Transport processes in the aspect of metabolic engineering.

1. BY,
RAJESWARI R
SUJANI SATHYA KEERTHANA S
JEYA CRESSIDA J

3. TRANSPORT ACROSS
MEMBRANES
 Every living cell must acquire raw materials from its
surroundings for biosynthesis and for energy production, and
must release the byproducts of metabolism to its environment.
 Few nonpolar compounds can dissolve in the lipid bilayer and
cross the membrane unassisted.
 But for polar or charged compounds or ions, a membrane
protein is essential for transmembrane movement.
 In some cases a membrane protein simply facilitates the
diffusion of a solute down its concentration gradient.
 If the transport occurs against a gradient of concentration,
requires energy.

4. CONTD..,
 The energy may come directly from ATP hydrolysis or may
be supplied in the form of movement of another solute down
its electrochemical gradient with enough energy to carry
another solute up its gradient.

5. TYPES OF TRANSPORT ACROSS
MEMBRANES
Transport process
Passive transport Active transport
Simple diffusion
Facilitated diffusion
Osmosis
Primary active
transport
Secondary
active transport

6. PASSIVE TRANSPORT
 In passive transport process a specific molecule flows from
high concentration to low concentration.
 Some materials diffuse readily through the membrane, but
others require specialized proteins, such as channels and
transporters, to carry them into or out of the cell.
 A movement of biochemicals and other atomic or molecular
substances across membranes that does not require an input of
chemical energy.

7. PASSIVE TRANSPORT
Simple diffusion
• Diffusion is a process of passive transport in which molecules
move from an area of higher concentration to one of lower
concentration.
• The diffusion continues till the gradient has been vanished.

8. PASSIVE TRANSPORT
Facilitated diffusion
• Facilitated diffusion is a process by which molecules are
transported across the plasma membrane with the help of
membrane proteins.
• A concentration gradient exists that would allow ions and
polar molecules to diffuse into the cell, but these materials are
repelled by the hydrophobic parts of the cell membrane.
• Facilitated diffusion uses integral membrane proteins to move
polar or charged substances across the hydrophobic regions of
the membrane.
• 2 types of protein involved in facilitated diffusion are channel
and carrier proteins.

9. PASSIVE TRANSPORT
 Channel protein
• Channel proteins can aid in the facilitated diffusion
of substances by forming a hydrophilic passage
through the plasma membrane through which polar
and charged substances can pass.
• Channel proteins can be open at all times,
constantly allowing a particular substance into or
out of the cell, depending on the concentration
gradient; or they can be gated and can only be
opened by a particular biological signal.

10. PASSIVE TRANSPORT
 Carrier protein
• Another type of protein embedded in the plasma membrane is
a carrier protein.
• This protein binds a substance and, in doing so, triggers a
change of its own shape, moving the bound molecule from
the outside of the cell to its interior; depending on the
gradient, the material may move in the opposite direction.
• Carrier proteins are typically specific for a single substance.
• Example: Glucose transport proteins, or GLUTs, are
involved in transporting glucose

11. PASSIVE TRANSPORT

12. PASSIVE TRANSPORT
 Osmosis
• Osmosis is the movement of water through a semipermeable
membrane according to the concentration gradient of water
across the membrane, which is inversely proportional to the
concentration of solutes.
• Semipermeable membranes, also termed selectively
permeable membranes or partially permeable membranes.
• In osmosis, water always moves from an area of higher water
concentration to one of lower concentration. In the diagram
shown, the solute cannot pass through the selectively
permeable membrane, but the water can.

13. PASSIVE TRANSPORT
 Osmosis

14. ACTIVE TRANSPORT
 Active transport is the movement of molecules across a
membrane from a region of their lower concentration to a
region of their higher concentration—in the direction against
the concentration gradient.
 Active transport requires cellular energy to achieve this
movement.
 There are two types of active transport: primary active
transport that uses ATP, and secondary active transport that
uses an electrochemical gradient.

15. ACTIVE TRANSPORT
 Primary active transport
• Primary active transport, also called direct active transport,
directly uses energy to transport molecules across a membrane.
• Example: Sodium-potassium pump, which helps to maintain
the cell potential.

16. ACTIVE TRANSPORT
 Secondary active transport
• Secondary active transport or co-transport, also uses energy to
transport molecules across a membrane
• It uses the electrochemical potential difference created by
pumping ions out of the cell
• The two main forms of secondary active transport are antiport
and symport.
• In secondary active transport, the two molecules being
transported may move either in the same direction (i.e., both
into the cell), or in opposite directions (i.e., one into and one
out of the cell).

17. ACTIVE TRANSPORT
 CONTD..,
 When they move in the same direction, the protein that
transports them is called a symporter, while if they move in
opposite directions, the protein is called an antiporter.

18. ENDOCYTOSIS AND EXOCYTOSIS
 Endocytosis
 Endocytosis is the process by which cells absorb larger
molecules and particles from the surrounding by engulfing
them.
 It is used by most of the cells because large and polar
molecules cannot cross the plasma membrane.
 Endocytosis consists of phagocytosis and pinocytosis
 Phagocytosis: Phagocytosis or “cell eating,” is the process by
which large particles, such as cells or relatively large particles,
are taken in by a cell.

19. ENDOCYTOSIS AND EXOCYTOSIS
 Contd..,
 For example, when microorganisms invade the human body, a
type of white blood cell called a neutrophil will remove the
invaders through this process, surrounding and engulfing the
microorganism, which is then destroyed by the neutrophil.

20. ENDOCYTOSIS AND EXOCYTOSIS
 Contd..,
 Pinocytosis: Pinocytosis, or “cell drinking,” is a process that
takes in molecules, including water, which the cell needs from
the extracellular fluid.

21. ENDOCYTOSIS AND EXOCYTOSIS
 Exocytosis: The process by which the cells direct the contents
of secretory vesicles out of the cell membrane is known as
exocytosis.
 These vesicles contain soluble proteins to be secreted to the
extracellular environment, as well as membrane proteins and
lipids that are sent to become components of the cell
membrane.

22. FUELING REACTIONS

23.  Fueling reactions serves 3 purposes
• Generation of Gibbs free energy in the form of ATP in, which is
used to fuel other cellular reactions
• Production of reducing power (or reducing equivalents), in the
form of cofactor NADPH required in biosynthesis reactions
• Formation of precursor metabolites required in the biosynthesis
of building blocks
 The carbon and energy source are required for biosynthesis of
building blocks.
 Some of the most frequently used substrate (act as both carbon
and energy source) are sugars(Glucose, fructose, lactose, etc.)
 The catabolism of sugars starts with glycolysis, and the end
product is pyruvate.
 Pyruvate is further processed in fermentative pathways,
anaplerotic pathways, TCA cycle, transamination pathways for
aminoacid formation.

24. Contd..,
 Thus many organisms have the ability to catabolize amino
acids, organic acids or fats either in the presence of sugar or
as sole carbon and energy source
 Due to the importance of fueling pathways in central carbon
metabolism and biosynthesis we are going to discuss about
various pathways.

25. GLYCOLYSIS

26. INTRODUCTION
 In glycolysis a molecule of glucose is degraded in a series of
enzyme-catalyzed reactions to yield two molecules of the
three-carbon compound pyruvate.
 During the sequential reactions of glycolysis, some of the free
energy released from glucose is conserved in the form of ATP
and NADH.
 The breakdown of the six-carbon glucose into two molecules
of the three-carbon pyruvate occurs in ten steps, the first five
of which constitute the preparatory phase
 The energy gain comes in the payoff phase of glycolysis(from
6th step to 10th step)

28. Contd..,

29. FERMENTATIVE PATHWAYS

30. Fermentation
Features of fermentation pathways
• Pyruvic acid is reduced to form reduced organic acids or
alcohols.
• The final electron acceptor is a reduced derivative of pyruvic
acid.
• NADH is oxidized to form NAD: Essential for continued
operation of the glycolytic pathways.
• O2 is not required.

31. Glycolysis
Glucose + 2NAD + 2ADP
2 pyruvate +2NADH + 2H + 2ATP +2H2O

32. Fermentation Pathways
• Glycolysis is the first stage of fermentation Forms 2 pyruvate,
2 NADH, and 2 ATP
• Pyruvate is converted to other molecules, but is not fully broken
down to CO2 and water
• Regenerates NAD+ but doesn’t produce ATP
• Provides enough energy for some single-celled anaerobic
species

33. Two Pathways of Fermentation
• Alcoholic fermentation
Pyruvate is split into acetaldehyde and CO2
Acetaldehyde receives electrons and hydrogen from
NADH, forming NAD+ and ethanol
• Lactate fermentation
Pyruvate receives electrons and hydrogen from
NADH,forming NAD+ and lactate

34. Two Pathways of Fermentation

35. Lactate Fermentation
lactic acid fermentation
 Vigorously contracting skeletal muscle must function under
lowoxygen conditions (hypoxia), NADH cannot be
reoxidized to NAD
 But NAD is required as an electron acceptor for the further
oxidation of pyruvate.
 Under these conditions pyruvate is reduced to lactate,
accepting electrons from NADH and thereby regenerating the
NAD necessary for glycolysis to continue.

36. CONTD..,
 Certain tissues and cell types (retina and erythrocytes,
for example)
 convert glucose to lactate even under aerobic
conditions.
 lactate is also the product of glycolysis
 Under anaerobic conditions in some microorganisms
also .

37. Examples of fermentation pathways
1) Lactic acid fermentation
Found in many bacteria;
e.g. Streptococcus sp, Lactobacillus acidophilus
2) Mixed acid fermentation
e.g. Escherichia coli
3) 2,3-Butanediol fermentation
e.g. Enterobacter aerogenes

38. Alcohol fermentation
 Humans cannot ferment alcohol in their own bodies, we lack
the genetic information to do so

39. Examples of alcoholic pathways
1) Ethanol
e.g. (Saccharomyces cerevesiae)
2) Butanol
e.g. (Clostridium acetobutylicum)

40. Real Life Example
Humans ferment lactic acid in muscles where oxygen becomes
depleted, resulting in localized anaerobic conditions. This
lactic acid causes the muscle stiffness couch-potatoes feel
after beginning exercise programs. The stiffness goes away
after a few days allows aerobic conditions to return to the
muscle, and the lactic acid can be converted into ATP via the
normal aerobic respiration pathways.

41. Fermentation Pathway as a whole

43. INTRODUCTION
 Citric acid cycle is also known as TCA(tricarboxylic acid)
cycle or the Krebs cycle
 Series of chemical reactions by aerobic organisms- generate
energy through the oxidation of acetate
 Eukaryotic cell-mitochondria
 Prokaryotic cell-cytosol
 It is the key metabolic pathway connects carbohydrate ,fat and
protein metabolism

44. CONTD..,
 The reactions are carried out by eight enzymes- completely
oxidize acetate in the form of acetyl-CoA – 2 molecules of
carbon dioxide and water.
 Primary source of acetyl-CoA – break down of sugars by
glycolysis – pyruvate – decarboxylated by pyruvate
dehydrogenase generates acetyl-CoA

45. CONTD..,
 The cycle consumes acetate (in the form of acetyl-CoA) and
water, reduces NAD+ to NADH, and produces carbon dioxide.
 The NADH generated by the TCA cycle is fed into the
oxidative phosphorylation pathway.
 The net result of these two closely linked pathways is the
oxidation of nutrients to produce usable energy in the form of
ATP.

46. BREAKDOWN OF PYRUVATE

47. CONTD..,
 Each pyruvate molecule loses a carboxylic group in the form
of carbon dioxide.
 The remaining two carbons are then transferred to the
enzyme CoA to produce Acetyl CoA.

49.  One complete cycle gives
3 NAD+ 3NADH
1Q 1QH2
1 GDP + Pi 1GTP
This NADH and QH2 are used in Oxidative
phosphorylation to generate ATP

50. OXIDATIVE PHOSPHORYLATION
It is the metabolic pathway – cells uses enzyme
to oxidize nutrients – releases energy which is
used to produce ATP.

52. COMPLEX 1
 NADH dehydrogenase or complex 1
 Reaction starts with binding of NADH molecule to comlplex1
and donation of 2 electrons
 As electrons pass through this complex four H+ are pumped
from matrix to intermembrane space.

53. COMPLEX 11
 Succinate dehydrogenase or complx 11
 It oxidizes succinate to fumerate and reduces ubiquinone
 As this reaction releases less energy than oxidation of
NADH, it does not transport protons across membrane.

54. COMPLEX III
 Cytochrome c reductase or complex III
 Cytochrome is a kind of electron transfering
protein contains atleast one heme group.
COMPLEX IV
 This mediates final reaction in electron transport
chain and transfers electros to oxygen.

55. COMPLEX V
 ATP synthase uses the energy stored in a
proton gradient across a membrane to drive
the synthesis of ATP, ADP and Pi.
NADH 3ATP
SUCCINATE 2ATP

56. THANK YOU