Biotechnological applications of biochemical pathways

 potential uses and applications of CRISPR-Cas9 technologies to improve photosynthetic efficiencies and crop
yields

Practical Applications
Gene editing via the CRISPR-Cas9 system has been used to address a variety of agricultural
issues associated with food production
Certain metabolic pathways have been enhanced to improve nutritional content (e.g. higher
starch production)
Plant absorption spectra have been modified to increase photosynthetic efficiencies (e.g. new
pigments introduced)
Higher tolerances to biotic pathogens (viral, bacterial, fungal) or abiotic stresses (cold, drought,
salt) have been achieved
Resistance to particular herbicides have been incorporated into crops to allow for elimination of
competing weed species
Improvements in photorespiration efficiencies has resulted in increased food production in a
variety of common C3 plants

 uses and applications of anaerobic fermentation of biomass for biofuel production

Key Knowledge:
 The role of Rubisco in photosynthesis, including adaptations of C3, C4 and CAM
plants to maximise the efficiency of photosynthesis

The light independent reactions use the enzyme Rubisco to fix carbon (from CO2) in
order to make carbohydrates (such as glucose)
 This process (called the Calvin cycle) involves the production of a 3C
intermediate called GP (glycerate-3-phosphate)
 As each cycle produces one GP molecule, it takes two cycles to synthesise
glucose and more to produce polysaccharides (like starch)

Plants that exclusively fix carbon dioxide this way are called C3 plants – as the initial
product (GP) is a 3C compound

Photorespiration
Rubisco can alternatively use oxygen (O2) as a substrate to undergo a different series of
reactions known as photorespiration
 Photorespiration creates a product that cannot be used to make sugars and
hence reduces the efficiency of the Calvin cycle
 Photorespiration reduces levels of photosynthesis by up to ~25% in C3 plants,
reducing energy yield in these plants
C4 and CAM Plants
Because oxygen acts as a competitive inhibitor for Rubisco, photosynthesis in C3 plants
is reduced in the presence of oxygen
 C3 plants are less efficient in hot and dry regions, as the stomata must remain
closed in order to prevent excessive water loss
 When the stomata are closed, oxygen cannot diffuse out of the leaf, increasing
oxygen concentration relative to CO2 levels

In an effort to maximise photosynthesis, some plants have evolved mechanisms to limit

the exposure of Rubisco to oxygen
 C4 and CAM plants use an alternate enzyme called PEP carboxylase to initially
fix the carbon and make a 4C compound
 PEP carboxylase has a higher affinity for carbon dioxide than Rubisco and
doesn’t bind to oxygen at all
 These plants can then store and transfer the 4C compounds to regions with
lower oxygen concentrations

C4 Pathway
In the C4 pathway, carbon dioxide is physically separated from oxygen in order to
improve CO2 binding to Rubisco
 The CO2 is converted to the 4C compound in the mesophyll and then
sequestered to a deeper tissue layer where less O2 is present
 In this deeper tissue layer (the bundle sheath), the CO2 is released and can enter
the Calvin cycle without competition from oxygen

CAM Pathway
In the CAM pathway, carbon reserves are created at night and then released for use
during the day (temporal isolation)
 CAM plants are suited to hot and arid environments where water loss is high and
stomata must therefore remain closed during the day
 The CO2 is converted into the 4C compound during the night, when stomata are
open and the CO2 is able to diffuse into the leaf
 The stored CO2 is then released for use during the day, when closed stomata
would otherwise prevent photosynthesis from proceeding
Comparison of the Carbon Fixation Pathways
Key Knowledge:

 The location, inputs and the difference in outputs of anaerobic fermentation in

animals and yeasts
 Uses and applications of anaerobic fermentation of biomass for biofuel
production

The anaerobic breakdown of glucose into pyruvate (via glycolysis) results in the production of a
small amount of hydrogen carrier (NADH)
 In the presence of oxygen, this hydrogen carrier will be oxidised as part of the electron
transport chain (i.e. becomes unloaded NAD)
 In the absence of oxygen, the carrier cannot be oxidised by mitochondria and stocks of
the unloaded coenzyme will become depleted

Fermentation
Fermentation involves the conversion of pyruvate into an alternative carbon compound via a
reaction that oxidises the hydrogen carrier
 This restores the stocks of unloaded coenzyme needed for glycolysis, allowing ATP
production to continue in the absence of oxygen
 Fermentation is therefore considered to be a vital component of anaerobic respiration, as
glycolysis could not otherwise be sustained

The products of fermentation differ between animals and plants or yeast:

 In animals, pyruvate is converted into lactic acid (lactate) – this reaction is reversible
(pyruvate can be reformed when oxygen is present)
 In plants or yeast, pyruvate is converted into ethanol and carbon dioxide – this reaction is
irreversible (the pyruvate cannot be reformed)

Anaerobic fermentation has many practical applications:

 In animals, fermentation is used to maximise the power of muscle contractions (allows
energy capacity to exceed oxygen levels)
 In yeasts, fermentation is used in the production of alcoholic beverages and allows for
dough to rise (leavening) when baking
Biofuels
Biofuels are an energy source produced from the anaerobic fermentation of biomass (organic
material from plants and animals)
 Whereas fossil fuels are non-renewable as they form via exceedingly slow geological
processes (over several million years), biofuels are derived from rapid biological
processes and are therefore functionally renewable (provided there is a constant stock of
reagents)
 Because the biomass is typically derived from the anabolic conversion of inorganic
sources of carbon (i.e. carbon dioxide), there is a lower carbon footprint associated with
its production (making it arguably better for the environment)

Biofuel production involves the growth of anaerobic microbes in an enclosed and sterilised
vessel called a fermenter
 When provided with a source of biomass, the microbes will anerobically ferment this
organic feedstock to produce the biofuel
 The fermenter distributes materials evenly within the chamber and controls the
temperature, pH, levels of nutrients and wastes
 The biofuel can be collected from the fermenter after a fixed amount of time (batch
cultivation) or ongoing (continuous cultivation)

The biomass used to produce biofuels (via anaerobic fermentation) can be sourced in a number
of different ways:
 It can be produced from agricultural feedstocks (i.e. edible crops), however this requires
the use of large amounts of arable land to produce the feedstock and can potentially drive
up regional food costs (as less crops are being used as a traditional food source)
 It can also be produced from non-edible plant components and certain municipal wastes,
however these have higher production costs

Algae is an alternative biomass source for anaerobic fermentation that does not require arable
land and can operate at low costs
 Algae can photosynthesise and hence produce a stock of biomass (starch, glucose,
cellulose) within contained photobioreactors
Bioethanol is a common biofuel that is used to supplement or replace traditional fossil fuels (e.g.
gasoline) in fuel tanks
 Drawbacks of bioethanol include the fact that it has a lower energy content than fossil
fuels, is harder to vaporise (so may be more difficult to use in colder temperatures) and is
more likely to corrode materials (such as car engines) upon extended exposure