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Microbiology II > Topic 2 - Bacterial respiration, fermentation, growth, and metabolic engineering > Flashcards

Topic 2 - Bacterial respiration, fermentation, growth, and metabolic engineering Flashcards

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1
Q

what do Glycolysis and the Krebs cycle produce (and why?)

A

ATP (substrate-level phosphorylation) and reduced electron carriers for
the ETC, which is localised to the cytoplasmic (inner) membrane

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2
Q

what are the two different NADH dehydrogenases in e. coli and what are their differences?

A

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

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3
Q

why would the Ndh enzyme be favoured over Nuo

A

NADH oxidation by Nuo conserves more energy but the simpler Ndh enzyme has a higher turnover rate and is
favoured under aerobic conditions

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4
Q

why are Ndh and Cyd good targets for antimicrobials

A

they are are not present in humans

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5
Q

what is the ATP yield of aerobic respiration in E. coli under vigorous aeration

A

~20 ATP per mol of glucose

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6
Q

differences between Cyo and Cyd

A

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)

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7
Q

why would Cyd be favoured over Cyo

A

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

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8
Q

what are the only oxygenic photosynthetic prokaryotes called

A

cyanobacteria

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9
Q

where is the thylakoid in cyanobacteria

A

centric around the cell membrane and then surrounded by ribosomes

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10
Q

what are carboxysomes, what is in them and why?

A

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

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11
Q

what are the photosynthetic pigments in cyanobacteria

A

Main = Chlorophyll A
+ carotenoids and antioxidants to expand the spectrum
+ bilins pink/blue(or cyan?)

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12
Q

what are far-red chlorophylls, why are they useful

A

extend the red limit of photosynthesis, useful in soil

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13
Q

what are anoxygenic chlorophototropic bacteria

A

don’t evolve oxygen as part of photosynthetic reactions

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14
Q

what is different about anoxygenic chlorophototropic bacteria compared to cyanobacteria

A

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

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15
Q

what complex generates ATP in mitochondria

A

complex v

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16
Q

what i the ATP yield for aerobic respiration in mitochondria (per mol glucose)

A

~ 30

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17
Q

e. coli is metabolically versatile?

A

it is a facultative anaerobe with 3 modes:
aerobic respiration
anaerobic respiration
fermentation

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18
Q

what is a change e. coli might go through in terms of respiration

A

aerobic outside the host to anaerobic in the lower intestine

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19
Q

what is the difference between aerobic respiration, anaerobic respiration and fermentation

A

aerobic has O2 as the terminal e acceptor
anaerobic has an alternative respiratory e acceptor
fermentation does not

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20
Q

what are the electron donors in e. coli (aerobic respiration)

A

Nuo (boot shape, like complex 1, only proton pump)
Ndh
Sdh

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21
Q

what is the electron transport chain combo with the highest potential to make ATP

A

Nuo –> Cyo –> ATP synthase

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22
Q

what are the terminal oxidases in e. coli (aerobic respiration)

A

Cyo (proton pump, 2H+)
Cyd

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23
Q

Why would Cyd be preferential over Cyo

A

it has a higher affinity for oxygen (can work in microoxic) conditions
it is more resistant to sulphide, hydrogen peroxide and nitric oxide

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24
Q

why would Ndh be preferential over Nuo

A

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

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25
Q

what do the terminal oxidases do in e. coli (aerobic respiration)

A

both Cyo and Cyd oxidise quinones to quinols
and reduce O2 producing H2O

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26
Q

which components in e. coli (aerobic respiration) are goof targets for antimicrobials and why

A

Ndh and Cyd
present in many pathogenic bacteria and important during infection but are not present in mitochondria

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27
Q

definition of anaerobic respiration

A

use of a membrane embedded e- transport chain to generate a pmf but with a terminal acceptor other than O2

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28
Q

what can be used as an e donor in e. coli (aerobic respiration)

A

NADH/NAD+
Succinate/fumarate (Sdh)

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29
Q

what are the inorganic terminal e acceptors in e. coli (anaerobic respiration)

A

NO3 - nitrate
NO2 - nitrite
these are the best anaerobic

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30
Q

what are the organic terminal e acceptors in e. coli (anaerobic respiration)

A

fumarate
trimethyl-N-oxide
dimethyl sulfoxide

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31
Q

what is the quinone species in e. coli (anaerobic respiration)
why is it different

A

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

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32
Q

is there a krebs cycle in anaerobic conditions

A

yes but not as a cycle, as a oxidative branch and reductive branch
needed for biosynthesis

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33
Q

what is important about NADH in e. coli (anaerobic respiration)

A

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

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34
Q

which e- donor complex is favoured in e. coli (anaerobic respiration)

A

Nuo because it has a higher H+/e- ratio so it can contribute to the pmf, increased ATP production

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35
Q

succinate/fumarate complex in e. coli (anaerobic respiration)

A

Frd fumarate reductase

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36
Q

what is industrial microbiology

A

large scale low value commercial products made by microorganisms usually by native metabolism

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37
Q

what is microbial biotechnology

A

engineering microbes to produce high-value non-native compounds (often a lesser quantity)

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38
Q

what makes good industrial microbe

A

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

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39
Q

how can you improve yield of a natural product

A

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

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40
Q

batch fermentations

A

the initial culture medium contains all the nutrients required for fermentation
when these run out the grown ceases and fermentation stops

41
Q

continuous fermentations

A

continually supplying the culture with fresh medium
subsequent removal of the the same amount of culture, resulting in a stead state in the fermenter

42
Q

fed-batch fermentations

A

nutrients in initial medium once consumed a feed is initiated to provide the culture with additional nutrients to allow for further growth

43
Q

which complexes are involved in formate dependant nitrate reduction, anaerobic respiration in e. coli

A

formate dehydrogenase (Fdn) reduces MK
Nitrate reductase - Nar or Nap oxidises MKH2 back

44
Q

Fdn

A

formative dehydrogenase used in anaerobic respiration in e. coli
oxidises formate to CO2 and 2H+
reduces MK

45
Q

Nar

A

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

46
Q

Nap

A

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

47
Q

what are the benefits of a branched electron transport chain

A

lots of donors and acceptors, lots of versatility

48
Q

examples of bacteria with branched electron transport chains

A

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

49
Q

definition of microbial fermentation

A

use endogenous organic molecules as electron acceptors in the absence of oxygen and a respiratory electron
transport chain
this is usually pyruvate

50
Q

why is fermentation needed (to keep the microbe alive)

A

glycolysis is an incomplete metabolic pathway
redox balance must be restored so NAD+ must be regenerated by oxidising NADH

51
Q

what is the general yield of fermentation

A

1-3 mols ATP per mol glucose

52
Q

fermentation in muscle tissue

A

pyruvate –> lactate, uses lactate dehydrogenase (Ldh)
same as homolactic in bacteria

53
Q

homolactic fermentation

A

pyruvate –> lactate, uses lactate dehydrogenase (Ldh)
same as in muscle tissue

54
Q

yield of homolactic fermentation

A

2 ATP/mol glucose

55
Q

yield of heterolactic fermentation

A

1 ATP/mol glucose

56
Q

yield of alcoholic fermentation

A

2 ATP/mol glucose

57
Q

yield of bacterial alcoholic fermentation

A

1 ATP/mol glucose, uses different glycolytic pathway

58
Q

yield of mixed acid fermentation

A

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

59
Q

yield of acetone-butanol-ethanol (ABE) fermentation

A

~ 3 ATP/mol glucose but different pathways and different flux

60
Q

heterolactic fermentation

A

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

61
Q

Yeast alcoholic fermentation

A

2x pyruvate –>2x acetaldehyde (losing 2x CO2) –> 2x ethanol (oxidising 2 NADH)

62
Q

Bacterial alcoholic fermentation

A

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)

63
Q

mixed acid fermentation

A

name comes from the mixture of organic acids produced
varied stoichiometry due to the different pathways and varied flux

64
Q

Acetone-butanol-ethanol (ABE) fermentation

A

ABE ratio of 3:6:1 but can be engineered
very industrially relevant for generating butanol as a renewable biofuel

65
Q

How does continuous culture differ to batch culture?

A

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

66
Q

Explain what is meant by a fed-batch culture and why this is often the preferred growth setup in industrial fermentations.

A

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?

67
Q

Give some examples of products produced by microorganisms and the microbes that
produce them.

A

bioethanol - saccharomyces cerevisiae
alcoholic drinks - yeasts
penicillin - penicillium chrysogenum

68
Q

What properties make a microorganism well suited to use in commercial applications.

A

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

69
Q

What is metabolic engineering? List some general ways that product yield can be enhanced using metabolic engineering approaches.

A

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

70
Q

Explain how allosteric feedback inhibition can be overcome with anti-metabolites using LysC as an example.

A

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

71
Q

Explain how lysine production in Corynebacterium glutamicum was improved by: (a) branchpoint engineering

A

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

72
Q

Explain how lysine production in Corynebacterium glutamicum was improved by: (b) optimising cofactor supply

A

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

73
Q

Explain how lysine production in Corynebacterium glutamicum was improved by: (c) increasing product efflux.

A

lysine is toxic to the cell at high concentrations
overexpressing the transporter protein (LysE) increases production

74
Q

Give a brief overview of the photosynthetic electron transport chain in oxygenic
chlorophototrophs. What are the two main outputs of this process?

A

photosystem II H2O as source of electrons –> O2
cytochrome b6f
photosystem I
ATP synthase
generates ATP and NADPH for carbon fixation

75
Q

Draw a labelled sketch of a cyanobacterial cell, highlighting key features.

A

thylakoid membranes (ETC) around the outer membrane -
carboxysomes

76
Q

Describe the cyanobacterial carbon concentrating mechanism.

A

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

77
Q

how to extend the far limit of photosynthesis

A

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)

78
Q

What is the FaRLiP response? How does this benefit some species of cyanobacteria?

A

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

79
Q

Describe the role of heterocysts in filamentous cyanobacteria.

A

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

80
Q

List the main ways that oxygenic and anoxygenic photosynthesis are different. Give one
example of an anoxygenic phototroph to demonstrate your answer.

A

8 phyla
only one photosystem - I or II
not enough energy to split water and produce O2
bacteriochlorophyll based
eg. proteobacteria

80
Q

What are the core features of electron transport chians:

A

-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)

81
Q

What components of the ETC does E.coli lack?

A

Complex III and c oxidase (complex IV) -> so instead uses terminal oxidases Cyo and Cyd

82
Q

What is the difference between the structure of menaquinone and ubiquinone:

A

Menaquinone has a naphthoquinone rather than a benzoquinone.

83
Q

E.coli Metabolism During anaerobic respiration:

A

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.

84
Q

What inhibits the action of pyruvate formate lyase?

A

Oxygen, therefore the formation of formate and Acetyl CoA by the enzyme only occurs in anaerobic conditions.

85
Q

What are the key feature of the Paracoccus dentrificans (gram -ve)ETC?

A

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.

86
Q

What are the key components of E.coli anaerobic metabolism?

A

Donor Complexes:
- NADH dehydrogenase
-Formate Dehydrogenase
-Succinate Dehydrogenase

Acceptor Complexes:
-Fumarate Reductase
-Nar and Nap Nitrate reductases
-Cytochrome oxidases bo3 or bd

87
Q

Product of the Reductive Branch of E.coli anaerobic metabolism (krebs cycle):

A

Succinate -> in its synthesis fumarate acts as terminal electron pair acceptor by fumarate reductase in the ETC.

88
Q

Product of the Oxidative Branch of E.coli anaerobic metabolism (krebs cycle):

A

Alpha-ketoglutarate

89
Q

What are the benefits of a branched non-cyclic krebs cycle?

A

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.

90
Q

Naturally, in what organism does Acetone-Butanol-Ethanol (ABE) fermentation take place?

A

A Gram +ve Clostridium Species.

91
Q

What is unique about ABE fermentation?

A

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+.

92
Q

In mixed acid fermentation why do the proportions of the end products vary dependent on growth conditions?

A

To balance ATP production and Redox Balance (compared to other fermentation methods where product stoichiometry is fixed)

93
Q

What are the products of mixed acid fermentation?

A

Mixture of organic acids:

94
Q

What is the type of fermentation used in wine production? (and why is it strange)

A

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!!

95
Q

Why do cells not grow much when using fermentation?

A

Most carbon substrates are routed to form fermentation
products which are excreted as wastr products -> therefore little biomass is generated.

96
Q

What determines the extent to which specific transport chains are associated with different generation PMF?

A

The orientation of the active site at respiratory complexes with respect to the periplasmic and cytoplasmic side of the membrane.

97
Q

Outline Penicillin production:

A

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)

98
Q

what is a phycobilisome

A

major LHC of cyanobacteria
large complex antenna system
on thylakoid membrane

Microbiology II (8 decks)

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