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Metabolism & Bioengineering: FULL COURSE GUIDE

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  • Course
  • Metabolism and Bioengineering (MCB2022S)
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  • University Of Cape Town (UCT)
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  • Biochemistry

This is a pack of notes designed and used by a student who obtained 94% for the course. It includes everything you need to succeed, including extensive and easy-to-understand lecture notes (re-written and combined with textbook and online sources) and the questions and answers to tutorials run in 2...

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  • February 10, 2022
  • 98
  • 2020/2021
  • Class notes
  • Ramona hurdayal
  • All classes

Subjects

  • biochemistry
  • genetics
  • science
  • metabolism
  • protein
  • biotechnology
  • microbiology
  • genes
  • uct

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Metabolism and Bioengineering
MCB2022S
Full course guide



Lecture notes
• Paul Meyers: Microbial Metabolism (pg 2-29)
• Sandiswa Mbewana: Microbial Biotechnology (pg 30-38)
• Ramona Hurdayal: Mammalian Metabolism (pg 39-69)

Tutorials (questions and answers)
• Microbial Metabolism 1 and 2 (pg 70-80)
• Microbial Biotechnology 1, 2, and 3 (pg 81-88)
• Mammalian Metabolism 1, 2, and 3 (89-98)

,1. Introduction to Microbial Metabolism

Thermodynamics: the energy changes in a system, ∆G = ∆H -T ∆S
• 1st Law: energy can be neither created nor destroyed, total energy in universe remains
constant.
• 2nd Law: physical and chemical processes proceed in such a way that the entropy of the
universe increases. (Though the entropy of any given system within the universe can
increase/decrease/remain unchanged).
• Free energy change (∆G): the maximum amount of energy in a system (cell) available to
do useful work at constant temperature and pressure.
◦ We use ∆G° (standard conditions of concentration, pressure, pH, temperature) and
∆G°' (standard biological conditions) in order to be able to compare different systems/
processes.
◦ The sign indicates spontaneity: <0 spontaneous and exergonic, >0 nonspontaneous
and endergonic, =0 equilibrium.

Oxidation-reduction: the movement of electrons from a donor to an acceptor.
• Metabolic energy is derived from redox reactions; energy used to fuel cell functions is in
the form of high-energy electrons. Transfer of energy in the form of
eletrons allows the cell to transfer/use energy incrementally rather than in
a destructive burst.
• Half reactions are written as reductions by convention (acceptor on left).
• Standard reduction potential (E°'): a measurement of the tendency of a
substance to be reduced, measured in volts (since electron flow
represents current).
◦ More negative potentials indicate a strong reducing agent (donor), while more positive
potentials indicate a strong oxidising agent (acceptor).
◦ The sum of the half reaction potentials gives the standard electrode potential for the
whole reaction under standard conditions.
• Redox towers list the reduction potentials (measured relative to the standard half-reaction
of reduction) vertically.
◦ The reduced substance with the greatest tendency to donate at the top right and
oxidised substance with greatest tendency to accept
at the bottom left.
◦ Those in the middle serve as either a donor (in
reduced form) or acceptor (in oxidised form), depending on the other substances in
the reaction.
• The standard free energy change under biological conditions is directly related to the
magnitude of the difference between the two reduction potentials: ∆G°' =
-n*F*∆E°' (where ∆E°' = acceptor ∆E°' - donor ∆E°', n = number of electrons transferred,
F = 96 400 J/V/mole).
◦ Spontaneous reactions occur when ∆E°'> 0; electrons flow from a donor to an
acceptor with a more positive reduction potential (ie., down the tower).
‣ For example, electron transport (see tower and pic).
‣ Note that if you want to add the potentials as in picture, you must change the sign
when the reaction is flipped.
◦ Non-spontaneous reactions occur when ∆E°'< 0. This involves the flow of electrons to

, an acceptor with a more negative potential (ie., up the tower).
◦ The greater the ∆E°', the more free energy made available/required; directly
proportional.
• Oxidation number or state: the total number of electrons that an atom gains or loses to
form a chemical bond with another atom, ie., the degree of oxidation of that atom in a
compound.
◦ These bear no relation to actual charges and are used to determine if an atom is being
oxidised or reduced in a chemical reaction; an increasingly positive ON indicates
oxidation and an increasingly negative ON indicates reduction.
◦ The sum of all the oxidation numbers of atoms in a compound/ion is equal to the total
charge of the compound/ion.
‣ An atom in the elemental state (Cl2, O2, Cu) has an ON of zero. Ie. an uncombined
element.
‣ A cationic/anionic single-atom ion (K+, Ca2+) has an ON equal to the
charge on the ion.
‣ H atoms in organic molecules have ON of +1.
‣ O atoms in peroxides have ON of -1; all other O atoms have ON of -2.
‣ Group one elements have +1
‣ Group 2 elements have +2

Metabolism: the set of life-sustaining chemical reactions in organisms; ordered
transformation of nutrients acquired from the environment via enzyme-catalysed pathways.
• Central pathways are amphibolic (serve both anabolism and catabolism) - the
intermediates which lie upon EMP, ED, and TCA pathways are
indispensable to both energy production and to the provision of carbon
skeletons for cell components.
• Cells are continually balancing catabolism with anabolism
◦ Catabolism involves processes that harvest energy released from
the oxidative, exergonic breakdown of compounds into smaller
molecules.
‣ These pathways are oxidative overall (every step is not necessarily oxidative).
‣ Produces (1) precursors required for synthesis of cell components, and (2) the
energy required for such processes (and other endergonic processes such as
nutrient uptake, flagella rotation).
◦ Anabolism involves processes that utilise the energy harvested by catabolism (and
stored in the form of ATP) for reductive, endergonic synthesis of monomers and
subsequent assembly of polymers/macromolecules that make up the cell.
• Adenosine triphosphate (ATP) acts to couple the energy of exergonic and endergonic
processes, thereby allowing energetically unfavourable chemical reactions to proceed.
◦ Energy released from exergonic reactions is (usually) conserved as ATP.
◦ Subsequent endergonic reactions are driven by the energy released from ATP
hydrolysis (highly exergonic, ∆G0' = -30.5 kJ/mole).
◦ Can power various types of cellular work: chemical work (anabolism), transport work
(nutrient uptake, waste elimination, iron balance), and mechanical work (cell motility,
structure movement).
• Pathways are the stepwise, interconnected chemical reactions that breakdown or build

,Microbial metabolic diversity
• All microbes need water and nutrients (macro-elements
CHONSPKCaMgFe in large amounts and micro-elements
MnMoZnCuCoNiV in trace amounts), carbon/energy/electron
source(s), and growth factors (amino acids, purine and pyrimidine
bases, vitamins/enzyme co-factors).
• Nutritional modes
◦ Photoautotrophy/photolithotrophy/photolithotrophic autotrophy:
energy is captured from sunlight and used to oxidise water;
transformed to chemical energy. The released electrons reduce CO2
to reduced carbon compounds (carbohydrates).
◦ Chemoautotrophy/chemolithotrophy/chemolithotrophic autotrophy: oxidation of
inorganic substances provides energy and electrons for biosynthetic
processes, fixation of CO2 provides carbon source. Methanogens,
sulphur-oxidising bacteria, hydrogen-oxidising bacteria, nitrifying
bacteria, iron-oxidising bacteria.
◦ Photoheterotrophy/photoorganotrophy/photoorganotrophic
heterotrophy: energy is captured from sunlight, and pre-formed organic molecules to
satisfy their carbon and electron requirements.
◦ Chemoheterotrophy/chemoorganotrophy/chemoorganotrophic heterotrophy: energy,
carbon and electrons are obtained from reduced organic compounds made by
autotrophs (carbohydrates, lipids, and proteins). Most non-photosynthetic microbes
including most pathogens, fungi, protozoa.
‣ Photosynthetic production is linked to the mineralisation (oxidation) of organic
compounds (see pic).
• Classification by
◦ Energy source (for driving endergonic processes): phototrophs absorb light in
photoreceptors and transform it into chemical energy, chemotrophs use the energy
released from oxidation of chemical compounds.
◦ Electron source/donor (for reducing power in biosynthesis): lithotrophs use reduced
inorganic substances (often also autotrophic), organotrophs use reduced organic
compounds (often also heterotrophic).
◦ Carbon source (for synthesis of organic precursor molecules, growth, development):
heterotrophs use reduced, pre-formed organic molecules (precursor metabolites
arise from central metabolism), autotrophs use carbon dioxide (precursor metabolites
arise from CO2 fixation and related pathways). Mixotrophs have the ability to combine
or switch between autotrophic and heterotrophic modes.
‣ Note, the carbon source is often also the energy source.


2. Chemoorganotrophic heterotrophy

Chemoheterotrophs use reduced organic compounds as sources of energy/
electrons/carbon.
• The energy-conserving oxidation of the energy source also provides the
carbon and electrons required for anabolism. There are two general
approaches for catabolising the energy source:

, ◦ In respiration, the electrons pass through an electron transport chain, generating a
PMF used for oxidative phosphorylation and the bulk of ATP generation.
‣ In aerobic respiration, the terminal electron acceptor is oxygen (O2).
‣ In anaerobic respiration, exogenous oxidised molecules other than
O2 serve as the terminal acceptor (NO³⁻, SO₄²⁻, CO₂², Fe³⁺, or
SeO₄²⁻).
‣ Both use a glycolytic pathway, TCA, and ETC.
◦ In fermentation, there is no ETC involvement, and ATP synthesis is generally
exclusively by substrate-level phosphorylation.
‣ Endogenous organic molecules (usually intermediates of the oxidative catabolic
pathway used) act as the terminal electron acceptors.
‣ The energy source is only partially catabolised; involves only a subset of the
reactions in respiration.
• Described with glucose as the energy source by convention, since
◦ (1) Glucose is a common energy source for COTs.
◦ (2) Catabolic pathways are organised such that a variety of complex organic
molecules can be degraded by pathways that either generate glucose or
intermediates of the pathways used in glucose metabolism (these simpler nutrients
are funnelled into the pathways of central metabolism).
‣ Having few very flexible metabolic pathways greatly increases metabolic
efficiency.
‣ Proteins, polysaccharides, and lipids are broken down enzymatically to their
monomeric constituents, and then processed/simplified into a carbon skeleton
that can be fed into a central pathway (deamination of amino acids, oxidation of
fatty acids, breakdown of monosaccharides).

(1) Glucose to Pyruvate: the Glycolytic Pathways
• Organisms use either EMP or ED (similar pathways, same function).
• Organisms may use PPP in addition to above, or exclusively.
• Relative participation of each pathway in hexose catabolism reflects the
needs of the cell under conditions of growth (see table): most carbon flow directed
towards energy-generating pathways, with the rest sent to the PPP.
• Also note that aerobic organisms get most of their ATP from oxiphos.

The Emden-Meyerhof-Parnas (EMP) pathway
• Net reaction: Glucose + 2ADP + 2Pi + 2NAD⁺ → 2 pyruvate + 2ATP + 2NADH
+ 2H⁺
◦ Involvement of NADH indicates the pathway is important for energy-conservation
(catabolism).
• Function: amphibolic.
◦ Energy-conservation; ∆G°' = -73.3 kJ/mole (73.3 kJ of energy released as heat for
every mole of glucose that passes through).
◦ Generates reducing power and biosynthetic precursors (eg.,
pyruvate).
• Key points:
◦ 6C preparatory phase: input of two ATP is used to phosphorylate
glucose twice.

, ◦ 3C energy-conserving phase: production of NADH and
two ATP per pyruvate, which happens twice per
glucose (since F-1,6-BP is cleaved into two halves;
DHAP and G3P, but DHAP is immediately converted to
G3P).
◦ G6P serves as a branch point at which carbon can be
sent down PPP or EMP..
• Prevalence: plants, animals, all major groups of microbes.

The Entner-Doudoroff (ED) pathway
• Net reaction: Glucose + ADP + Pi + NADP+ + NAD⁺ → 2
pyruvate* + 1 ATP* + NADPH + NADH + H2O
◦ Strictly, the ED pathway yields pyruvate and G3P, but organisms can couple ED with
lower EMP to catabolise G3P to pyruvate.
◦ NADPH can donate its phosphate to ADP (generating NADH). Thus effectively 2 ATP
molecules produced per glucose.
• Function: as for EMP.
• Key points:
◦ 2-keto-3-deoxy-6-phosphogluconate (KDPG) is formed from glucose (in
three reactions) and is cleaved to pyruvate and G3P.
◦ G6P branch-point to continue down ED or to PPP.
• Prevalence: unique to bacteria; mainly in gram-negative soil bacteria.

The Pentose Phosphate pathway (PPP)
• Net reaction: 3 G6P* + 6 NADP⁺ + 3 H2O → 2 F6P + G3P + 3CO2 + 6 NADPH + 6 H⁺.
◦ Involvement of NADPH here indicates the pathway is important for biosynthesis
(anabolism).
◦ The products can be used in two ways
‣ F6P isomerised back to G6P to continue on in PPP, EMP (as F6P or G6P), or ED
(as G6P). G3P is converted to pyruvate by EMP enzymes (generates two ATP).
‣ 2 x G3P molecules can combine to form F-1,6-BP, converted back into G6P.
• Function: biosynthesis rather than energy generation (although bugs using only PPP can
generate two ATP per G3P converted to pyruvate by EMP pathway enzymes).
◦ Generates reduced electron carriers/reducing power (two NADPH per glucose) for the
reduction of molecules during biosynthesis.
◦ Generates precursor metabolites that can be used in biosynthetic reactions to make
cellular molecules, eg., ribose-5-phosphate for RNA (and deoxyribose-phosphate for
DNA), erythrose-4-phosphate for aromatic amino acids.
• Key points: carbon shuffling reactions (conversion of ribulose-5-phosphate to a mixture of
3C-7C sugar phosphates).
◦ Transketolase catalyses the transfer of 2C groups
‣ Ribose-5-phosphate (5C) + Xu5P (5C) → Glyceraldehyde-3-phosphate (3C) +
Sedoheptulose-7-phosphate (7C)
‣ Erythrose-4-phosphate (4C) + Xu5P (5C) → G3P + F6P (6C)
◦ Transaldolase catalyses the transfer of 3C groups
‣ Sedoheptulose 7-phosphate (7C) + G3P (3C) → F6P + E4P (4C)
• Prevalence: used in all organisms, simultaneously with EMP or ED.

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