Topic 2 - Bacterial respiration, fermentation, growth, and metabolic engineering Flashcards
what do Glycolysis and the Krebs cycle produce (and why?)
ATP (substrate-level phosphorylation) and reduced electron carriers for
the ETC, which is localised to the cytoplasmic (inner) membrane
what are the two different NADH dehydrogenases in e. coli and what are their differences?
Nuo (similar to complex 1, boot shape) and Ndh they have different H+/e- ratios as Nuo is a proton pump (pumping 4H+ into the periplasm) but Ndh is not
why would the Ndh enzyme be favoured over Nuo
NADH oxidation by Nuo conserves more energy but the simpler Ndh enzyme has a higher turnover rate and is
favoured under aerobic conditions
why are Ndh and Cyd good targets for antimicrobials
they are are not present in humans
what is the ATP yield of aerobic respiration in E. coli under vigorous aeration
~20 ATP per mol of glucose
differences between Cyo and Cyd
Cyo: A proton pump -> has lower affinity for oxygen
Cyd: Doesn’t effect the pmf therefore no redox loop has higher affinity for oxygen so used in microoxic conditions + more resistant to chemicals (e.g. sulfides)
why would Cyd be favoured over Cyo
Cyd conserves less energy but allows oxygen reduction at very low
oxygen tensions and is more resistant to some toxic compounds encountered in the host
what are the only oxygenic photosynthetic prokaryotes called
cyanobacteria
where is the thylakoid in cyanobacteria
centric around the cell membrane and then surrounded by ribosomes
what are carboxysomes, what is in them and why?
a CO2 concentrating mechanism in cyanobacteria
they encapsulate enzymes from the cytoplasm
pore mediates metabolites in/out
lets in HCO3- (converted to CO2 in the carboxysome and charged = easier to remove) and rubisco
separates rubisco from O2
what are the photosynthetic pigments in cyanobacteria
Main = Chlorophyll A
+ carotenoids and antioxidants to expand the spectrum
+ bilins pink/blue(or cyan?)
what are far-red chlorophylls, why are they useful
extend the red limit of photosynthesis, useful in soil
what are anoxygenic chlorophototropic bacteria
don’t evolve oxygen as part of photosynthetic reactions
what is different about anoxygenic chlorophototropic bacteria compared to cyanobacteria
they don’t evolve oxygen as part of photosynthetic reactions
they have 1 type of RC
they are bacteriochlorophyll based which can absorb lower energy photons - not enough energy to split water
what complex generates ATP in mitochondria
complex v
what i the ATP yield for aerobic respiration in mitochondria (per mol glucose)
~ 30
e. coli is metabolically versatile?
it is a facultative anaerobe with 3 modes:
aerobic respiration
anaerobic respiration
fermentation
what is a change e. coli might go through in terms of respiration
aerobic outside the host to anaerobic in the lower intestine
what is the difference between aerobic respiration, anaerobic respiration and fermentation
aerobic has O2 as the terminal e acceptor
anaerobic has an alternative respiratory e acceptor
fermentation does not
what are the electron donors in e. coli (aerobic respiration)
Nuo (boot shape, like complex 1, only proton pump)
Ndh
Sdh
what is the electron transport chain combo with the highest potential to make ATP
Nuo –> Cyo –> ATP synthase
what are the terminal oxidases in e. coli (aerobic respiration)
Cyo (proton pump, 2H+)
Cyd
Why would Cyd be preferential over Cyo
it has a higher affinity for oxygen (can work in microoxic) conditions
it is more resistant to sulphide, hydrogen peroxide and nitric oxide
why would Ndh be preferential over Nuo
it is a simpler enzyme and quicker to synthesise than Nuo
it has quicker NADH turnover –> high metabolic flux –> increased growth rate
in high PMF
what do the terminal oxidases do in e. coli (aerobic respiration)
both Cyo and Cyd oxidise quinones to quinols
and reduce O2 producing H2O
which components in e. coli (aerobic respiration) are goof targets for antimicrobials and why
Ndh and Cyd
present in many pathogenic bacteria and important during infection but are not present in mitochondria
definition of anaerobic respiration
use of a membrane embedded e- transport chain to generate a pmf but with a terminal acceptor other than O2
what can be used as an e donor in e. coli (aerobic respiration)
NADH/NAD+
Succinate/fumarate (Sdh)
what are the inorganic terminal e acceptors in e. coli (anaerobic respiration)
NO3 - nitrate
NO2 - nitrite
these are the best anaerobic
what are the organic terminal e acceptors in e. coli (anaerobic respiration)
fumarate
trimethyl-N-oxide
dimethyl sulfoxide
what is the quinone species in e. coli (anaerobic respiration)
why is it different
MK/MKH2
menaquinol/menaquinone
it is more negative (further up the diagram) than UQ/UQH2 so it allows for the variety of e- acceptors to still be downhill especially fumarate
is there a krebs cycle in anaerobic conditions
yes but not as a cycle, as a oxidative branch and reductive branch
needed for biosynthesis
what is important about NADH in e. coli (anaerobic respiration)
it is produced by the branches of the krebs cycle and glycolysis(?)
but must be reduced back to NAD+ to restore redox balance if this cannot be done by an alternative e- acceptor, fermentation will occur
which e- donor complex is favoured in e. coli (anaerobic respiration)
Nuo because it has a higher H+/e- ratio so it can contribute to the pmf, increased ATP production
succinate/fumarate complex in e. coli (anaerobic respiration)
Frd fumarate reductase
what is industrial microbiology
large scale low value commercial products made by microorganisms usually by native metabolism
what is microbial biotechnology
engineering microbes to produce high-value non-native compounds (often a lesser quantity)
what makes good industrial microbe
high yields
rapid and reproducible production
large scale culture
simple and cheap growth media
metabolic flexibility/adaptability
non-toxic/pathogenic
genetically stable, possible to engineer
can be stocked/stored eg. frozen/spores
ideal if product is secreted into the media
how can you improve yield of a natural product
mutation + selection
metabolic engineering
- redirection of metabolism to a specific pathway
- engineer transport system eg. increase efflux
- increase cellular tolerance for product or substrate
- decrease feedback inhibition
- decouple growth and product formation
batch fermentations
the initial culture medium contains all the nutrients required for fermentation
when these run out the grown ceases and fermentation stops
continuous fermentations
continually supplying the culture with fresh medium
subsequent removal of the the same amount of culture, resulting in a stead state in the fermenter
fed-batch fermentations
nutrients in initial medium once consumed a feed is initiated to provide the culture with additional nutrients to allow for further growth
which complexes are involved in formate dependant nitrate reduction, anaerobic respiration in e. coli
formate dehydrogenase (Fdn) reduces MK
Nitrate reductase - Nar or Nap oxidises MKH2 back
Fdn
formative dehydrogenase used in anaerobic respiration in e. coli
oxidises formate to CO2 and 2H+
reduces MK
Nar
more energetically favourable nitrate reductase complex in anaerobic respiration in e. coli
reduces nitrite (NO3-) to nitrate (NO2-), oxidising MKH2
Nitrite + 2H+ –> Nitrate + H2O
active site in cytoplasm but still releases protons from menaquinone pool to periplasm
Nap
less energetically favourable nitrate reductase complex in anaerobic respiration in e. coli
reduces nitrite (NO3-) to nitrate (NO2-), oxidising MKH2
Nitrite + 2H+ –> Nitrate + H2O
active site in the periplasm so the protons liberated by the formate dehydrogenase are cancelled out
used when high pmf is not wanted
what are the benefits of a branched electron transport chain
lots of donors and acceptors, lots of versatility
examples of bacteria with branched electron transport chains
P. denitrificans - soil bac, flexibility, similar to mitochondria in aerobic conditions, can use H2O2 as an e acceptor
helicobacter pylori - microaerophilic = likes small amount of O2
campylobacter jejuni - highly branched, the most frequent cause of food-borne bacterial gastroenteritis worldwide, used methylated MK/MKH2 as well as normal
definition of microbial fermentation
use endogenous organic molecules as electron acceptors in the absence of oxygen and a respiratory electron
transport chain
this is usually pyruvate
why is fermentation needed (to keep the microbe alive)
glycolysis is an incomplete metabolic pathway
redox balance must be restored so NAD+ must be regenerated by oxidising NADH
what is the general yield of fermentation
1-3 mols ATP per mol glucose
fermentation in muscle tissue
pyruvate –> lactate, uses lactate dehydrogenase (Ldh)
same as homolactic in bacteria
homolactic fermentation
pyruvate –> lactate, uses lactate dehydrogenase (Ldh)
same as in muscle tissue
yield of homolactic fermentation
2 ATP/mol glucose
yield of heterolactic fermentation
1 ATP/mol glucose
yield of alcoholic fermentation
2 ATP/mol glucose
yield of bacterial alcoholic fermentation
1 ATP/mol glucose, uses different glycolytic pathway
yield of mixed acid fermentation
3 ATP/mol glucose in theory but actually ~ 2.3 due to different pathways + different flux
extra ATP from substrate level phosphorylation in the acetate producing pathway
yield of acetone-butanol-ethanol (ABE) fermentation
~ 3 ATP/mol glucose but different pathways and different flux
heterolactic fermentation
uses pentose phosphoketolase pathway which produces a 5C molecule
this is split into a 3C and 2C molecule
2C –> ethanol
3C –> pyruvate –> lactate via ldh
hetero refers to the mixture of products also including CO2
Yeast alcoholic fermentation
2x pyruvate –>2x acetaldehyde (losing 2x CO2) –> 2x ethanol (oxidising 2 NADH)
Bacterial alcoholic fermentation
comes from a different glycolytic pathway
produces by-products eg. esters which are unwanted in the beverage industry but bac. have a higher tolerance for ethanol (16%) so could be engineered to make bioethanol (e.g. Zymomonas mobilis in production of pulque - net yield 1 ATP and 2 NADH per glucose)
mixed acid fermentation
name comes from the mixture of organic acids produced
varied stoichiometry due to the different pathways and varied flux
Acetone-butanol-ethanol (ABE) fermentation
ABE ratio of 3:6:1 but can be engineered
very industrially relevant for generating butanol as a renewable biofuel
How does continuous culture differ to batch culture?
continuous culture is kept running by addition of new medium and removal of old medium - steady state
whereas in batch culture all the nutrients are provided in the initial medium and once this runs out fermentation stops
Explain what is meant by a fed-batch culture and why this is often the preferred growth setup in industrial fermentations.
fed-batch: once the initial nutrients are consumed a feed is set up.
easy to let run once set up? easy to keep it in the steady state?
Give some examples of products produced by microorganisms and the microbes that
produce them.
bioethanol - saccharomyces cerevisiae
alcoholic drinks - yeasts
penicillin - penicillium chrysogenum
What properties make a microorganism well suited to use in commercial applications.
makes the product of interest in high yield in reactor conditions (or engineerable)
quickly replicate in reactor conditions, no need for special media/vitamins
easy to store (eg. spores)
non-pathogenic and do not produce toxic by-products
stable genome and metabolism open to engineering
secrete product/easy to obtain
What is metabolic engineering? List some general ways that product yield can be enhanced using metabolic engineering approaches.
Metabolic Engineering is the modification of the metabolic pathway to redirect metabolism to produce specific products.
- select for mutations with no feedback inhibition
- increase tolerance to (toxic) product/substrate
- transport systems
- decouple growth and product formation
- increase energy by engineering the central metabolism
- overexpress cofactor
Explain how allosteric feedback inhibition can be overcome with anti-metabolites using LysC as an example.
antimetabolite - an analog of the metabolite - binds to allosteric feedback inhibition site on LysC blocking metabolism
organism must mutate to stop uptake of the antimetabolite (not useful) or mutate the enzyme so it doesn’t bind the antimetabolite
this also stops the metabolite product (lysine) binding - stopping feedback inhibition
Explain how lysine production in Corynebacterium glutamicum was improved by: (a) branchpoint engineering
DapA is the first enzyme specific to the lysine pathway out of the a pathway that can be used for multiple amino acids
point mutations can be introduced in the promoter which increases activity
Explain how lysine production in Corynebacterium glutamicum was improved by: (b) optimising cofactor supply
the increase in flux leads to an increase in NADP+ so redox balance in needed
NADH + NADP+ <–> NADPH + NAD+ is catalysed by PntAB
overexpression of PntAB increases production
Explain how lysine production in Corynebacterium glutamicum was improved by: (c) increasing product efflux.
lysine is toxic to the cell at high concentrations
overexpressing the transporter protein (LysE) increases production
Give a brief overview of the photosynthetic electron transport chain in oxygenic
chlorophototrophs. What are the two main outputs of this process?
photosystem II H2O as source of electrons –> O2
cytochrome b6f
photosystem I
ATP synthase
generates ATP and NADPH for carbon fixation
Draw a labelled sketch of a cyanobacterial cell, highlighting key features.
thylakoid membranes (ETC) around the outer membrane -
carboxysomes
Describe the cyanobacterial carbon concentrating mechanism.
carboxysomes
cytosolic bicarbonate (HCO3-) is taken up along with RuBP
inside the carboxysome is Rubisco and CA - carbonic anhydrase (HCO3- –> CO2)
this makes 3-phosphogylcerate with leaves the carboxysome
separates O2 from rubisco (and RuBP) stops photorespiration
how to extend the far limit of photosynthesis
chlorophyll d and/or f
formyl group at C3 or C2 position shifts absorption
l = 700-800nm
extends absorption into far red light region (lower energy)
What is the FaRLiP response? How does this benefit some species of cyanobacteria?
only produce lower energy pigments (chlorophyll d and f) when grown in environments rich in far red light where viable light (l = 400-700nm) is attenuated eg. soil
needs remodelling of photosystems
enlarge absorbance cross section into far red, competitive and niche specific advantage
Describe the role of heterocysts in filamentous cyanobacteria.
specific sites of nitrogen fixation - differentiate in nitrogen starvation
separates fixation to stop futile cycling
make microoxic environment by degrading photosystem II
thick wall to prevent O2 getting in
List the main ways that oxygenic and anoxygenic photosynthesis are different. Give one
example of an anoxygenic phototroph to demonstrate your answer.
8 phyla
only one photosystem - I or II
not enough energy to split water and produce O2
bacteriochlorophyll based
eg. proteobacteria
What are the core features of electron transport chians:
-Made of 5 complexes
-Electrons enter the chain from NADH at complex I (or succinate CII) to reduce quinones into quinols -> these are then oxidised by Complex II reducing soluble cytochrome C in the IMS which then acts as an electron donor for complex IV wherein oxygen is used to reduce water and generate a PMF (for ATP synthesis by ATP synthase)
What components of the ETC does E.coli lack?
Complex III and c oxidase (complex IV) -> so instead uses terminal oxidases Cyo and Cyd
What is the difference between the structure of menaquinone and ubiquinone:
Menaquinone has a naphthoquinone rather than a benzoquinone.
E.coli Metabolism During anaerobic respiration:
In anaerobic conditions pyruvate dehydrogenase is inhibited, pyruvate forms formate and acetyl-CoA by pyruvate formate lyase (PFL) -> the Acetyl-CoA is converted to acetate by generating ATP by substrate level phosphorylation and the formate acts as an electron donor to the anaerobic ETC via formate dehydrogenase.
What inhibits the action of pyruvate formate lyase?
Oxygen, therefore the formation of formate and Acetyl CoA by the enzyme only occurs in anaerobic conditions.
What are the key feature of the Paracoccus dentrificans (gram -ve)ETC?
It’s highly branched, under aerobic conditions its very similar to the mitochondrial ETC, however it has two additional terminal oxidases . It can use one carbon compounds as electron donors and Hydrogen Peroxide as an electron acceptor. It can also denitrify nitrates.
What are the key components of E.coli anaerobic metabolism?
Donor Complexes:
- NADH dehydrogenase
-Formate Dehydrogenase
-Succinate Dehydrogenase
Acceptor Complexes:
-Fumarate Reductase
-Nar and Nap Nitrate reductases
-Cytochrome oxidases bo3 or bd
Product of the Reductive Branch of E.coli anaerobic metabolism (krebs cycle):
Succinate -> in its synthesis fumarate acts as terminal electron pair acceptor by fumarate reductase in the ETC.
Product of the Oxidative Branch of E.coli anaerobic metabolism (krebs cycle):
Alpha-ketoglutarate
What are the benefits of a branched non-cyclic krebs cycle?
You can make many biosynthetic intermediates without needing oxygen; this allows for continued growth under a variety of conditions, by maximising PMF in favourable conditions, and biosynthesis in others.
Naturally, in what organism does Acetone-Butanol-Ethanol (ABE) fermentation take place?
A Gram +ve Clostridium Species.
What is unique about ABE fermentation?
It can generate extra ATP and re-assimilate the acetic and butyric acid products generating acetone and re-generating acetyl-CoA and butyryl-CoA, which can alternative be converted to ethanol and butanol to reform NAD+.
In mixed acid fermentation why do the proportions of the end products vary dependent on growth conditions?
To balance ATP production and Redox Balance (compared to other fermentation methods where product stoichiometry is fixed)
What are the products of mixed acid fermentation?
Mixture of organic acids:
What is the type of fermentation used in wine production? (and why is it strange)
Malolactic ‘Fermentation’ -> a form of fermentation by lactic acid bacteria e.g. O. oeni -> takes up malic acid and decarboxylases it forms lactic acid -> excreted and increases pH (deacidifies wine) -> Method: Antiport of malate (2-) and lactic acid (1-) by MleP generates a membrane potential and decarboxylation of malate by MleA consumes a proton in the cytoplasm contributing to the pH gradient -> ATP synthesis is therefore chemiosmotic
and no redox balancing!!
Why do cells not grow much when using fermentation?
Most carbon substrates are routed to form fermentation
products which are excreted as wastr products -> therefore little biomass is generated.
What determines the extent to which specific transport chains are associated with different generation PMF?
The orientation of the active site at respiratory complexes with respect to the periplasmic and cytoplasmic side of the membrane.
Outline Penicillin production:
Penicillin production is performed by the fed-batch fermentation of Penicillium chrysogenum -> carried out in bioreactors of up to 300,000litres. In the initial growth phase a small fermenter is inoculated with freeze-dried spores -> these are cultured and scaled with two successively larger fermenters. During the fermentation production phase, the fed-batch culture is maintained at high oxygen levels with C and N feeding (carefully monitored for optimal production) -> penicillin is excreted into medium, extracted and purified at the end of fermentation (120-200hours)
what is a phycobilisome
major LHC of cyanobacteria
large complex antenna system
on thylakoid membrane
