°' 
(kJ/mol)����
ΔΔΔΔG 
(kJ/mol)����
1� Hexokinase� -16.7� -33.5�
2� Phosphoglucoisomerase� +1.7� -2.5�
3� Phosphofructokinase� -14.2� -22.2�
4� Aldolase� +23.9� -1.3�
5� Triose phosphate isomerase� +7.6� +2.5�
6� Glyceraldehyde-3-phosphate 
dehydrogenase� +12.6� -3.4�
7� Phosphoglycerate kinase� -37.6� +2.6�
8� Phosphoglycerate mutase� +8.8� +1.6�
9� Enolase� +3.4� -6.6�
10� Pyruvate kinase� -62.8� -33.4�
         
  
Os valores de ΔG foram calculados com base nas concentrações dos intermediários da via glicolítica de músculo de coelho
2 vezes 




 
    
   
De Embden-Meyerhof
route voor glycolyse
2 ATP per glucose
2 NADH per glucose
 


  
Gibbs vrije energie veranderingen in de glycolyse
variação de 
energia-livre das 
reações
2 vezes
Resumo:
A via glicolítica de 
Embden-Meyerhof 
(“clássica”)
2 ATP/glicose
2 NADH/glicose
reoxidação de 
NADH é essencial!!!
© 2004 Pearson Education, Inc.
FIGURA 5.14 B
Fermentação alcoólica 
(leveduras)
© 2004 Pearson Education, Inc.
FIGURA 5.14 B
Fermentação láctica 
(bactérias lácticas)
Pergunta?
• Por que não é necessário gerar CO2 na 
fermentação láctica (ao contrário do que 
ocorre na fermentação etanólica)?
Dissimilação de açúcares 
por respiração


































  
VIAS METABÓLICAS CENTRAIS (RESUMO!!!) 
 
 
 
 
 
coenzimas reduzidas (NAD+ -> NADH)
precursores (AKG,AcCOA,OAA)
ATP (por fosforilação ao nível de substrato)
GLC
G6P
PYR
via PP
ciclo TCA
CO2
vias fermentativas PRODUTOS DE
FERMENTAÇÃO
poder redutor (NADPH)
precursores (R5P, E4P)
coenzimas reduzidas (NAD+ -> NADH)
precursores (G6P,F6P,G3P,3PG,PEP,PYR)
ATP (por fosforilação ao nível de substrato)
reoxidação de coenzimas (NADH -> NAD+)
ATP (por fosforilação oxidativa)
reoxidação de coenzimas (NADH -> NAD+)
 (FADH2 -> FAD)
cadeia transporte
de é
O2 H2O
via EMP
vias respiratórias
CATABOLISMO